**Acidified Foods: Food Safety Considerations for Food Processors**

Felix H. Barron and Angela M. Fraser

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55161

## **1. Introduction**

The food processing industry is one of the United States' largest manufacturing sectors, accounting for more than 10 percent of all manufacturing shipments. Concerns over food safety have increased as the industry has been hit by several high profile and large-scale food recalls. Thus, commercial food processors must be vigilant about ensuring the safety of their products. If inadequate or improper manufacturing, processing or packaging procedures are used in the production of low-acid or acidified canned foods serious health hazards, especially *Clostridium botulinum*, could result. To prevent this, processors must be in compliance with regulations established by the U.S. Food and Drug Administration (F.D.A., U.S. Department of Agricul‐ ture) and state agriculture and health departments across the United States (Barron, 2000).

## **2. Acidified foods**

The term "acidified foods" means low-acid foods to which acid(s) or acid food(s) are added. These products include, but are not limited to:


© 2013 Barron and Fraser; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**•** Tomato salsa made from tomatoes with a pH of 4.6 or below and low-acid ingredients, when the amount of low-acid ingredients is not a small amount and/or the resultant finished equilibrium pH differs significantly from that of the predominant acid or acid food; and

**•** Tomatoes and tomato products having a finished equilibrium pH less than 4.7

**•** Any food prepared under the continuous inspection of the meat and poultry inspection program of the Animal and Plant Health Inspection Service of the Department of Agricul‐ ture under the Federal Meat Inspection Act and the Poultry Products Inspection Act

Acidified Foods: Food Safety Considerations for Food Processors

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Because these foods are not recognized as acidified foods, commercial processors do NOT have to file and register their processing information for these products with the Food and Drug

In 1979, the Code of Federal Regulations (CFR) published the acidified regulations identified today as 21 CFR Part 114. Since then, new food processing technologies and methodologies have been developed and are frequently used in the industry. Furthermore, pathogens, such as *E. coli* 0157:H7 and *Salmonella* spp. have been shown to survive and grow in acidic envi‐ ronments. As a result of changing technologies and emerging pathogens, actions by federal agencies have motivated researchers to investigate new ways to eliminate pathogens, such as *E. coli* and *Salmonella* spp. The following are several research citations that provide a brief

In 1996, an outbreak of *E. coli* 0157:H7 was identified when an individual contracted hemolytic uremic syndrome after drinking apple juice packaged in sealed containers. The outbreak affected 45 individuals across the USA and Canada. The product was voluntarily recalled by

In 1999, an outbreak of *Salmonella* Muenchen serotype in the United States and Canada caused 298 cases of illness, which were attributed to unpasteurized orange juice. The out‐ break affected 17 states, primarily in the Midwest, as well as regions of Canada. The product was voluntarily recalled after unopened product tested positive for the causative

A study performed on the relative safety of pickled cucumbers from *Clostridium botulinum* infection, as a response to a 1976 study in which the organism was found in sealed containers previously believed to be safe. The study involved introducing *C. botulinum* spores into experimentally packed pickles artificially adjusted to a target pH and checking for growth of the organism. It was reported that any pH less acidic than 4.8 was insufficient to effectively kill *C. botulinum* spores, thus establishing a minimum safe pH for pickled cucumbers. (Ito et

**•** Foods that are NOT packaged in hermetically sealed containers

**•** Foods that are stored distributed and retailed under refrigeration

history of developments related to pathogens in acidified foods.

serotype (*Centers for Disease Control and Prevention,* 1999).

the manufacturing company (*Centers for Disease Control and Prevention,* 1996)

**•** Foods with water activity of 0.85 or below

**•** Food that are not thermally processed

Administration (FDA 2010b).

**3. Pathogens of concern**

al, 1996).

**•** Cold-pack pickles that are subjected to the action of acid-producing microorganisms but require the addition of acid or an acid food to achieve a pH of 4.6 or below.

All acidified foods must have a water activity (aw) greater than 0.85 and a finished equilibrium pH of 4.6 or below within the time designated in the scheduled process. These parameters must be maintained in all finished foods as outlined in 21 CFR 114.80(a). These foods may be called, or may purport to be, "pickles" or "pickled." However, some barriers exist in the preparation of acidified foods, including inadequate acid in the cover brine to overcome buffering capacity of the food, the presence of alkaline compounds from peeling or other processing aids, and the peels, waxing, piece size or oil in the product which can cause a barrier to penetration of the acid. These barriers may cause the failure to achieve the final equilibrium of a pH value of 4.6 and raise concerns about the growth of pathogens and production of toxins in the finished product.

After proper acidification, all acidified foods must then be heat processed to destroy the vegetative cells of pathogenic microorganisms or other microorganisms that cause spoilage and to inactivate enzymes that might affect color, flavor, or texture of the product. Acidified foods can be heat processed in a boiling water canner or by low-temperature pasteurization. The processing time, temperature, and procedure necessary to safely preserve acidified foods are determined by factors such as level of acidity (pH), size of food pieces (density) and percentage salt. An FDA recognized process authority must review the product and process and make the appropriate recommendations about time and temperature requirements. Processing temperatures higher than 185°F (85°C) could break down pectin and cause unnecessary softening of acidified foods (FDA 2010a).

All commercial establishments engaged in the manufacture of Acidified Foods and Low-Acid Canned Foods (LACF) offered for interstate commerce in the United States are required by 21CFR Parts 108, 113 and 114 to register their facility with form FDA 2541, "Food Canning Establishment Registration," and file scheduled processes for their products with forms FDA 2541a, "Food Process Filing for all Methods Except Low-Acid Aseptic," and FDA 2541c, "Process Filing for Low-Acid Aseptic Systems." The following items are not considered to be acidified foods or low-acid foods.


Because these foods are not recognized as acidified foods, commercial processors do NOT have to file and register their processing information for these products with the Food and Drug Administration (FDA 2010b).

## **3. Pathogens of concern**

**•** Tomato salsa made from tomatoes with a pH of 4.6 or below and low-acid ingredients, when the amount of low-acid ingredients is not a small amount and/or the resultant finished equilibrium pH differs significantly from that of the predominant acid or acid food; and **•** Cold-pack pickles that are subjected to the action of acid-producing microorganisms but

All acidified foods must have a water activity (aw) greater than 0.85 and a finished equilibrium pH of 4.6 or below within the time designated in the scheduled process. These parameters must be maintained in all finished foods as outlined in 21 CFR 114.80(a). These foods may be called, or may purport to be, "pickles" or "pickled." However, some barriers exist in the preparation of acidified foods, including inadequate acid in the cover brine to overcome buffering capacity of the food, the presence of alkaline compounds from peeling or other processing aids, and the peels, waxing, piece size or oil in the product which can cause a barrier to penetration of the acid. These barriers may cause the failure to achieve the final equilibrium of a pH value of 4.6 and raise concerns about the growth of pathogens and production of toxins

After proper acidification, all acidified foods must then be heat processed to destroy the vegetative cells of pathogenic microorganisms or other microorganisms that cause spoilage and to inactivate enzymes that might affect color, flavor, or texture of the product. Acidified foods can be heat processed in a boiling water canner or by low-temperature pasteurization. The processing time, temperature, and procedure necessary to safely preserve acidified foods are determined by factors such as level of acidity (pH), size of food pieces (density) and percentage salt. An FDA recognized process authority must review the product and process and make the appropriate recommendations about time and temperature requirements. Processing temperatures higher than 185°F (85°C) could break down pectin and cause

All commercial establishments engaged in the manufacture of Acidified Foods and Low-Acid Canned Foods (LACF) offered for interstate commerce in the United States are required by 21CFR Parts 108, 113 and 114 to register their facility with form FDA 2541, "Food Canning Establishment Registration," and file scheduled processes for their products with forms FDA 2541a, "Food Process Filing for all Methods Except Low-Acid Aseptic," and FDA 2541c, "Process Filing for Low-Acid Aseptic Systems." The following items are not considered to be

**•** Acid foods (including such foods as standardized and non-standardized food dressings and condiment sauces) that contain small amounts of low-acid food(s) and have a resultant finished equilibrium pH that does not significantly differ from that of the predominant acid

require the addition of acid or an acid food to achieve a pH of 4.6 or below.

in the finished product.

232 Food Industry

unnecessary softening of acidified foods (FDA 2010a).

**•** Acid foods (naturally acid foods have a pH of 4.6 or less)

**•** Standardized jams, jellies and preserves (21 CFR 150)

acidified foods or low-acid foods.

or acid food

**•** Alcoholic beverages **•** Carbonated beverages In 1979, the Code of Federal Regulations (CFR) published the acidified regulations identified today as 21 CFR Part 114. Since then, new food processing technologies and methodologies have been developed and are frequently used in the industry. Furthermore, pathogens, such as *E. coli* 0157:H7 and *Salmonella* spp. have been shown to survive and grow in acidic envi‐ ronments. As a result of changing technologies and emerging pathogens, actions by federal agencies have motivated researchers to investigate new ways to eliminate pathogens, such as *E. coli* and *Salmonella* spp. The following are several research citations that provide a brief history of developments related to pathogens in acidified foods.

In 1996, an outbreak of *E. coli* 0157:H7 was identified when an individual contracted hemolytic uremic syndrome after drinking apple juice packaged in sealed containers. The outbreak affected 45 individuals across the USA and Canada. The product was voluntarily recalled by the manufacturing company (*Centers for Disease Control and Prevention,* 1996)

In 1999, an outbreak of *Salmonella* Muenchen serotype in the United States and Canada caused 298 cases of illness, which were attributed to unpasteurized orange juice. The out‐ break affected 17 states, primarily in the Midwest, as well as regions of Canada. The product was voluntarily recalled after unopened product tested positive for the causative serotype (*Centers for Disease Control and Prevention,* 1999).

A study performed on the relative safety of pickled cucumbers from *Clostridium botulinum* infection, as a response to a 1976 study in which the organism was found in sealed containers previously believed to be safe. The study involved introducing *C. botulinum* spores into experimentally packed pickles artificially adjusted to a target pH and checking for growth of the organism. It was reported that any pH less acidic than 4.8 was insufficient to effectively kill *C. botulinum* spores, thus establishing a minimum safe pH for pickled cucumbers. (Ito et al, 1996).

A study investigating the effects of acetic acid on *E. coli* O157:H7 in apple juice and pick‐ le brine found that increasing the pH of the food product yielded an increased inhibitory effect on pathogen growth. The study also demonstrated that acetic acid, a key compo‐ nent in vinegar, had a significant effect on the aforementioned inhibition over other methods of manipulating pH. (Breidt et al, 2004).

**•** Containers for acidified foods should be such that a hermetic seal is obtained. Vacuum is a

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235

Probably the most comprehensive guide to assist food processors in determining what constitutes an acidified food is a document prepared by the FDA in 2010 titled "Guidance for the Food Industry: Acidified Foods." This guidance document provides nonbinding recom‐ mendations but nevertheless presents step by step guidelines to determine if a food can be classified as an acidified food. In this document standardized and non-standardized food dressings, such as mayonnaise, and condiment sauces, such as ketchup, are considered acid

Processors who are not sure if a particular food is classified as an acidified or not, can volun‐ tarily submit the respective FDA forms for a preliminary evaluation. The draft guidance reminds processors that jams, jellies and preserves are excluded from the 21CFR114 as long as these products meet the applicable standard of identity under 21CFR150; otherwise, the nonstandardized products are covered by 21CFR114 based on the pH of the fruit, the pH of the

Another important aspect to be considered by a food processor is the use of acid foods and small amounts of low amounts of low acid foods as ingredients to produce an acidified food. There are two basic criteria needed to exclude any food from being subject to 21 CFR Part 114. The first is that acid foods contain small amounts of low acid foods and the second is that acid foods have a resultant finished equilibrium pH that does not significantly differ from that of

Fermented foods with a water activity level above 0.85, such as cucumber pickles and green olives, are considered low acid foods subject to the action of acid producing microorganisms to reduce the pH of the food to 4.6 or below. As such, these products are subject to the requirements of 21CFR114. Processors repacking and reprocessing previously acidified foods

Common questions of food processors new to the food processing industry are precisely related to this matter of reprocessing or repacking a previously acidified food and to procedures to determine a finished equilibrium pH. The draft guidance reminds process‐ ors about the meaning of equilibrium pH. It is recommended to use a reference tempera‐

fully diffused throughout the food (especially solid particles) and any successive meas‐ urements produce the same results. Further recommendations about food preparation for pH measurements and indicated to follow 21CFR114.90 and to ensure the pH of an in process batch to be reduced and reach the 4.6 within 24 consecutive hours. The likeli‐ hood that spores of *C. botulinum* will germinate and grow increases with the length of

C, commonly used in laboratory measurements. Equilibrium means the acid is

good indicator of a hermetic seal and helps to keep the quality of the product.

**5. Acified food guidance**

foods, which have a natural pH of 4.6 or below.

the predominant acid or acid food.

are also subject to 21CFR114.

ture of 25o

final product and the water activity level of the finished product.

time it takes to reduce the equilibrium pH of a food to 4.6

A study investigating the thermal resistance of *E. coli* O157:H7 found evidence for the phe‐ nomenon known as cross-protection, or the ability of a bacterium to apply resistance to one negative condition against another. These authors reported that microorganisms grown in an acidic environment display increased resistance to killing via thermal methods, indicating an increased threat by these types of organisms against current food safety methods involving both heat and acid (Buchanan and Edelson, 1999).

Recently, a study found that the Breidt model could be used to measure five-log reduction times in a less conservative manner, allowing for a more encompassing approach to deter‐ mining safe preparation times for various foods. Acidified vegetable products with a pH above 3.3 must be pasteurized to assure the destruction of acid resistant pathogenic bacteria. The times and temperatures needed to assure a five log reduction by pasteurization have previ‐ ously been determined using a non-linear (Weibull) model. Recently, the Food and Drug Administration has required that linear models be used with online electronic process filing forms for acidified foods. A linear model was developed that is based on the existing safe processing data. The processing times and temperatures meet or exceed the established heat processing conditions needed to assure safety (Breidt et al, 2010).

## **4. Control measures for ensuring food safety of acidified foods**

Control measures for ensuring the safety of acidified foods are well documented in the scientific literature. A simple overview of appropriate measures includes:


**•** Containers for acidified foods should be such that a hermetic seal is obtained. Vacuum is a good indicator of a hermetic seal and helps to keep the quality of the product.

## **5. Acified food guidance**

A study investigating the effects of acetic acid on *E. coli* O157:H7 in apple juice and pick‐ le brine found that increasing the pH of the food product yielded an increased inhibitory effect on pathogen growth. The study also demonstrated that acetic acid, a key compo‐ nent in vinegar, had a significant effect on the aforementioned inhibition over other

A study investigating the thermal resistance of *E. coli* O157:H7 found evidence for the phe‐ nomenon known as cross-protection, or the ability of a bacterium to apply resistance to one negative condition against another. These authors reported that microorganisms grown in an acidic environment display increased resistance to killing via thermal methods, indicating an increased threat by these types of organisms against current food safety methods involving

Recently, a study found that the Breidt model could be used to measure five-log reduction times in a less conservative manner, allowing for a more encompassing approach to deter‐ mining safe preparation times for various foods. Acidified vegetable products with a pH above 3.3 must be pasteurized to assure the destruction of acid resistant pathogenic bacteria. The times and temperatures needed to assure a five log reduction by pasteurization have previ‐ ously been determined using a non-linear (Weibull) model. Recently, the Food and Drug Administration has required that linear models be used with online electronic process filing forms for acidified foods. A linear model was developed that is based on the existing safe processing data. The processing times and temperatures meet or exceed the established heat

methods of manipulating pH. (Breidt et al, 2004).

234 Food Industry

both heat and acid (Buchanan and Edelson, 1999).

to a pH of 4.2 or below.

acid before being filled into the final container.

authority and be monitored, controlled and documented.

processing conditions needed to assure safety (Breidt et al, 2010).

**4. Control measures for ensuring food safety of acidified foods**

scientific literature. A simple overview of appropriate measures includes:

Control measures for ensuring the safety of acidified foods are well documented in the

**•** Acidified foods must be properly acidified to a pH below 4.6, but most foods are acidified

**•** To assure quick and proper acidification, the food is normally cooked or heated with the

**•** A thermal process or heating step is required to kill all pathogens and any other nonpathogenic microorganisms that could grow during storage of the product. Thermal processing must be completed by hot-filling the product or by the boiling water bath process. The heating temperature and time must be validated by an FDA recognized process control

**•** The final equilibrium pH must be checked, controlled and documented after the product has completed the thermal processing step. A pH meter with two decimal places accuracy must be used to measure the pH if the final pH is 4.0 or above; other methods can be used such as pH paper or a pH meter with one decimal place, if the final pH is below 4.0.

Probably the most comprehensive guide to assist food processors in determining what constitutes an acidified food is a document prepared by the FDA in 2010 titled "Guidance for the Food Industry: Acidified Foods." This guidance document provides nonbinding recom‐ mendations but nevertheless presents step by step guidelines to determine if a food can be classified as an acidified food. In this document standardized and non-standardized food dressings, such as mayonnaise, and condiment sauces, such as ketchup, are considered acid foods, which have a natural pH of 4.6 or below.

Processors who are not sure if a particular food is classified as an acidified or not, can volun‐ tarily submit the respective FDA forms for a preliminary evaluation. The draft guidance reminds processors that jams, jellies and preserves are excluded from the 21CFR114 as long as these products meet the applicable standard of identity under 21CFR150; otherwise, the nonstandardized products are covered by 21CFR114 based on the pH of the fruit, the pH of the final product and the water activity level of the finished product.

Another important aspect to be considered by a food processor is the use of acid foods and small amounts of low amounts of low acid foods as ingredients to produce an acidified food.

There are two basic criteria needed to exclude any food from being subject to 21 CFR Part 114. The first is that acid foods contain small amounts of low acid foods and the second is that acid foods have a resultant finished equilibrium pH that does not significantly differ from that of the predominant acid or acid food.

Fermented foods with a water activity level above 0.85, such as cucumber pickles and green olives, are considered low acid foods subject to the action of acid producing microorganisms to reduce the pH of the food to 4.6 or below. As such, these products are subject to the requirements of 21CFR114. Processors repacking and reprocessing previously acidified foods are also subject to 21CFR114.

Common questions of food processors new to the food processing industry are precisely related to this matter of reprocessing or repacking a previously acidified food and to procedures to determine a finished equilibrium pH. The draft guidance reminds process‐ ors about the meaning of equilibrium pH. It is recommended to use a reference tempera‐ ture of 25o C, commonly used in laboratory measurements. Equilibrium means the acid is fully diffused throughout the food (especially solid particles) and any successive meas‐ urements produce the same results. Further recommendations about food preparation for pH measurements and indicated to follow 21CFR114.90 and to ensure the pH of an in process batch to be reduced and reach the 4.6 within 24 consecutive hours. The likeli‐ hood that spores of *C. botulinum* will germinate and grow increases with the length of time it takes to reduce the equilibrium pH of a food to 4.6

There are three very important terms embedded in the definition of acidified foods (21 CFR par 114): (1) small amount of low acid food(s), (2) predominant acid or acid food, and (3) pH that does not significantly differ. Regarding the small amount of low acid food(s), it has been recommended to be no more than 10% by weight in the finished product. This recommendation is based on FDA experience when evaluating filed processed. This recommendation has been identified by FDA as the 'small amount provision" which means that acid foods that contain small amounts of low acid food(s) AND have a resultant finished equilibrium pH that does not significantly differ from that of the predominant acid or acid food are excluded from complying with 21CFR114. Some examples under this provision may be products such as tomato puree with added spices, or a salad dressing where the predominant acid is the mixture of all acid ingredients, such as mayonnaise, lemon juice, vinegar and tomato paste, and the small amount of low acid foods are red peppers, onion and garlic.

of acid tolerant spoilage microorganisms such as *B. licheniformis* need to be destroyed, while at a pH range below 4.0, the vegetative cells of yeasts, molds and non spore forming bacteria

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237

The thermal destruction of spores and microorganisms can be expressed in terms of heat resistance parameters. The adequate combination of time and temperature (extent of thermal processing) to safely manufacture a commercial food product and is also resistant to spoilage is called thermal process lethality. The draft guidance document provides a table demonstrat‐ ing relationships between finished equilibrium pH of products and the thermal process lethality of acidified foods. For example, for a pH range between 3.3 and 3.5 the F value of 1 minute is recommended. F being the destruction time desired at reference temperature of 195 F and a Z value of 10 F. This is typically written as F 10/195 =1.0 minutes. The thermal process lethality is part of a scheduled process required by FDA to prevent the growth of microor‐ ganisms of public health significance in the thermally processed food. This process need to be

The draft guidance also includes final recommendations to address spoilage problems through quality control procedures such as systematically implementing written plans to investigate

A commercial processor engaged in the processing of acidified foods is also required by 21CFR108.25 to prepare and maintain a written recall plan. Guidelines for product re‐ calls are contained in 21CFR7. This plan will provide a current procedure for implemen‐

**•** a procedure for distributors to follow to recall products which may be injurious to health

**•** a procedure for identifying, collecting, warehousing and controlling products and a method

Recall is a voluntary action taken by manufacturers and distributors to remove food that is in violation of laws administered by the FDA and USDA. These agencies may request a recall, but cannot order one without a court order. Product recovery is only classified as a recall when

Product Identification. Each batch or production lot must be properly coded. This code will allow the product lot to be identified as to date, batch product personnel production records,

Records. Records are key to the recall plan and must be maintained for three years.

signs of spoilage and their causes, as well as corrective actions to solve the problem.

established by a competent process authority as defined in 21CFR114.3(e).

such as lactobacillus need to be destroyed.

**6. Recalls**

tation, including:

**•** notifying FDA of any recalls

the product is violative.

and ingredient records.

They include:

for determining the effectiveness of any recalls.

The acid ingredient, such as vinegar has a pH of 4.6 or below; the acid food such as tomatoes has a natural pH of 4.6 or below. These acid ingredients need to be at least 90% of the total weight of the finished product to be considered predominant.

**If the equilibrium pH of the predominant acid or acid food is: Then one should consider a shift in pH to be significant when:** >4.2 Any shift in pH is present 4.2 The shift in pH is >0.2 ≥ 3.8 and < 4.2 The shift in pH is >0.3

<3.8 The shift in pH is >0.4

Regarding the term pH that does not significantly differ from that of the predominantly acid or acid foods, FDA recommends the following criteria:

It is important to consider variability factors, such as the accuracy of the pH meter and variations in the finished equilibrium pH of the food itself. Also, as a reminder to processors, water, being an important ingredient in many acidified foods, it is a low acid food and if it is a predominant ingredient in the finished product, this product is considered a water-based acidified food. Apple juice, bended juices, reconstituted juices and vegetable juices are all considered to be water-based liquids. When the finished equilibrium pH of a water-based liquid that contains acid(s) or acid food(s) is 4.6 or below, the product is subject to 21CFR114, unless the liquid is a carbonated beverage.

The draft guidelines recommend the use of decision tables to determine if a given food, including fermented foods to which low acid foods are added fall under the coverage of 21CFR114. These tables are a step-by-step series of questions that lead to the most probable correct answer about a food being an acidified or not product; however, it is recommended to consider other factors related to the product and the manufacturing process to make the final decision. The guidelines indicate that most acidified foods would require a heat treatment step. This thermal process is to be developed based on the most resistant microorganism that must be controlled under the given pH conditions. For example for a pH range of 4.0 to 4.6 the spores of acid tolerant spoilage microorganisms such as *B. licheniformis* need to be destroyed, while at a pH range below 4.0, the vegetative cells of yeasts, molds and non spore forming bacteria such as lactobacillus need to be destroyed.

The thermal destruction of spores and microorganisms can be expressed in terms of heat resistance parameters. The adequate combination of time and temperature (extent of thermal processing) to safely manufacture a commercial food product and is also resistant to spoilage is called thermal process lethality. The draft guidance document provides a table demonstrat‐ ing relationships between finished equilibrium pH of products and the thermal process lethality of acidified foods. For example, for a pH range between 3.3 and 3.5 the F value of 1 minute is recommended. F being the destruction time desired at reference temperature of 195 F and a Z value of 10 F. This is typically written as F 10/195 =1.0 minutes. The thermal process lethality is part of a scheduled process required by FDA to prevent the growth of microor‐ ganisms of public health significance in the thermally processed food. This process need to be established by a competent process authority as defined in 21CFR114.3(e).

The draft guidance also includes final recommendations to address spoilage problems through quality control procedures such as systematically implementing written plans to investigate signs of spoilage and their causes, as well as corrective actions to solve the problem.

## **6. Recalls**

There are three very important terms embedded in the definition of acidified foods (21 CFR par 114): (1) small amount of low acid food(s), (2) predominant acid or acid food, and (3) pH that does not significantly differ. Regarding the small amount of low acid food(s), it has been recommended to be no more than 10% by weight in the finished product. This recommendation is based on FDA experience when evaluating filed processed. This recommendation has been identified by FDA as the 'small amount provision" which means that acid foods that contain small amounts of low acid food(s) AND have a resultant finished equilibrium pH that does not significantly differ from that of the predominant acid or acid food are excluded from complying with 21CFR114. Some examples under this provision may be products such as tomato puree with added spices, or a salad dressing where the predominant acid is the mixture of all acid ingredients, such as mayonnaise, lemon juice, vinegar and tomato paste, and the

The acid ingredient, such as vinegar has a pH of 4.6 or below; the acid food such as tomatoes has a natural pH of 4.6 or below. These acid ingredients need to be at least 90% of the total

Regarding the term pH that does not significantly differ from that of the predominantly acid

>4.2 Any shift in pH is present 4.2 The shift in pH is >0.2 ≥ 3.8 and < 4.2 The shift in pH is >0.3 <3.8 The shift in pH is >0.4

It is important to consider variability factors, such as the accuracy of the pH meter and variations in the finished equilibrium pH of the food itself. Also, as a reminder to processors, water, being an important ingredient in many acidified foods, it is a low acid food and if it is a predominant ingredient in the finished product, this product is considered a water-based acidified food. Apple juice, bended juices, reconstituted juices and vegetable juices are all considered to be water-based liquids. When the finished equilibrium pH of a water-based liquid that contains acid(s) or acid food(s) is 4.6 or below, the product is subject to 21CFR114,

The draft guidelines recommend the use of decision tables to determine if a given food, including fermented foods to which low acid foods are added fall under the coverage of 21CFR114. These tables are a step-by-step series of questions that lead to the most probable correct answer about a food being an acidified or not product; however, it is recommended to consider other factors related to the product and the manufacturing process to make the final decision. The guidelines indicate that most acidified foods would require a heat treatment step. This thermal process is to be developed based on the most resistant microorganism that must be controlled under the given pH conditions. For example for a pH range of 4.0 to 4.6 the spores

**Then one should consider a shift in pH to be significant when:**

small amount of low acid foods are red peppers, onion and garlic.

weight of the finished product to be considered predominant.

or acid foods, FDA recommends the following criteria:

**If the equilibrium pH of the predominant acid or acid food is:**

236 Food Industry

unless the liquid is a carbonated beverage.

A commercial processor engaged in the processing of acidified foods is also required by 21CFR108.25 to prepare and maintain a written recall plan. Guidelines for product re‐ calls are contained in 21CFR7. This plan will provide a current procedure for implemen‐ tation, including:


Recall is a voluntary action taken by manufacturers and distributors to remove food that is in violation of laws administered by the FDA and USDA. These agencies may request a recall, but cannot order one without a court order. Product recovery is only classified as a recall when the product is violative.

Product Identification. Each batch or production lot must be properly coded. This code will allow the product lot to be identified as to date, batch product personnel production records, and ingredient records.

Records. Records are key to the recall plan and must be maintained for three years. They include:

**•** Records of examination of raw materials, packaging materials, and finished product along with any supplier guarantees or certifications.

[4] Buchanan, R. L., and S. G. Edelson. 1999. pH-dependent Stationary-phase Acid Resistance Response of Enterohemorrhagic *Escherichia Coli* in the Presence of Various

Acidified Foods: Food Safety Considerations for Food Processors

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239

[5] *Centers for Disease Control and Prevention*. Outbreak of *Salmonella* Serotype Muenchen Infections Associated with Unpasteurized Orange Juice -- United States and Canada, June 1999. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm4827a2.htm. Accessed

[6] *Centers for Disease Control and Prevention*. 8 Nov. 1996. Outbreak of *Escherichia coli* O157:H7 Infections Associated with Drinking Unpasteurized Commercial Apple Juice -- British Columbia, California, Colorado, and Washington, October 1996. http:// www.cdc.gov/mmwr/preview/mmwrhtml/00044358.htm. Accessed October 7, 2012

[7] Food and Drug Administration (FDA), 2010a. Acidified and Low-Acid Canned Foods (LCAF). http://www.fda.gov/Food/FoodSafety/Product-SpecificInformation/Acidi‐

[8] Food and Drug Administration (FDA), 2010b. Guidance for the Food Industry: Acidified Foods. http://www.fda.gov/Food/Guidance compliance regulatory informa‐ tion/guidance documents/acidified and low acid foods/ucm222618.htm. Accessed

[9] Ito, K. A., J. K. Chen, P. A. Lerke, M. L. Seeger, and J. A. Unverferth. 1976. Effect of Acid and Salt Concentration in Fresh-pack Pickles on Growth of *Clostridium botulinum*

fiedLow-AcidCannedFoods/default.htm. Accessed October 11, 2012.

Spores. *Applied Environmental Microbiology.* 32(1):121-24.

Acidulants." *Journal of Food Protection.* 62(3):211-18.

October 7, 2012.

September 10, 2012.


Notification. Persons to be notified in the event of a recall include FDA and USDA, key company personnel, and distributors. The notification should include the product, container size, and code of affected lots. The extent of the hazard and the level of the recall will be as determined by FDA and USDA. Based on this determination, FDA will approve the recall strategy. The notification will include instructions for consumers and distributors for product recovery and information feedback. The contact person should be listed on all notification forms.

Product Recovery. Plans for recovery include procedures for segregation of affected lots, storage, warehousing, and control. Procedures in place shall allow determination of the effectiveness of the recall. The recall is concluded when FDA and USDA determine that recovery is adequate and there is no longer any threat to the public.

## **Author details**

Felix H. Barron and Angela M. Fraser

Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC, USA

## **References**


[4] Buchanan, R. L., and S. G. Edelson. 1999. pH-dependent Stationary-phase Acid Resistance Response of Enterohemorrhagic *Escherichia Coli* in the Presence of Various Acidulants." *Journal of Food Protection.* 62(3):211-18.

**•** Records of examination of raw materials, packaging materials, and finished product along

**•** Processing and production records showing adherence to scheduled processes, including

**•** A log of all departures from scheduled processes, actions taken to rectify them, and

**•** Records of initial distribution of the finished product adequate to facilitate separation of

Notification. Persons to be notified in the event of a recall include FDA and USDA, key company personnel, and distributors. The notification should include the product, container size, and code of affected lots. The extent of the hazard and the level of the recall will be as determined by FDA and USDA. Based on this determination, FDA will approve the recall strategy. The notification will include instructions for consumers and distributors for product recovery and information feedback. The contact person should be listed on all notification

Product Recovery. Plans for recovery include procedures for segregation of affected lots, storage, warehousing, and control. Procedures in place shall allow determination of the effectiveness of the recall. The recall is concluded when FDA and USDA determine that

Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC, USA

[1] Barron, F. H. 2000. Acid, Acidified and Low-acid Foods Canning Guidelines for Food Processors. Bulletin EC 705, Food Nutrition and Packaging Science Department,

[2] Breidt F., Sandeep, K.P., and Arrit D.M., 2010. Use of Linear Models for Thermal

[3] Breidt, F., Jr., J. S. Hayes, and R. F. McFeeters. 2004. Independent Effects of Acetic Acid and PH on Survival of Escherichia Coli in Simulated Acidified Pickle Products." *Journal*

Processing of Acidified Foods. *Food Protections Trends*, 30(5), 268-272.

food lots which may have become contaminated or otherwise unfit for use.

with any supplier guarantees or certifications.

forms.

238 Food Industry

**Author details**

**References**

Felix H. Barron and Angela M. Fraser

Clemson University, Clemson, SC, USA.

*of Food Protection* 67(1):12-18.

records of pH measurement and other critical factors.

disposition records of the portion of product involved.

recovery is adequate and there is no longer any threat to the public.


**Chapter 12**

**Microbiological Contamination of Homemade Food**

The consumption of healthy food is a consumer's right and the duty of the manufacturing industry. Health authorities are duty bound to prepare and enforce laws to protect the popu‐ lation's health. The supply of food free from health risks to the population is actually a chal‐ lenge. In fact, contaminated food may cause serious infections and jeopardize the health of the

Owing to their frequency, food-caused infections are a very grave issue to public health. They may cause hazards ranging from a simple intestine discomfort to cases that are more serious, such as neurological disorders and death, because of the high number of microorganisms in‐

Fresh or processed animal-derived food may harbor several pathogenic microorganisms that cause physiological disorders in people who consume them. When food eventually contami‐ nated by disease-causing microorganisms is consumed, pathogens or their metabolites invade the host's fluids or tissues and trigger serious types of diseases, such as tuberculosis. They are conveyed by non pasteurized milk or by cheese contaminated by bacterial populations of *Mycobacterium bovis* and *M*. *tubercolosis* or by *Brucella abortus*, gram negative bacteria, intracel‐

Bacteria, fungi, protozoa and viruses are the main microorganism groups that cause food dis‐ orders. Due to their diversity and pathogenesis, bacteria are by far the most important micro‐ bial group commonly associated with food-transmitted diseases. High rated agents in food

and reproduction in any medium, provided the original work is properly cited.

© 2013 Baroni et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

lular pathogen that cause undulant fever and arthritis in human beings.

Suzymeire Baroni, Izabel Aparecida Soares,

Carmem Lucia de Mello Sartori Cardoso da Rocha

Additional information is available at the end of the chapter

Rodrigo Patera Barcelos,

http://dx.doi.org/10.5772/53170

volved in a simple epidemic event.

**1. Introduction**

population.

Alexandre Carvalho de Moura, Fabiana Gisele da Silva Pinto and

## **Microbiological Contamination of Homemade Food**

Suzymeire Baroni, Izabel Aparecida Soares, Rodrigo Patera Barcelos, Alexandre Carvalho de Moura, Fabiana Gisele da Silva Pinto and Carmem Lucia de Mello Sartori Cardoso da Rocha

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53170

## **1. Introduction**

The consumption of healthy food is a consumer's right and the duty of the manufacturing industry. Health authorities are duty bound to prepare and enforce laws to protect the popu‐ lation's health. The supply of food free from health risks to the population is actually a chal‐ lenge. In fact, contaminated food may cause serious infections and jeopardize the health of the population.

Owing to their frequency, food-caused infections are a very grave issue to public health. They may cause hazards ranging from a simple intestine discomfort to cases that are more serious, such as neurological disorders and death, because of the high number of microorganisms in‐ volved in a simple epidemic event.

Fresh or processed animal-derived food may harbor several pathogenic microorganisms that cause physiological disorders in people who consume them. When food eventually contami‐ nated by disease-causing microorganisms is consumed, pathogens or their metabolites invade the host's fluids or tissues and trigger serious types of diseases, such as tuberculosis. They are conveyed by non pasteurized milk or by cheese contaminated by bacterial populations of *Mycobacterium bovis* and *M*. *tubercolosis* or by *Brucella abortus*, gram negative bacteria, intracel‐ lular pathogen that cause undulant fever and arthritis in human beings.

Bacteria, fungi, protozoa and viruses are the main microorganism groups that cause food dis‐ orders. Due to their diversity and pathogenesis, bacteria are by far the most important micro‐ bial group commonly associated with food-transmitted diseases. High rated agents in food

infections are *Salmonella* sp., *Campylobacter* sp and *Listeria monocytogenes* due to their impor‐ tance in eventual sequelae. The microbiological health risks in fowl consumption and its raw products include contamination by the above food pathogens.

teristics that go against health norms and legislation [3]. The quality of milk and that of its products is a highly relevant factor for positive industrialization success since both the dairy and the consumer are interested in the outcome. In some case, however, a significant increase in the price of milk ensues. Milk is a product that should come from healthy herds, with good meals and managements, and from farms with proper technical installations that guarantee

Microbiological Contamination of Homemade Food

http://dx.doi.org/10.5772/53170

243

Since the number of milk contaminants increases at a slow rate from the moment of their introduction, the importance of adequate conservation of recently obtained milk should be underpinned as a basic practice for the maintenance of its quality. Milk should be submitted at low temperatures immediately after the milking process, with the consequent avoidance of

As a milk-derived product, cheese is frequently a food-originating pathogen vector. This is especially true for handmade fresh cheese manufactured from raw milk, lacking any matura‐ tion process. The product's microbial contamination is relevant for the industry because of financial liabilities, and for public health because of the risks in food-transmitted diseases.

Several studies [6] have shown that a product's quality and durability largely depend on the prime matter used in manufacturing. It is practically impossible to improve the qualities of a derived product, such as cheese, with a high number of microorganisms

Fresh Minas cheese (traditionally manufactured in the state of Minas Gerais, Brazil, whence its name) is defined by the Brazilian Ministry of Health (Decree 146) as fresh cheese obtained by enzyme coagulation of milk with curds and other appropriate coagulant enzymes, supple‐ mented or not by the activity of specific lactic bacteria. According to the Technical Rules for the Identification and Quality of Milk Products [7], fresh Minas cheese may be classified as cheese with low moisture or semi-hard cheese with moisture ranging between 36 and 45.9%; cheese with high moisture or moderate mass cheese with 46 to 54.9% moisture; and very high

The processing of fresh Minas cheese comprises the following stages: milk pasteurization, coagulation, cutting, draining, milling, salting, packing and cooling [8]. Since the manufac‐ turing of this type of cheese is highly simple, many small, medium-sized and large dairies are interested in its fabrication. In fact, it is the most common type of cheese found in fairs, bars and grocers. The cheese is normally placed in a common non-vacuum plastic bag and closed

According to the Brazilian Association of Cheese Industry (ABIQ), Brazil produces 400,000 tons of cheese per year, of which 240,000 tons are produced under federal, state and municipal

moisture cheese or soft mass cheese, with not less than 55% moisture.

inspection. Most production (95%) is consumed by common people [10].

conservation during transport up to the dairy factory [4].

the proliferation of unwanted microorganisms [5].

present in raw milk.

by a metal seal [9].

**3. Fresh Minas cheese**

Besides being one of the principal causes of food-derived diseases since its attack generally involves a great number of people, the genus *Salmonella* is associated with economic liabilities, commercial damage and decrease in production due to its frequency and extension. These facts occur because of the great number of food products that may be contaminated by this bacte‐ rium, namely, food with high humidity, protein and carbohydrate rates, such as beef, pork, chicken, eggs, milk and their derived products, highly liable to deteriorate. The contamination process by pathogenic bacteria in humans may be caused by poor hygiene conditions during processing involving sick people and animals or involving feces from infected agents. Bacteriacontaminated food may also be hazardous to public health due to the excessive growth in bacteria populations at food surface or within the food. These bacteria may come from the environment and cause toxins that develop into serious health problems on intake.

Hand-manipulated meat, sausages, salamis and cheese are among the most consumed prod‐ ucts worldwide. They are also liable to high microbiological contamination due to their man‐ ufacturing process.

The World Health Organization and the Food and Agriculture Organization of the United Nations have published reports and studies developed in several regions of the planet high‐ lighting the pathogen risks to populations and suggested the protection of food consumers through special industrial, operational, commercial and residence care. The need for great attention in food safety is a self-evident topic. In fact, improvements in food processing meth‐ ods and conscience-awareness with regard to food safety by all involved in the food production chain will surely reduce the incidence of food-originated diseases.

## **2. Microbiological contaminants of milk and homemade fresh cheese**

Milk is one of the most complete food featuring high levels of protein and mineral salts. How‐ ever, due to the availability of nutrients and almost neutral pH, milk is highly perishable. It is highly liable to microbial growth and requires thermal treatment for its conservation [1]. Pas‐ teurization prolongs milk conservation time, conserves its natural characteristics and pre‐ serves it safe for human consumption. High temperatures are involved so that the product's pathogenic microbiota are eliminated with no changes in its physical and chemical constitu‐ tion. However, people in rural regions still drink milk in natura and use it thus as prime matter for the manufacture of derived products.

The hygienic obtaining of milk is the first critical factor within the manufacturing process of cheese and other products. In fact, the animal, equipments and environment at milking may be an important contamination source by microorganisms [2]. Faults during milking and processing coupled to inadequate conservation temperatures at the selling outlets are factors that contribute towards the commercialization of milk products with microbiological charac‐ teristics that go against health norms and legislation [3]. The quality of milk and that of its products is a highly relevant factor for positive industrialization success since both the dairy and the consumer are interested in the outcome. In some case, however, a significant increase in the price of milk ensues. Milk is a product that should come from healthy herds, with good meals and managements, and from farms with proper technical installations that guarantee conservation during transport up to the dairy factory [4].

Since the number of milk contaminants increases at a slow rate from the moment of their introduction, the importance of adequate conservation of recently obtained milk should be underpinned as a basic practice for the maintenance of its quality. Milk should be submitted at low temperatures immediately after the milking process, with the consequent avoidance of the proliferation of unwanted microorganisms [5].

As a milk-derived product, cheese is frequently a food-originating pathogen vector. This is especially true for handmade fresh cheese manufactured from raw milk, lacking any matura‐ tion process. The product's microbial contamination is relevant for the industry because of financial liabilities, and for public health because of the risks in food-transmitted diseases.

Several studies [6] have shown that a product's quality and durability largely depend on the prime matter used in manufacturing. It is practically impossible to improve the qualities of a derived product, such as cheese, with a high number of microorganisms present in raw milk.

## **3. Fresh Minas cheese**

infections are *Salmonella* sp., *Campylobacter* sp and *Listeria monocytogenes* due to their impor‐ tance in eventual sequelae. The microbiological health risks in fowl consumption and its raw

Besides being one of the principal causes of food-derived diseases since its attack generally involves a great number of people, the genus *Salmonella* is associated with economic liabilities, commercial damage and decrease in production due to its frequency and extension. These facts occur because of the great number of food products that may be contaminated by this bacte‐ rium, namely, food with high humidity, protein and carbohydrate rates, such as beef, pork, chicken, eggs, milk and their derived products, highly liable to deteriorate. The contamination process by pathogenic bacteria in humans may be caused by poor hygiene conditions during processing involving sick people and animals or involving feces from infected agents. Bacteriacontaminated food may also be hazardous to public health due to the excessive growth in bacteria populations at food surface or within the food. These bacteria may come from the

environment and cause toxins that develop into serious health problems on intake.

chain will surely reduce the incidence of food-originated diseases.

for the manufacture of derived products.

Hand-manipulated meat, sausages, salamis and cheese are among the most consumed prod‐ ucts worldwide. They are also liable to high microbiological contamination due to their man‐

The World Health Organization and the Food and Agriculture Organization of the United Nations have published reports and studies developed in several regions of the planet high‐ lighting the pathogen risks to populations and suggested the protection of food consumers through special industrial, operational, commercial and residence care. The need for great attention in food safety is a self-evident topic. In fact, improvements in food processing meth‐ ods and conscience-awareness with regard to food safety by all involved in the food production

**2. Microbiological contaminants of milk and homemade fresh cheese**

Milk is one of the most complete food featuring high levels of protein and mineral salts. How‐ ever, due to the availability of nutrients and almost neutral pH, milk is highly perishable. It is highly liable to microbial growth and requires thermal treatment for its conservation [1]. Pas‐ teurization prolongs milk conservation time, conserves its natural characteristics and pre‐ serves it safe for human consumption. High temperatures are involved so that the product's pathogenic microbiota are eliminated with no changes in its physical and chemical constitu‐ tion. However, people in rural regions still drink milk in natura and use it thus as prime matter

The hygienic obtaining of milk is the first critical factor within the manufacturing process of cheese and other products. In fact, the animal, equipments and environment at milking may be an important contamination source by microorganisms [2]. Faults during milking and processing coupled to inadequate conservation temperatures at the selling outlets are factors that contribute towards the commercialization of milk products with microbiological charac‐

products include contamination by the above food pathogens.

ufacturing process.

242 Food Industry

Fresh Minas cheese (traditionally manufactured in the state of Minas Gerais, Brazil, whence its name) is defined by the Brazilian Ministry of Health (Decree 146) as fresh cheese obtained by enzyme coagulation of milk with curds and other appropriate coagulant enzymes, supple‐ mented or not by the activity of specific lactic bacteria. According to the Technical Rules for the Identification and Quality of Milk Products [7], fresh Minas cheese may be classified as cheese with low moisture or semi-hard cheese with moisture ranging between 36 and 45.9%; cheese with high moisture or moderate mass cheese with 46 to 54.9% moisture; and very high moisture cheese or soft mass cheese, with not less than 55% moisture.

The processing of fresh Minas cheese comprises the following stages: milk pasteurization, coagulation, cutting, draining, milling, salting, packing and cooling [8]. Since the manufac‐ turing of this type of cheese is highly simple, many small, medium-sized and large dairies are interested in its fabrication. In fact, it is the most common type of cheese found in fairs, bars and grocers. The cheese is normally placed in a common non-vacuum plastic bag and closed by a metal seal [9].

According to the Brazilian Association of Cheese Industry (ABIQ), Brazil produces 400,000 tons of cheese per year, of which 240,000 tons are produced under federal, state and municipal inspection. Most production (95%) is consumed by common people [10].

The intake of fresh cheese may be risky for the consumer's health. However, Decree 861/1984 basically prohibits the sale of fresh cheese manufactured from the raw milk of cows, goats or sheep, pure or mixed. Milk should undergo pasteurization or other equivalent thermal treat‐ ment. Current legislation was published after several registers of human brucellosis caused by fresh cheese. In defiance of the law, the homemade manufacture of cheese in certain regions of Brazil is not done with pasteurized milk. Consequently, the consumption of homemade cheese brings to the fore old dangers such as brucellosis (Maltese fever) and other infectious diseases.

Problems in the manufacture of cheese in Brazil are related to precarious conditions of milk, bad conditions during the manhandling of cheese and the lack or deficiency of refrigeration throughout the production chain. These factors worsen the situation and establish contami‐ nation conditions which favor the development of microorganisms at several places [18].

Microbiological Contamination of Homemade Food

http://dx.doi.org/10.5772/53170

245

Whereas some microorganisms contribute beneficently towards the processing, safety and quality of certain food products, other organisms are involved in processes with unwanted effects in food and for the consumers' health. There are two categories of food-transmitted microbial diseases: food intoxication and infection by food. In food intoxication, the person ingests toxins that are pre-formed by microorganisms in the food. The toxin causes damage to the organism. Examples comprise botulinum toxin that binds itself to the nerve terminals at the muscle level and impedes the release of acetylcholine neurotransmitter, and staphylococ‐ cus toxin that acts on the brain's vomiting-center [19]. Infection by food occurs when the pathogen, such as by *Salmonella typhy* and other serotypes, is ingested and multiplies itself,

The sale of animal-derived food in fair stalls without any refrigeration and without any pro‐ tection against dust and insects may alter their quality. In the case of cheese, it is sold in portions or slices and thus the external incorporation of biological or non-biological foreign matter is dangerous due to faults in the handling of the product during commercialization, poor hygiene of the stalls and utensils used, and crossed contamination between exposed products [21].

Food microbial contamination is unwanted and dangerous within food microbiology. This aspect should be faced with great strictness. The acknowledgement of possible hygiene defi‐ ciency implying in food contamination brings to the fore microorganism groups, comprising indicators, and pathogenic microorganisms that find an excellent environment in food for their development and even for the release of toxic substances [22]. Total and thermotolerant coli‐ forms, such as *Staphylococcus aureus,* fungi, yeasts and even *Salmonella* spp., should be high‐ lighted among the microorganisms whose presence and numbers indicate the quality of the

The above mentioned microorganisms, causes of several types of pathogenesis, are transmitted to humans because of lack of hygiene, bad habits of handlers, inefficient production processes, maintenance or re-heating of food at inadequate temperatures and also by non-adequate con‐

Most microorganisms, whose pathogenicity in humans depends on their variegated presence in food, are relatively sensitive to high temperatures. In fact, they are destroyed by the adequate

The Brazilian Agency for Health Vigilance (ANVISA) established, by Decree RDC 12 of the 2nd January 2001[24], the microbiological Standards for several types of food, descri‐

So that food-caused disease cases and events could be characterized, the populations should be informed on the symptoms of each, such as mild diarrheas and vomiting since these are considered as a "passing illness" and not necessarily associated with food consumption [25].

cooking of eventually contaminated food or by pasteurization processes.

causing diseases in the intestine tract and often in other organs [20].

ditions in industries where the food is produced [23].

product.

bed in Table 1.

In spite of the legal prohibition against the commercialization of fresh and tender cheese man‐ ufactured from raw milk, the sale of homemade fresh Minas cheese occurs openly and every‐ where in Brazil [11]. This is partially due to a greater yield, simpler processing and lack of product's maturation in the fabrication of this type of cheese, with low costs for the consumer and a fast return of expenditure to the manufacturer [12].

Food protection authorities classify microbial biological contamination as a main danger to public health. Who has constantly raised its voice on the need to restrict food contamination by health-impairing biological agents. Although microbial quality of food is of paramount importance, registration at the Federal Inspection Service does not guarantee lack of pathogens in food [13].

Food-derived diseases may be caused by several microorganism groups that include bacteria, fungi, yeasts, protozoa and viruses. Due to their diversity and pathogenesis, bacteria are by far the most important microbial group and commonly associated with food-transmitting dis‐ eases

Bacteria are microorganisms largely spread throughout the natural world and may be found in every type of environment [14]. They cause diseases in humans, animals and plants and deteriorate food and other materials. On the other hand, they may be useful too when they compose the human being's normal microbiota and are used in the production of food as symbiotic in agriculture and medicine.

In spite of certain unreliable Brazilian statistics, it is believed that food-derived diseases in Brazil are high [15]. In fact, several studies estimate that 12% of hospitalization cases in Brazil occur because of infectious intestinal diseases [16].

Occurrences of food-derived diseases are normally associated with certain risk factors, or rather, procedures that benefit toxin infections. The following may be highlighted: faults in food refrigeration; conservation of warm food at room temperature; food prepared many hours earlier for later consumption with inadequate conditioning during the interval; faults in the cooking process; handling of food by people with inadequate personal hygiene practices, or with lesions or with contaminating diseases; usage of contaminated prime matter; faults in the hygiene of utensils and other equipments in food preparation; favorable environmental con‐ ditions for the growth of etiological agents; food obtained from unreliable sources; inadequate storage; use of utensils which release toxic residues; intentional or accidental addition of toxic chemicals to the food; usage of water with uncontrolled drinkability features; water contam‐ ination from damages in the supply system [17].

Problems in the manufacture of cheese in Brazil are related to precarious conditions of milk, bad conditions during the manhandling of cheese and the lack or deficiency of refrigeration throughout the production chain. These factors worsen the situation and establish contami‐ nation conditions which favor the development of microorganisms at several places [18].

The intake of fresh cheese may be risky for the consumer's health. However, Decree 861/1984 basically prohibits the sale of fresh cheese manufactured from the raw milk of cows, goats or sheep, pure or mixed. Milk should undergo pasteurization or other equivalent thermal treat‐ ment. Current legislation was published after several registers of human brucellosis caused by fresh cheese. In defiance of the law, the homemade manufacture of cheese in certain regions of Brazil is not done with pasteurized milk. Consequently, the consumption of homemade cheese brings to the fore old dangers such as brucellosis (Maltese fever) and other infectious

In spite of the legal prohibition against the commercialization of fresh and tender cheese man‐ ufactured from raw milk, the sale of homemade fresh Minas cheese occurs openly and every‐ where in Brazil [11]. This is partially due to a greater yield, simpler processing and lack of product's maturation in the fabrication of this type of cheese, with low costs for the consumer

Food protection authorities classify microbial biological contamination as a main danger to public health. Who has constantly raised its voice on the need to restrict food contamination by health-impairing biological agents. Although microbial quality of food is of paramount importance, registration at the Federal Inspection Service does not guarantee lack of pathogens

Food-derived diseases may be caused by several microorganism groups that include bacteria, fungi, yeasts, protozoa and viruses. Due to their diversity and pathogenesis, bacteria are by far the most important microbial group and commonly associated with food-transmitting dis‐

Bacteria are microorganisms largely spread throughout the natural world and may be found in every type of environment [14]. They cause diseases in humans, animals and plants and deteriorate food and other materials. On the other hand, they may be useful too when they compose the human being's normal microbiota and are used in the production of food as

In spite of certain unreliable Brazilian statistics, it is believed that food-derived diseases in Brazil are high [15]. In fact, several studies estimate that 12% of hospitalization cases in Brazil

Occurrences of food-derived diseases are normally associated with certain risk factors, or rather, procedures that benefit toxin infections. The following may be highlighted: faults in food refrigeration; conservation of warm food at room temperature; food prepared many hours earlier for later consumption with inadequate conditioning during the interval; faults in the cooking process; handling of food by people with inadequate personal hygiene practices, or with lesions or with contaminating diseases; usage of contaminated prime matter; faults in the hygiene of utensils and other equipments in food preparation; favorable environmental con‐ ditions for the growth of etiological agents; food obtained from unreliable sources; inadequate storage; use of utensils which release toxic residues; intentional or accidental addition of toxic chemicals to the food; usage of water with uncontrolled drinkability features; water contam‐

and a fast return of expenditure to the manufacturer [12].

symbiotic in agriculture and medicine.

occur because of infectious intestinal diseases [16].

ination from damages in the supply system [17].

diseases.

244 Food Industry

in food [13].

eases

Whereas some microorganisms contribute beneficently towards the processing, safety and quality of certain food products, other organisms are involved in processes with unwanted effects in food and for the consumers' health. There are two categories of food-transmitted microbial diseases: food intoxication and infection by food. In food intoxication, the person ingests toxins that are pre-formed by microorganisms in the food. The toxin causes damage to the organism. Examples comprise botulinum toxin that binds itself to the nerve terminals at the muscle level and impedes the release of acetylcholine neurotransmitter, and staphylococ‐ cus toxin that acts on the brain's vomiting-center [19]. Infection by food occurs when the pathogen, such as by *Salmonella typhy* and other serotypes, is ingested and multiplies itself, causing diseases in the intestine tract and often in other organs [20].

The sale of animal-derived food in fair stalls without any refrigeration and without any pro‐ tection against dust and insects may alter their quality. In the case of cheese, it is sold in portions or slices and thus the external incorporation of biological or non-biological foreign matter is dangerous due to faults in the handling of the product during commercialization, poor hygiene of the stalls and utensils used, and crossed contamination between exposed products [21].

Food microbial contamination is unwanted and dangerous within food microbiology. This aspect should be faced with great strictness. The acknowledgement of possible hygiene defi‐ ciency implying in food contamination brings to the fore microorganism groups, comprising indicators, and pathogenic microorganisms that find an excellent environment in food for their development and even for the release of toxic substances [22]. Total and thermotolerant coli‐ forms, such as *Staphylococcus aureus,* fungi, yeasts and even *Salmonella* spp., should be high‐ lighted among the microorganisms whose presence and numbers indicate the quality of the product.

The above mentioned microorganisms, causes of several types of pathogenesis, are transmitted to humans because of lack of hygiene, bad habits of handlers, inefficient production processes, maintenance or re-heating of food at inadequate temperatures and also by non-adequate con‐ ditions in industries where the food is produced [23].

Most microorganisms, whose pathogenicity in humans depends on their variegated presence in food, are relatively sensitive to high temperatures. In fact, they are destroyed by the adequate cooking of eventually contaminated food or by pasteurization processes.

The Brazilian Agency for Health Vigilance (ANVISA) established, by Decree RDC 12 of the 2nd January 2001[24], the microbiological Standards for several types of food, descri‐ bed in Table 1.

So that food-caused disease cases and events could be characterized, the populations should be informed on the symptoms of each, such as mild diarrheas and vomiting since these are considered as a "passing illness" and not necessarily associated with food consumption [25].


production of fresh Minas cheese. *Staphylococcus aureus* bacteria rates higher than those per‐ mitted by current legislation are rife. The need for more sanitary surveillance and orientation

Microbiological Contamination of Homemade Food

http://dx.doi.org/10.5772/53170

247

Research work in the southeastern region of Brazil [28] (Salotti et al 2006) evaluated the mi‐ crobiological quality of fresh Minas cheese samples. Results from the hinterlands of the state of São Paulo, Brazil, showed non-compliance to rules established by the Brazilian Agency for Sanitary Vigilance (ANVISA) for 83.4% of homemade products and 66.7% for industrial sam‐ ples with regard to thermotolerant coliforms. In the case of positive coagulase *Staphylococcus*, 20% of homemade samples and 10% of industrial products failed to comply with the ANVISA regulations. Microbiological results revealed the potential risk of the product for consumers.

After analyzing samples of fresh Minas cheese in Minas Gerais for coliforms and *E. coli*, a recent study [29] showed the presence of microorganisms, above the rates allowed by current legis‐ lation, in 30% of cheese with certificate; 70% of cheese without certificate and 61.4% of mild cheese. Since *E. coli, Proteus*, *Providencia*, *Serratia, Klebsiella* and *Enterobacter* were identified within the Enterobacteriaceae isolated in fresh Minas cheese, the risk to public health when

Was reported [30] on the risk in the consumption of fresh Minas cheese by the population of the state of Paraná, southern Brazil. Samples inspected by the Federal Inspection Service of Santa Helena PR Brazil revealed that only 15% were in accord to ANVISA standards. All homemade cheese samples and 70% of inspected ones were not according to legislation. Stud‐ ies [31] confirmed the above results and reported that 50% of samples of analyzed cheese had thermotolerant coliforms, 100% had positive coagulase *Staphyloccocus* and 12.5% had *Salmo‐ nella* sp. These samples were inadequate for human consumption since they were not conso‐

One of the most traditional products of the northeastern region of Brazil is jerked beef which may be characterized as a nutrition food with high calorie rates and widely accepted by con‐ sumers for its peculiar sensorial features. Jerked beef is produced from cuts derived from all parts of cattle carcass, salted and dried, with longer durability when compared to that of fresh

Due to different nomenclature in Brazil, such as 'carne-de-sertão', 'carne serenada', 'carne deviagem', 'carne-mole', 'carne-do-vento', 'cacina' or more simple still, dehydrated meat, jerk beef is often confused with another type of salted beef, albeit industrialized, called 'charque'

Jerked beef was first used in the northeastern region of Brazil as an alternative to preserve beef surplus which could not be consumed immediately and so that the meat would not deteriorate quickly due to difficulties in its preservation especially among the poor population with no

by government authorities is urgent.

the products are consumed is amply demonstrated.

nant to cheese microbiological standards.

meat [32].

or dried salted meat [33].

**4. Microbiological contaminants of jerked beef**

**Table 1.** Microbiological Standards for Food: cheese with high moisture (55%).

According with registers, more than a billion cases of acute diarrhea are detected in lessthan-5-year-old children in developing countries yearly, with 5 million deaths. Between 1999 and 2001, in the state of Paraná, Brazil, 67.1% of food epidemics were caused by bacteria. Moreover, out of 1389 notified epidemics, 38.6 were confirmed in the laborato‐ ry; 29/7% were confirmed clinically or epidemiologically suspect and 31.6% were of un‐ known etiology [25].

World cheese production is slightly above 19 million tons. Cheese production increased more than 76.3% during the last thirty years, or rather, from approximately 10.8 million tons in 1978 to more than 19 millions in 2008. The expansion of milk-producing regions and production increase throughout recent years provided a highly relevant presence of Brazilian production within the world market of milk-derived exports. Concern is therefore high with regard to the quality of commercialized goods for internal and external consumption.

Family-run agriculture in Brazil has an important share in the milk production chain, with approximately 86% of milk producers. However, the production and management of these milk producers are foregrounded on a homemade basis with scanty technical assistance and high influence of cultural factors that may put to risk consumers' health. Technical and edu‐ cational orientation through the introduction of healthy manufacturing practices are deemed necessary to minimize contamination risks and food intoxication by the product.

Research in all Brazilian regions, where the production and commercialization of cheese is undertaken mainly by small producers, has demonstrated the risk of toxin infections in the consumption of these products by the population.

The curd-cheese is the most produced and consumed milk-derived product in the northeastern region of Brazil. Several investigations [26] have shown that the handling and carelessness in hygiene within the production system have made it foremost as a contamination source. The manufacturers are transmission vectors of the pathogen *Staphylococcus aureus* and others that may cause food intoxication. The presence of positive coagulase staphylococcus witnesses the lack of hygiene and sanitary conditions during the production, processing, distribution, stor‐ ing and commercialization stages of samples of curd-cheese. Sanitary education of the pro‐ ducers and the spreading of processing techniques based on good manufacturing practices are mandatory.

Researches in the state of Mato Grosso, in the Mid-Western region of Brazil, (Loguercio & Aleixo 2001) [27] have shown the poor hygiene and sanitary conditions that characterize the production of fresh Minas cheese. *Staphylococcus aureus* bacteria rates higher than those per‐ mitted by current legislation are rife. The need for more sanitary surveillance and orientation by government authorities is urgent.

Research work in the southeastern region of Brazil [28] (Salotti et al 2006) evaluated the mi‐ crobiological quality of fresh Minas cheese samples. Results from the hinterlands of the state of São Paulo, Brazil, showed non-compliance to rules established by the Brazilian Agency for Sanitary Vigilance (ANVISA) for 83.4% of homemade products and 66.7% for industrial sam‐ ples with regard to thermotolerant coliforms. In the case of positive coagulase *Staphylococcus*, 20% of homemade samples and 10% of industrial products failed to comply with the ANVISA regulations. Microbiological results revealed the potential risk of the product for consumers.

After analyzing samples of fresh Minas cheese in Minas Gerais for coliforms and *E. coli*, a recent study [29] showed the presence of microorganisms, above the rates allowed by current legis‐ lation, in 30% of cheese with certificate; 70% of cheese without certificate and 61.4% of mild cheese. Since *E. coli, Proteus*, *Providencia*, *Serratia, Klebsiella* and *Enterobacter* were identified within the Enterobacteriaceae isolated in fresh Minas cheese, the risk to public health when the products are consumed is amply demonstrated.

Was reported [30] on the risk in the consumption of fresh Minas cheese by the population of the state of Paraná, southern Brazil. Samples inspected by the Federal Inspection Service of Santa Helena PR Brazil revealed that only 15% were in accord to ANVISA standards. All homemade cheese samples and 70% of inspected ones were not according to legislation. Stud‐ ies [31] confirmed the above results and reported that 50% of samples of analyzed cheese had thermotolerant coliforms, 100% had positive coagulase *Staphyloccocus* and 12.5% had *Salmo‐ nella* sp. These samples were inadequate for human consumption since they were not conso‐ nant to cheese microbiological standards.

## **4. Microbiological contaminants of jerked beef**

**Microorganism Quantity** Coliforms at 45ºC 5x102 MPN/g *Staphylococcus aureus* 5x102 CFU/g *Salmonella* sp. Absence in 25g

known etiology [25].

246 Food Industry

mandatory.

\* MPN (most probable number), CFU (colony forming unit). Source: ANVISA/2001[24]

quality of commercialized goods for internal and external consumption.

consumption of these products by the population.

necessary to minimize contamination risks and food intoxication by the product.

According with registers, more than a billion cases of acute diarrhea are detected in lessthan-5-year-old children in developing countries yearly, with 5 million deaths. Between 1999 and 2001, in the state of Paraná, Brazil, 67.1% of food epidemics were caused by bacteria. Moreover, out of 1389 notified epidemics, 38.6 were confirmed in the laborato‐ ry; 29/7% were confirmed clinically or epidemiologically suspect and 31.6% were of un‐

World cheese production is slightly above 19 million tons. Cheese production increased more than 76.3% during the last thirty years, or rather, from approximately 10.8 million tons in 1978 to more than 19 millions in 2008. The expansion of milk-producing regions and production increase throughout recent years provided a highly relevant presence of Brazilian production within the world market of milk-derived exports. Concern is therefore high with regard to the

Family-run agriculture in Brazil has an important share in the milk production chain, with approximately 86% of milk producers. However, the production and management of these milk producers are foregrounded on a homemade basis with scanty technical assistance and high influence of cultural factors that may put to risk consumers' health. Technical and edu‐ cational orientation through the introduction of healthy manufacturing practices are deemed

Research in all Brazilian regions, where the production and commercialization of cheese is undertaken mainly by small producers, has demonstrated the risk of toxin infections in the

The curd-cheese is the most produced and consumed milk-derived product in the northeastern region of Brazil. Several investigations [26] have shown that the handling and carelessness in hygiene within the production system have made it foremost as a contamination source. The manufacturers are transmission vectors of the pathogen *Staphylococcus aureus* and others that may cause food intoxication. The presence of positive coagulase staphylococcus witnesses the lack of hygiene and sanitary conditions during the production, processing, distribution, stor‐ ing and commercialization stages of samples of curd-cheese. Sanitary education of the pro‐ ducers and the spreading of processing techniques based on good manufacturing practices are

Researches in the state of Mato Grosso, in the Mid-Western region of Brazil, (Loguercio & Aleixo 2001) [27] have shown the poor hygiene and sanitary conditions that characterize the

**Table 1.** Microbiological Standards for Food: cheese with high moisture (55%).

One of the most traditional products of the northeastern region of Brazil is jerked beef which may be characterized as a nutrition food with high calorie rates and widely accepted by con‐ sumers for its peculiar sensorial features. Jerked beef is produced from cuts derived from all parts of cattle carcass, salted and dried, with longer durability when compared to that of fresh meat [32].

Due to different nomenclature in Brazil, such as 'carne-de-sertão', 'carne serenada', 'carne deviagem', 'carne-mole', 'carne-do-vento', 'cacina' or more simple still, dehydrated meat, jerk beef is often confused with another type of salted beef, albeit industrialized, called 'charque' or dried salted meat [33].

Jerked beef was first used in the northeastern region of Brazil as an alternative to preserve beef surplus which could not be consumed immediately and so that the meat would not deteriorate quickly due to difficulties in its preservation especially among the poor population with no refrigeration equipments. Favorable climate conditions and availability of seawater salt, fresh meat could be preserved by being dehydrated and salted.

between 3.73% and 9.79%. Consequently, NaCl employed in the process was insufficient to decrease water activity in the product and thus it did not have a significant inhibito‐ ry action in the development of most microorganisms in the beef [46]. Lack of standard‐ ization in the quality of jerked beef was also assessed in samples collected at inspected shops. Mean rates of water activity were 0.94±0.02. The same average was obtained for samples collected in shops without any health inspection [47]. Variations in sodium chloride rates were also registered in the samples. Techniques for more efficient conser‐ vation are required to decrease such risks since it is a type of food with contamination

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http://dx.doi.org/10.5772/53170

249

With regard to the microbiological contamination of jerked beef, the transformation by which meat in natura is processed into jerked beef requires that technological alterations modify the initial microbiota by which the salting and dehydration process selects more tolerant microorganisms for such conditions [48]. Pathogens that may contaminate jerked beef comprise *Clostridium perfringens*, *Staphilococus aureus*, *Salmonella*, verotoxin-producing *Escherichia coli*, *Campylobacter*, *Yersinia enterocolítica*, *Listeria monocytogenes*, *Aeromonas hydro‐ fila*, and other deteriorating bacteria [49]. However, low NaCl rates used in jerked beef is one of the factors that trigger microbiological development since decrease in water activi‐ ty is insufficient to hinder the development of deterioration-producing bacteria of the ge‐ nus *Pseudomona*. It also provides proper conditions for the growth of gram-positive

Samples of jerked beef from the north of the state of Minas Gerais, Brazil, showed that the amount of mesophile aerobic bacteria, an index of food hygiene quality, was between 2.0x104 UFC/g and 8.9x108 UFC/g. Psichrotrophic bacteria were found in 93.33% of sam‐ ples, between 5.4x103 UFC/g and 2.9x106 UFC/g. Results show poor hygiene in the manu‐ facture of jerked beef [50]. Similar results were reported in samples of jerked beef commercialized in João Pessoa where the number of mesophile bacteria ranged between 1.8x105 and 7.5x107 UFC/g, with a clear correlationship between mesophile contamina‐

High thermotolerant coliform rates, which also demonstrate unsatisfactory hygiene and san‐ itary conditions during the processing stages in the manufacture of jerked beef, were also registered in most jerked beef samples sold in butcheries and supermarkets in João Pessoa PB Brazil [46]. However, total coliforms in food did not report recent fecal contamination or the occurrence of enteropathogens [51,52]. However, Brazilian sanitary laws did not regulate the

The commercialization of jerked beef in health inspected or not in the region of João Pessoa PB Brazil has been evaluated and results showed high rates in both groups. Ninety-six samples were analyzed and high contamination by feces-derived microorgan‐ isms was reported. *Staphylococcus ssp*. rates were high in both groups, with a low fre‐ quency for *S. aureus* [47]. *Staphylococcus aureus* rates were higher than 5logUFC/cm2 in 50% of jerked beef samples commercialized in butcheries and supermarkets in João Pes‐ soa PB Brazil. The above amounts demonstrate high contamination causing gastrointesti‐

possibilities throughout the manufacturing process.

bacteria as those of the genus *Staphylococcus* [38].

tion and hygiene and sanitary standards [42].

presence of this microorganism group in meat.

nal disorders in consumers [53].

Currently the above-mentioned preservation process is less relevant due to the introduction of refrigeration. However, many people from different regions of Brazil, especially from the northeast, became accustomed to the produce's characteristic taste and continued to produce jerked beef will less amounts of salt and frequently without exposure to the sun.

Each Brazilian state developed its own technology and thus produced jerked beef with different characteristics with regard to aspect, taste, color, amount of salt and shelf life. The states of Rio Grande do Norte and Ceará are the greatest producers of jerked beef mainly due to climatic conditions that favor the food's dehydration. In fact, jerked beef passed from a locally consumed product and used in certain food receipts to wider con‐ ditions. In fact, it is appreciated throughout Brazil and in several meal preparations. Jerked beef may be found in big city centers such as São Paulo and Rio de Janeiro, in homes and restaurants, outside the restricted circle of northeastern cuisine [34], and in the menu of the poorest worker [35,36].

Owing to the popularization of homemade salting technique, jerked beef production follows typically regional norms. Consequently, it is produced in a highly rudimentary way under inadequate sanitary conditions [37,38]. Analysis of the hygiene conditions in the production and commercialization of jerked beef in the region of Itapetinga BA Brazil may be brought forward as an example of the popularization of the technique. In fact, 73.3% of the shopkeepers interviewed admitted that they themselves produced the jerked beef on sale in their shops. Whereas 63.6% used non-inspected meat, 27.3% used meat inspected by municipal health of‐ ficers and only 0.1% was inspected by federal health officers. Jerked beef was stored and com‐ mercialized in 71% of the shops at room temperature, which favored the multiplication of contaminant microorganisms and flies. These facts bring health risks to consumers and jeop‐ ardize the product's physical aspects [39].

Salting technique consists in the removal of water from the meat tissues; decrease in water activity ensues, inhibits microbial development and the speed of unwanted reactions of the final product. When salted beef is conserved without any type of refrigeration, its shelf life is higher than that of fresh meat [40]. However, jerked beef has low sodium chloride (NaCl) rates, between 5 and 6%, high moisture, between 65 and 70% [35,41,42] and water activity of 0.92. It may be characterized as partially dehydrated meat in which water activity is not sufficient decreased to avoid microbial development (and consequently degradation) or the production of microbial toxins [43,44].

Although the literal translation of the jerked beef in Portuguese is 'meat exposed to the sun', it is actually only rarely exposed to the sunrays during the dehydration process. The end product is a semi-dehydrated homemade product with four-day shelf-life at room temperature and up to eight days under refrigeration [43,45,41].

Data on the physical and chemical qualities of jerked beef sold in butcheries and super‐ markets in João Pessoa PB Brazil showed that water activity in all samples was relative‐ ly high, between 0.898 and 0.967, and that the rates of sodium chloride (NaCl) ranged between 3.73% and 9.79%. Consequently, NaCl employed in the process was insufficient to decrease water activity in the product and thus it did not have a significant inhibito‐ ry action in the development of most microorganisms in the beef [46]. Lack of standard‐ ization in the quality of jerked beef was also assessed in samples collected at inspected shops. Mean rates of water activity were 0.94±0.02. The same average was obtained for samples collected in shops without any health inspection [47]. Variations in sodium chloride rates were also registered in the samples. Techniques for more efficient conser‐ vation are required to decrease such risks since it is a type of food with contamination possibilities throughout the manufacturing process.

refrigeration equipments. Favorable climate conditions and availability of seawater salt, fresh

Currently the above-mentioned preservation process is less relevant due to the introduction of refrigeration. However, many people from different regions of Brazil, especially from the northeast, became accustomed to the produce's characteristic taste and continued to produce

Each Brazilian state developed its own technology and thus produced jerked beef with different characteristics with regard to aspect, taste, color, amount of salt and shelf life. The states of Rio Grande do Norte and Ceará are the greatest producers of jerked beef mainly due to climatic conditions that favor the food's dehydration. In fact, jerked beef passed from a locally consumed product and used in certain food receipts to wider con‐ ditions. In fact, it is appreciated throughout Brazil and in several meal preparations. Jerked beef may be found in big city centers such as São Paulo and Rio de Janeiro, in homes and restaurants, outside the restricted circle of northeastern cuisine [34], and in

Owing to the popularization of homemade salting technique, jerked beef production follows typically regional norms. Consequently, it is produced in a highly rudimentary way under inadequate sanitary conditions [37,38]. Analysis of the hygiene conditions in the production and commercialization of jerked beef in the region of Itapetinga BA Brazil may be brought forward as an example of the popularization of the technique. In fact, 73.3% of the shopkeepers interviewed admitted that they themselves produced the jerked beef on sale in their shops. Whereas 63.6% used non-inspected meat, 27.3% used meat inspected by municipal health of‐ ficers and only 0.1% was inspected by federal health officers. Jerked beef was stored and com‐ mercialized in 71% of the shops at room temperature, which favored the multiplication of contaminant microorganisms and flies. These facts bring health risks to consumers and jeop‐

Salting technique consists in the removal of water from the meat tissues; decrease in water activity ensues, inhibits microbial development and the speed of unwanted reactions of the final product. When salted beef is conserved without any type of refrigeration, its shelf life is higher than that of fresh meat [40]. However, jerked beef has low sodium chloride (NaCl) rates, between 5 and 6%, high moisture, between 65 and 70% [35,41,42] and water activity of 0.92. It may be characterized as partially dehydrated meat in which water activity is not sufficient decreased to avoid microbial development (and consequently degradation) or the production

Although the literal translation of the jerked beef in Portuguese is 'meat exposed to the sun', it is actually only rarely exposed to the sunrays during the dehydration process. The end product is a semi-dehydrated homemade product with four-day shelf-life at room temperature

Data on the physical and chemical qualities of jerked beef sold in butcheries and super‐ markets in João Pessoa PB Brazil showed that water activity in all samples was relative‐ ly high, between 0.898 and 0.967, and that the rates of sodium chloride (NaCl) ranged

jerked beef will less amounts of salt and frequently without exposure to the sun.

meat could be preserved by being dehydrated and salted.

248 Food Industry

the menu of the poorest worker [35,36].

ardize the product's physical aspects [39].

and up to eight days under refrigeration [43,45,41].

of microbial toxins [43,44].

With regard to the microbiological contamination of jerked beef, the transformation by which meat in natura is processed into jerked beef requires that technological alterations modify the initial microbiota by which the salting and dehydration process selects more tolerant microorganisms for such conditions [48]. Pathogens that may contaminate jerked beef comprise *Clostridium perfringens*, *Staphilococus aureus*, *Salmonella*, verotoxin-producing *Escherichia coli*, *Campylobacter*, *Yersinia enterocolítica*, *Listeria monocytogenes*, *Aeromonas hydro‐ fila*, and other deteriorating bacteria [49]. However, low NaCl rates used in jerked beef is one of the factors that trigger microbiological development since decrease in water activi‐ ty is insufficient to hinder the development of deterioration-producing bacteria of the ge‐ nus *Pseudomona*. It also provides proper conditions for the growth of gram-positive bacteria as those of the genus *Staphylococcus* [38].

Samples of jerked beef from the north of the state of Minas Gerais, Brazil, showed that the amount of mesophile aerobic bacteria, an index of food hygiene quality, was between 2.0x104 UFC/g and 8.9x108 UFC/g. Psichrotrophic bacteria were found in 93.33% of sam‐ ples, between 5.4x103 UFC/g and 2.9x106 UFC/g. Results show poor hygiene in the manu‐ facture of jerked beef [50]. Similar results were reported in samples of jerked beef commercialized in João Pessoa where the number of mesophile bacteria ranged between 1.8x105 and 7.5x107 UFC/g, with a clear correlationship between mesophile contamina‐ tion and hygiene and sanitary standards [42].

High thermotolerant coliform rates, which also demonstrate unsatisfactory hygiene and san‐ itary conditions during the processing stages in the manufacture of jerked beef, were also registered in most jerked beef samples sold in butcheries and supermarkets in João Pessoa PB Brazil [46]. However, total coliforms in food did not report recent fecal contamination or the occurrence of enteropathogens [51,52]. However, Brazilian sanitary laws did not regulate the presence of this microorganism group in meat.

The commercialization of jerked beef in health inspected or not in the region of João Pessoa PB Brazil has been evaluated and results showed high rates in both groups. Ninety-six samples were analyzed and high contamination by feces-derived microorgan‐ isms was reported. *Staphylococcus ssp*. rates were high in both groups, with a low fre‐ quency for *S. aureus* [47]. *Staphylococcus aureus* rates were higher than 5logUFC/cm2 in 50% of jerked beef samples commercialized in butcheries and supermarkets in João Pes‐ soa PB Brazil. The above amounts demonstrate high contamination causing gastrointesti‐ nal disorders in consumers [53].

Mesophile microorganisms *Salmonella sp*. and *Staphylococcus aureus* in jerked beef commer‐ cialized at room temperature and under refrigeration in Campina Grande PB Brazil showed no significant difference in *S. aureus* counts for samples commercialized at room temperature and under refrigeration. *Salmonella* ssp. was detected in 40% of jerked beef samples commer‐ cialized at room temperature and in 30% of samples under refrigeration.

throughout the country. Sausages have great acceptance in the southern and southeast‐

Microbiological Contamination of Homemade Food

http://dx.doi.org/10.5772/53170

251

Brazilian swine breeding has a very important role in several sectors of Brazilian econo‐ my. It produces jobs and intensifies demand of agricultural products in the industrializa‐ tion and commercialization of animal-derived products. Besides providing excellent animal protein to the population, the meat industry exports meat and important eco‐

Data by the Brazilian Association of Production and Exportation Industry of Pork (ABIPECS) showed that approximately 65% of the Brazilian pork production is directed towards the in‐ ternal market through industrialized products. Among the processed products, the fresh Tus‐ can-type sausage, made exclusively from pork, uses the less important animal parts as food,

Pork and its derived products undergo bacterial alterations owing to several factors such as animal health and fecal contamination by *Escherichia coli* highly relevant worldwide as a mi‐ croorganism hazardous to animal and population health involving hygiene and sanitary issues [57]. The same author evaluated the occurrence of *E.coli* in swine in the abattoirs of Rio de Janeiro, Brazil, from which the Tuscun-type sausages were made. Different parts of the animal used in the stuffing process were examined and concluded that, depending on the meat and

*Toxoplasma gondhii* in fresh pork sausages commercialized in Botucatu SP Brazil was evaluated by researches [58]. Pork represents one of the main sources of infection by *T. gondii* in humans. Swine were the most important animals in the process of toxoplasmo‐ sis transmission [59,60,58,61]. Mendonça's data did not show any evidence of *T. gondii* in the samples, perhaps due to salt, used in the manufacturing process, which eliminated

The occurrence of food infection by pork sausages contaminated with *Salmonella sp*. has been suggested [62]. Brazilian sanitary laws [63] make it mandatory that the microorgan‐ ism should be lacking in 25% so that human intoxication may occur. However, such pos‐ sibility may vary since it depends on serotype and the person's health conditions and tolerance. Mürmann's results [62] showed that 24% of pork sausages samples were conta‐

Contamination by *Salmonella sp* in pork may occur in pens through contact with feces, lack of hygiene and sanitation in the installations and by other animals during the transport, waiting or pre-finishing period. A high increase of *S. enteriditis*in food toxin infections in humans and

Fecal coliforms, positive coagulase staphylococcus, *Salmonella* spp and *Campylobacter* spp in fresh sausages were evaluated [65]. When the hygiene and sanitary quality among the different types of fresh sausages was compared, pork sausages had the worst scores with regard to risks

ern regions due to a more Europeanized culture.

nomical assets are aggregated [57].

the microorganism.

minated by *Salmonella enterica*.

with great acceptance among the population.

the manufacturing process, sausages were not fit for consumption.

in aviary products has been reported in Brazil since the 1990 [64].

in public health, as ruled by the RDC n.12 of Anvisa [63].

Another source of contamination in the commercialization of jerked beef may be found in supermarkets, open market stalls and butcheries. Data reveal that the utensils used in 75% of these outlets were not exclusively for meat cutting and that the handling of money and food was common practice in 25% of the businesses. Aprons, disposable caps and clean closed shoes were only found in 25% of the shops.

The inadequate washing of hands and other habits such as talking during the handling and commercialization of food were also reported in all commercial enterprises [54]. It has been verified that in João Pessoa, supermarkets had the best hygiene and sanitary profile in jerked beef quality, whereas open markets and stalls in fairs had the worst [42]. In the latter case, meat is exposed without any type of protection and any passerby may handle it at will.

Investigations were carried out with regard to alien matter, such as flies, acarids, larvae, insects, feathers and others, found in jerked beef sold in 20 (90.9%) shops in Diadema SP Brazil, specialized in typical products from the northeastern region of Brazil. Expo‐ sure of products without any wrappings is an excellent condition for attacks by insects, especially flies, and rodents, making it improper for human consumption in the wake of health-hazard matter [55].

Almost all jerked beef is manufactured and sold in small shops and specifically prepared for people who appreciate the product. Consequently, lack of sanitary rules for its production, precarious conditions in its commercialization, storage without refrigeration and its exposure without any protection characterize jerked beef in such conditions as haphazard to public health.

## **5. Microbiological contaminants in meat fillings (sausages made from beef and fowl meat, salami)**

Animal-derived food conveys a host of microorganisms dangerous to human health. The incidence of toxin infections in Brazil is high, although statistics are rather lacking on the matter. Bacteria causing toxin infections are widely distributed although their main natural habitat is the human or animal intestine tract [14]. The most common bacteria in food contamination are of the genera *Escherichia, Salmonella, Shigella, Yersinia, Vibrio, Bru‐ cella, Clostridium, Listeria, Campylobacter, Bacillus cereus* and *Staphylococcus aureus* [56]. Sau‐ sages, widely used in Europe, is a type of food stuffed with meat from swine, fowls, goats, cattle and fish, seasoned with several types of spicy ingredients. Sausages are a highly popular food in Brazil, easily accessible to all classes of people and consumed throughout the country. Sausages have great acceptance in the southern and southeast‐ ern regions due to a more Europeanized culture.

Mesophile microorganisms *Salmonella sp*. and *Staphylococcus aureus* in jerked beef commer‐ cialized at room temperature and under refrigeration in Campina Grande PB Brazil showed no significant difference in *S. aureus* counts for samples commercialized at room temperature and under refrigeration. *Salmonella* ssp. was detected in 40% of jerked beef samples commer‐

Another source of contamination in the commercialization of jerked beef may be found in supermarkets, open market stalls and butcheries. Data reveal that the utensils used in 75% of these outlets were not exclusively for meat cutting and that the handling of money and food was common practice in 25% of the businesses. Aprons, disposable caps and clean closed shoes

The inadequate washing of hands and other habits such as talking during the handling and commercialization of food were also reported in all commercial enterprises [54]. It has been verified that in João Pessoa, supermarkets had the best hygiene and sanitary profile in jerked beef quality, whereas open markets and stalls in fairs had the worst [42]. In the latter case, meat

Investigations were carried out with regard to alien matter, such as flies, acarids, larvae, insects, feathers and others, found in jerked beef sold in 20 (90.9%) shops in Diadema SP Brazil, specialized in typical products from the northeastern region of Brazil. Expo‐ sure of products without any wrappings is an excellent condition for attacks by insects, especially flies, and rodents, making it improper for human consumption in the wake of

Almost all jerked beef is manufactured and sold in small shops and specifically prepared for people who appreciate the product. Consequently, lack of sanitary rules for its production, precarious conditions in its commercialization, storage without refrigeration and its exposure without any protection characterize jerked beef in such conditions as haphazard to public

**5. Microbiological contaminants in meat fillings (sausages made from beef**

Animal-derived food conveys a host of microorganisms dangerous to human health. The incidence of toxin infections in Brazil is high, although statistics are rather lacking on the matter. Bacteria causing toxin infections are widely distributed although their main natural habitat is the human or animal intestine tract [14]. The most common bacteria in food contamination are of the genera *Escherichia, Salmonella, Shigella, Yersinia, Vibrio, Bru‐ cella, Clostridium, Listeria, Campylobacter, Bacillus cereus* and *Staphylococcus aureus* [56]. Sau‐ sages, widely used in Europe, is a type of food stuffed with meat from swine, fowls, goats, cattle and fish, seasoned with several types of spicy ingredients. Sausages are a highly popular food in Brazil, easily accessible to all classes of people and consumed

is exposed without any type of protection and any passerby may handle it at will.

cialized at room temperature and in 30% of samples under refrigeration.

were only found in 25% of the shops.

health-hazard matter [55].

**and fowl meat, salami)**

health.

250 Food Industry

Brazilian swine breeding has a very important role in several sectors of Brazilian econo‐ my. It produces jobs and intensifies demand of agricultural products in the industrializa‐ tion and commercialization of animal-derived products. Besides providing excellent animal protein to the population, the meat industry exports meat and important eco‐ nomical assets are aggregated [57].

Data by the Brazilian Association of Production and Exportation Industry of Pork (ABIPECS) showed that approximately 65% of the Brazilian pork production is directed towards the in‐ ternal market through industrialized products. Among the processed products, the fresh Tus‐ can-type sausage, made exclusively from pork, uses the less important animal parts as food, with great acceptance among the population.

Pork and its derived products undergo bacterial alterations owing to several factors such as animal health and fecal contamination by *Escherichia coli* highly relevant worldwide as a mi‐ croorganism hazardous to animal and population health involving hygiene and sanitary issues [57]. The same author evaluated the occurrence of *E.coli* in swine in the abattoirs of Rio de Janeiro, Brazil, from which the Tuscun-type sausages were made. Different parts of the animal used in the stuffing process were examined and concluded that, depending on the meat and the manufacturing process, sausages were not fit for consumption.

*Toxoplasma gondhii* in fresh pork sausages commercialized in Botucatu SP Brazil was evaluated by researches [58]. Pork represents one of the main sources of infection by *T. gondii* in humans. Swine were the most important animals in the process of toxoplasmo‐ sis transmission [59,60,58,61]. Mendonça's data did not show any evidence of *T. gondii* in the samples, perhaps due to salt, used in the manufacturing process, which eliminated the microorganism.

The occurrence of food infection by pork sausages contaminated with *Salmonella sp*. has been suggested [62]. Brazilian sanitary laws [63] make it mandatory that the microorgan‐ ism should be lacking in 25% so that human intoxication may occur. However, such pos‐ sibility may vary since it depends on serotype and the person's health conditions and tolerance. Mürmann's results [62] showed that 24% of pork sausages samples were conta‐ minated by *Salmonella enterica*.

Contamination by *Salmonella sp* in pork may occur in pens through contact with feces, lack of hygiene and sanitation in the installations and by other animals during the transport, waiting or pre-finishing period. A high increase of *S. enteriditis*in food toxin infections in humans and in aviary products has been reported in Brazil since the 1990 [64].

Fecal coliforms, positive coagulase staphylococcus, *Salmonella* spp and *Campylobacter* spp in fresh sausages were evaluated [65]. When the hygiene and sanitary quality among the different types of fresh sausages was compared, pork sausages had the worst scores with regard to risks in public health, as ruled by the RDC n.12 of Anvisa [63].

The authors also registered that most samples were not in accordance to microbiological standards and thus hazardous to consumer's health. Another datum refers to the absence of *Campylobacter* spp in the samples, perhaps due to sodium chloride concentrations over 1.5% that may have inhibited these microorganisms.

exposure of the meat to several contamination sources or to already contaminated chickens

The above authors researched the microbiological quality of industrialized avian products and their derivates in another region of the state of São Paulo. Research determined the presence of *Campylobacter jejuni* and *Salmonella sp.* Sausages samples analyzed were 42.8% positive for

The presence of microorganisms in the above research works suggests the need for greater care during the handling and preparation of sausages that may be eaten in natura, without any heating treatment that would reduce the number of microorganisms causing toxin infec‐

Vienna sausage may be defined as an industrialized meat-stuffed product obtained from the emulsion of animal meat to which are added a variety of ingredients and condiments, filling a natural or artificial casing, and submitted to proper thermal process [76]. Vienna sausages are highly popular in Brazil due to their low costs and for the manufacturing of the ubiquitous

The physical and chemical characteristics of Vienna sausages should contain a maximum of 65% moisture, 30% fat, 2% starch, 7% total carbohydrates, 12% protein. Fresh sausages should

Vienna sausages samples of the hot-dog type were analyzed in Niterói and Rio de Janeiro RJ Brazil to detect thermotolerant coliforms, positive coagulase *Staphylococcus*, *Clostridium* spp and *Salmonella* spp by conventional methods with the necessary modifications [78]. When compared to health norms, results showed that 33% of samples were inadequate for con‐

Salami is another highly appreciated product in southern Brazil. Its homemade manufacture started in the early 20th century with an enormous variety of industrialized types that dif‐ fered in composition, casing, size of meat and fats, spices, smoking process and maturation period prior to commercialization. Researchers revaluated the various characteristics [79] of salamis produced by small- and medium-sized agro-industries in the southern state of Santa Catarina, Brazil. Bacteria *Staphylococcus aureus*, *Salmonella* spp, *Listeria monocystogenes* and *E.coli* were researched in the products. Although results did not identify contamination by *Salmonella* spp, the *E. coli* and *S. aureus* counts were significant, but within the reliability pa‐

Was analyzed the quality [80] of salami in the interior of the state of São Paulo, Brazil, and verified that, despite samples with *E. coli* and fecal coliforms, all samples were within health standards. Nevertheless, 60% of samples were contaminated by *Staphylococcus aureus* and 22%

C) from manufacture until consumption, with ex‐

Microbiological Contamination of Homemade Food

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253

C to 5o

sumption due to the presence of their isolated microorganisms.

that provided the contamination of the final product.

*C. jejuni* and 28.5% for *Salmonella sp*.

be under permanent refrigeration (0o

piry period after 48 hours [77].

were unhealthy for consumption.

tions [75].

hot dog.

rameters.

Was analyzed [66] the presence of *Listeria* spp, principally *L. monocytogenes*, during the man‐ ufacture of fresh mixed-meat sausages in three abattoirs, supervised by state health authorities, in Pelotas RS Brazil. Results showed that all samples from the three abattoirs were contami‐ nated by *Listeria* spp, of which the most frequent species was *L. innocua* (97.6), followed by *L. monocytogenes* (29.3%) and *L. welshimeri* (24.4%).

When the hygiene and sanitary conditions in the manufacture of fresh sausages in the north‐ western region of the state of Paraná, Brazil, were analyzed [67] data failed to show any mi‐ crobiological contamination that would jeopardize the health of the consumer. The manufacture of these samples followed strict handling and processing procedures.

On the other hand, another authors [68] studied the prevalence of antimicrobial resistance by serotypes of *Salmonella* isolated from fresh pork sausages and found significant quantities of the above in samples collected in the southern state of Santa Catarina, Brazil. These serotypes resisted the antimicrobial products sulfonamide and tetracycline (81%); ampicillin (50%) and chloramphenicol (31.25%). Was evaluated the microbiological quality of fresh sausages in two towns of the state of Minas Gerais, Brazil [69]. Results confirmed positive coagulase *Staphylo‐ coccus* in 35% of samples which made them improper for human consumption. The same au‐ thor also demonstrated that 35% of samples were contaminated by thermotolerant fecal coliforms above the maximum limits.

The consumption of chicken meat and its derivates has recently increased considerably in Brazil due to price decrease, good quality and practical cuttings provided [70]. Per capita con‐ sumption increased from 10 kg to 35.4 kg, only slightly lower than beef consumption (União Brasileira de Avicultura) [71]. The products' quality is highly important and a great concern to health authorities, food industry and consumers. Chickens bred for human consumption may host several pathogenic microorganisms such as *Campylobacter jejuni, Salmonella* sp and *E. coli* [72,73].

Rall investigated [70] the hygiene and sanitary conditions of chicken meat and several types of sausages commercialized in the interior of the state of São Paulo, Brazil, by determining the Most Probable Number of coliforms at 45o C. The same authors also analyzed the presence of *Samonella sp* by the traditional method and by PCR. Data showed that 40% of the 75 sausage samples analyzed were improper for human consumption due to excess in coliforms and 7 samples (9.3%) were positive for *Salmonella* sp. (9.3%). Research by PCR increased to 56% *Salmonella*-positive samples. When the frequency rate of *Salmonella* was added to the micro‐ biological limits for coliforms, it might be concluded that 86.7% of sausages were improper for human consumption.

In their research in the northwestern region of the state of São Paulo, Brazil, others authors [74] found contamination by *Salmonella* in 16% of chicken sausages samples. The most relevant item in the above result may be the handling of the product during processing, coupled to the exposure of the meat to several contamination sources or to already contaminated chickens that provided the contamination of the final product.

The authors also registered that most samples were not in accordance to microbiological standards and thus hazardous to consumer's health. Another datum refers to the absence of *Campylobacter* spp in the samples, perhaps due to sodium chloride concentrations over 1.5%

Was analyzed [66] the presence of *Listeria* spp, principally *L. monocytogenes*, during the man‐ ufacture of fresh mixed-meat sausages in three abattoirs, supervised by state health authorities, in Pelotas RS Brazil. Results showed that all samples from the three abattoirs were contami‐ nated by *Listeria* spp, of which the most frequent species was *L. innocua* (97.6), followed by *L.*

When the hygiene and sanitary conditions in the manufacture of fresh sausages in the north‐ western region of the state of Paraná, Brazil, were analyzed [67] data failed to show any mi‐ crobiological contamination that would jeopardize the health of the consumer. The

On the other hand, another authors [68] studied the prevalence of antimicrobial resistance by serotypes of *Salmonella* isolated from fresh pork sausages and found significant quantities of the above in samples collected in the southern state of Santa Catarina, Brazil. These serotypes resisted the antimicrobial products sulfonamide and tetracycline (81%); ampicillin (50%) and chloramphenicol (31.25%). Was evaluated the microbiological quality of fresh sausages in two towns of the state of Minas Gerais, Brazil [69]. Results confirmed positive coagulase *Staphylo‐ coccus* in 35% of samples which made them improper for human consumption. The same au‐ thor also demonstrated that 35% of samples were contaminated by thermotolerant fecal

The consumption of chicken meat and its derivates has recently increased considerably in Brazil due to price decrease, good quality and practical cuttings provided [70]. Per capita con‐ sumption increased from 10 kg to 35.4 kg, only slightly lower than beef consumption (União Brasileira de Avicultura) [71]. The products' quality is highly important and a great concern to health authorities, food industry and consumers. Chickens bred for human consumption may host several pathogenic microorganisms such as *Campylobacter jejuni, Salmonella* sp and

Rall investigated [70] the hygiene and sanitary conditions of chicken meat and several types of sausages commercialized in the interior of the state of São Paulo, Brazil, by determining the

*Samonella sp* by the traditional method and by PCR. Data showed that 40% of the 75 sausage samples analyzed were improper for human consumption due to excess in coliforms and 7 samples (9.3%) were positive for *Salmonella* sp. (9.3%). Research by PCR increased to 56% *Salmonella*-positive samples. When the frequency rate of *Salmonella* was added to the micro‐ biological limits for coliforms, it might be concluded that 86.7% of sausages were improper for

In their research in the northwestern region of the state of São Paulo, Brazil, others authors [74] found contamination by *Salmonella* in 16% of chicken sausages samples. The most relevant item in the above result may be the handling of the product during processing, coupled to the

C. The same authors also analyzed the presence of

manufacture of these samples followed strict handling and processing procedures.

that may have inhibited these microorganisms.

*monocytogenes* (29.3%) and *L. welshimeri* (24.4%).

coliforms above the maximum limits.

Most Probable Number of coliforms at 45o

*E. coli* [72,73].

252 Food Industry

human consumption.

The above authors researched the microbiological quality of industrialized avian products and their derivates in another region of the state of São Paulo. Research determined the presence of *Campylobacter jejuni* and *Salmonella sp.* Sausages samples analyzed were 42.8% positive for *C. jejuni* and 28.5% for *Salmonella sp*.

The presence of microorganisms in the above research works suggests the need for greater care during the handling and preparation of sausages that may be eaten in natura, without any heating treatment that would reduce the number of microorganisms causing toxin infec‐ tions [75].

Vienna sausage may be defined as an industrialized meat-stuffed product obtained from the emulsion of animal meat to which are added a variety of ingredients and condiments, filling a natural or artificial casing, and submitted to proper thermal process [76]. Vienna sausages are highly popular in Brazil due to their low costs and for the manufacturing of the ubiquitous hot dog.

The physical and chemical characteristics of Vienna sausages should contain a maximum of 65% moisture, 30% fat, 2% starch, 7% total carbohydrates, 12% protein. Fresh sausages should be under permanent refrigeration (0o C to 5o C) from manufacture until consumption, with ex‐ piry period after 48 hours [77].

Vienna sausages samples of the hot-dog type were analyzed in Niterói and Rio de Janeiro RJ Brazil to detect thermotolerant coliforms, positive coagulase *Staphylococcus*, *Clostridium* spp and *Salmonella* spp by conventional methods with the necessary modifications [78]. When compared to health norms, results showed that 33% of samples were inadequate for con‐ sumption due to the presence of their isolated microorganisms.

Salami is another highly appreciated product in southern Brazil. Its homemade manufacture started in the early 20th century with an enormous variety of industrialized types that dif‐ fered in composition, casing, size of meat and fats, spices, smoking process and maturation period prior to commercialization. Researchers revaluated the various characteristics [79] of salamis produced by small- and medium-sized agro-industries in the southern state of Santa Catarina, Brazil. Bacteria *Staphylococcus aureus*, *Salmonella* spp, *Listeria monocystogenes* and *E.coli* were researched in the products. Although results did not identify contamination by *Salmonella* spp, the *E. coli* and *S. aureus* counts were significant, but within the reliability pa‐ rameters.

Was analyzed the quality [80] of salami in the interior of the state of São Paulo, Brazil, and verified that, despite samples with *E. coli* and fecal coliforms, all samples were within health standards. Nevertheless, 60% of samples were contaminated by *Staphylococcus aureus* and 22% were unhealthy for consumption.

## **6. Final considerations**

Owing to their importance for public health, the correct handling of meat and milk products required greater attention, care and supervision from the competent health authorities. Since there is great cultural diversity in food manufactured in Brazil, the direct intervention of all the sectors involved within the food production chain is mandatory to warrant healthy and reliable products and thus a decrease in diseases caused by food contamination.

[5] Olivieri D. de A. Avaliação da qualidade microbiológica de amostras de mercado de queijo mussarela, elaborado a partir do leite de búfala (Bubalus bubalis). 61 p. Disser‐ tação (Mestrado) - Escola Superior de Agricultura Luiz de Queiroz, Piracicaba, SP, 2004.

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## **Author details**

Suzymeire Baroni1 , Izabel Aparecida Soares2 , Rodrigo Patera Barcelos1 , Alexandre Carvalho de Moura2 , Fabiana Gisele da Silva Pinto3 and Carmem Lucia de Mello Sartori Cardoso da Rocha4


## **References**


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**6. Final considerations**

254 Food Industry

**Author details**

Suzymeire Baroni1

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[51] Franco BDGM, Landgraf, M. Microrganismos patogênicos de importância em alimen‐ tos. In: Microbiologia dos alimentos. São Paulo: Atheneu, 2008. cap. 4, p. 33-82. [52] Silva N, Junqueira VCA, Silveira NFA, Tanawaki MH, Dos Santos, R S, Gomes R A R. Manual de Métodos de Análise Microbiológica de Alimentos. 4° edição. São Paulo. Ed.

[53] Costa EL, Silva JA. Qualidade sanitária da carne de sol comercializada em açougues e supermercados de João Pessoa – PB. Bol. CEPPA Curitiba 1999; 17(2): 137-44.

[54] Miranda PC, Barreto NSE. Avaliação Higiênico-Sanitária de diferentes Estabelecimen‐ tos de Comercialização da Carne-de sol no Município de Cruz Das Almas-Ba. Revista

[55] Mennucci TA, Marciano MAM, ATUI, MB, Polineto A, Germano PML. Study on con‐ taminant materials within "sun dried meat (jerked beef)" at the "Northern Houses.

[56] Pinto A. Doenças de origem microbiana transmitidas pelos alimentos. Millenium 1996;

[57] Franco R. *E.coli*: ocorrência em suínos abatidos na grande Rio e sua viabilidade exper‐ imental em linguiça frescal tipo toscana.Tese doutorado. Universidade Federal Flumi‐

[58] Mendonça A O. Detecção de *Toxoplasma gondii* em linguiças comercializadas no muni‐ cípio de Botucatu- SP. Tese doutorado. Universidade Estadual Paulista Julio Mesquita

[59] Durbey J P. Refinemente of pepsin digestion method for isolation of *Toxoplasma gon‐*

[60] Gamble H R, Murrel K D. Detection of parasites in food. Parasitology 1998; (117): 97-111. [61] Tenter A M. Current knowledge on the epidemiology of infections with *Toxoplasma*. The Tokai Journal of Experimental and Clinical Medicine 1998; 23 (6): 391.

[62] Mürmann L. Avaliação do risco de infecção por *Salmonella* sp em consumidores de linguiça frescal de carne suína em Porto Alegre –RS. Tese doutorado. Universidade

[63] Brasil. Agência Nacional de Vigilância Veterinária. Resolução nº12 de 12 de janeiro 2001. Regulamento técnico sobre os padrões microbiológicos para alimentos. 2001. Dis‐

*dii* from infected tissues. Veterinary Parasitology 1998; 74: 75-77.

demic & Professional, 1998. cap. 4, p. 119-157

Mestrado,Montes Claros, MG: ICA/UFMG, 2010.

Caatinga, Mossoró, 2012; 25(2): 166-172.

Federal do Rio Grande do Sul; 2008.

Revista Instituto Adolfo Lutz 2010; 69(1): 47-54.

Livraria Varela, 2010.

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(4): 91-100.

nense; 2002.

Filho; 2003.


[75] Carvalho A C F B, Cortes A L L. Contaminação de produtos avícolas industrialização e seus derivados por *Campylobacter jejuni* e *Salmonella* sp. Arquivo Veterinária 2003; 19 (1): 057-062.

**Chapter 13**

**Occurrence of Organochlorine Pesticides**

S. Panseri, P.A. Biondi, D. Vigo, R. Communod and

Additional information is available at the end of the chapter

L. M. Chiesa

**1. Introduction**

http://dx.doi.org/10.5772/54182

**Residues in Animal Feed and Fatty Bovine Tissue**

Nowadays, more than 800 different kinds of pesticides are used for the control of insects, rodents, fungi and unwanted plants in the process of agricultural production. Although most of them leave the products or degrade in soil, water and atmosphere, some trace amounts of pesticide residues can be transferred to humans via the food chain, being poten‐ tially harmful to human health. [1] Pest control in intensive agriculture involves treatment of crops (fruits, vegetables, cereals, etc) pre and post harvest stages, rodenticides are em‐ ployed in the post-harvest storage stage, and fungicides are applied at any stage of the proc‐ ess depending on the crop. These chemicals can be transferred from plant to animal via the food chain. Furthermore, breeding animals and their accommodation can themselves be sprayed with pesticide solution to prevent pest infestations. Consequently, both these con‐ tamination routes can lead to bioaccumulation of persistent pesticides in food products of animal origin such as meat, fat, fish, eggs and milk. [2,3] During the last decades much at‐ tention has been given to this group of substances and the international level after it became apparent that they are transported through the environment and critical concentrations have been reached in some areas even in places where they have never been produced or used. Several countries banned the use of Organochlorine Pesticides (OCPs) during the 1970s and 1980s, although many of them continue to been used by other countries. OCPs have been identified as one of the major classes of environmental contaminants because of their persis‐ tence, long-range transport ability and human and animal toxic effects. OCPs are carcino‐ genic in animals as well as in human (International Agency for Research on Cancer, 1987). The immunotoxicity of selected OCPs has been also documented in vitro [4], in vivo [5], as

well as in animals, in human fetal, neonatal and infant immune systems [6,7,8,9].

© 2013 Panseri et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.


## **Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue**

S. Panseri, P.A. Biondi, D. Vigo, R. Communod and

L. M. Chiesa

[75] Carvalho A C F B, Cortes A L L. Contaminação de produtos avícolas industrialização e seus derivados por *Campylobacter jejuni* e *Salmonella* sp. Arquivo Veterinária 2003; 19

[76] Brasil, Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agro‐ pecuária – MAPA/SDA. Instrução Normativa Nº 4 de 31 de março de 2000.-Aprova os Regulamentos Técnicos de Identidade e Qualidade de Carne Mecanicamente Separada, de Mortadela, de Lingüiça e de Salsicha -Diário Oficial da União, Brasília, DF, p.6, de

[77] Ferreira M C, Fraqueza M J, Barreto A S.Avaliação do prazo de vida útil da salsicha fresca. Revista Portuguesa de Ciências Veterinárias 2007; 102 (561): 141-143.

[78] Martins L L, Santos J F, Franco R M, Oliveira L A T, Bezz J. Bacteriological study in bovine and chikem hot dog type- sausages sold in vacuumed packing-case and retail commercialized in Rio de Janeiro city and Niterói, RJ/ Brazil supermarkets. Revista.

[79] D'Agostini F P, Campana P, Degenhart R. Qualidade e identidade de embutidos pro‐ duzidos no baixo Vale do Rio do peixe, Santa Catarina- Brasil. E. Tech Tecnologias para

[80] Hoffmann F L, Garcia-Cruz C H, Vinturim T M, Carmello M T. Qualidade microbio‐

(1): 057-062.

260 Food Industry

05 de abril de 2000. Seção 1.

Instituto. Adolfo Lutz 2008; 67 (3): 215-220.

Competitividade Industrial 2009; 2 (2): 1-13.

lógica do salame. B. Ceppa 1997; 15 (1): 57-64.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54182

## **1. Introduction**

Nowadays, more than 800 different kinds of pesticides are used for the control of insects, rodents, fungi and unwanted plants in the process of agricultural production. Although most of them leave the products or degrade in soil, water and atmosphere, some trace amounts of pesticide residues can be transferred to humans via the food chain, being poten‐ tially harmful to human health. [1] Pest control in intensive agriculture involves treatment of crops (fruits, vegetables, cereals, etc) pre and post harvest stages, rodenticides are em‐ ployed in the post-harvest storage stage, and fungicides are applied at any stage of the proc‐ ess depending on the crop. These chemicals can be transferred from plant to animal via the food chain. Furthermore, breeding animals and their accommodation can themselves be sprayed with pesticide solution to prevent pest infestations. Consequently, both these con‐ tamination routes can lead to bioaccumulation of persistent pesticides in food products of animal origin such as meat, fat, fish, eggs and milk. [2,3] During the last decades much at‐ tention has been given to this group of substances and the international level after it became apparent that they are transported through the environment and critical concentrations have been reached in some areas even in places where they have never been produced or used. Several countries banned the use of Organochlorine Pesticides (OCPs) during the 1970s and 1980s, although many of them continue to been used by other countries. OCPs have been identified as one of the major classes of environmental contaminants because of their persis‐ tence, long-range transport ability and human and animal toxic effects. OCPs are carcino‐ genic in animals as well as in human (International Agency for Research on Cancer, 1987). The immunotoxicity of selected OCPs has been also documented in vitro [4], in vivo [5], as well as in animals, in human fetal, neonatal and infant immune systems [6,7,8,9].

© 2013 Panseri et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A growing number of epidemiological studies have investigated blood or adipose levels of OCPs and their metabolites in relation with cancer, neurodevelopmental effects, immuno‐ toxicity and reproductive efficiency [10,11,12]. The main sources of OCPs in the human diet are foods of animal origin and environmental exposure. It has been concluded that humans are exposed to toxic compounds via diet in a much higher degree compared to other expo‐ sure routes such as inhalation and dermal exposure. Low volatility and high stability, to‐ gether with lipophilic behaviour, are responsible critical factor for their persistence in the environment (air, water and soil) and subsequent concentration in fatty tissues through the food chain. Therefore, it's important to identify and to monitor levels of OCPs in foodstuff of animal origin (meat and tissues that contain fat, milk and dairy products, eggs, honey and fish). The main pathway for the OCPs contamination of animal food is the ingestion of the contaminated food and/or water by the animals. [13,14,15] Breeding animals can accumulate persistent organic pollutants from contaminated feed and water, and/or from pesticides ap‐ plication in livestock areas (treatment of cowshed, pigsties, sheepfold etc.).[16,17,18] The use of feedstuffs in farms has become indispensable for animal diet in developed countries be‐ cause of increasingly higher production requirements. Animal feed plays an important part in the food chain and has implication for the composition and quality of the livestock prod‐ ucts that people consume. Therefore, the control of OCPs residues in animal feed is manda‐ tory as well as the control in fatty tissues.

been used for monitoring the residuals levels of OCPs. As regards food of animal origin, one efficient way to avoid large-scale contamination is to control and monitor the levels of OCPs residues present in animal feeds before being fed to the husbandry animals. [28,29,30]

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

http://dx.doi.org/10.5772/54182

263

At the same time, public health safety authorities should constantly monitor the OCPs in animal food commodities as the major source of human background exposure to OCPs is through food of animal origin. Most persistent organic pollutant (POPs) are OCPs, name‐ ly, aldrin, endrin, chlordane, DDT and hexachlorobenzene (HCB). They have been ban‐ ned for agricultural or domestic use in Europe, North America and many countries of South America, in accordance with Stockholm Convention in 1980s. However, some OCPs are still used, e.g. DDT is used to control the growth of mosquito that spread ma‐ laria or as antifouling agent in some developing countries. [31,32] Residues of OCPs have been detected in breast milk (including DDT, HCB and HCH isomers) in contami‐ nated areas. Recently, the scope of POPs was extended to include nine plus one chemi‐ cals. Among these new POPs, chlordecone, lindane, α-HCH, β-HCH, pentachlorobenzene (PeCB) and endosulfan, also belong to OCPs. [33,34] In order to fulfil the requirements of the Stockholm convention, the participating countries have to develop their own im‐ plementation plant to monitor the background level and collate the exposure data. To ensure the pesticide residues are not found in food of feed at levels presenting an unac‐ ceptable risk for human consumption, maximum residue levels (MRLs) have therefore been set by the European Commission. [35,36,37] MRLs are the upper legal concentration limits for pesticides in or on food or feed. They are set for a wide range of food com‐ modities of plant and animal origin, and they usually apply to the product as placed in the market. MRLs are not simply set as toxicological threshold levels; they are derived after a comprehensive assessment of the properties of the active substance and the resi‐ dues behaviour on treated crops. Both the periodic estimation of human exposure to per‐ sistent organic pollutants and the establishment by the EU authorities of MRLs in foods have required the development of analytical methods suitable for research purposes and inspection programmes. As an example, the European Union has established maximum contents for these compounds in animal feed which can be as low as 5 µg Kg-1 for some OCPs in fish feed and β-HCH in cattle feed. In the rest of feed materials these values can be as low as 10 µg Kg-1 relative to feedstuff with moisture content of 12%. [38,39,40]

Animal feed as well as animal fat are considered a very complex matrices with large number of components especially lipids. Consequently, the development od sensitive methods for its analysis with elimination of interferent compounds and enough efficien‐ cy in term of analyte recovery represents an interesting task. [41,42] The most intricate step in these procedures is represented by the sample extraction and clean-up that should be efficient enough to allow a reliable screening of contaminated samples. The se‐ lection of suitable solvent (s) and extraction method is critical for obtaining satisfactory recovery of OCPs from the food matrix. Of course, if co-extracted materials are mini‐ mised in the extract, the clean-up procedure would became simpler. Owing to the lipo‐

**1.2. Extraction methods and clean-up of OCPs**

#### **1.1. Organochlorine Pesticides (OCPs)**

Organochlorine pesticides (OCPs) were intensively used in agriculture to protect cultivated plants in mid-twentieth century. 1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), one of the common OCPs, was used to prevent spreading of malaria and other vector-borne diseas‐ es such as dengue, leishmaniasis and Japanese encephalitis through the prevention of growth of mosquito.[19,20] After OCPs were used widely in soil and plants for some years and due to their relative stability and bioaccumulation property, these persistent chemicals can be transferred and magnified to higher trophic level through the food chain. Conse‐ quently, OCP residues are present in fatty foods, both foods of animal origin such as meat, eggs and milk, and of plant origin such as vegetable oil, nuts, oat and olives. Besides, these chemicals are widely distributed in the environment, which provides another route of un‐ wanted intake in human. [21,22,23] Nevertheless, human exposure occurs still primarily via low level food contamination. Since their mode of action is by targeting system or enzymes in the pest which may be identical or very similar to system or enzymes in human beings, these OCPs pose risks to human health and the environment. [24,25] Thus, monitoring of OCPs residues in food becomes a routine analysis of pesticides monitoring laboratories. All US government pesticides datasets showed that persistent OCP residues were surprisingly common in certain foods despite being off the market for over 30 years. Residues of dieldrin, in particular, posed substantial risks in certain root crops. About one quarter of samples of organically labelled fresh produce contained pesticides residues, compared with about three quarters of conventional samples. [26,27] Among the contaminated organic vegetable sam‐ ples, about 60% of them were contaminated with OCPs. After some OCPs were banned for use since the 80s, common daily food items such as eggs, milk, poultry, meat and fish have been used for monitoring the residuals levels of OCPs. As regards food of animal origin, one efficient way to avoid large-scale contamination is to control and monitor the levels of OCPs residues present in animal feeds before being fed to the husbandry animals. [28,29,30]

At the same time, public health safety authorities should constantly monitor the OCPs in animal food commodities as the major source of human background exposure to OCPs is through food of animal origin. Most persistent organic pollutant (POPs) are OCPs, name‐ ly, aldrin, endrin, chlordane, DDT and hexachlorobenzene (HCB). They have been ban‐ ned for agricultural or domestic use in Europe, North America and many countries of South America, in accordance with Stockholm Convention in 1980s. However, some OCPs are still used, e.g. DDT is used to control the growth of mosquito that spread ma‐ laria or as antifouling agent in some developing countries. [31,32] Residues of OCPs have been detected in breast milk (including DDT, HCB and HCH isomers) in contami‐ nated areas. Recently, the scope of POPs was extended to include nine plus one chemi‐ cals. Among these new POPs, chlordecone, lindane, α-HCH, β-HCH, pentachlorobenzene (PeCB) and endosulfan, also belong to OCPs. [33,34] In order to fulfil the requirements of the Stockholm convention, the participating countries have to develop their own im‐ plementation plant to monitor the background level and collate the exposure data. To ensure the pesticide residues are not found in food of feed at levels presenting an unac‐ ceptable risk for human consumption, maximum residue levels (MRLs) have therefore been set by the European Commission. [35,36,37] MRLs are the upper legal concentration limits for pesticides in or on food or feed. They are set for a wide range of food com‐ modities of plant and animal origin, and they usually apply to the product as placed in the market. MRLs are not simply set as toxicological threshold levels; they are derived after a comprehensive assessment of the properties of the active substance and the resi‐ dues behaviour on treated crops. Both the periodic estimation of human exposure to per‐ sistent organic pollutants and the establishment by the EU authorities of MRLs in foods have required the development of analytical methods suitable for research purposes and inspection programmes. As an example, the European Union has established maximum contents for these compounds in animal feed which can be as low as 5 µg Kg-1 for some OCPs in fish feed and β-HCH in cattle feed. In the rest of feed materials these values can be as low as 10 µg Kg-1 relative to feedstuff with moisture content of 12%. [38,39,40]

#### **1.2. Extraction methods and clean-up of OCPs**

A growing number of epidemiological studies have investigated blood or adipose levels of OCPs and their metabolites in relation with cancer, neurodevelopmental effects, immuno‐ toxicity and reproductive efficiency [10,11,12]. The main sources of OCPs in the human diet are foods of animal origin and environmental exposure. It has been concluded that humans are exposed to toxic compounds via diet in a much higher degree compared to other expo‐ sure routes such as inhalation and dermal exposure. Low volatility and high stability, to‐ gether with lipophilic behaviour, are responsible critical factor for their persistence in the environment (air, water and soil) and subsequent concentration in fatty tissues through the food chain. Therefore, it's important to identify and to monitor levels of OCPs in foodstuff of animal origin (meat and tissues that contain fat, milk and dairy products, eggs, honey and fish). The main pathway for the OCPs contamination of animal food is the ingestion of the contaminated food and/or water by the animals. [13,14,15] Breeding animals can accumulate persistent organic pollutants from contaminated feed and water, and/or from pesticides ap‐ plication in livestock areas (treatment of cowshed, pigsties, sheepfold etc.).[16,17,18] The use of feedstuffs in farms has become indispensable for animal diet in developed countries be‐ cause of increasingly higher production requirements. Animal feed plays an important part in the food chain and has implication for the composition and quality of the livestock prod‐ ucts that people consume. Therefore, the control of OCPs residues in animal feed is manda‐

Organochlorine pesticides (OCPs) were intensively used in agriculture to protect cultivated plants in mid-twentieth century. 1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), one of the common OCPs, was used to prevent spreading of malaria and other vector-borne diseas‐ es such as dengue, leishmaniasis and Japanese encephalitis through the prevention of growth of mosquito.[19,20] After OCPs were used widely in soil and plants for some years and due to their relative stability and bioaccumulation property, these persistent chemicals can be transferred and magnified to higher trophic level through the food chain. Conse‐ quently, OCP residues are present in fatty foods, both foods of animal origin such as meat, eggs and milk, and of plant origin such as vegetable oil, nuts, oat and olives. Besides, these chemicals are widely distributed in the environment, which provides another route of un‐ wanted intake in human. [21,22,23] Nevertheless, human exposure occurs still primarily via low level food contamination. Since their mode of action is by targeting system or enzymes in the pest which may be identical or very similar to system or enzymes in human beings, these OCPs pose risks to human health and the environment. [24,25] Thus, monitoring of OCPs residues in food becomes a routine analysis of pesticides monitoring laboratories. All US government pesticides datasets showed that persistent OCP residues were surprisingly common in certain foods despite being off the market for over 30 years. Residues of dieldrin, in particular, posed substantial risks in certain root crops. About one quarter of samples of organically labelled fresh produce contained pesticides residues, compared with about three quarters of conventional samples. [26,27] Among the contaminated organic vegetable sam‐ ples, about 60% of them were contaminated with OCPs. After some OCPs were banned for use since the 80s, common daily food items such as eggs, milk, poultry, meat and fish have

tory as well as the control in fatty tissues.

262 Food Industry

**1.1. Organochlorine Pesticides (OCPs)**

Animal feed as well as animal fat are considered a very complex matrices with large number of components especially lipids. Consequently, the development od sensitive methods for its analysis with elimination of interferent compounds and enough efficien‐ cy in term of analyte recovery represents an interesting task. [41,42] The most intricate step in these procedures is represented by the sample extraction and clean-up that should be efficient enough to allow a reliable screening of contaminated samples. The se‐ lection of suitable solvent (s) and extraction method is critical for obtaining satisfactory recovery of OCPs from the food matrix. Of course, if co-extracted materials are mini‐ mised in the extract, the clean-up procedure would became simpler. Owing to the lipo‐ philicity of OCPs, organic solvent (s) normally can extract OCPs form food efficiently but lipids are also co-extracted. Solid-liquid extraction method was applicable for extracting OCPs from various types of food samples including vegetables, meats and its products, fish, eggs and animal fats. In addition, several standardised methods, including AOAC 970.52, EN 1528 and EN 12393, have employed such solid-liquid or liquid-liquid extrac‐ tion techniques for the determination of OCPs in both fatty and non-fatty foods. [43,44,45] In some occasions, sonication or Polytron was also applied to improve the ex‐ traction efficiency and recoveries.

showed that Florisil had better cleaning efficiency of fatty acids in fish extract when compared with C18. Besides, recoveries of some OCPs were poor with hexane as eluent and these more polar OCPs could be eluted out from the column with acetone. Bazlic et al., reported also that the quality of Florisil was important in avoiding possible interfer‐ ence and misinterpretation of results. Even though GC-MS was employed as the detec‐ tion system, poor quality Florisil could introduce false positive results for lindane and dieldrin. [53,54] To sum up, the combination of sorbent(s) and eluting solvent(s) have to be chosen very carefully. Otherwise, some OCPs or their metabolites/derivatives would be lost during the clean-up step. [55,56,57] These OCPs could either break down or ad‐ here to the sorbent material, leading to low or even no recovery. Finding of the optima clean-up conditions is an art itself. As the targeted OCPs might cover a wide range of polarities, it is quite difficult to find the best combination of SPE column material and eluting solvent, which permits recovering the polar OCPs (but leaving the polar interfer‐ ents behind on the column), as well as recovering the non-polar OCPs (without eluting

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

http://dx.doi.org/10.5772/54182

265

A number of different selective detectors can be coupled with GC for analyzing OCPs, in‐ cluding electron capture detector (ECD), halogen specific detector (XSD), electrolytic con‐ ductivity detector (ELCD) and atomic emission detector (AED). GC-ECD is the most commonly used detection method with low detection limits. It is particularly useful for de‐ tecting halogen containing molecules. However, other organic molecules, such as aromatic compounds, would also give positive signal. Users have to confirm the presence of OCPs by another confirmative technique. Even though the above-mentioned selective detector can be used for quantification, it is unlike to fulfil the European Commission's stringest require‐ ments as set for pesticides analysis. Confirmation with GC-hyphenated with mass spectro‐ metric (MS) detector is normally required. Single quadrupole MS detector running in electron ionisation (EI) mode with target analytes monitored by selective ion monitoring (SIM) becomes a routine monitoring tool for OCPs nowadays. Since some OCPs are electro‐ negative in nature, GC-MS detector under negative chemical ionisation mode with methane as reagent gas could provide better sensitivity. [58,59,60,61] To further increase confidence in confirmative analysis, GC coupled with tandem Ms is one of suitable techniques. Besides providing a more definitive detection tool, tandem MS also decrease matrix interferences, improves selectivity and achieves higher signal-to-noise ratio and subsequently improves the detection limit. Both tandem-in-time (ion-trap) and tandem-in-space (triple quadru‐ poles) detector have been applied for OCPs residues analysis in different matrices. The de‐ termination of pesticides residues in the environment and in food is necessary for ensuring that human exposure to contaminants, especially by dietary intake, does not exceed accepta‐ ble level for health. Consequently, robust analytical methods have to be validated for carry‐ ing out both research and monitoring programmes, and thus for defining limitations and supporting enforcement of regulations. In this field, reproducible analytical methods are re‐ quired to allow the effective separation, selective identification and accurate quantification

of pesticides analyses at low levels in food-stuff including food of animal origin.

any residual oil present in the extract from the column).

**1.4. Detection techniques of OCPs**

#### **1.3. Clean-up methods**

Matrix constituents can be co-extracted and later co-eluted with analysed components and can consequently interfere with analyte identification and quantification. Moreover, co-extracted compounds, especially lipids, tend to adsorb in GC system such as injection port and column, resulting in poor chromatographic performance. A through clean-up minimised such matrix issues, improves sensitivity, permits more consistent and repeata‐ ble results, and extend the capillary column lifetime. Several approaches have been at‐ tempted to eliminated co-extracted interferences from extracts, including freezing centrifugation or filtration, liquid-liquid partitioning, gel permeation chromatography (GPC), solid phase extraction (SPE) and solid-phase microextraction (SPME). The sim‐ plest approach to remove the fatty co-extracted is by freezing centrifugation. [46,47] The logic behind is that fatty substances (mainly lipids) have lower melting point than the solvent so that frozen lipids can be removed by centrifugation or filtering while OCPs remain dissolved in the solvent. Different freezing temperatures ranged from -24 °C to -70 °C have been used. However, the solubility of lipids in solvent not only depends on the temperature but also the solubility product. Therefore this technique can remove sig‐ nificant amount of lipids for some food matrix but not for every matrix. Certain amount of lipids would remain in the solvent after the freezing centrifugation step and hence further cleanup is required. Using materials with large surfaces area for absorption of lipids have been employed since early 1970s. These materials include, Florisil, Lipid Re‐ moval Agent (LRA) media from Supelco, micro Cel E and Calflo E from Johns-Manville. Micro Cel E and Calflo E and LRA are synthetic calcium silicate while Florisil is a mag‐ nesium silicate with high specific surface area. [48,49] They can be applied to remove lip‐ ids either in sample preparation, solid phase extraction step or during sample clean-up step, with minimal effect on non-lipid chemicals. When food sample is mixed with these lipids absorbing materials, edible fat could be removed. Therefore it is common to con‐ duct a clean-up step by solid phase extraction (SPE) nowadays. Both, conventional glass column packed with sorbent(s) and ready-to-use cartridges have been utilised and the common used phases are silica, Florisil, alumina and C18-bounded silica. Doong and Lee compared the cleaning efficiency of ready-to-use cartridge filled with three different ad‐ sorbents for shellfish extract. [50,51,52] Their results demonstrated that out of 14 OCPs tested, two were retained in the C18-cartridge. As for alumina and Florisil SPE, though all 14 pesticides tested could be recovered, Florisil provide better results in term of re‐ coveries, repeatability and removal of interfering substances. Similarly, Hong et al., also showed that Florisil had better cleaning efficiency of fatty acids in fish extract when compared with C18. Besides, recoveries of some OCPs were poor with hexane as eluent and these more polar OCPs could be eluted out from the column with acetone. Bazlic et al., reported also that the quality of Florisil was important in avoiding possible interfer‐ ence and misinterpretation of results. Even though GC-MS was employed as the detec‐ tion system, poor quality Florisil could introduce false positive results for lindane and dieldrin. [53,54] To sum up, the combination of sorbent(s) and eluting solvent(s) have to be chosen very carefully. Otherwise, some OCPs or their metabolites/derivatives would be lost during the clean-up step. [55,56,57] These OCPs could either break down or ad‐ here to the sorbent material, leading to low or even no recovery. Finding of the optima clean-up conditions is an art itself. As the targeted OCPs might cover a wide range of polarities, it is quite difficult to find the best combination of SPE column material and eluting solvent, which permits recovering the polar OCPs (but leaving the polar interfer‐ ents behind on the column), as well as recovering the non-polar OCPs (without eluting any residual oil present in the extract from the column).

#### **1.4. Detection techniques of OCPs**

philicity of OCPs, organic solvent (s) normally can extract OCPs form food efficiently but lipids are also co-extracted. Solid-liquid extraction method was applicable for extracting OCPs from various types of food samples including vegetables, meats and its products, fish, eggs and animal fats. In addition, several standardised methods, including AOAC 970.52, EN 1528 and EN 12393, have employed such solid-liquid or liquid-liquid extrac‐ tion techniques for the determination of OCPs in both fatty and non-fatty foods. [43,44,45] In some occasions, sonication or Polytron was also applied to improve the ex‐

Matrix constituents can be co-extracted and later co-eluted with analysed components and can consequently interfere with analyte identification and quantification. Moreover, co-extracted compounds, especially lipids, tend to adsorb in GC system such as injection port and column, resulting in poor chromatographic performance. A through clean-up minimised such matrix issues, improves sensitivity, permits more consistent and repeata‐ ble results, and extend the capillary column lifetime. Several approaches have been at‐ tempted to eliminated co-extracted interferences from extracts, including freezing centrifugation or filtration, liquid-liquid partitioning, gel permeation chromatography (GPC), solid phase extraction (SPE) and solid-phase microextraction (SPME). The sim‐ plest approach to remove the fatty co-extracted is by freezing centrifugation. [46,47] The logic behind is that fatty substances (mainly lipids) have lower melting point than the solvent so that frozen lipids can be removed by centrifugation or filtering while OCPs remain dissolved in the solvent. Different freezing temperatures ranged from -24 °C to -70 °C have been used. However, the solubility of lipids in solvent not only depends on the temperature but also the solubility product. Therefore this technique can remove sig‐ nificant amount of lipids for some food matrix but not for every matrix. Certain amount of lipids would remain in the solvent after the freezing centrifugation step and hence further cleanup is required. Using materials with large surfaces area for absorption of lipids have been employed since early 1970s. These materials include, Florisil, Lipid Re‐ moval Agent (LRA) media from Supelco, micro Cel E and Calflo E from Johns-Manville. Micro Cel E and Calflo E and LRA are synthetic calcium silicate while Florisil is a mag‐ nesium silicate with high specific surface area. [48,49] They can be applied to remove lip‐ ids either in sample preparation, solid phase extraction step or during sample clean-up step, with minimal effect on non-lipid chemicals. When food sample is mixed with these lipids absorbing materials, edible fat could be removed. Therefore it is common to con‐ duct a clean-up step by solid phase extraction (SPE) nowadays. Both, conventional glass column packed with sorbent(s) and ready-to-use cartridges have been utilised and the common used phases are silica, Florisil, alumina and C18-bounded silica. Doong and Lee compared the cleaning efficiency of ready-to-use cartridge filled with three different ad‐ sorbents for shellfish extract. [50,51,52] Their results demonstrated that out of 14 OCPs tested, two were retained in the C18-cartridge. As for alumina and Florisil SPE, though all 14 pesticides tested could be recovered, Florisil provide better results in term of re‐ coveries, repeatability and removal of interfering substances. Similarly, Hong et al., also

traction efficiency and recoveries.

**1.3. Clean-up methods**

264 Food Industry

A number of different selective detectors can be coupled with GC for analyzing OCPs, in‐ cluding electron capture detector (ECD), halogen specific detector (XSD), electrolytic con‐ ductivity detector (ELCD) and atomic emission detector (AED). GC-ECD is the most commonly used detection method with low detection limits. It is particularly useful for de‐ tecting halogen containing molecules. However, other organic molecules, such as aromatic compounds, would also give positive signal. Users have to confirm the presence of OCPs by another confirmative technique. Even though the above-mentioned selective detector can be used for quantification, it is unlike to fulfil the European Commission's stringest require‐ ments as set for pesticides analysis. Confirmation with GC-hyphenated with mass spectro‐ metric (MS) detector is normally required. Single quadrupole MS detector running in electron ionisation (EI) mode with target analytes monitored by selective ion monitoring (SIM) becomes a routine monitoring tool for OCPs nowadays. Since some OCPs are electro‐ negative in nature, GC-MS detector under negative chemical ionisation mode with methane as reagent gas could provide better sensitivity. [58,59,60,61] To further increase confidence in confirmative analysis, GC coupled with tandem Ms is one of suitable techniques. Besides providing a more definitive detection tool, tandem MS also decrease matrix interferences, improves selectivity and achieves higher signal-to-noise ratio and subsequently improves the detection limit. Both tandem-in-time (ion-trap) and tandem-in-space (triple quadru‐ poles) detector have been applied for OCPs residues analysis in different matrices. The de‐ termination of pesticides residues in the environment and in food is necessary for ensuring that human exposure to contaminants, especially by dietary intake, does not exceed accepta‐ ble level for health. Consequently, robust analytical methods have to be validated for carry‐ ing out both research and monitoring programmes, and thus for defining limitations and supporting enforcement of regulations. In this field, reproducible analytical methods are re‐ quired to allow the effective separation, selective identification and accurate quantification of pesticides analyses at low levels in food-stuff including food of animal origin.

## **2. Aim of the research**

The aims of the present work were:


## **3. Experimental**

#### **3.1. Feed and subcutaneous bovine fat samples**

25 feed samples used for bovine with different composition were obtained from intensive livestock farming. An example of feed mixture was shown in figure 1. 35 fat samples were obtained from bovine for slaughter (18-24 month age) and presented in figure 2.

**Figure 2.** Subcutaneous fatty tissue sample

All OCPs were purchased from Supelco Inc.: mix 32094 and 32412 (Bellefonte, PA, USA). The figure 3 displays the structures of these OCPs considered in this study. Purities of pesti‐ cides standards were greater than 99%. Working standard solution was prepared at concen‐ tration of 0.1-5 µg mL-1 by volume, dilution with acetone and hexane. Organic solvents (hexane, acetone and acetonitrile) were of pesticide residue analysis grade (Sigma Aldrich, USA). All glassware was cleaned with laboratory reagent, sequentially rinsed with distilled water, acetone and methanol and finally baked in a oven at 300 °C. Distilled water was ob‐ tained with a Milli-Q system (Millipore, Bedford, MA, USA). For SPE, Florisil 5 g was pur‐

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

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267

Ultrasonic bath (Branson) was used for the extraction of chlorinated pesticides form feed and fat samples. The generator of ultrasonic bath has an output of 150 W and a frequency of 35 kHz. Rotary evaporator (Buchi, Swiss) was used for the concentration of organic solvent. High intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan)

was used to obtain representative aliquots of feed samples powder.

**3.2. Chemicals and reagents**

chased from Supelco.

**3.3. Equipments**

**Figure 1.** Feed sample mixture

#### **Figure 2.** Subcutaneous fatty tissue sample

#### **3.2. Chemicals and reagents**

**2. Aim of the research**

266 Food Industry

fat bovine tissue).

**3. Experimental**

**Figure 1.** Feed sample mixture

The aims of the present work were:

mation of a large number of pesticides at trace level.

concentration phenomena of these persistent pollutants.

**3.1. Feed and subcutaneous bovine fat samples**

**•** To monitor the OCPs level in animal feed samples used in bovine farm.

**•** To develop and optimise a simple extraction and clean-up method to quantify non-polar chlorinated compounds in high lipid containing samples (animal feed and subcutaneous

**•** To validate a multiresidues method for the simultaneous determination of 20 OCPs by us‐ ing GC-MS/MS in term of repeatability, precision, limit of detection (LOD), limit of quan‐ tification (LOQ) etc. The coupling of this detection mode is very useful for the analysis of these complex samples allowing the separation, identification, quantification and confir‐

**•** To monitor the OCPs level in subcutaneous fat bovine tissue to asses and to verify the

25 feed samples used for bovine with different composition were obtained from intensive livestock farming. An example of feed mixture was shown in figure 1. 35 fat samples were

obtained from bovine for slaughter (18-24 month age) and presented in figure 2.

All OCPs were purchased from Supelco Inc.: mix 32094 and 32412 (Bellefonte, PA, USA). The figure 3 displays the structures of these OCPs considered in this study. Purities of pesti‐ cides standards were greater than 99%. Working standard solution was prepared at concen‐ tration of 0.1-5 µg mL-1 by volume, dilution with acetone and hexane. Organic solvents (hexane, acetone and acetonitrile) were of pesticide residue analysis grade (Sigma Aldrich, USA). All glassware was cleaned with laboratory reagent, sequentially rinsed with distilled water, acetone and methanol and finally baked in a oven at 300 °C. Distilled water was ob‐ tained with a Milli-Q system (Millipore, Bedford, MA, USA). For SPE, Florisil 5 g was pur‐ chased from Supelco.

#### **3.3. Equipments**

Ultrasonic bath (Branson) was used for the extraction of chlorinated pesticides form feed and fat samples. The generator of ultrasonic bath has an output of 150 W and a frequency of 35 kHz. Rotary evaporator (Buchi, Swiss) was used for the concentration of organic solvent. High intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan) was used to obtain representative aliquots of feed samples powder.

cation of mechanochemistry deal with the physical changes of substances in all state of ag‐ gregation, for instance occurring with the combined action of pressure and shear in energyintensive grinding mills. Mechanochemical technology has been developed and applied in different fields (synthesis of superfine powder, surface modification and drug modification) and could represent a novel tool of research. [62,63,64] The procedure is presented in Fig.4.

Sample 10 g

Superfine grinding extraction (SGE)

*3.4.2. Samples extraction*

rotary evaporator to follow Florisil-SPE clean-up.

70 mL acetone/hexane (5:2), v/v

filtering

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

filtering

acetonitrile 50mL

X 2

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269

Elution 13 mL 10% acetone/hexane (1:9, v/v)

Sonication 20 min

Rotary evaporation

Freezing 30 min, -24 C

Rotary evaporation

Clean-up with Florisil

Concentration

GC-MS/MS

**Figure 4.** Analytical procedure for the extraction and purification of OCPs from feed and subcutaneous fatty tissue samples

10 g of subcutaneous fat tissue (homogenised in a cooled mixer) or feed sample finely ground‐ ed and prepared with the procedure described above (SFG) were extracted by ultrasonic agita‐ tion with a mixed solvent of 70 mL of acetone-*n*-hexane (5:2, v/v) for 20 min. Extract was filtered to remove traces of water with filter paper containing 5 g of sodium sulphate, and then transfer‐ red into a 250 mL round flask. The extraction was repeated one more time. Extracted solvent was dried and redissolved in 50 mL of acetonitrile that has low solubility for lipids. Acetoni‐ trile extract was stored in the freezer at -24 °C for 30 min to freeze lipids. Most of the lipids were precipitated as pale yellow, condensed lump on glassware surface. Cold extract at -24 °C was immediately filtered with filter paper to remove frozen lipids. The precipitated lipid on glass‐ ware surface was redissolved in 50 mL of acetonitrile to perform filtration again by same proce‐ dure. The filtered extracts were combined and concentrated to a final volume of 1 mL by a

Figure 3. Chemical structures of chlorinated pesticides investigated in this study (19 OCPs) **Figure 3.** Chemical structures of chlorinated pesticides investigated in this study (19 OCPs)

#### **3.3. Equipments 3.4. Sample extraction, delipidation and clean-up procedure**

#### Ultrasonic bath (Branson) was used for the extraction of chlorinated pesticides form feed and fat samples. The generator of *3.4.1. Superfine Grinding (SFG) of feed sample*

procedure is presented in Fig.4.

ultrasonic bath has an output of 150 W and a frequency of 35 kHz. Rotary evaporator (Buchi, Swiss) was used for the concentration of organic solvent. High intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan) was used to obtain representative aliquots of feed samples powder. **3.4. Sample extraction, delipidation and clean-up procedure 3.4.1. Superfine grinding (SFG) of feed sample**  In order to obtain a representative feed sample a superfine powder was prepared from feed using mechanical grinding-activation in an energy intensive vibrational mill. 50 g of differ‐ ent feed sample were ground in a high intensity planetary mill. The mill was vibrating at a frequency of 25 Hz for 4 min using two 50 mL jars with 20 mm stainless steel balls. Pre cool‐ ing of jars were carried out with liquid nitrogen in order to prevent temperature increasing during the grinding process. The speed differences between balls and jar resulted in the in‐ teraction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces resulted in the very effective energy input of planetary ball mills. The appli‐

In order to obtain a representative feed sample a superfine powder was prepared from feed using mechanical grinding-activation in an energy intensive vibrational mill. 50 g of different feed sample were ground in a high intensity planetary mill. The mill was vibrating at a frequency of 25 Hz for 4 min using two 50 mL jars with 20 mm stainless steel balls. Pre cooling of jars were carried out with liquid nitrogen in order to prevent temperature increasing during the grinding process. The speed differences between balls and jar resulted in the interaction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces resulted in the very effective energy input of planetary ball mills. The application of mechanochemistry deal with the physical changes of substances in all state of aggregation, for instance occurring with the combined action of pressure and shear in energy-intensive grinding mills. Mechanochemical technology has been developed and applied in different fields (synthesis of superfine powder, surface modification and drug modification) and could represent a novel tool of research. [62,63,64] The cation of mechanochemistry deal with the physical changes of substances in all state of ag‐ gregation, for instance occurring with the combined action of pressure and shear in energyintensive grinding mills. Mechanochemical technology has been developed and applied in different fields (synthesis of superfine powder, surface modification and drug modification) and could represent a novel tool of research. [62,63,64] The procedure is presented in Fig.4.

**Figure 4.** Analytical procedure for the extraction and purification of OCPs from feed and subcutaneous fatty tissue samples

#### *3.4.2. Samples extraction*

Figure 3. Chemical structures of chlorinated pesticides investigated in this study (19 OCPs)

*3.4.1. Superfine Grinding (SFG) of feed sample*

**Figure 3.** Chemical structures of chlorinated pesticides investigated in this study (19 OCPs)

**3.4. Sample extraction, delipidation and clean-up procedure**

**3.4. Sample extraction, delipidation and clean-up procedure** 

**3.4.1. Superfine grinding (SFG) of feed sample** 

Ultrasonic bath (Branson) was used for the extraction of chlorinated pesticides form feed and fat samples. The generator of ultrasonic bath has an output of 150 W and a frequency of 35 kHz. Rotary evaporator (Buchi, Swiss) was used for the concentration of organic solvent. High intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan) was used to obtain

In order to obtain a representative feed sample a superfine powder was prepared from feed using mechanical grinding-activation in an energy intensive vibrational mill. 50 g of differ‐ ent feed sample were ground in a high intensity planetary mill. The mill was vibrating at a frequency of 25 Hz for 4 min using two 50 mL jars with 20 mm stainless steel balls. Pre cool‐ ing of jars were carried out with liquid nitrogen in order to prevent temperature increasing during the grinding process. The speed differences between balls and jar resulted in the in‐ teraction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces resulted in the very effective energy input of planetary ball mills. The appli‐

‐HCH ‐HCH ‐HCH(lindane) ‐HCH

heptachlor heptachlor epoxide methoxychlor endrine

endrine aldehyde ‐chlordane ‐chlordane endosulfan I

endosulfan II endosulfan sulfate aldrin dieldrin

p‐p'DDT p‐p'DDE p‐p'DDD

In order to obtain a representative feed sample a superfine powder was prepared from feed using mechanical grinding-activation in an energy intensive vibrational mill. 50 g of different feed sample were ground in a high intensity planetary mill. The mill was vibrating at a frequency of 25 Hz for 4 min using two 50 mL jars with 20 mm stainless steel balls. Pre cooling of jars were carried out with liquid nitrogen in order to prevent temperature increasing during the grinding process. The speed differences between balls and jar resulted in the interaction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces resulted in the very effective energy input of planetary ball mills. The application of mechanochemistry deal with the physical changes of substances in all state of aggregation, for instance occurring with the combined action of pressure and shear in energy-intensive grinding mills. Mechanochemical technology has been developed and applied in different fields (synthesis of superfine powder, surface modification and drug modification) and could represent a novel tool of research. [62,63,64] The

**3.3. Equipments** 

268 Food Industry

procedure is presented in Fig.4.

representative aliquots of feed samples powder.

10 g of subcutaneous fat tissue (homogenised in a cooled mixer) or feed sample finely ground‐ ed and prepared with the procedure described above (SFG) were extracted by ultrasonic agita‐ tion with a mixed solvent of 70 mL of acetone-*n*-hexane (5:2, v/v) for 20 min. Extract was filtered to remove traces of water with filter paper containing 5 g of sodium sulphate, and then transfer‐ red into a 250 mL round flask. The extraction was repeated one more time. Extracted solvent was dried and redissolved in 50 mL of acetonitrile that has low solubility for lipids. Acetoni‐ trile extract was stored in the freezer at -24 °C for 30 min to freeze lipids. Most of the lipids were precipitated as pale yellow, condensed lump on glassware surface. Cold extract at -24 °C was immediately filtered with filter paper to remove frozen lipids. The precipitated lipid on glass‐ ware surface was redissolved in 50 mL of acetonitrile to perform filtration again by same proce‐ dure. The filtered extracts were combined and concentrated to a final volume of 1 mL by a rotary evaporator to follow Florisil-SPE clean-up.

#### *3.4.3. Sample clean-up*

The SPE cartridge was cleaned with 12 mL of n-hexane and air dried by positive pressure prior sample application. 5 mL of hexane were used to condition the cartridge. After sample loading, the cartridge was air dried for 10 min. Desorption of the OCPs, which had been concentrated on the Florisil sorbent, was carried out using 13 mL of acetone-*n*-hexane (1:9, v/v) mixture at a flow of 1 mL min-1 and collected in a 50 mL round flask. The eluate was then concentrated at 45 °C under nitrogen stream until just the disappearance of the last drop of solution. Finally, the residue was redissolved in 1 mL hexane Pestanal prior to its injection in GC-MS/MS system.

**4. Results and disussion**

from bovine.

**4.1. Key results about extraction and clean-up method**

ters are the major components in meat fats.

cient for the clean-up of food samples.

**4.2. Optimisation of MS/MS transitions**

summarise in table 1.

Two extraction and clean-up methods have been developed, tested and optimised for the extraction of 20 OCPs from animal feed sample and subcutaneous fatty tissue samples

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

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271

Large amounts of lipids were extracted when n-hexane or acetone was used as extraction solvents. In general, complex mixtures of of several types of lipids were co-extracted during the extraction of chlorinated pesticides from biological sample. Triglycerides and sterol es‐

The key point of the extraction method take advantage of significant difference of melting points between lipids (below about 40 C) and chlorinated pesticides (above 260 C), so that lipid components can be easily separated from chlorinated compounds. After extraction, lip‐ ids in organic extracts were precipitated as frozen at -24 C in the freezer, while chlorinated compounds were still dissolved in cold organic solvents. Thus frozen-lipids can be removed just by filtering extracts. During overall process, approximately 90% of lipids were eliminat‐ ed without any significant loss of pesticides. After freezing-lipid filtration, the remaining in‐

Sample clean-up was necessary for the removal of polar coextracted substances. Florisil car‐ tridges have been employed for that purpose since that adsorbent has proved to be very effi‐

From full scan spectra, the most intense higher mass precursor ions were selected for devel‐ opment of MRM method. For the most of the analytes these were the base peak ions in the mass spectra, but in some cases higher mass ions of lower intensity were selected to mini‐ mise the possibility of matrix interferences. Precursor ions were examined using different collision energies (automated method development) and the most intense product ions were selected for each precursor ion. The products ions for all OCPs determined in this study are

For quantification of the target analytes linear calibration curves for all pesticides over six calibration levels (0.005 mg kg-1-1.5 mg kg -1) using a feed and fat blank samples were pre‐ pared taking also in consideration the MRLs levels for each compounds. In quantitative analysis one of the main problems is the suppression/enhancement of the analyte response caused by sample matrix components. Calibration curves were performed by using matrixmatched (in each matrix) because the feed and fat samples contain many compounds that are co-extracted in the extraction organic solvent. The use of Florisil-SPE tries to avoid ma‐ trix effect using a clean-up step, but this not eliminates completely the problem. A matrix

effect on the analytical signal due to the matrix was noticed for most pesticides.

terferences were successfully removed by a solid-phase (SPE) Florisil cartridge.

#### **3.5. GC-MS/MS analysis and detection**

A Varian GC 3800 gas chromatograph coupled to a Varian Saturn 2000 ion trap mass spec‐ trometer was used for the analysis and detection of the OCPs. The gas chromatograph was equipped with a Rtx-5 fused-silica capillary column (30 m x 0.25 mm i.d., 0.25 um film thick‐ ness) obtained from Restek. Helium (purity 99,99%) was the carrier gas at constant flow of 1 mL min-1. The GC injector temperature was maintained at 280 °C. The oven program tem‐ perature was: initial temperature 120 °C increased by 5 °C min -1 to 280 °C and held for 10 min. The ion trap spectrometer was operated in electron ionisation (EI) mode.

The ionization energy was set at 70eV. The detector range was *m/z* 40-650. The transfer line and trap temperature were 250 °C and 170 °C respectively.

**Figure 5.** GC-MS chromatogram (TIC mode) of a standard OCPs mixture (MRL 0.5 mg kg-1) used in the present study. 1:α-HCH, 2:β-HCH, 3:γ-HCH, 4:δ-HCH, 5:Heptachlor, 6:Aldrin, 7: Heptachlor epoxide, 8:Endosulfan I, 9:Diel‐ drin, 10:p-p'DDE, 11:Endrin, 12:Endosulfan II, 13:p-p'DDD, 14:Endrin Aldheyde, 15: Endosulfan Sulphate, 16:pp'DDT, 17:Methoxychlor

## **4. Results and disussion**

*3.4.3. Sample clean-up*

270 Food Industry

injection in GC-MS/MS system.

p'DDT, 17:Methoxychlor

**3.5. GC-MS/MS analysis and detection**

The SPE cartridge was cleaned with 12 mL of n-hexane and air dried by positive pressure prior sample application. 5 mL of hexane were used to condition the cartridge. After sample loading, the cartridge was air dried for 10 min. Desorption of the OCPs, which had been concentrated on the Florisil sorbent, was carried out using 13 mL of acetone-*n*-hexane (1:9, v/v) mixture at a flow of 1 mL min-1 and collected in a 50 mL round flask. The eluate was then concentrated at 45 °C under nitrogen stream until just the disappearance of the last drop of solution. Finally, the residue was redissolved in 1 mL hexane Pestanal prior to its

A Varian GC 3800 gas chromatograph coupled to a Varian Saturn 2000 ion trap mass spec‐ trometer was used for the analysis and detection of the OCPs. The gas chromatograph was equipped with a Rtx-5 fused-silica capillary column (30 m x 0.25 mm i.d., 0.25 um film thick‐ ness) obtained from Restek. Helium (purity 99,99%) was the carrier gas at constant flow of 1 mL min-1. The GC injector temperature was maintained at 280 °C. The oven program tem‐ perature was: initial temperature 120 °C increased by 5 °C min -1 to 280 °C and held for 10

The ionization energy was set at 70eV. The detector range was *m/z* 40-650. The transfer line

**Figure 5.** GC-MS chromatogram (TIC mode) of a standard OCPs mixture (MRL 0.5 mg kg-1) used in the present study. 1:α-HCH, 2:β-HCH, 3:γ-HCH, 4:δ-HCH, 5:Heptachlor, 6:Aldrin, 7: Heptachlor epoxide, 8:Endosulfan I, 9:Diel‐ drin, 10:p-p'DDE, 11:Endrin, 12:Endosulfan II, 13:p-p'DDD, 14:Endrin Aldheyde, 15: Endosulfan Sulphate, 16:p-

min. The ion trap spectrometer was operated in electron ionisation (EI) mode.

and trap temperature were 250 °C and 170 °C respectively.

### **4.1. Key results about extraction and clean-up method**

Two extraction and clean-up methods have been developed, tested and optimised for the extraction of 20 OCPs from animal feed sample and subcutaneous fatty tissue samples from bovine.

Large amounts of lipids were extracted when n-hexane or acetone was used as extraction solvents. In general, complex mixtures of of several types of lipids were co-extracted during the extraction of chlorinated pesticides from biological sample. Triglycerides and sterol es‐ ters are the major components in meat fats.

The key point of the extraction method take advantage of significant difference of melting points between lipids (below about 40 C) and chlorinated pesticides (above 260 C), so that lipid components can be easily separated from chlorinated compounds. After extraction, lip‐ ids in organic extracts were precipitated as frozen at -24 C in the freezer, while chlorinated compounds were still dissolved in cold organic solvents. Thus frozen-lipids can be removed just by filtering extracts. During overall process, approximately 90% of lipids were eliminat‐ ed without any significant loss of pesticides. After freezing-lipid filtration, the remaining in‐ terferences were successfully removed by a solid-phase (SPE) Florisil cartridge.

Sample clean-up was necessary for the removal of polar coextracted substances. Florisil car‐ tridges have been employed for that purpose since that adsorbent has proved to be very effi‐ cient for the clean-up of food samples.

#### **4.2. Optimisation of MS/MS transitions**

From full scan spectra, the most intense higher mass precursor ions were selected for devel‐ opment of MRM method. For the most of the analytes these were the base peak ions in the mass spectra, but in some cases higher mass ions of lower intensity were selected to mini‐ mise the possibility of matrix interferences. Precursor ions were examined using different collision energies (automated method development) and the most intense product ions were selected for each precursor ion. The products ions for all OCPs determined in this study are summarise in table 1.

For quantification of the target analytes linear calibration curves for all pesticides over six calibration levels (0.005 mg kg-1-1.5 mg kg -1) using a feed and fat blank samples were pre‐ pared taking also in consideration the MRLs levels for each compounds. In quantitative analysis one of the main problems is the suppression/enhancement of the analyte response caused by sample matrix components. Calibration curves were performed by using matrixmatched (in each matrix) because the feed and fat samples contain many compounds that are co-extracted in the extraction organic solvent. The use of Florisil-SPE tries to avoid ma‐ trix effect using a clean-up step, but this not eliminates completely the problem. A matrix effect on the analytical signal due to the matrix was noticed for most pesticides.


**Subcutaneous fat tissue Animal feed**

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

MRL\*\* (mg kg-1)

LOD (mg kg-1)

http://dx.doi.org/10.5772/54182

LOQ (mg kg-1) 273

LOQ (mg kg-1)

α-BHC 0.5 0.007 0.024 0.02 0.002 0.016

β-BHC 0.1 0.012 0.041 0.01 0.003 0.010 γ-BHC 0.02 0.001 0.006 0.2 0.012 0.04 δ-BHC 0.5 0.007 0.023 0.02 - - Heptachlor 0.2 0.004 0.010 0.01 0.001 0.005 Aldrin 0.5 0.003 0.010 0.01 0.004 0.015

Hexachlorobenzene 0.2 0.007 0.026 0.01 0.003 0.011

Heptachlor epoxide 0.2 0.002 0.008 0.01 0.002 0.009 γ-Clordane 0.05 0.005 0.019 0.02 0.003 0.013 Endosulfan I 0.05 0.003 0.012 0.1 0.007 0.024 α-Clordane 0.05 0.005 0.019 0.02 0.005 0.017 Dieldrin 0.2 0.002 0.008 0.01 0.002 0.007 p-p' DDE 1 0.001 0.005 0.05 0.003 0.012 Endrin 0.05 0.002 0.007 0.01 0.004 0.013 Endosulfan II 0.05 0.003 0.010 0.1 0.004 0.016 p-p' DDD 1 0.002 0.008 0.05 0.002 0.008 Endrin aldehyde 0.05 0.003 0.011 0.02 - - Endosulfan sulphate 0.05 0.002 0.006 0.1 0.007 0.023 p-p' DDT 1 0.004 0.001 0.05 0.006 0.02 Methoxychlor 0.01 0.002 0.008 0.5 0.002 0.007

\*= MRLs of EU regulation guidelines (CE 32/2002); \*\* = MRLs of EU regulation guidelines (CE 396/2005)

**4.3. Occurrence of OCPs in animal feed samples and subcutaneous fat samples**

methoxychlor and aldrin. The frequency of detection is presented in figure. 6.

**Table 2.** MRLs, limits of detection (LOD) and limits of quantification (LOQ) for OCPs in fat and feed samples.

The OCPs residues may concentrate in the adipose tissue and in blood serum of animals lead‐ ing to environmental persistence, bioconcentration and biomagnifications through the food chain. Pesticides contamination of meat as well as chicken resulting from feeding a diet con‐ taining a low concentration of pesticides is a well established fact. [63,64] OCPs residues in feed may be ingested bi herbivores and eventually find their way into the animal body which ulti‐ mately results in contamination of milk, meat eggs, etc. consumed by human being. [65,66]

The most pesticides detected in animal feed were p-p' DDT, heptachlor followed by lindane,

**OCPs**

MRL\* (mg kg-1)

LOD (mg kg-1)

**Table 1.** Summary of precursor ions and products ions selected for analysis of OCPs n EI mode and linearity for fat and feed sample calibration curves.

The linearity of the curves was studied for each pesticide considering the area of the peak relative to the internal standard. The calibration data are given in table 2, showing a good linearity of the response for all pesticides at concentration within the interval tested.

LOD and LOQ were evaluated taking into account the baseline noise variations in the chro‐ matogram obtained from the analysis of blank feed and blank fat samples (n=10). The LOD and LOQ were defined as the concentration of the analyte that produced a signal-to-noise ratio of 3 times and 10 times the standard deviation respectively above the blank signal. Ta‐ ble 2 shows the values in mg kg-1 of feed and fat sample calculated with blank sample ex‐ tracts. The values are similar to those obtained by other authors for the LOD and LOQ in feed animal samples. LOD and LOQ values for subcutaneous fat sample are not present in literature. Our results are very similar to that obtained in fish muscle and meat.


**OCPs**

272 Food Industry

feed sample calibration curves.

**R.T. (min)** **Precursor ion (m/z)**

**Product ions (m/z)**

α-BHC 18.01 181 109, 142 1.0 0,9997 0,9974

β-BHC 19.82 181 109, 145 1.0 0,9980 0,9950 γ-BHC 20.12 181 109, 145 1.0 0,9984 0,9985 δ-BHC 21.73 181 109, 145 1.0 0,9994 - Heptachlor 24.54 272 100, 237 0.4 0,9987 0,9984 Aldrin 26.63 293 220, 255 0.8 0,9992 0,9951

Hexachlorobenzene 18.41 286 214, 249 1.0 0,9974 0,9951

Heptachlor epoxide 29.21 353 263, 334 0.7 0,9982 0,9977 γ-Clordane 30.66 375 266, 301 0.8 0,9944 0,9945 Endosulfan I 31.37 241 170, 260 0.9 0,9993 0,9978 α-Clordane 31.61 375 266, 301 0.8 0,9935 0,9910 Dieldrin 32.96 263 193, 228 0.7 0,9988 0,9979 p-p' DDE 33.16 318 246, 283 0.7 0,9974 0,9987 Endrin 34.23 263 193, 228 0.7 0,9982 0,9975 Endosulfan II 34.84 241 170, 260 0.9 0,9951 0,9973 p-p' DDD 35.66 235 165, 199 0.6 0,9958 0,9976 Endrin aldheyde 36.03 345 243, 279 0.7 0,9968 - Endosulfan sulphate 37.43 387 251, 289 0.6 0,9988 0,9925 p-p' DDT 37.84 235 165, 199 0.6 0,9996 0,9971 Methoxychlor 41.04 227 196, 212 0.7 0,9982 0,9991

**Table 1.** Summary of precursor ions and products ions selected for analysis of OCPs n EI mode and linearity for fat and

The linearity of the curves was studied for each pesticide considering the area of the peak relative to the internal standard. The calibration data are given in table 2, showing a good

LOD and LOQ were evaluated taking into account the baseline noise variations in the chro‐ matogram obtained from the analysis of blank feed and blank fat samples (n=10). The LOD and LOQ were defined as the concentration of the analyte that produced a signal-to-noise ratio of 3 times and 10 times the standard deviation respectively above the blank signal. Ta‐ ble 2 shows the values in mg kg-1 of feed and fat sample calculated with blank sample ex‐ tracts. The values are similar to those obtained by other authors for the LOD and LOQ in feed animal samples. LOD and LOQ values for subcutaneous fat sample are not present in

linearity of the response for all pesticides at concentration within the interval tested.

literature. Our results are very similar to that obtained in fish muscle and meat.

**Excitation voltage (V)** **Linearity fat ( r2)**

**Linearity Feed ( r2)**

**Table 2.** MRLs, limits of detection (LOD) and limits of quantification (LOQ) for OCPs in fat and feed samples.

#### **4.3. Occurrence of OCPs in animal feed samples and subcutaneous fat samples**

The OCPs residues may concentrate in the adipose tissue and in blood serum of animals lead‐ ing to environmental persistence, bioconcentration and biomagnifications through the food chain. Pesticides contamination of meat as well as chicken resulting from feeding a diet con‐ taining a low concentration of pesticides is a well established fact. [63,64] OCPs residues in feed may be ingested bi herbivores and eventually find their way into the animal body which ulti‐ mately results in contamination of milk, meat eggs, etc. consumed by human being. [65,66]

The most pesticides detected in animal feed were p-p' DDT, heptachlor followed by lindane, methoxychlor and aldrin. The frequency of detection is presented in figure. 6.

In subcutaneous fat sample the most detected OCPs were heptachlor, hexachlorobenzene detected in all samples followed by p-p' DDE, p-p'DDT, methoxychlor, lindane and p-p' DDD as shown in figure 7. Aldrin was detected both in feed samples and animal fat. The presence of aldrin in meat indicates the need for concern from the public health point of view because of its much higher toxicity than other OCPs. [67,68] These results are in ac‐ cordance with other author that found HCHS and DDTs the most compounds detected in meat samples. In general, it was observed that the p-p' isomers of DDE, DDT and DDDwere detected in samples. All detected pesticides in feed samples and fat samples did not exceed the MRLs established by the European Union for each compounds (Fig 8, 9). The concentra‐ tion of detected pesticides in the samples are summarised in table 3.

<sup>2</sup> <sup>1</sup> <sup>0</sup> <sup>0</sup>

**Figure 6.** OCPs detection frequency in feed samples

9

**number of samples**

6 6

10

**Figure 7.** OCPs detection frequency in subcutaneous fat samples

29

23

33 32 35

16

35

**OCP detection frequency in subcutaneous adipose tissue samples**

30

2

12

3 0 10

0 0 0

0

5

10

15

**number of samples**

20

25

3

1 1

9 8

0 0

**OCP detection frequency in feed samples**

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

3

<sup>1</sup> <sup>1</sup> <sup>0</sup> <sup>0</sup> <sup>0</sup> <sup>0</sup> <sup>0</sup>

http://dx.doi.org/10.5772/54182

275


**Figure 6.** OCPs detection frequency in feed samples

In subcutaneous fat sample the most detected OCPs were heptachlor, hexachlorobenzene detected in all samples followed by p-p' DDE, p-p'DDT, methoxychlor, lindane and p-p' DDD as shown in figure 7. Aldrin was detected both in feed samples and animal fat. The presence of aldrin in meat indicates the need for concern from the public health point of view because of its much higher toxicity than other OCPs. [67,68] These results are in ac‐ cordance with other author that found HCHS and DDTs the most compounds detected in meat samples. In general, it was observed that the p-p' isomers of DDE, DDT and DDDwere detected in samples. All detected pesticides in feed samples and fat samples did not exceed the MRLs established by the European Union for each compounds (Fig 8, 9). The concentra‐

**OCPs mean sd mean sd**

Σ-Heptachlor 4.11 1.15 2.16 1.02

Σ-DDT 38.68 6.60 4.12 1.79

Σ-Aldrin 8.46 6.01 4.53 1.12

Σ-Endosulfan 9.30 1.36 nd -

α-HCH 1.32 0.07 nd -

β-HCH 3.07 0.69 nd -

δ-HCH 5.67 1.51 nd -

γ-HCH 11.27 1.21 5.17 1.29

Endrin 16.91 2.82 4.15 0.63

Endrin aldheyde 6.89 1.60 12.99 1.57

Methoxychlor 3.78 1.08 nd -

Hexachlorobenzene 11.73 1.20 nd -

**Table 3.** Mean organochlorine residues levels (µg kg-1) in subcutaneous fat and feed samples

nd= not detected; sd=standard deviation

**Fat samples Feed samples**

(n=35) (±) (n=25) (±)

tion of detected pesticides in the samples are summarised in table 3.

274 Food Industry

**Figure 7.** OCPs detection frequency in subcutaneous fat samples

**Page No.** 

Notice:

corrections form.

with' column.

L. M. Chiesa

**and Fatty Bovine Tissue** 

16 **Figure 8** The compound's name are not legible in the fig (f.e. -BHC)

fig (f.e. -BHC)

**Proof Corrections Form** 

**Chapter Title: Occurrence of Organochlorine Pesticides Residues in Animal Feed** 

**SAMPLE PROOF CORRECTIONS FORM** 

With figure add below

With figure add below

**Line No. Comments Replace with** 

**Author(s) Name(s):** S. Panseri, P.A. Biondi, D. Vigo, R. Communod and

**Fig. 8.** Radar plot of detected OCPs content in (mg kg -1) feed samples in relation to MRLs (red line) **Figure 8.** Radar plot of detected OCPs content in (mg kg -1) feed samples in relation to MRLs (red line)

1

In conclusion a rapid extraction, freezing lipid filtration and GC-MS/MS measurement methods were developed and used to measure chlorinated pesticide levels in animal feed sample and subcutaneous fatty tissue in order to assess the possible concentration phe‐ nomena of these persistent compounds. The freezing lipid filtration combined with Flori‐ sil-SPE cartridge enabled efficient removal of lipids extracted from feed and fat samples without significant loss of pesticides. Hence, the method offers a rapid and valid screen‐ ing tool with high sensitivity for determination of organochlorine pesticides based on

Occurrence of Organochlorine Pesticides Residues in Animal Feed and Fatty Bovine Tissue

http://dx.doi.org/10.5772/54182

277

The subcutaneous fatty bovine tissue has been confirmed as target organ able to concentrate pesticides with lipophilic behaviour like organochlorine residues. The feed could also repre‐ sent a possible source for contamination of OCPs through the food-chain. Therefore, the de‐ termination of pesticides residues in feed and food is today necessary for ensuring that human exposure to contaminants, especially by dietary intake, does not exceed acceptable levels for heath. One analytical challenge in the food safety is to present reliable results with

, R. Communod1

1 Department of Veterinary Science and Public Health, Faculty of Veterinary Medicine, Uni‐

2 Department of Department of Health, Animal Science and Food Safety, Faculty of Veteri‐

[1] Benbrook C.M. Organochlorine Residues Pose Surprisingly High Dietary Risks. J.

[2] Lehotay S.J., Mastovska K., Yun S.J. Evaluation of Two Fast And Easy Methods For Pesticide Residue Analysis In Fatty Food Matrices. J. Aoac Int. 2005; 88 630.

[3] Qiu X., Zhu T., Yao B., Hu, J. Hu, S. Contribution of Dicofol To The Current Ddt Pol‐

[4] Bilrha, H., Roy, R., Moreau, B., Belles-Isles, M., Dewailly, E., Ayotte, P.,. In vitro acti‐ vation of cord blood mononuclear cells and cytokine production in a remote coastal population exposed to organochlorines and methyl mercury. Environ. Health Per‐

and L. M. Chiesa 1

GC-MS/MS detection.

respect to official guidelines.

, P.A. Biondi2

versity of Milan, Milan, Italy

spect. 2003; 111.

, D. Vigo1

Epidemiol. Community Health 2002; 56 822.

lution In China. Environ.Sci. Technol. 2005; 39 4385.

nary Medicine, University of Milan Milan, Italy

**Author details**

S. Panseri1

**References**

2

**Fig. 9.** Radar plot of detected OCPs content (mg kg -1) in subcutaneous fat tissue samples in relation to MRLs (red line) **Figure 9.** Radar plot of detected OCPs content (mg kg -1) in subcutaneous fat tissue samples in relation to MRLs (red line)





why such formatting was removed from the original manuscript.



new, corrected references list after the proofreading form table.

In conclusion a rapid extraction, freezing lipid filtration and GC-MS/MS measurement methods were developed and used to measure chlorinated pesticide levels in animal feed sample and subcutaneous fatty tissue in order to assess the possible concentration phe‐ nomena of these persistent compounds. The freezing lipid filtration combined with Flori‐ sil-SPE cartridge enabled efficient removal of lipids extracted from feed and fat samples without significant loss of pesticides. Hence, the method offers a rapid and valid screen‐ ing tool with high sensitivity for determination of organochlorine pesticides based on GC-MS/MS detection.

The subcutaneous fatty bovine tissue has been confirmed as target organ able to concentrate pesticides with lipophilic behaviour like organochlorine residues. The feed could also repre‐ sent a possible source for contamination of OCPs through the food-chain. Therefore, the de‐ termination of pesticides residues in feed and food is today necessary for ensuring that human exposure to contaminants, especially by dietary intake, does not exceed acceptable levels for heath. One analytical challenge in the food safety is to present reliable results with respect to official guidelines.

## **Author details**

S. Panseri1 , P.A. Biondi2 , D. Vigo1 , R. Communod1 and L. M. Chiesa 1

1 Department of Veterinary Science and Public Health, Faculty of Veterinary Medicine, Uni‐ versity of Milan, Milan, Italy

2 Department of Department of Health, Animal Science and Food Safety, Faculty of Veteri‐ nary Medicine, University of Milan Milan, Italy

## **References**

1

2

**Proof Corrections Form** 

**Chapter Title: Occurrence of Organochlorine Pesticides Residues in Animal Feed** 

**SAMPLE PROOF CORRECTIONS FORM** 

0.01

0.02

0.2 1

Σ-DDT

0.01

0.000

ENDRIN α-BHC

**Fig. 8.** Radar plot of detected OCPs content in (mg kg -1) feed samples in relation to MRLs (red line) **Figure 8.** Radar plot of detected OCPs content in (mg kg -1) feed samples in relation to MRLs (red line)

0.001

0.010

0.100

1.000 Σ-HEPTACHLOR

With figure add below

With figure add below

0.05

Σ-DDT

0.5

Σ-ALDRIN

Σ-ENDOSULFAN

0.05

0.5

α-BHC

0.1

β-BHC

0.5

δ-BHC

**Fig. 9.** Radar plot of detected OCPs content (mg kg -1) in subcutaneous fat tissue samples in relation to MRLs (red line)

**Figure 9.** Radar plot of detected OCPs content (mg kg -1) in subcutaneous fat tissue samples in relation to MRLs (red line)





0.000

0.001

0.010

0.100

1.000 Σ-HEPTACHLOR

0.02

0.01

0.05

ENDRIN

ENDRIN ALDEHIDE

Notice:

corrections form.

with' column.

HEXACHLOROBENZENE

γ-BHC (Lindane)

why such formatting was removed from the original manuscript.



new, corrected references list after the proofreading form table.

0.05

0.2

METHOXYCHLOR

Σ-ALDRIN

0.01

**Line No. Comments Replace with** 

**Author(s) Name(s):** S. Panseri, P.A. Biondi, D. Vigo, R. Communod and

L. M. Chiesa

**Page No.** 

276 Food Industry

**and Fatty Bovine Tissue** 

16 **Figure 8** The compound's name are not legible in the fig (f.e. -BHC)

16 **Figure 9** The compound's name are not legible in the fig (f.e. -BHC)

0.2

γ-BHC (Lindane)

METHOXYCHLOR

0.5


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[67] Gillespie, A.M. Walters, S.M. Semi-Preparative Reverse Phase Hplc Fractionation Of

[68] Van Der Hoff G.R., Van Beuzekom, A.C. Brinkman, U.A. Baumann, R.A. Van Zoo‐ nen, P. Determination Of Organochlorine Compounds In Fatty Matrices - Applica‐ tion Of Rapid Off-Line Normal-Phase Liquid Chromatographic Clean-Up. J.

Pesticides From Edible Fats And Oils. J. Liq. Chromatogr. 1989; 12 1687.

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[66] Guan, H. Brewer, W.E. Morgan, S.L. New Approach To Multiresidue Pesticide Deter‐ mination In Foods With High Fat Content Using Disposable Pipette Extraction (Dpx) And Gas Chromatography-Mass Spectrometry (Gc-Ms). J. Agric. Food Chem. 2009; 57 10531.

In Catfish (Ictalurus-Punctatus) Muscle-Tissue. J. Assoc. Off. Anal. Chem. 1991; 74

[54] Valsamaki, V.I. Boti, V.I. Sakkas, V.A. Albanis, T.A. Determination Of Organochlor‐ ine Pesticides And Polychlorinated Biphenyls In Chicken Eggs By Matrix Solid Phase

[55] Rogers, W.M. The Use Of A Solid Support For The Extraction Of Chlorinated Pesti‐ cides From Large Quantities Of Fats And Oils. J. Assoc. Off. Anal. Chem. 1972; 55

[56] Porter, M.L. Burke, J.A. An Isolation And Cleanup Procedure For Low Levels Of Or‐ ganochlorine Pesticide Residues In Fats And Oils. J. Assoc. Off. Anal. Chem. 1973; 56

[57] Bong, R.L. Determination Of Hexachlorobenzene And Mirex In Fatty Products. J. As‐

[58] Goodspeed, D.P. Chestnut, L.I. Determining Organohalides In Animal Fats Using Gel-Permeation Chromatographic Cleanup - Repeatability Study. J. Assoc. Off. Anal.

[59] Bazulic, D. Sapunar-Postruznik, J. Bilic, Arh. S. Significance Of The Quality Of Flori‐ sil In Organochlorine Pesticide Analysis; Hig. Rada Toksikol. 49 (1998) 319.

[60] Beyer, A. Biziuk, M. Comparison Of Efficiency Of Different Sorbents Used During Clean-Up Of Extracts For Determination Of Polychlorinated Biphenyls And Pesticide

[61] Determination Of Pesticides In Composite Dietary Samples By Gas Chromatogra‐ phy/Mass Spectrometry In The Selected Ion Monitoring Mode By Using A Tempera‐ ture-Programmable Large Volume Injector With Preseparation Column. Rosenblum,

[62] Xie J, Shi L, Zhu X, Wang P, Zhao Y and Su W, Mechanochemical-assisted efficient extraction of ruitn from Hibiscus mutabilis L. Innovative Food Science and Emerging

[63] Liu Y, Jin LJ, Li XY and Xu YP, Application of Mechanochemical Pretreatment to Aqueous Extraction of Isofraxidin from Eleutherococcus Senticosus. Ind. Eng. Chem.

[64] Zhu XY, Lin HM, Chen X, Xie J and Wang P, Mechanochemical-assisted Extraction and antioxidant Activities of Kaempferol Glycosides from Camellia oleifera Abel.

[65] Fillion, J. Hindle, R. Lacroix, M. Selwyn, J. Multiresidue Determination Of Pesticides In Fruit And Vegetables By Gas Chromatography Mass-Selective Detection And Liq‐

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282 Food Industry

1053.

733.


**Section 4**

**Food Processing**

**Section 4**

## **Food Processing**

**Chapter 14**

**Enzymes in Bakery:**

Ângelo Samir Melim Miguel, Tathiana Souza Martins-Meyer,

Bianca Waruar Paulo Lobo and Gisela Maria Dellamora-Ortiz

http://dx.doi.org/10.5772/53168

**1. Introduction**

ent areas.

**Current and Future Trends**

Érika Veríssimo da Costa Figueiredo,

Additional information is available at the end of the chapter

saccharification catalyzed by fungal glucoamylase.

The use of enzymes dates from much longer than their ability to catalyze reactions was rec‐ ognized and their chemical nature was known. The first completely enzymatic industrial process was developed in the years 1960 [1]. Starch processing, which is undertaken in two steps, involves liquefaction of the polysaccharide using bacterial α-amylase, followed by

After the Second World War, enzyme applications rose due to advances in industrial micro‐ biology and biochemical engineering [1]. Nowadays, enzymes are employed in many differ‐ ent areas such as food, feed, detergent, textiles, laundry, tanning, as well as pharmaceuticals, cosmetics, and fine-chemicals industries. Industrial applications account for over 80% of the global market of enzymes [2]. At least 50% of the enzymes marketed today are obtained from genetically modified organisms, employing genetic and protein engineering. Food en‐ zymes are the most widely used and still represent the major share in enzyme market.

Developments in process technology allied to the use of recombinant techniques during the last decades allowed for considerably improved yields by fermentation, increased stability, and altered specificity and selectivity of enzymes [3-5]. Those techniques thrust forward and are continuing to broaden the applications of enzymes in food technology and many differ‐

> © 2013 Miguel et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Chapter 14**

## **Enzymes in Bakery: Current and Future Trends**

Ângelo Samir Melim Miguel, Tathiana Souza Martins-Meyer, Érika Veríssimo da Costa Figueiredo, Bianca Waruar Paulo Lobo and Gisela Maria Dellamora-Ortiz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53168

## **1. Introduction**

The use of enzymes dates from much longer than their ability to catalyze reactions was rec‐ ognized and their chemical nature was known. The first completely enzymatic industrial process was developed in the years 1960 [1]. Starch processing, which is undertaken in two steps, involves liquefaction of the polysaccharide using bacterial α-amylase, followed by saccharification catalyzed by fungal glucoamylase.

After the Second World War, enzyme applications rose due to advances in industrial micro‐ biology and biochemical engineering [1]. Nowadays, enzymes are employed in many differ‐ ent areas such as food, feed, detergent, textiles, laundry, tanning, as well as pharmaceuticals, cosmetics, and fine-chemicals industries. Industrial applications account for over 80% of the global market of enzymes [2]. At least 50% of the enzymes marketed today are obtained from genetically modified organisms, employing genetic and protein engineering. Food en‐ zymes are the most widely used and still represent the major share in enzyme market.

Developments in process technology allied to the use of recombinant techniques during the last decades allowed for considerably improved yields by fermentation, increased stability, and altered specificity and selectivity of enzymes [3-5]. Those techniques thrust forward and are continuing to broaden the applications of enzymes in food technology and many differ‐ ent areas.

© 2013 Miguel et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

There are two scenarios regarding the use of enzymes, either the enzymes are used to con‐ vert the raw material into the main product, or the enzymes are used as additives to alter a functional characteristic of the product. In the first case, the enzymatic process is undertaken in optimized and controlled conditions to enhance the catalytic potential of the enzyme, whereas in the second situation it is more difficult to assure optimal conditions and to con‐ trol the enzymatic reaction [1]. An example of the first case is the use of immobilized glucose isomerase for the production of high-fructose syrups (HFS), and an example of the second scenario is the use of fungal proteases in dough making [1,6,7].

Enzymes are an important ingredient used in most bakery products. More recently enzymes have assumed an even greater importance in baking, due to the restrictions on the use of chemical additives, especially in the manufacture of bread and other fermented products [8].

The aim of this review is to discuss current applications of enzymes in the bakery industry and to explore future trends in this sector of food industry.

## **2. Bakery enzymes market**

The development of bread process was an important event in mankind. After the 19th centu‐ ry, with the agricultural mechanization, bread's quality was increased while its price was re‐ duced; thereby white bread became a commodity within almost everyone´s reach [9]. An important aspect that contributed to evolution of the baking market was the introduction of industrial enzymes in the baking process, where bakery enzymes represent a relevant seg‐ ment of the industry.

Source: Adapted from The Freedonia Group Inc., World Enzymes to 2015.

**Item**

Source: Adapted from The Freedonia Group Inc., World Enzymes to 2015.

**3. Main constituents of baked products**

lion dollars from 2000 to 2020.

2020.

**Figure 1.** Estimated world food and beverage enzyme demand participation on the world industrial enzymes in mil‐

World food and beverage enzyme demand 520 760 1220 1770 2520 Baked goods 140 250 420 625 900 Dairy 180 260 360 465 610 Other foods and beverage 200 250 440 680 1010

**Table 1.** Estimated demand of baked goods, dairy and other food & beverage enzymes in million dollars from 2000 to

Baking is a common name for the production of baked goods, such as bread, cake, pastries, bis‐ cuits, crackers, cookies, pies and tortillas, where wheat flour is both the most essential ingredi‐ ent and key source of enzyme substrates for the product [12]. Even though based on cereals other than wheat, baked goods such as gluten-free products or rye bread are also considered to be baked products [8]. Baked goods formulations vary significantly depending on the desired

**Years** 2000 2005 2010 2015 2020

Enzymes in Bakery: Current and Future Trends

http://dx.doi.org/10.5772/53168

289

Among the main industrial enzyme producers, according to Novozymes S/A report 2011 [10], Novozymes S/A occupies 47% of the market, DuPont 21%, DSM 6% and the rest is oc‐ cupied by other players. Furthermore, in that year, food and beverage enzymes represented 29% of enzyme business and biobusiness sales by the industry [10].

The world enzyme market is in evolution and a growth of 6.8% per year is expected [11]. The world food and beverage enzymes demand requires attention, because it represented \$1,220 million dollars in 2010, around 36.5% of the total world industrial enzyme demand, estimated in \$3,345 million dollars. Moreover, the world food and beverages enzymes de‐ mand is expected to be responsible for 40.1% of the world industrial enzyme demand in 2020, accounting for \$2,520 of \$6,280 million dollars of the world industrial enzyme market (Figure 1) [11].

Table 1 summarizes the world bakery and enzyme demand between 2000 and 2020, seg‐ mented according to products. It is possible to observe that the enzymes market for baked goods is expected to increase from 420 million dollars in 2010 to 900 million dollars in 2020, although maintaining its representativeness in this segment, varying from 34.4 in 2010 to 35.7% in 2020 [11].

Source: Adapted from The Freedonia Group Inc., World Enzymes to 2015.

There are two scenarios regarding the use of enzymes, either the enzymes are used to con‐ vert the raw material into the main product, or the enzymes are used as additives to alter a functional characteristic of the product. In the first case, the enzymatic process is undertaken in optimized and controlled conditions to enhance the catalytic potential of the enzyme, whereas in the second situation it is more difficult to assure optimal conditions and to con‐ trol the enzymatic reaction [1]. An example of the first case is the use of immobilized glucose isomerase for the production of high-fructose syrups (HFS), and an example of the second

Enzymes are an important ingredient used in most bakery products. More recently enzymes have assumed an even greater importance in baking, due to the restrictions on the use of chemical additives, especially in the manufacture of bread and other fermented products [8].

The aim of this review is to discuss current applications of enzymes in the bakery industry

The development of bread process was an important event in mankind. After the 19th centu‐ ry, with the agricultural mechanization, bread's quality was increased while its price was re‐ duced; thereby white bread became a commodity within almost everyone´s reach [9]. An important aspect that contributed to evolution of the baking market was the introduction of industrial enzymes in the baking process, where bakery enzymes represent a relevant seg‐

Among the main industrial enzyme producers, according to Novozymes S/A report 2011 [10], Novozymes S/A occupies 47% of the market, DuPont 21%, DSM 6% and the rest is oc‐ cupied by other players. Furthermore, in that year, food and beverage enzymes represented

The world enzyme market is in evolution and a growth of 6.8% per year is expected [11]. The world food and beverage enzymes demand requires attention, because it represented \$1,220 million dollars in 2010, around 36.5% of the total world industrial enzyme demand, estimated in \$3,345 million dollars. Moreover, the world food and beverages enzymes de‐ mand is expected to be responsible for 40.1% of the world industrial enzyme demand in 2020, accounting for \$2,520 of \$6,280 million dollars of the world industrial enzyme market

Table 1 summarizes the world bakery and enzyme demand between 2000 and 2020, seg‐ mented according to products. It is possible to observe that the enzymes market for baked goods is expected to increase from 420 million dollars in 2010 to 900 million dollars in 2020, although maintaining its representativeness in this segment, varying from 34.4 in 2010 to

scenario is the use of fungal proteases in dough making [1,6,7].

and to explore future trends in this sector of food industry.

29% of enzyme business and biobusiness sales by the industry [10].

**2. Bakery enzymes market**

ment of the industry.

288 Food Industry

(Figure 1) [11].

35.7% in 2020 [11].

**Figure 1.** Estimated world food and beverage enzyme demand participation on the world industrial enzymes in mil‐ lion dollars from 2000 to 2020.


Source: Adapted from The Freedonia Group Inc., World Enzymes to 2015.

**Table 1.** Estimated demand of baked goods, dairy and other food & beverage enzymes in million dollars from 2000 to 2020.

## **3. Main constituents of baked products**

Baking is a common name for the production of baked goods, such as bread, cake, pastries, bis‐ cuits, crackers, cookies, pies and tortillas, where wheat flour is both the most essential ingredi‐ ent and key source of enzyme substrates for the product [12]. Even though based on cereals other than wheat, baked goods such as gluten-free products or rye bread are also considered to be baked products [8]. Baked goods formulations vary significantly depending on the desired final product, and typical ingredients, apart from starch, can include wheat flour (8-16% pro‐ tein, 71-79% carbohydrate), fats, sugars, eggs, emulsifiers, milk and/or water [13].

Bread is usually made from wheat flour as raw material, which is a mixture of starch, gluten, lipids, non-starch polysaccharides and enzymes. After flour, yeast and water are mixed, com‐ plex biochemical and biophysical processes begin, catalyzed by the wheat enzymes and by the yeast, characterizing the dough phase. These processes go on in the baking phase, giving rise to bread. Extra enzymes added to the dough improve control of the baking process, allowing the use of different baking processes, reducing process time, slowing-down staling, compensat‐ ing for flour variability and substituting chemical additives [14]. Starch is the main component of products such as bread and other bakery goods and is added to different foods, acting as a thickener, water binder, emulsion stabilizer, gelling agent and fat substitute [15]. It is the most abundant constituent and most important reserve polysaccharide of many plants, including cereals, occurring as intracellular, semi-crystalline granules. On a molecular level, its major components are the glucose polymers amylose and amylopectin [16]. Amylose is an essential‐ ly linear molecule, consisting of up to 6000 glucose units with α-(1,4)-glycosidic bonds (Figure 2). On the other hand, amylopectin is a highly branched polysaccharide constituted of short α-1,4 linked linear chains of 10–60 glucose units and α-1,6 linked side chains with 15–45 glu‐ cose units (Figure 3), containing on average 2 million glucose units [17].

**Figure 3.** Partial structure of amylopectin with amylolytic enzymes action sites represented by arrows: (a) α-amylases; (b) amyloglucosidases; (c) β-amylases; (d) isoamylases and pullulanases. Both α(1→4) glycosidic linkages between the glucose units in the linear chain and one α(1→6) glycosidic linkage to a side chain of the polysaccharide are represent‐

Enzymes in Bakery: Current and Future Trends

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**Figure 4.** Schematic drawing of gluten proteins structure, where gliadins are represented by spheres and glutenins by filaments. The bulkier structure in the upper part shows the gas retained in the gluten network and consequent dough volume expansion observed in the baking process. The slimmer structure in the lower part represents plasticity,

Gluten proteins can be divided into monomeric gliadins and polymeric glutenins, based on solubility in 70% aqueous ethanol solutions [23]. Gliadins are globular proteins with molec‐

extensibility and viscous properties of the gluten matrix.

ed.

**Figure 2.** Structure of amylose chain, assumed as a left-handed spiral due to α(1→4) glycosidic bonds (n = 500 - 6000 α-D-glucopyranosyl units).

Even though many flour components such as starch, arabinoxylans and lipids affect dough rheological properties [16,18-20], gluten provides dough with extensibility, viscosity, elastici‐ ty, cohesiveness and contributes to its water absorption capacity [21]. The unique ability of wheat flour to form visco-elastic dough with gas-holding properties is mostly due to the gluten proteins, the major storage proteins of wheat, which have an essential role in breadmaking [22].

final product, and typical ingredients, apart from starch, can include wheat flour (8-16% pro‐

Bread is usually made from wheat flour as raw material, which is a mixture of starch, gluten, lipids, non-starch polysaccharides and enzymes. After flour, yeast and water are mixed, com‐ plex biochemical and biophysical processes begin, catalyzed by the wheat enzymes and by the yeast, characterizing the dough phase. These processes go on in the baking phase, giving rise to bread. Extra enzymes added to the dough improve control of the baking process, allowing the use of different baking processes, reducing process time, slowing-down staling, compensat‐ ing for flour variability and substituting chemical additives [14]. Starch is the main component of products such as bread and other bakery goods and is added to different foods, acting as a thickener, water binder, emulsion stabilizer, gelling agent and fat substitute [15]. It is the most abundant constituent and most important reserve polysaccharide of many plants, including cereals, occurring as intracellular, semi-crystalline granules. On a molecular level, its major components are the glucose polymers amylose and amylopectin [16]. Amylose is an essential‐ ly linear molecule, consisting of up to 6000 glucose units with α-(1,4)-glycosidic bonds (Figure 2). On the other hand, amylopectin is a highly branched polysaccharide constituted of short α-1,4 linked linear chains of 10–60 glucose units and α-1,6 linked side chains with 15–45 glu‐

**Figure 2.** Structure of amylose chain, assumed as a left-handed spiral due to α(1→4) glycosidic bonds (n = 500 - 6000

Even though many flour components such as starch, arabinoxylans and lipids affect dough rheological properties [16,18-20], gluten provides dough with extensibility, viscosity, elastici‐ ty, cohesiveness and contributes to its water absorption capacity [21]. The unique ability of wheat flour to form visco-elastic dough with gas-holding properties is mostly due to the gluten proteins, the major storage proteins of wheat, which have an essential role in breadmaking [22].

tein, 71-79% carbohydrate), fats, sugars, eggs, emulsifiers, milk and/or water [13].

cose units (Figure 3), containing on average 2 million glucose units [17].

α-D-glucopyranosyl units).

290 Food Industry

**Figure 3.** Partial structure of amylopectin with amylolytic enzymes action sites represented by arrows: (a) α-amylases; (b) amyloglucosidases; (c) β-amylases; (d) isoamylases and pullulanases. Both α(1→4) glycosidic linkages between the glucose units in the linear chain and one α(1→6) glycosidic linkage to a side chain of the polysaccharide are represent‐ ed.

**Figure 4.** Schematic drawing of gluten proteins structure, where gliadins are represented by spheres and glutenins by filaments. The bulkier structure in the upper part shows the gas retained in the gluten network and consequent dough volume expansion observed in the baking process. The slimmer structure in the lower part represents plasticity, extensibility and viscous properties of the gluten matrix.

Gluten proteins can be divided into monomeric gliadins and polymeric glutenins, based on solubility in 70% aqueous ethanol solutions [23]. Gliadins are globular proteins with molec‐ ular weights ranging from 30,000 to 80,000, and are further classified into three groups: α-, γ- and ω-gliadins [24,25]. Except for the ω-gliadins which lack cysteine residues, gliadins contain intramolecular disulfide bonds [21]. Glutenins consist of a heterogeneous mixture of linear polymers with a broad molecular weight range from ca. 80,000 up into the millions [22], made up of disulfide cross-linked glutenin subunits which are biochemically related to the gliadins. The intermolecular disulfide bonds stabilize the glutenin polymers [21]. Glia‐ dins mainly impart the plasticity, extensibility and viscous properties to wheat flour dough whereas glutenins are mostly responsible for the elasticity and cohesive strength of dough (Figure 4) [21,22]. Aspects such as the glutenin polymer structure, size distribution and sub‐ unit composition, as well as the gliadin/glutenin ratio are important to determine gluten quality and, consequently, the breadmaking potential of wheat flour [25-29].

In addition to starch, gluten proteins and wheat flour non-starch polysaccharides such as arabinoxylans, lipids and enzymes can considerably improve the breadmaking performance [16,18,22,36,37]. Lipids are important components in breadmaking because they provide a variety of beneficial properties during processing and storage. In bread, lipids come from multiple ingredients, largely wheat flour, shortening and surfactants in a typical bread for‐ mula [38]. Wheat flour contains about 2% lipids [23], which occur free and bound to other wheat constituents. They are classified as starch lipids and free and bound non-starch lipids, based on their solubility in solvents of different polarities [39]. The bound non-starch lipids are mainly associated with flour protein and consist predominantly of non-polar lipids,

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Bread processing can be divided into three basic operations mixing, fermentation (resting and proofing) and baking. Through baking the mainly fluid dough or batter is transformed into a predominantly solid baked product. Indirectly, baking alters the sensory properties, improving palatability, and extending the range of tastes, aromas and textures of foods pro‐

Although baking has been practiced for a very long time, the whole process is not complete‐ ly understood, possibly due to the occurrence of several coupled complex physical [42] and molecular processes [43]. The baking process therefore results in a series of physical, chemi‐ cal and biochemical changes in the product. These changes include volume expansion, evap‐ oration of water, formation of a porous structure, denaturation of protein, gelatinization of

Bread consists of an unstable, elastic, solid foam structure, containing a continuous phase made up of an elastic network of cross-linked gluten protein molecules and of leached starch polymer molecules, mainly amylose, uncomplexed and complexed with polar lipid molecules, and also a discontinuous phase of entrapped, gelatinised, swollen, deformed starch granules [45]. The nature and properties of the final product are influenced by physi‐ cal and mechanical mixing, chemical reactions (including enzyme-catalyzed reactions), and

The simplest breadmaking procedure is a straight-dough system where all bread formula in‐ gredients are mixed into developed dough [46]. A second process is the sponge and dough method where mixing of ingredients is performed in two steps. Leavening agent is prepared in the first step, by mixing together the yeast and certain quantity of water and flour. The mixture is left to develop for a few hours and then it is mixed with the other ingredients [42]. A third procedure is the Chorleywood method in which all the ingredients are mixed

In conventional breadmaking, the most commonly used leavening agent is the yeast *Saccharo‐ myces cerevisiae*, although other *Saccharomyces* species such as *S. cariocanus*, *S. mikatae, S. para‐*

while free non-starch lipids comprise mostly polar glyco- and phospholipids [40].

**4. Baking process**

duced from raw materials [41].

starch, crust formation and browning reactions [44].

thermal effects (baking time and temperature).

for a few minutes in an ultrahigh mixer [47].

Cereal non-starch polysaccharides are dietary fibre constituents, mostly composed of arabi‐ noxylans, β-glucan and arabinogalactan-peptides. Arabinoxylans make up the largest nonstarch polysaccharide fraction of cell walls of many cereals, such as wheat and rye [22,30]. They are polydisperse polyssacharides with similar structural properties, which are present in water-extractable (WE-AX) and water-unextractable (WN-AX) forms [16]. Arabinoxylans consist of a β-1,4 linked D-xylopyranosyl backbone substituted with α-L-arabinofuranose residues at the C(O)3 and/or C(O)2 positions [31-33]. Arabinose residues can be further cou‐ pled at the C(O)5 to ferulic acid through an ester linkage [34] (Figure 5). Even though minor flour constituents, arabinoxylans have the capacity to significantly affect the properties of dough and the final baked product [18]. Arabinoxylans and arabinogalactans possess impor‐ tant functional properties for the cereal industry. They can improve dough development and dough stability, by enhancing the water absorption capacity of the dough. These poly‐ saccharides also confer viscosity and may increase gas permeability by contributing to the elasticity of the protein film around them. Additionally, during breadmaking they improve loaf volume, crumb firmness, reduce retrogradation and therefore, enhance the shelf life and storage stability of bread [35].

**Figure 5.** Partial structure of an arabinoxylan: a linear main chain formed by xylan (a pentosan consisting of D-xylose units connected by β(1→4) linkages), randomly attached to L-arabinofuranose residues by α(1→3) or α(1→2) linkages.

In addition to starch, gluten proteins and wheat flour non-starch polysaccharides such as arabinoxylans, lipids and enzymes can considerably improve the breadmaking performance [16,18,22,36,37]. Lipids are important components in breadmaking because they provide a variety of beneficial properties during processing and storage. In bread, lipids come from multiple ingredients, largely wheat flour, shortening and surfactants in a typical bread for‐ mula [38]. Wheat flour contains about 2% lipids [23], which occur free and bound to other wheat constituents. They are classified as starch lipids and free and bound non-starch lipids, based on their solubility in solvents of different polarities [39]. The bound non-starch lipids are mainly associated with flour protein and consist predominantly of non-polar lipids, while free non-starch lipids comprise mostly polar glyco- and phospholipids [40].

## **4. Baking process**

ular weights ranging from 30,000 to 80,000, and are further classified into three groups: α-, γ- and ω-gliadins [24,25]. Except for the ω-gliadins which lack cysteine residues, gliadins contain intramolecular disulfide bonds [21]. Glutenins consist of a heterogeneous mixture of linear polymers with a broad molecular weight range from ca. 80,000 up into the millions [22], made up of disulfide cross-linked glutenin subunits which are biochemically related to the gliadins. The intermolecular disulfide bonds stabilize the glutenin polymers [21]. Glia‐ dins mainly impart the plasticity, extensibility and viscous properties to wheat flour dough whereas glutenins are mostly responsible for the elasticity and cohesive strength of dough (Figure 4) [21,22]. Aspects such as the glutenin polymer structure, size distribution and sub‐ unit composition, as well as the gliadin/glutenin ratio are important to determine gluten

Cereal non-starch polysaccharides are dietary fibre constituents, mostly composed of arabi‐ noxylans, β-glucan and arabinogalactan-peptides. Arabinoxylans make up the largest nonstarch polysaccharide fraction of cell walls of many cereals, such as wheat and rye [22,30]. They are polydisperse polyssacharides with similar structural properties, which are present in water-extractable (WE-AX) and water-unextractable (WN-AX) forms [16]. Arabinoxylans consist of a β-1,4 linked D-xylopyranosyl backbone substituted with α-L-arabinofuranose residues at the C(O)3 and/or C(O)2 positions [31-33]. Arabinose residues can be further cou‐ pled at the C(O)5 to ferulic acid through an ester linkage [34] (Figure 5). Even though minor flour constituents, arabinoxylans have the capacity to significantly affect the properties of dough and the final baked product [18]. Arabinoxylans and arabinogalactans possess impor‐ tant functional properties for the cereal industry. They can improve dough development and dough stability, by enhancing the water absorption capacity of the dough. These poly‐ saccharides also confer viscosity and may increase gas permeability by contributing to the elasticity of the protein film around them. Additionally, during breadmaking they improve loaf volume, crumb firmness, reduce retrogradation and therefore, enhance the shelf life and

**Figure 5.** Partial structure of an arabinoxylan: a linear main chain formed by xylan (a pentosan consisting of D-xylose units connected by β(1→4) linkages), randomly attached to L-arabinofuranose residues by α(1→3) or α(1→2) linkages.

quality and, consequently, the breadmaking potential of wheat flour [25-29].

storage stability of bread [35].

292 Food Industry

Bread processing can be divided into three basic operations mixing, fermentation (resting and proofing) and baking. Through baking the mainly fluid dough or batter is transformed into a predominantly solid baked product. Indirectly, baking alters the sensory properties, improving palatability, and extending the range of tastes, aromas and textures of foods pro‐ duced from raw materials [41].

Although baking has been practiced for a very long time, the whole process is not complete‐ ly understood, possibly due to the occurrence of several coupled complex physical [42] and molecular processes [43]. The baking process therefore results in a series of physical, chemi‐ cal and biochemical changes in the product. These changes include volume expansion, evap‐ oration of water, formation of a porous structure, denaturation of protein, gelatinization of starch, crust formation and browning reactions [44].

Bread consists of an unstable, elastic, solid foam structure, containing a continuous phase made up of an elastic network of cross-linked gluten protein molecules and of leached starch polymer molecules, mainly amylose, uncomplexed and complexed with polar lipid molecules, and also a discontinuous phase of entrapped, gelatinised, swollen, deformed starch granules [45]. The nature and properties of the final product are influenced by physi‐ cal and mechanical mixing, chemical reactions (including enzyme-catalyzed reactions), and thermal effects (baking time and temperature).

The simplest breadmaking procedure is a straight-dough system where all bread formula in‐ gredients are mixed into developed dough [46]. A second process is the sponge and dough method where mixing of ingredients is performed in two steps. Leavening agent is prepared in the first step, by mixing together the yeast and certain quantity of water and flour. The mixture is left to develop for a few hours and then it is mixed with the other ingredients [42]. A third procedure is the Chorleywood method in which all the ingredients are mixed for a few minutes in an ultrahigh mixer [47].

In conventional breadmaking, the most commonly used leavening agent is the yeast *Saccharo‐ myces cerevisiae*, although other *Saccharomyces* species such as *S. cariocanus*, *S. mikatae, S. para‐* *doxus* and *S. kudriavzevii* can be also employed [48]. Furthermore, lactic acid bacteria, mainly *Lactobacillus* species are used as leavening agents for sourdough bread production [49].

Fresh bread consists mainly of a continuous gluten network, which forms a compressed ma‐ trix between the swollen, gelatinised starch granules, and the starch network, consisting of entangled, gelatinised starch polymers [22]. It usually presents an appealing brownish and crunchy crust, a pleasant aroma, fine slicing characteristics, a soft and elastic crumb texture, and a moist mouthfeel [47]. However, when a loaf of bread is removed from the oven after baking, a series of undesirable changes called staling starts, eventually leading to deteriora‐

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Staling implies a relatively short shelf life for fresh bakery products. The loss of freshness is paralleled by an increase in crumb firmness and a decrease in flavour and aroma, leading to loss of consumer acceptance. Loss of moisture and starch retrogradation are accepted as two of the basic mechanisms in the firming of the crumb [58]. This subject has been extensively reviewed and discussed in [16,22,45]. In this context, mechanization, large scale production and increase in consumer demand for consistent product quality and longer shelf life of baked goods have led to the use of a wide range of additives (bread improvers) in the bak‐ ing industry, which include emulsifiers, soy flour, chemical redox agents and enzymes

Baking comprises the use of enzymes from three sources: the endogenous enzymes in flour, enzymes associated with the metabolic activity of the dominant microorganisms and exoge‐

The supplementation of flour and dough with enzyme improvers is a usual practice for flour standardization and also as baking aids. Enzymes are usually added to modify dough rheology, gas retention and crumb softness in bread manufacture, to modify dough rheolo‐ gy in the manufacture of pastry and biscuits, to change product softness in cake making and to reduce acrylamide formation in bakery products [8]. The enzymes can be added individu‐ ally or in complex mixtures, which may act in a synergistic way in the production of baked

Enzymes as technological aids are usually added to flour, during the mixing step of the breadmaking process. The enzymes most frequently used in breadmaking are the α-amylas‐

The industrial processing of starch is usually started by α-amylases (α-1,4-glucanohydro‐ lase). Most of the starch-converting enzymes belong to the α-amylase family or family 13 glycosyl hydrolases (GH), based on amino acid sequence and structural similarities

tion of quality [46].

[29,42,59].

**5.1. Hydrolases**

[64,65,66,67].

es from different origins [63].

**5. Enzymes used in baked products**

nous enzymes which are added in the dough [60].

goods [60-62], and their levels are usually very low.

*5.1.1. Amylases and other starch-converting enzymes*

The breadmaking process begins with the formation of dough through mixing of flour, water, yeast, sugar, salt, shortening and other ingredients. Flour particles are hydrated and sheared during mixing, and dough develops when gluten proteins form a continuous cohesive net‐ work in which the starch granules are dispersed [40]. Depolymerisation and polymerisation re‐ actions possibly give rise to the gluten network, mostly made up of glutenin [50]. Incorporation of air during dough mixing is extremely important, affecting the final crumb structure because the carbon dioxide produced by yeast during fermentation diffuses to pre-existing air bubbles [40,51]. An optimal gluten network confers dough machinability, good gas retention, high bread volume and fine crumb structure [29]. After resting, the dough is divided into loaf-sized pieces, rounded, moulded, placed on a baking tray, proofed and baked.

The combined effects of heat, moisture and time induce starch gelatinisation and pasting which together with heatsetting of gluten proteins occur during baking, giving rise to the typical solid foam structure of baked bread [22]. The partially crystalline starch is converted into amorphous, transient, gelatinised starch networks. The swollen gelatinised starch gran‐ ules are deformed, part of the starch polymers leach out of the granules and form a continu‐ ous network in the bread crumb [40,52]. Besides accumulation of amylose outside the granules, the presence of an amylose-rich region in the centre of gelatinised starch granules was found after baking [22,52].

During baking the transient gluten network formed in dough is transformed into a continu‐ ous, permanent network probably due to modifications in protein surface hydrophobicity, sulfhydryl/disulfide interchanges and formation of new disulfide cross-links [22,38,50,53]. Moreover, heat-induced sulfhydryl-disulfide exchange reactions can lead to incorporation of α- and γ-gliadins into the glutenin network [54]. Gas cell opening occurs, and besides be‐ coming gluten continuous the bread is also gas-continuous [38,40].

Macroscopic changes during baking include further expansion of the dough and crust for‐ mation and browning [40]. The oven spring is due to continued production of carbon diox‐ ide by yeast, its expansion by heating and vaporisation of ethanol and water. The bread bakes from the outside to the inside, resulting in a baked crumb [38].

The crust browning is directly related to the reducing sugars (glucose, fructose, maltose, etc.) formed by hydrolysis of starch and complex sugars of the flour, during dough making and leavening. Under heating, the sugars can undergo caramelisation, and/or the reducing sugars can react with the free amino acid groups of proteins in the Maillard reaction [54,55]. Besides, different flavour compounds are produced, giving bread its appealing smell and taste [55].

Additional interactions between biopolymers in the bread crumb occur during cooling. Amylose chains form helices, self-associate and crystallise [22,52,57]. Moreover, amylose may form more inclusion complexes with polar lipids. As a consequence, a permanent and in part crystalline amylose network is formed, providing a soft crumb in fresh bread. The gluten network organized during baking and the amylose network developed while cooling thus account for the plasticity of freshly baked bread [22].

Fresh bread consists mainly of a continuous gluten network, which forms a compressed ma‐ trix between the swollen, gelatinised starch granules, and the starch network, consisting of entangled, gelatinised starch polymers [22]. It usually presents an appealing brownish and crunchy crust, a pleasant aroma, fine slicing characteristics, a soft and elastic crumb texture, and a moist mouthfeel [47]. However, when a loaf of bread is removed from the oven after baking, a series of undesirable changes called staling starts, eventually leading to deteriora‐ tion of quality [46].

Staling implies a relatively short shelf life for fresh bakery products. The loss of freshness is paralleled by an increase in crumb firmness and a decrease in flavour and aroma, leading to loss of consumer acceptance. Loss of moisture and starch retrogradation are accepted as two of the basic mechanisms in the firming of the crumb [58]. This subject has been extensively reviewed and discussed in [16,22,45]. In this context, mechanization, large scale production and increase in consumer demand for consistent product quality and longer shelf life of baked goods have led to the use of a wide range of additives (bread improvers) in the bak‐ ing industry, which include emulsifiers, soy flour, chemical redox agents and enzymes [29,42,59].

## **5. Enzymes used in baked products**

Baking comprises the use of enzymes from three sources: the endogenous enzymes in flour, enzymes associated with the metabolic activity of the dominant microorganisms and exoge‐ nous enzymes which are added in the dough [60].

The supplementation of flour and dough with enzyme improvers is a usual practice for flour standardization and also as baking aids. Enzymes are usually added to modify dough rheology, gas retention and crumb softness in bread manufacture, to modify dough rheolo‐ gy in the manufacture of pastry and biscuits, to change product softness in cake making and to reduce acrylamide formation in bakery products [8]. The enzymes can be added individu‐ ally or in complex mixtures, which may act in a synergistic way in the production of baked goods [60-62], and their levels are usually very low.

#### **5.1. Hydrolases**

*doxus* and *S. kudriavzevii* can be also employed [48]. Furthermore, lactic acid bacteria, mainly

The breadmaking process begins with the formation of dough through mixing of flour, water, yeast, sugar, salt, shortening and other ingredients. Flour particles are hydrated and sheared during mixing, and dough develops when gluten proteins form a continuous cohesive net‐ work in which the starch granules are dispersed [40]. Depolymerisation and polymerisation re‐ actions possibly give rise to the gluten network, mostly made up of glutenin [50]. Incorporation of air during dough mixing is extremely important, affecting the final crumb structure because the carbon dioxide produced by yeast during fermentation diffuses to pre-existing air bubbles [40,51]. An optimal gluten network confers dough machinability, good gas retention, high bread volume and fine crumb structure [29]. After resting, the dough is divided into loaf-sized

The combined effects of heat, moisture and time induce starch gelatinisation and pasting which together with heatsetting of gluten proteins occur during baking, giving rise to the typical solid foam structure of baked bread [22]. The partially crystalline starch is converted into amorphous, transient, gelatinised starch networks. The swollen gelatinised starch gran‐ ules are deformed, part of the starch polymers leach out of the granules and form a continu‐ ous network in the bread crumb [40,52]. Besides accumulation of amylose outside the granules, the presence of an amylose-rich region in the centre of gelatinised starch granules

During baking the transient gluten network formed in dough is transformed into a continu‐ ous, permanent network probably due to modifications in protein surface hydrophobicity, sulfhydryl/disulfide interchanges and formation of new disulfide cross-links [22,38,50,53]. Moreover, heat-induced sulfhydryl-disulfide exchange reactions can lead to incorporation of α- and γ-gliadins into the glutenin network [54]. Gas cell opening occurs, and besides be‐

Macroscopic changes during baking include further expansion of the dough and crust for‐ mation and browning [40]. The oven spring is due to continued production of carbon diox‐ ide by yeast, its expansion by heating and vaporisation of ethanol and water. The bread

The crust browning is directly related to the reducing sugars (glucose, fructose, maltose, etc.) formed by hydrolysis of starch and complex sugars of the flour, during dough making and leavening. Under heating, the sugars can undergo caramelisation, and/or the reducing sugars can react with the free amino acid groups of proteins in the Maillard reaction [54,55]. Besides, different flavour compounds are produced, giving bread its appealing smell and taste [55].

Additional interactions between biopolymers in the bread crumb occur during cooling. Amylose chains form helices, self-associate and crystallise [22,52,57]. Moreover, amylose may form more inclusion complexes with polar lipids. As a consequence, a permanent and in part crystalline amylose network is formed, providing a soft crumb in fresh bread. The gluten network organized during baking and the amylose network developed while cooling

*Lactobacillus* species are used as leavening agents for sourdough bread production [49].

pieces, rounded, moulded, placed on a baking tray, proofed and baked.

coming gluten continuous the bread is also gas-continuous [38,40].

bakes from the outside to the inside, resulting in a baked crumb [38].

thus account for the plasticity of freshly baked bread [22].

was found after baking [22,52].

294 Food Industry

Enzymes as technological aids are usually added to flour, during the mixing step of the breadmaking process. The enzymes most frequently used in breadmaking are the α-amylas‐ es from different origins [63].

#### *5.1.1. Amylases and other starch-converting enzymes*

The industrial processing of starch is usually started by α-amylases (α-1,4-glucanohydro‐ lase). Most of the starch-converting enzymes belong to the α-amylase family or family 13 glycosyl hydrolases (GH), based on amino acid sequence and structural similarities [64,65,66,67].

α-Amylases (EC 3.2.1.1) are endoenzymes that catalyze the cleavage of α-1,4-glycosidic bonds in the inner part of the amylose or amylopectin chain. The end products of α-amylase action are oligosaccharides, with an α-configuration and varying lengths, and α-limit dex‐ trins, which are branched oligosaccharides [17]. These enzymes can be obtained from cereal, fungal, bacterial and biotechnologically altered bacterial sources. Differences in the number of binding sites and location of catalytic regions determine substrate specificity of α-amylas‐ es, the length of the oligosaccharide fragments released after hydrolysis and, consequently, the carbohydrate profile of the final product. The different forms of α-amylases also have diverse thermal stability profiles [15].

amylases with intermediate thermostability [16,22]. In this context, one of the most effective an‐ ti-staling amylases is the *Bacillus stearothermophilus* maltogenic α-amylase [22]. The anti-staling action of amylases has been attributed to the modified retrogradation behaviour of the hydro‐ lysed starch [72-74]. Yet, other researchers ascribe the effect to the interference of the low molec‐

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Proteases can be subdivided into two major groups according to their site of action: exopep‐ tidases and endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate [77]. Most of the proteolytic activity of wheat and rye flours corresponds to aspartic proteases and carboxypeptidases, which are both active in acid pH. Additionally, aspartic proteases of wheat are partly associated with gluten [78]. Neverthe‐

Proteases are used on a large commercial scale in the production of bread, baked goods, crackers and waffles [80]. These enzymes can be added to reduce mixing time, to decrease dough consistency, to assure dough uniformity, to regulate gluten strength in bread, to con‐ trol bread texture and to improve flavour [16,60]. In addition, proteases have largely re‐ placed bisulfite, which was previously used to control consistency through reduction of gluten protein disulfide bonds, while proteolysis breaks down peptide bonds. In both cases,

In bread production, a fungal acid protease is used to modify mixtures containing high glu‐ ten content. When proteases are mixed in the blend, it undergoes partial hydrolysis becom‐ ing soft and easy to pull and knead [7,60]. Proteases are also frequently added to dough preparations. These enzymes have great impact on dough rheology and the quality of bread

Proteases are also applied in the manufacture of pastries, biscuits and cookies. They act on the proteins of wheat flour, reducing gluten elasticity and therefore reducing shrinkage of dough or paste after moulding and sheeting [8,81]; for instance, hydrolysis of glutenin pro‐ teins, which are responsible for the elasticity of dough, has considerable improving effects

Hemicellulases are a diverse class of enzymes that hydrolyse hemicelluloses, a group of pol‐ ysaccharides comprising xylan, xylobiose, arabinoxylan and arabinogalactan [82]. This group includes xylanase or endo-1,4-β-xylanase (4-β-D-xylan xylanohydrolase, EC 3.2.1.8), a glycosidase that catalyses the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan and ara‐

Xylanase, also designated endoxylanase, was originally termed pentosanase [83]. A wide va‐ riety of xylanases have been reported from a plethora of microorganisms including bacteria,

ular weight dextrins with starch-starch and/or gluten-starch interactions [74-76].

less, the proteolytic activity of sound, ungerminated grain is normally low [79].

the final effect is a similar weakening of the gluten network [79].

possibly due to effects on the gluten network or on gliadin [7].

on the spread ratio of cookies [81].

*5.1.3. Hemicellulases*

binoxylan.

*5.1.2. Proteases*

Also part of the GH13 family are the exoenzymes maltogenic α-amylase (glucan 1,4-α-glu‐ canhydrolase, EC 3.2.1.133) and other maltooligosaccharide forming amylases (EC 3.2.1.60, for instance). While maltogenic α-amylase mainly releases maltose from starch, maltooligo‐ saccharide producing amylases give rise to maltotetraose or maltohexaose, among others. On the other hand, debranching enzymes, such as pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), grouped as well in the GH13 family, hydrolyse α-(1,6)-bonds removing the side-chains from amylopectin [16,17].

β-Amylases (EC 3.2.1.2) and glucoamylases (EC 3.2.1.3) are encompassed in the GH14 and GH15 families, respectively. Both are exoamylases that employ the inverting mechanism to cleave α-glycosidic bonds at the non-reducing ends of amylose and amylopectin, producing low molecular weight carbohydrates in the β-anomeric form [15,68]. β-Amylases are unable to cleave α-1,6-linkages and the final products consist of maltose and β-limit dextrin. There‐ fore hydrolysis of amylopectin is incomplete, resulting in only 50-60% conversion to mal‐ tose. In the case of amylose, the maximum degree of hydrolysis is 75-90% due to the slightly branched structure of this polysaccharide [15]. On the other hand, glucoamylase has a limit‐ ed activity on α-1,6-linkages and would possibly be able to catalyse total conversion of starch into β-glucose [16].

Malt and microbial α-amylases have been widely used in the baking industry. The malt preparation led the way for the commercial use of many other enzymes in baking [69]. Fun‐ gal α-amylases or malt are usually added to optimize amylase activity of the flour, initially aiming to increase the levels of fermentable and reducing sugars. In view of their lower ther‐ mostability, fungal α-amylases are more appropriate than malt amylases for flour standardi‐ zation. The α- and β-amylases have different but complementary functions during the breadmaking process [70]. The supplemented α-amylases break down damaged starch par‐ ticles into low molecular weight dextrins during the dough stage, while endogenous β-amy‐ lase converts these oligosaccharides into maltose which is used as fermentable sugar by the yeast or sourdough microorganisms [15,16]. The increased levels of reducing sugars lead to the formation of Maillard reaction products, intensifying bread flavour and crust colour. In addition, these enzymes can improve the gas-retention properties of fermented dough and reduce dough viscosity during starch gelatinization, with consequent improvements in product volume and softness [8,22,71].

Certain amylases are able to decrease the firming rate of bread crumb, acting as anti-staling agents. Amylase-containing anti-staling products typically consist of bacterial or fungal αamylases with intermediate thermostability [16,22]. In this context, one of the most effective an‐ ti-staling amylases is the *Bacillus stearothermophilus* maltogenic α-amylase [22]. The anti-staling action of amylases has been attributed to the modified retrogradation behaviour of the hydro‐ lysed starch [72-74]. Yet, other researchers ascribe the effect to the interference of the low molec‐ ular weight dextrins with starch-starch and/or gluten-starch interactions [74-76].

### *5.1.2. Proteases*

α-Amylases (EC 3.2.1.1) are endoenzymes that catalyze the cleavage of α-1,4-glycosidic bonds in the inner part of the amylose or amylopectin chain. The end products of α-amylase action are oligosaccharides, with an α-configuration and varying lengths, and α-limit dex‐ trins, which are branched oligosaccharides [17]. These enzymes can be obtained from cereal, fungal, bacterial and biotechnologically altered bacterial sources. Differences in the number of binding sites and location of catalytic regions determine substrate specificity of α-amylas‐ es, the length of the oligosaccharide fragments released after hydrolysis and, consequently, the carbohydrate profile of the final product. The different forms of α-amylases also have

Also part of the GH13 family are the exoenzymes maltogenic α-amylase (glucan 1,4-α-glu‐ canhydrolase, EC 3.2.1.133) and other maltooligosaccharide forming amylases (EC 3.2.1.60, for instance). While maltogenic α-amylase mainly releases maltose from starch, maltooligo‐ saccharide producing amylases give rise to maltotetraose or maltohexaose, among others. On the other hand, debranching enzymes, such as pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), grouped as well in the GH13 family, hydrolyse α-(1,6)-bonds removing the

β-Amylases (EC 3.2.1.2) and glucoamylases (EC 3.2.1.3) are encompassed in the GH14 and GH15 families, respectively. Both are exoamylases that employ the inverting mechanism to cleave α-glycosidic bonds at the non-reducing ends of amylose and amylopectin, producing low molecular weight carbohydrates in the β-anomeric form [15,68]. β-Amylases are unable to cleave α-1,6-linkages and the final products consist of maltose and β-limit dextrin. There‐ fore hydrolysis of amylopectin is incomplete, resulting in only 50-60% conversion to mal‐ tose. In the case of amylose, the maximum degree of hydrolysis is 75-90% due to the slightly branched structure of this polysaccharide [15]. On the other hand, glucoamylase has a limit‐ ed activity on α-1,6-linkages and would possibly be able to catalyse total conversion of

Malt and microbial α-amylases have been widely used in the baking industry. The malt preparation led the way for the commercial use of many other enzymes in baking [69]. Fun‐ gal α-amylases or malt are usually added to optimize amylase activity of the flour, initially aiming to increase the levels of fermentable and reducing sugars. In view of their lower ther‐ mostability, fungal α-amylases are more appropriate than malt amylases for flour standardi‐ zation. The α- and β-amylases have different but complementary functions during the breadmaking process [70]. The supplemented α-amylases break down damaged starch par‐ ticles into low molecular weight dextrins during the dough stage, while endogenous β-amy‐ lase converts these oligosaccharides into maltose which is used as fermentable sugar by the yeast or sourdough microorganisms [15,16]. The increased levels of reducing sugars lead to the formation of Maillard reaction products, intensifying bread flavour and crust colour. In addition, these enzymes can improve the gas-retention properties of fermented dough and reduce dough viscosity during starch gelatinization, with consequent improvements in

Certain amylases are able to decrease the firming rate of bread crumb, acting as anti-staling agents. Amylase-containing anti-staling products typically consist of bacterial or fungal α-

diverse thermal stability profiles [15].

296 Food Industry

side-chains from amylopectin [16,17].

starch into β-glucose [16].

product volume and softness [8,22,71].

Proteases can be subdivided into two major groups according to their site of action: exopep‐ tidases and endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate [77]. Most of the proteolytic activity of wheat and rye flours corresponds to aspartic proteases and carboxypeptidases, which are both active in acid pH. Additionally, aspartic proteases of wheat are partly associated with gluten [78]. Neverthe‐ less, the proteolytic activity of sound, ungerminated grain is normally low [79].

Proteases are used on a large commercial scale in the production of bread, baked goods, crackers and waffles [80]. These enzymes can be added to reduce mixing time, to decrease dough consistency, to assure dough uniformity, to regulate gluten strength in bread, to con‐ trol bread texture and to improve flavour [16,60]. In addition, proteases have largely re‐ placed bisulfite, which was previously used to control consistency through reduction of gluten protein disulfide bonds, while proteolysis breaks down peptide bonds. In both cases, the final effect is a similar weakening of the gluten network [79].

In bread production, a fungal acid protease is used to modify mixtures containing high glu‐ ten content. When proteases are mixed in the blend, it undergoes partial hydrolysis becom‐ ing soft and easy to pull and knead [7,60]. Proteases are also frequently added to dough preparations. These enzymes have great impact on dough rheology and the quality of bread possibly due to effects on the gluten network or on gliadin [7].

Proteases are also applied in the manufacture of pastries, biscuits and cookies. They act on the proteins of wheat flour, reducing gluten elasticity and therefore reducing shrinkage of dough or paste after moulding and sheeting [8,81]; for instance, hydrolysis of glutenin pro‐ teins, which are responsible for the elasticity of dough, has considerable improving effects on the spread ratio of cookies [81].

### *5.1.3. Hemicellulases*

Hemicellulases are a diverse class of enzymes that hydrolyse hemicelluloses, a group of pol‐ ysaccharides comprising xylan, xylobiose, arabinoxylan and arabinogalactan [82]. This group includes xylanase or endo-1,4-β-xylanase (4-β-D-xylan xylanohydrolase, EC 3.2.1.8), a glycosidase that catalyses the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan and ara‐ binoxylan.

Xylanase, also designated endoxylanase, was originally termed pentosanase [83]. A wide va‐ riety of xylanases have been reported from a plethora of microorganisms including bacteria, archaea and fungi [84]. These enzymes are mainly classified in the glycosyl hydrolase (GH) families 10 and 11 [16,64,65], although putative xylanase activities have been reported in GH families 5, 7, 8 and 43 [84,85]. GH10 xylanases are regarded to have broader substrate specif‐ icity and release shorter fragments compared to GH11 xylanases, while the latter enzymes are more susceptible to steric hindrance by arabinose substituents [86,87]. In addition, differ‐ ent endogenous xylanase inhibitors occur in cereals: *Triticum aestivum* L. xylanase inhibitor (TAXI) [88,89], xylanase inhibitor proteins (XIP-type inhibitors) [90] and TLXI-type (thauma‐ tin-like endoxylanase inhibitors) [91].

*5.1.4. Lipases*

[71,79,98].

Lipases (EC 3.1.13) or triacylglycerol acylhydrolases hydrolyse triacylglycerols (TAG) pro‐ ducing monoacylglycerols (MAG), diacylglycerols (DAG), glycerol and free fatty acids. These enzymes are widely found in nature [97]. Besides TAG lipases there are phospholi‐ pases A1 (EC 3.1.1.32), A2 (EC 3.1.1.4), C (EC 3.1.4.3), D (EC 3.1.4.4) and galactolipases (EC 3.1.1.26). Even though they are present in all cereal grains; lipase activity of white flour is usually low enough to avoid rancidity due to hydrolysis of native lipids and of baking fat

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The use of lipases in the baking segment is much more recent in comparison to α-amylases and proteases. The first generation of commercial lipase preparations was introduced to the market in the years 1990 and recently a third generation became available [59]. The latter are protein engineered enzymes, claimed to give a better effect in high speed mixing and notime dough processes. Moreover, third generation lipases have lower affinity for short chain fatty acids, which reduces the risk for off-flavour formation on account of prolonged storage

Lipases (TAG lipases) of the first generation are 1,3-specific, removing preferentially fatty acids from positions 1 and 3 in TAG. These enzymes can improve dough rheology, increase dough strength and stability, thus improving dough machinability [62,99,100]. In addition, lipases lead to an increase in volume which results in an improved, more uniform crumb

The second generation lipases act simultaneously on TAG, diacylgalactolipids and phospho‐ lipids, producing more polar lipids, providing a greater increase in volume, better stability to mechanical stress on the dough, and a fine, uniform bread crumb structure compared to the first generation lipases [43,59,101]. Moreover, a third generation lipase was found to in‐ crease expansion of the gluten network, increase the wall thickness and reduce cell density,

The surface active properties of the hydrolysis reaction products (MAG, DAG, monoacylga‐ lactolipids and lysophospholipids), along with modifications on the interactions between lipids and gluten proteins caused by the lipases, as well as the effect of these enzymes on the incorporation of air during mixing are possible mechanisms by which they affect bread vol‐ ume [101]. In this context, the roles of lipids and surfactants in breadmaking have been ex‐

The addition of lipases has been claimed to retard the rate of staling in baked products [8,103,104]. The effect of these enzymes has been attributed to *in situ* production of MAG fol‐ lowing TAG hydrolysis, although this mechanism is not completely accepted because the amount of MAG would be insufficient to account for the antistaling effect [45,99]. Lipases

The effect of a third generation lipase on the quality of high-fibre enriched brewer's spent grain breads has been evaluated. The enzyme produced beneficial effects during bread mak‐

may also be used for the development of particular flavors in bakery products [100].

ing, positively affecting loaf volume, staling rate and crumb structure [102].

of the baked goods and the use of butter or milk fat in baked products [12].

enhancing volume and crumb structure of high fibre white bread [102].

structure; hence a softer crumb is obtained [99].

tensively reviewed elsewhere [38,45].

The complete hydrolysis of arabinoxylans requires the concerted action of different en‐ zymes. The xylan backbone will be cleaved randomly by endo-1,4-β-xylanases, the main ara‐ binoxylan hydrolysing enzymes, yielding arabinoxylo-oligosaccharides. β-D-xylosidases (EC 3.2.1.37) cleave xylose monomers from the non-reducing end of arabinoxylo-oligosac‐ charides. The arabinose residues are removed by α-L-arabinofuranosidases (EC 3.2.1.55), while ferulic acid esterases (EC 3.1.1.73) cleave ester linkages between arabinose residues and ferulic acid [30,83].

Xylanases were introduced to the baking segment in the years 1970 and are most often used combined with amylases, lipases and many oxidoreductases to attain specific effects on the rheological properties of dough and organoleptic properties of bread [85]. These enzymes have also been used to improve the quality of biscuits, cakes and other baked products [71].

The most favourable xylanases for breadmaking are those that preferentially act on WU-AX and are poorly active on WE-AX, because they remove the insoluble arabinoxylans which interfere with the formation of the gluten network, giving rise to high molecular weight solubilised arabinoxylans, resulting in increased viscosity and thus enhancing dough stabili‐ ty [92-94]. As a consequence, a more stable, flexible and easy to handle dough is obtained, resulting in improved oven spring, larger loaf volume, as well as a softer crumb with im‐ proved structure [43]. Moreover, the addition of xylanases during dough processing is ex‐ pected to increase the concentration of arabinoxylo-oligosaccharides in bread, which have beneficial effects on human health [95].

The potential of GH family 8 xylanases as technological aids in baking was shown for a psy‐ chrophilic enzyme from *Pseudoalteromonas haloplanktis* and a mesophilic enzyme from *Bacil‐ lus halodurans*. Although both enzymes had a positive effect on loaf volume, psychrophilic GH8 xylanase was apparently much more efficient than the mesophilic enzyme from the same family, because much lower concentrations of the former enzyme were required to produce a similar increase in bread volume. Additionally, a psychrophilic GH10 xylanase from *Cryptococcus adeliae* was found to be ineffective [85].

Recently, a purified GH11 xylanase from *Penicillium occitanis* was evaluated as an additive during mixing of wheat flours. Significant improvements of bread characteristics, including higher final moisture content, volume and specific volume, were observed. Enhancements in sensory and textural properties were also obtained [96].

### *5.1.4. Lipases*

archaea and fungi [84]. These enzymes are mainly classified in the glycosyl hydrolase (GH) families 10 and 11 [16,64,65], although putative xylanase activities have been reported in GH families 5, 7, 8 and 43 [84,85]. GH10 xylanases are regarded to have broader substrate specif‐ icity and release shorter fragments compared to GH11 xylanases, while the latter enzymes are more susceptible to steric hindrance by arabinose substituents [86,87]. In addition, differ‐ ent endogenous xylanase inhibitors occur in cereals: *Triticum aestivum* L. xylanase inhibitor (TAXI) [88,89], xylanase inhibitor proteins (XIP-type inhibitors) [90] and TLXI-type (thauma‐

The complete hydrolysis of arabinoxylans requires the concerted action of different en‐ zymes. The xylan backbone will be cleaved randomly by endo-1,4-β-xylanases, the main ara‐ binoxylan hydrolysing enzymes, yielding arabinoxylo-oligosaccharides. β-D-xylosidases (EC 3.2.1.37) cleave xylose monomers from the non-reducing end of arabinoxylo-oligosac‐ charides. The arabinose residues are removed by α-L-arabinofuranosidases (EC 3.2.1.55), while ferulic acid esterases (EC 3.1.1.73) cleave ester linkages between arabinose residues

Xylanases were introduced to the baking segment in the years 1970 and are most often used combined with amylases, lipases and many oxidoreductases to attain specific effects on the rheological properties of dough and organoleptic properties of bread [85]. These enzymes have also been used to improve the quality of biscuits, cakes and other baked products [71].

The most favourable xylanases for breadmaking are those that preferentially act on WU-AX and are poorly active on WE-AX, because they remove the insoluble arabinoxylans which interfere with the formation of the gluten network, giving rise to high molecular weight solubilised arabinoxylans, resulting in increased viscosity and thus enhancing dough stabili‐ ty [92-94]. As a consequence, a more stable, flexible and easy to handle dough is obtained, resulting in improved oven spring, larger loaf volume, as well as a softer crumb with im‐ proved structure [43]. Moreover, the addition of xylanases during dough processing is ex‐ pected to increase the concentration of arabinoxylo-oligosaccharides in bread, which have

The potential of GH family 8 xylanases as technological aids in baking was shown for a psy‐ chrophilic enzyme from *Pseudoalteromonas haloplanktis* and a mesophilic enzyme from *Bacil‐ lus halodurans*. Although both enzymes had a positive effect on loaf volume, psychrophilic GH8 xylanase was apparently much more efficient than the mesophilic enzyme from the same family, because much lower concentrations of the former enzyme were required to produce a similar increase in bread volume. Additionally, a psychrophilic GH10 xylanase

Recently, a purified GH11 xylanase from *Penicillium occitanis* was evaluated as an additive during mixing of wheat flours. Significant improvements of bread characteristics, including higher final moisture content, volume and specific volume, were observed. Enhancements in

tin-like endoxylanase inhibitors) [91].

beneficial effects on human health [95].

from *Cryptococcus adeliae* was found to be ineffective [85].

sensory and textural properties were also obtained [96].

and ferulic acid [30,83].

298 Food Industry

Lipases (EC 3.1.13) or triacylglycerol acylhydrolases hydrolyse triacylglycerols (TAG) pro‐ ducing monoacylglycerols (MAG), diacylglycerols (DAG), glycerol and free fatty acids. These enzymes are widely found in nature [97]. Besides TAG lipases there are phospholi‐ pases A1 (EC 3.1.1.32), A2 (EC 3.1.1.4), C (EC 3.1.4.3), D (EC 3.1.4.4) and galactolipases (EC 3.1.1.26). Even though they are present in all cereal grains; lipase activity of white flour is usually low enough to avoid rancidity due to hydrolysis of native lipids and of baking fat [71,79,98].

The use of lipases in the baking segment is much more recent in comparison to α-amylases and proteases. The first generation of commercial lipase preparations was introduced to the market in the years 1990 and recently a third generation became available [59]. The latter are protein engineered enzymes, claimed to give a better effect in high speed mixing and notime dough processes. Moreover, third generation lipases have lower affinity for short chain fatty acids, which reduces the risk for off-flavour formation on account of prolonged storage of the baked goods and the use of butter or milk fat in baked products [12].

Lipases (TAG lipases) of the first generation are 1,3-specific, removing preferentially fatty acids from positions 1 and 3 in TAG. These enzymes can improve dough rheology, increase dough strength and stability, thus improving dough machinability [62,99,100]. In addition, lipases lead to an increase in volume which results in an improved, more uniform crumb structure; hence a softer crumb is obtained [99].

The second generation lipases act simultaneously on TAG, diacylgalactolipids and phospho‐ lipids, producing more polar lipids, providing a greater increase in volume, better stability to mechanical stress on the dough, and a fine, uniform bread crumb structure compared to the first generation lipases [43,59,101]. Moreover, a third generation lipase was found to in‐ crease expansion of the gluten network, increase the wall thickness and reduce cell density, enhancing volume and crumb structure of high fibre white bread [102].

The surface active properties of the hydrolysis reaction products (MAG, DAG, monoacylga‐ lactolipids and lysophospholipids), along with modifications on the interactions between lipids and gluten proteins caused by the lipases, as well as the effect of these enzymes on the incorporation of air during mixing are possible mechanisms by which they affect bread vol‐ ume [101]. In this context, the roles of lipids and surfactants in breadmaking have been ex‐ tensively reviewed elsewhere [38,45].

The addition of lipases has been claimed to retard the rate of staling in baked products [8,103,104]. The effect of these enzymes has been attributed to *in situ* production of MAG fol‐ lowing TAG hydrolysis, although this mechanism is not completely accepted because the amount of MAG would be insufficient to account for the antistaling effect [45,99]. Lipases may also be used for the development of particular flavors in bakery products [100].

The effect of a third generation lipase on the quality of high-fibre enriched brewer's spent grain breads has been evaluated. The enzyme produced beneficial effects during bread mak‐ ing, positively affecting loaf volume, staling rate and crumb structure [102].

A recent study compared three generations of lipase enzymes with the emulsifier, diacetyl tartaric esters of monoglycerides (DATEM), on white wheat flour bread. Lipases and DA‐ TEM improved most aspects of bread quality. In shorter fermentation times, DATEM, a sec‐ ond generation (Lipopan F-BG) and a third generation (Lipopan Xtra-BG) lipase were more effective. In longer fermentations, unlike the third generation lipase (Lipopan Xtra-BG), moderate amounts of the second generation lipase (Lipopan F-BG) significantly increased the bread volume [59].

Lipoxygenases are also employed to improve mixing tolerance and dough handling proper‐ ties [115]. In this case, the effect of these enzymes may be explained by oxidation of thiol groups of gluten proteins which can lead to rearrangement of intra- or inter-chain disulfide bonds [21] and also to formation of tyrosine cross-links [116], with consequent strengthening of the gluten network. As a result, improvement in dough rheology occurs, with increase in dough strength through proofing and baking, finally leading to improved loaf volume.

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On the other hand, the action of lipoxygenase can lead to undesirable flavors in bread [79,114]. These flavors are possibly due to some of the breakdown products (ketodienes)

Glucose oxidase (β-D-glucose:oxygen: 1-oxidoreductase; EC 1.1.3.4) catalyzes the oxidation of β-D-glucose to D-glucono-δ-lactone and hydrogen peroxide [118,119]. This enzyme has been obtained from different fungal sources, mainly from genus *Aspergillus* and *Penicillium*,

Glucose oxidase has been used successfully to remove residual glucose and oxygen in foods and beverages aiming to increase their shelf life. The hydrogen peroxide generated by this enzyme presents antimicrobial properties, and is easily removed by catalase utilization, which is an enzyme that catalyzes the conversion of hydrogen peroxide to oxygen and water [12,124-127]. Glucose oxidase can be used as alternative oxidizing agent instead of potassi‐ um bromate in breadmaking. Potassium bromate is an oxidizing agent that was traditionally used in baking, and its use was prohibited in many countries after it was recognized as car‐

Although the mechanism of action of glucose oxidase is still not completely understood, a possible explanation is that the hydrogen peroxide formed during catalysis promotes, indi‐ rectly, the formation of either disulfide bonds or dityrosine crosslinks, or both, in the gluten network [116,130,131]. Therefore, the increase in disulfide crosslinking and/or promotion of gelative oxidation on the gluten matrix confers dough machinability, good gas retention, high bread volume and fine crumb structure [54,132-134]. Addition of increasing glucose ox‐ idase concentrations to wheat flour dough produced significant changes on dough rheology and bread quality; and the extent of the effect was highly dependent on the amount of en‐ zyme and the original wheat flour quality [130]. Furthermore, glucose oxidase was able to recover the breadmaking ability of damaged gluten [135]. Another possibility to explain the improvements on crumb properties, in both bread and croissants, as a result of glucose oxi‐ dase catalysed reactions would be the crosslinking of the albumin/globulin fraction with both disulfide and non-disulfide bonds, and the slight occurrence of non-disulfide crosslink‐

Among the enzymes which have attracted attention for use in bakery is asparaginase. Different‐ ly from other enzymes, its use is not associated with improved bread volume, crumb softening

formed during the anaerobic reaction [117,112].

being *Aspergillus niger* the most commonly used [120-123].

*5.2.2. Glucose oxidase*

cinogenic [128,129].

ing in the gluten proteins [131].

**5.3. Other enzymes**

The application of lipase and MAG to produce fiber enriched pan bread using the straight dough method was assessed. The use of lipase dosages up to 50 ppm and MAG up to 2% indicated the possibility of replacement of MAG by lipases in fiber enriched pan bread [105].

Recently, the effects of two lipases and DATEM on the rheological and thermal properties of white and whole wheat flour doughs were compared. Lipases were able to cause modifica‐ tions in the dough components (gluten proteins and starch). The enzymes improved dough handling properties to a similar or greater extent than DATEM, increasing dough stability, maximum resistance to extension and hardness, and decreasing softening degree and sticki‐ ness. The possible role of lipases in delaying starch retrogradation was indicated by the greater extent of formation of amylose-lipid complexes promoted by lipases in comparison to DATEM [106].

#### **5.2. Oxidoreductases**

#### *5.2.1. Lipoxygenases*

Lipoxygenase (linoleate oxygen oxidoreductase, EC 1.13.11.12) is a non-heme iron-contain‐ ing dioxygenase, found in a wide variety of plant and animal tissues, which with molecular oxygen catalyses the oxidation of polyunsaturated fatty acids (PUFA) containing a *cis,cis*-1,4-pentadiene system, such as linoleic or linolenic acid, to form fatty acid hydroper‐ oxides [107,108]. These enzymes are abundant in grain legume seeds (beans and peas) and potato tubers, being minor constituents of wheat flour [107]. Multiple isoforms of lipoxyge‐ nases are found in plants; for example a multigene family encodes soybean lipoxygenases, three members of which encode the three major seed isoforms L1, L2 and L3 [109].

The main commercial sources of lipoxygenases are enzyme-active soybean flour and, to low‐ er extent, flour from other beans, such as fava beans [12]. Wheat lipoxygenase catalyses the oxidation of PUFA in the free or MAG forms [110] while soybean or horse bean lipoxygenas‐ es also catalyse the oxidation of PUFA present in TAG [111]. The transient alkyl, peroxyl and hydroxyl radicals formed during lipoxygenase catalysed reactions are able to oxidise carotenoid pigments and sulfhydryl groups in peptides and proteins present in the dough, mainly giving rise to hydroxyacids [112].

In fact, the initial application of lipoxygenases in doughs was based on their ability to bleach fat-soluble carotenoid flour pigments, through co-oxidation of carotenoids with PUFA [113,114]. However, since the endogenous lipoxygenase content of wheat flour is insufficient to give enough bleaching effect, enzyme-active soybean or fava bean flour is added [114].

Lipoxygenases are also employed to improve mixing tolerance and dough handling proper‐ ties [115]. In this case, the effect of these enzymes may be explained by oxidation of thiol groups of gluten proteins which can lead to rearrangement of intra- or inter-chain disulfide bonds [21] and also to formation of tyrosine cross-links [116], with consequent strengthening of the gluten network. As a result, improvement in dough rheology occurs, with increase in dough strength through proofing and baking, finally leading to improved loaf volume.

On the other hand, the action of lipoxygenase can lead to undesirable flavors in bread [79,114]. These flavors are possibly due to some of the breakdown products (ketodienes) formed during the anaerobic reaction [117,112].

## *5.2.2. Glucose oxidase*

A recent study compared three generations of lipase enzymes with the emulsifier, diacetyl tartaric esters of monoglycerides (DATEM), on white wheat flour bread. Lipases and DA‐ TEM improved most aspects of bread quality. In shorter fermentation times, DATEM, a sec‐ ond generation (Lipopan F-BG) and a third generation (Lipopan Xtra-BG) lipase were more effective. In longer fermentations, unlike the third generation lipase (Lipopan Xtra-BG), moderate amounts of the second generation lipase (Lipopan F-BG) significantly increased

The application of lipase and MAG to produce fiber enriched pan bread using the straight dough method was assessed. The use of lipase dosages up to 50 ppm and MAG up to 2% indicated the possibility of replacement of MAG by lipases in fiber enriched pan bread [105].

Recently, the effects of two lipases and DATEM on the rheological and thermal properties of white and whole wheat flour doughs were compared. Lipases were able to cause modifica‐ tions in the dough components (gluten proteins and starch). The enzymes improved dough handling properties to a similar or greater extent than DATEM, increasing dough stability, maximum resistance to extension and hardness, and decreasing softening degree and sticki‐ ness. The possible role of lipases in delaying starch retrogradation was indicated by the greater extent of formation of amylose-lipid complexes promoted by lipases in comparison

Lipoxygenase (linoleate oxygen oxidoreductase, EC 1.13.11.12) is a non-heme iron-contain‐ ing dioxygenase, found in a wide variety of plant and animal tissues, which with molecular oxygen catalyses the oxidation of polyunsaturated fatty acids (PUFA) containing a *cis,cis*-1,4-pentadiene system, such as linoleic or linolenic acid, to form fatty acid hydroper‐ oxides [107,108]. These enzymes are abundant in grain legume seeds (beans and peas) and potato tubers, being minor constituents of wheat flour [107]. Multiple isoforms of lipoxyge‐ nases are found in plants; for example a multigene family encodes soybean lipoxygenases,

The main commercial sources of lipoxygenases are enzyme-active soybean flour and, to low‐ er extent, flour from other beans, such as fava beans [12]. Wheat lipoxygenase catalyses the oxidation of PUFA in the free or MAG forms [110] while soybean or horse bean lipoxygenas‐ es also catalyse the oxidation of PUFA present in TAG [111]. The transient alkyl, peroxyl and hydroxyl radicals formed during lipoxygenase catalysed reactions are able to oxidise carotenoid pigments and sulfhydryl groups in peptides and proteins present in the dough,

In fact, the initial application of lipoxygenases in doughs was based on their ability to bleach fat-soluble carotenoid flour pigments, through co-oxidation of carotenoids with PUFA [113,114]. However, since the endogenous lipoxygenase content of wheat flour is insufficient to give enough bleaching effect, enzyme-active soybean or fava bean flour is added [114].

three members of which encode the three major seed isoforms L1, L2 and L3 [109].

the bread volume [59].

300 Food Industry

to DATEM [106].

**5.2. Oxidoreductases**

*5.2.1. Lipoxygenases*

mainly giving rise to hydroxyacids [112].

Glucose oxidase (β-D-glucose:oxygen: 1-oxidoreductase; EC 1.1.3.4) catalyzes the oxidation of β-D-glucose to D-glucono-δ-lactone and hydrogen peroxide [118,119]. This enzyme has been obtained from different fungal sources, mainly from genus *Aspergillus* and *Penicillium*, being *Aspergillus niger* the most commonly used [120-123].

Glucose oxidase has been used successfully to remove residual glucose and oxygen in foods and beverages aiming to increase their shelf life. The hydrogen peroxide generated by this enzyme presents antimicrobial properties, and is easily removed by catalase utilization, which is an enzyme that catalyzes the conversion of hydrogen peroxide to oxygen and water [12,124-127]. Glucose oxidase can be used as alternative oxidizing agent instead of potassi‐ um bromate in breadmaking. Potassium bromate is an oxidizing agent that was traditionally used in baking, and its use was prohibited in many countries after it was recognized as car‐ cinogenic [128,129].

Although the mechanism of action of glucose oxidase is still not completely understood, a possible explanation is that the hydrogen peroxide formed during catalysis promotes, indi‐ rectly, the formation of either disulfide bonds or dityrosine crosslinks, or both, in the gluten network [116,130,131]. Therefore, the increase in disulfide crosslinking and/or promotion of gelative oxidation on the gluten matrix confers dough machinability, good gas retention, high bread volume and fine crumb structure [54,132-134]. Addition of increasing glucose ox‐ idase concentrations to wheat flour dough produced significant changes on dough rheology and bread quality; and the extent of the effect was highly dependent on the amount of en‐ zyme and the original wheat flour quality [130]. Furthermore, glucose oxidase was able to recover the breadmaking ability of damaged gluten [135]. Another possibility to explain the improvements on crumb properties, in both bread and croissants, as a result of glucose oxi‐ dase catalysed reactions would be the crosslinking of the albumin/globulin fraction with both disulfide and non-disulfide bonds, and the slight occurrence of non-disulfide crosslink‐ ing in the gluten proteins [131].

#### **5.3. Other enzymes**

Among the enzymes which have attracted attention for use in bakery is asparaginase. Different‐ ly from other enzymes, its use is not associated with improved bread volume, crumb softening or reduced staling. Instead, asparaginase is claimed to have a high potential of reducing forma‐ tion of acrylamide during baking [136-138]. Asparaginase (L-asparagine amidohydrolases, EC 3.5.1.1) catalyses the hydrolysis of asparagine to aspartic acid and ammonium, removing the precursor of acrylamide formation [139]. Acrylamide, classified as a probable human carcino‐ gen, is formed in heated foods via Maillard reaction between asparagine and a carbonyl source [137,138,140,141]. Although asparaginase can be found among living organisms, including an‐ imals, plants and microorganisms, filamentous fungi as *Aspergillus oryzae* and *A. niger* have been explored for enzyme preparation aiming commercial purposes [142-144].

Transglutaminases (EC 2.3.2.13) from microbial sources also have potential for application in bakery products. Food proteins can be modified through cross-linking by transglutami‐ nases, resulting in textured products, protecting lysine in food proteins from undesired chemical reactions, encapsulating lipids and lipid-soluble materials, forming heat and water resistant films, improving elasticity and water-holding capacity, modifying solubility and functional properties, and producing food proteins of higher nutritive value [29,145-153].

Laccase (EC 1.10.3.2) is a copper containing enzyme that catalyses the oxidation of a wide vari‐ ety of phenolic compounds via one-electron removal, generating reactive phenolic radicals [29,154]. This enzyme is very interesting for baking due its ablity to cross-link the esterified ferulic acid on the arabinoxylan fraction of dough, resulting in a strong arabinoxylan network [155]. It was also reported that laccase may improve crumb structure and softness of baked products. Futhermore, increases in strength and stability, as well as reduced stickness of dough, which confers improvement of machinability, have been described [149,155-157].

A summary of the main applications of different classes of enzymes in the baking industry

[endo]

glycosidic

glycosidic

Terminal α-L-Arabinofuranoside residues

β(1→4)-D-xylosidic bonds (nonreducing end)

*cereals Hydrolysis of linkages*

Arabinoxylan β(1→4)-D-xylosidic bonds

**Applications in baked products**

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Removal of insoluble arabinoxylans, contributing to gluten network formation; Increase in dough viscosity, stability, with better moldable form; Improvements on rheological properties of dough; Reduction in fermentation time; Increase of bread volume; Synergistic action of glucanases on xylanolytic attack of cereals structure, providing more soluble dietary fiber in bread products; Production of prebiotic oligosaccharides in bread.

**References**

[30,71,83,95]

[71]

[30,71,83]

[71,83,85,95,165,1 76,177]

[71,83,95,102]

[83,95]

is presented in tables 2, 3 and 4.

*Cellulases and Hemicellulases*

Endo β(1,4)-D-xylanase (EC 3.2.1.8) or endoxylanase

α-L-Arabinosidase (EC

3.2.1.55) Arabinoxylan

**Table 3.** Applications of cellulases and hemicellulases in baking.

β-D-Xylosidase (EC 3.2.1.37) Arabinoxylan

**Enzyme (classification) Substrate in foods Reaction**

*Non-starch components of*

Cellulase (EC 3.2.1.4) Cellulose and β-glucan β(1→4)-D-glycosidic

Lamarinase (EC 3.2.1.6) β-glucans β(1→3)- and β(1→4)-D-

Lichenase (EC 3.2.1.73) β-glucans β(1→3)- and β(1→4)-D-


**Table 2.** Applications of starch modifying enzymes in baking.

Transglutaminases (EC 2.3.2.13) from microbial sources also have potential for application in bakery products. Food proteins can be modified through cross-linking by transglutami‐ nases, resulting in textured products, protecting lysine in food proteins from undesired chemical reactions, encapsulating lipids and lipid-soluble materials, forming heat and water resistant films, improving elasticity and water-holding capacity, modifying solubility and functional properties, and producing food proteins of higher nutritive value [29,145-153].

or reduced staling. Instead, asparaginase is claimed to have a high potential of reducing forma‐ tion of acrylamide during baking [136-138]. Asparaginase (L-asparagine amidohydrolases, EC 3.5.1.1) catalyses the hydrolysis of asparagine to aspartic acid and ammonium, removing the precursor of acrylamide formation [139]. Acrylamide, classified as a probable human carcino‐ gen, is formed in heated foods via Maillard reaction between asparagine and a carbonyl source [137,138,140,141]. Although asparaginase can be found among living organisms, including an‐ imals, plants and microorganisms, filamentous fungi as *Aspergillus oryzae* and *A. niger* have

**Reaction Applications in baked**

**products**

Generation of fermentable compounds; Increase in bread volume; Reduction in fermentation time; Improvement in dough viscosity, rheology and bread softness; Improvement in bread texture; Formation of reducing sugars and subsequent Maillard reaction products, intensifying bread flavor and color; Decrease of bread crumb firming rate; Anti-staling effects.

liberating maltose [16,17,22,74,175]

**References**

[16,17,22,48,74,174]

[16,17,22,175]

[16,17,22]

[16,17,22,175]

[17]

been explored for enzyme preparation aiming commercial purposes [142-144].

α(1→4)-D-glycosidic [endo], liberating α*dextrins*

α(1→4)-D-glycosidic [exo], liberating βdextrins and β-maltose

α(1→4)- and α(1→6)-Dglucosidic, liberating β*glucose*

Pullulanase (EC 3.2.1.41) Amylopectin α(1→6)-D-glycosidic [16,17,22] Isoamylase (EC 3.2.1.68) Amylopectin α(1→6)-D-glycosidic [16,17,22]

α(1→4)-D-glycosidic,

Liberation of maltotetraose or maltohexaose

Hydrolysis of α(1→4) glycosidic bonds and transference of a reducing group to a non-reducing acceptor (monosaccharide unit)

**Substrate in foods**

*Amylolytic enzymes Starch Hydrolysis of linkages*

Amylose and amylopectin

amylopectin

Amylose and amylopectin

Amylose and amylopectin

Amylose and amylopectin

Amylose, amylopectin and dextrins

**Table 2.** Applications of starch modifying enzymes in baking.

**Enzyme (classification)**

302 Food Industry

α-Amylases (EC 3.2.1.1) or α-(1,4) glucanhydrolases

> Glucoamylase (EC 3.2.1.3) or amyloglucosidase

Maltogenic α-amylase (EC 3.2.1.133)

Maltooligosaccharides forming amylases ( glucan 1,4-αmaltotetraohydrolase) (ex., EC.3.2.1.60)

Transferases Amylomaltases (EC 2.4.1.25) Amylosucrases (EC 2.4.1.4) Ciclodextrin glycosyltransferases (EC 2.4.1.19)

β-Amylases (EC 3.2.1.2) Amylose and

Laccase (EC 1.10.3.2) is a copper containing enzyme that catalyses the oxidation of a wide vari‐ ety of phenolic compounds via one-electron removal, generating reactive phenolic radicals [29,154]. This enzyme is very interesting for baking due its ablity to cross-link the esterified ferulic acid on the arabinoxylan fraction of dough, resulting in a strong arabinoxylan network [155]. It was also reported that laccase may improve crumb structure and softness of baked products. Futhermore, increases in strength and stability, as well as reduced stickness of dough, which confers improvement of machinability, have been described [149,155-157].

A summary of the main applications of different classes of enzymes in the baking industry is presented in tables 2, 3 and 4.


**Table 3.** Applications of cellulases and hemicellulases in baking.


**Enzyme (classification)**

or benzenediol:oxygen oxidoreductase

sulfhydryl groups in gluten proteins

proteins

Sulfhydryl oxidase Sulfhydryl groups in

**5.4. Use of enzyme combinations**

**Substrate in foods Reaction Applications in baked products References**

Help gluten network formation and

increase dough stability. [79,162]

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Oxidation of sulfhydryl groups

necessary in order to provide answers to the more complicated questions [158].

but it may be reversed with opposite amylase, xylanase and protease effects.

**Table 4.** Applications of proteases, transglutaminases, lipases and esterases, and oxidoreductases in baking.

It is common practice to use mixtures of enzymes, some of which are commercially available. The enzymes may act individually or present a synergistic effect. The trend is to choose and control the use of complex mixtures of enzymes which may act in a synergistic way and can ex‐ ert a better effect (than the individually used) on the different flour components [60]. Recent advances in understanding of the dough forming and overall baking processes at the molecu‐ lar level have focused attention on improvements that can be achieved by application of more specially tailored enzymes alone or in combinations. Usually, integrated experimental design and optimization followed by chemical analyses, rheological experiments and baking trials are

The use of a combination of enzymatic preparations of amylases, xylanases and lipases has been reported by different authors [60,102,159]. This specific mixture is claimed to increase bread volume and shelf-life. The use of α-amylase and glucose oxidase to replace bromate led to a significant improvement in dough extensibility and bread volume [160]. Addition of commercial enzyme mixtures, containing α-amylase and lipase activities to produce bread samples, using the straight dough method, had a beneficial effect on bread keeping proper‐ ties and resulted in the formation of a more thermostable amylose-lipid complex compared to the control bread [161]. Amylopectin retrogradation was inhibited by the use of the en‐ zyme combinations and this effect was strongly related to a decrease in crumb-firming rates.

The combined use of different enzymes, classified as gluten degrading (like proteases) or ad‐ juvants, such as amylases and xylanases, with a group of crosslink promoting enzymes, such as transglutaminases and glucose oxidase, was also studied [149]. Better shaped bread could be obtained after the use of gluten degrading or adjuvant enzymes, and association with transglutaminase resulted in improvements on texture and rheological properties. The crumb firmness which can further lead to staling, can result from transglutaminase action,

In a similar way, combinations of enzymes classified as carbohydrate degrading, including amylases and xylanases (pentosanases), and crosslink promoting enzymes, like transgluta‐ minases and oxidases, including glucose oxidase, laccase [149], lipoxygenase and sulfhydryl oxidase [79,162] were evaluated. The most frequent associations contained xylanases and glucose oxidase, but addition of laccase and transglutaminase was also employed. The hy‐


**Table 4.** Applications of proteases, transglutaminases, lipases and esterases, and oxidoreductases in baking.

#### **5.4. Use of enzyme combinations**

**Enzyme (classification)**

304 Food Industry

Proteases (EC 3.4.)

Transglutaminases Protein-glutamine γ-glutamyltransferase (EC 2.3.2.13)

*Lipases and*

Glicose oxidase (EC 1.1.3.4) β-D-glucose:oxygen 1-oxidoreductase

Lipoxygenase (EC 1.13.11.12) linoleate:oxygen 13-oxidoreductase

> Laccase (EC 1.10.3.2)

Lipase (EC 3.1.1.3) Triacylglycerols

Gluten proteins Gliadin and glutenin

*Gluten Proteins*

*esterases Lipids Hydrolysis of ester*

*Oxidoreductases Various Oxi-reductions*

β-D-glucose

Polyunsaturated fatty acids

Feruloyl esters of arabinoxylans;

*Hydrolysis of peptide bonds*

Acyl-transfer reaction between γ-carboxyamide and primary amines

*bonds*

Liberation of free fatty acids

Oxidation of β-D-glucose to gluconic acid

Oxidation of fatty acids

Oxidation of phenol groups

**Substrate in foods Reaction Applications in baked products References**

Reduction of dough mixing time; Control of dough rheology or viscoelastic properties of gluten strength in bread; Enhance dough extensibility; Increase loaf or bread volumes; Formation of aminoacids and flavors; Crispness feature on bread crust; Production of gluten-free products.

Cross-link between gluten and other peptides, forming a new protein network; Increase volume and improve structure of breads, better retention of gas; Improve bread crumb strength, height increase in puff pastry and croissants volume; Improve dough stability; Improve properties of gluten-free breads; Protect frozen doughs from damage.

Improvement in bread volume and dough stability; Formation of emulsifiers; Retard staling; Development of flavors.

Control on browning for Maillard reaction; Improvements in crumb properties.

Bleaching of fat-soluble flour pigments; Hydroperoxides formed can oxidize sulfhydryl groups in proteins.

Dough strength, stability and reduced stickiness; Increase in volume; Improved crumb structure and softness.

[78,79,101,178, 179,180]

[29,145-150]

[59,106,158]

[130,131,181]

[79,182]

[155,163]

It is common practice to use mixtures of enzymes, some of which are commercially available. The enzymes may act individually or present a synergistic effect. The trend is to choose and control the use of complex mixtures of enzymes which may act in a synergistic way and can ex‐ ert a better effect (than the individually used) on the different flour components [60]. Recent advances in understanding of the dough forming and overall baking processes at the molecu‐ lar level have focused attention on improvements that can be achieved by application of more specially tailored enzymes alone or in combinations. Usually, integrated experimental design and optimization followed by chemical analyses, rheological experiments and baking trials are necessary in order to provide answers to the more complicated questions [158].

The use of a combination of enzymatic preparations of amylases, xylanases and lipases has been reported by different authors [60,102,159]. This specific mixture is claimed to increase bread volume and shelf-life. The use of α-amylase and glucose oxidase to replace bromate led to a significant improvement in dough extensibility and bread volume [160]. Addition of commercial enzyme mixtures, containing α-amylase and lipase activities to produce bread samples, using the straight dough method, had a beneficial effect on bread keeping proper‐ ties and resulted in the formation of a more thermostable amylose-lipid complex compared to the control bread [161]. Amylopectin retrogradation was inhibited by the use of the en‐ zyme combinations and this effect was strongly related to a decrease in crumb-firming rates.

The combined use of different enzymes, classified as gluten degrading (like proteases) or ad‐ juvants, such as amylases and xylanases, with a group of crosslink promoting enzymes, such as transglutaminases and glucose oxidase, was also studied [149]. Better shaped bread could be obtained after the use of gluten degrading or adjuvant enzymes, and association with transglutaminase resulted in improvements on texture and rheological properties. The crumb firmness which can further lead to staling, can result from transglutaminase action, but it may be reversed with opposite amylase, xylanase and protease effects.

In a similar way, combinations of enzymes classified as carbohydrate degrading, including amylases and xylanases (pentosanases), and crosslink promoting enzymes, like transgluta‐ minases and oxidases, including glucose oxidase, laccase [149], lipoxygenase and sulfhydryl oxidase [79,162] were evaluated. The most frequent associations contained xylanases and glucose oxidase, but addition of laccase and transglutaminase was also employed. The hy‐ drogen peroxide formed by glucose oxidase catalysis may interfere in gluten network, via oxidized glutathione reaction, leading to gluten disulfide bonds formation [43], and it also interferes in the formation of a soluble pentosan gel (from xylans) that increased dough con‐ sistency [146]. Because both oxidases and xylanases influence the xylan properties, xylanas‐ es and oxidases could be used advantageously in combination, resulting in a mesh of gluten and gelified xylans matrix, which increases gas retention, dough stability and bread volume. Laccase is reported to catalyze dimerization of feruloylated esters in feruloylated arabinoxy‐ lans in doughs [163,164], forming a xylan network, contributing to increase strength of dough and volume.

ages of psychrophylic xylanases than of the mesophilic enzymes could be used to attain

Enzymes in Bakery: Current and Future Trends

http://dx.doi.org/10.5772/53168

307

Directed evolution is a powerful tool of protein engineering to design and modify the prop‐ erties of enzymes [171]. This technology can be employed for a wide range of proteins, most of which are of interest for biocatalytic processes. Within a decade, directed evolution has become a standard methodology in protein engineering and can be used in combination with rational protein design and other standard techniques to meet the demands for indus‐ trially applicable biocatalysts capable of withstanding process conditions such as high sub‐ strate concentrations, high temperatures and long-term stability, as well as presenting desired specificity and/or selectivity [172]. For instance, a recent study reported the com‐ bined use of directed evolution and high-throughput screening to improve the perfomance of a maltogenic α-amylase from *Bacillus* sp. for low pH bread applications. One of the result‐ ing variants showed an important increase in thermal stability at pH 4.5 and a considerable

maximal bread volumes [85,169,170].

antistaling effect in low pH breads [173].

Ângelo Samir Melim Miguel, Tathiana Souza Martins-Meyer,

\*Address all correspondence to: gisela@pharma.ufrj.br

cations. Springer; 2008. p. 19-56.

technology Advances 1991;9(4) 643-658.

New York: Marcel Dekker; 2003. Chapter 2.

Érika Veríssimo da Costa Figueiredo, Bianca Waruar Paulo Lobo and

Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

[1] Illanes A. Introduction. In; Illanes A. (ed.) Enzyme Biocatalysis. Principles and Appli‐

[2] van Oort, M. Enzymes in food technology – introduction. In: Whitehurst RJ, van Oort M. (eds.) Enzymes in Food Technology, second ed. Chichester: Wiley-Blackwell;

[3] Falch E.A. Industrial Enzymes – Developments in Production and Application. Bio‐

[4] Poulsen PB, Bucholz K. History of Enzymology with Emphasis on Food Production. In: Whitaker JR, Voragen AGJ, Wong DWS. (eds.) Handbook of Food Enzymology.

**Author details**

**References**

Gisela Maria Dellamora-Ortiz\*

2010. p. 1-17.

Lipoxygenases oxidize polyunsaturated fatty acids during dough mixing. The hydroperox‐ ides formed can oxidize the sulfhydryl groups of gluten proteins and thus be advantageous in the formation of the gluten network of dough. Sulfhydryl oxidase combined to glucose oxidase and xylanases has been used to strengthen weak doughs [79,162].

The use of a combination of commercial preparations of glucolipase, hemicellulase and hex‐ ose oxidase in formulations of frozen pre-baked French bread, substituted with whole wheat flour, improved parameters such as proofing time, oven spring and cut opening and cut height [158]. An interaction among the three enzymes was observed for most of the parame‐ ters, because the responses of each enzyme to variations in dosing were influenced by the doses of the other two.

## **6. Future trends**

Besides the demand for replacement of chemical additives by others from natural sources, there is an increasing concern among the consumers and consequently an increased demand for preservation and/or enrichment of foods with products that have beneficial effects on human health. Regarding baked goods, the use of enzymes to obtain dietary fiber enriched bread [102,165], for the development of gluten free products [145], to obtain products with increased contents of arabinoxylan oligosaccharides with prebiotic potential [165], has been reported.

Several aspects can be pointed out for the development of enzyme preparations able to pro‐ vide the desired effects or with adequate characteristics for use under process conditions. Some of the possible strategies include selection of novel enzymes from different sources [166], especially from microorganisms obtained from the vast biodiversity of the planet, pro‐ duction of recombinant proteins from genetically modified organisms [167], as well as pro‐ tein engineering.

Psychrophilic enzymes usually have higher optimal activity and stability at lower tempera‐ tures than their mesophilic counterparts [168]. Due to the fact that the temperatures most frequently used in dough mixing and proofing are around or below 35 °C, it has been sug‐ gested that psychrophilic enzymes would be advantageous candidates for use as additives in the baking industry [83,85]. In this context, researchers have shown that much lower dos‐ ages of psychrophylic xylanases than of the mesophilic enzymes could be used to attain maximal bread volumes [85,169,170].

Directed evolution is a powerful tool of protein engineering to design and modify the prop‐ erties of enzymes [171]. This technology can be employed for a wide range of proteins, most of which are of interest for biocatalytic processes. Within a decade, directed evolution has become a standard methodology in protein engineering and can be used in combination with rational protein design and other standard techniques to meet the demands for indus‐ trially applicable biocatalysts capable of withstanding process conditions such as high sub‐ strate concentrations, high temperatures and long-term stability, as well as presenting desired specificity and/or selectivity [172]. For instance, a recent study reported the com‐ bined use of directed evolution and high-throughput screening to improve the perfomance of a maltogenic α-amylase from *Bacillus* sp. for low pH bread applications. One of the result‐ ing variants showed an important increase in thermal stability at pH 4.5 and a considerable antistaling effect in low pH breads [173].

## **Author details**

drogen peroxide formed by glucose oxidase catalysis may interfere in gluten network, via oxidized glutathione reaction, leading to gluten disulfide bonds formation [43], and it also interferes in the formation of a soluble pentosan gel (from xylans) that increased dough con‐ sistency [146]. Because both oxidases and xylanases influence the xylan properties, xylanas‐ es and oxidases could be used advantageously in combination, resulting in a mesh of gluten and gelified xylans matrix, which increases gas retention, dough stability and bread volume. Laccase is reported to catalyze dimerization of feruloylated esters in feruloylated arabinoxy‐ lans in doughs [163,164], forming a xylan network, contributing to increase strength of

Lipoxygenases oxidize polyunsaturated fatty acids during dough mixing. The hydroperox‐ ides formed can oxidize the sulfhydryl groups of gluten proteins and thus be advantageous in the formation of the gluten network of dough. Sulfhydryl oxidase combined to glucose

The use of a combination of commercial preparations of glucolipase, hemicellulase and hex‐ ose oxidase in formulations of frozen pre-baked French bread, substituted with whole wheat flour, improved parameters such as proofing time, oven spring and cut opening and cut height [158]. An interaction among the three enzymes was observed for most of the parame‐ ters, because the responses of each enzyme to variations in dosing were influenced by the

Besides the demand for replacement of chemical additives by others from natural sources, there is an increasing concern among the consumers and consequently an increased demand for preservation and/or enrichment of foods with products that have beneficial effects on human health. Regarding baked goods, the use of enzymes to obtain dietary fiber enriched bread [102,165], for the development of gluten free products [145], to obtain products with increased contents of arabinoxylan oligosaccharides with prebiotic potential [165], has been

Several aspects can be pointed out for the development of enzyme preparations able to pro‐ vide the desired effects or with adequate characteristics for use under process conditions. Some of the possible strategies include selection of novel enzymes from different sources [166], especially from microorganisms obtained from the vast biodiversity of the planet, pro‐ duction of recombinant proteins from genetically modified organisms [167], as well as pro‐

Psychrophilic enzymes usually have higher optimal activity and stability at lower tempera‐ tures than their mesophilic counterparts [168]. Due to the fact that the temperatures most frequently used in dough mixing and proofing are around or below 35 °C, it has been sug‐ gested that psychrophilic enzymes would be advantageous candidates for use as additives in the baking industry [83,85]. In this context, researchers have shown that much lower dos‐

oxidase and xylanases has been used to strengthen weak doughs [79,162].

dough and volume.

306 Food Industry

doses of the other two.

**6. Future trends**

reported.

tein engineering.

Ângelo Samir Melim Miguel, Tathiana Souza Martins-Meyer, Érika Veríssimo da Costa Figueiredo, Bianca Waruar Paulo Lobo and Gisela Maria Dellamora-Ortiz\*

\*Address all correspondence to: gisela@pharma.ufrj.br

Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

## **References**


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320 Food Industry

**Chapter 15**

**Grinding Characteristics of Wheat in Industrial Mills**

Grinding of cereal seeds is due to the mechanical action of several forces: compression, shearing, crushing, cutting, friction and collision, to which seeds are subjected, depending on the design if the mill used for grinding (roller mill, hammer mill, stones mill or ball mill). By applying these forces, when the mechanical resistance of the particles is exceeded, their division happens in a number of smaller particles of different sizes, geometric shapes, mass‐

An industrial wheat mill has several technological phases, starting with coarse grinding of seeds to fine grinding of the resulted milling products, after their sorting in fractions of dif‐ ferent sizes. The first technological phase of grinding process, in wheat mills, is gristing or

A technological passage consists of a grinding machine (roller mill), a machine for sifting and sorting of the resulted milling fractions (plansifter compartment) and, eventually, a ma‐ chine for the conditioning of semi-final product (semolina machine or bran finisher). In a technological passage, intermediate fractions are obtained, which, by a new grinding, lead

Wheat processing requires a long and gradual transformation into flour. This process takes place after a gradual crushing schedule, from fine to finer, from machine to machine, of wheat seed, respectively of the crushed particles resulting from it. Each grinding operation is immediately followed by a sorting operation by sifting (fig.1) because during grinding, a

> © 2013 Voicu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

coarse grinding phase, which also consists of several technological passages.

to the obtaining of high-quality flour at milling passages (fine grinding).

wide variety of grinded seed particles is obtained.

Gheorghe Voicu, Sorin-Stefan Biris,

Gabriel-Alexandru Constantin and

Additional information is available at the end of the chapter

Elena-Madalina Stefan,

http://dx.doi.org/10.5772/53160

Nicoleta Ungureanu

**1. Introduction**

es and volumes.

## **Grinding Characteristics of Wheat in Industrial Mills**

Gheorghe Voicu, Sorin-Stefan Biris, Elena-Madalina Stefan, Gabriel-Alexandru Constantin and Nicoleta Ungureanu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53160

## **1. Introduction**

Grinding of cereal seeds is due to the mechanical action of several forces: compression, shearing, crushing, cutting, friction and collision, to which seeds are subjected, depending on the design if the mill used for grinding (roller mill, hammer mill, stones mill or ball mill). By applying these forces, when the mechanical resistance of the particles is exceeded, their division happens in a number of smaller particles of different sizes, geometric shapes, mass‐ es and volumes.

An industrial wheat mill has several technological phases, starting with coarse grinding of seeds to fine grinding of the resulted milling products, after their sorting in fractions of dif‐ ferent sizes. The first technological phase of grinding process, in wheat mills, is gristing or coarse grinding phase, which also consists of several technological passages.

A technological passage consists of a grinding machine (roller mill), a machine for sifting and sorting of the resulted milling fractions (plansifter compartment) and, eventually, a ma‐ chine for the conditioning of semi-final product (semolina machine or bran finisher). In a technological passage, intermediate fractions are obtained, which, by a new grinding, lead to the obtaining of high-quality flour at milling passages (fine grinding).

Wheat processing requires a long and gradual transformation into flour. This process takes place after a gradual crushing schedule, from fine to finer, from machine to machine, of wheat seed, respectively of the crushed particles resulting from it. Each grinding operation is immediately followed by a sorting operation by sifting (fig.1) because during grinding, a wide variety of grinded seed particles is obtained.

© 2013 Voicu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Schematic diagram of a grinding passage

Before the grinding process is started, grains must undergo the cleansing process. This is fol‐ lowed by a conditioning process that ensures a uniform moisture content for the entire lot of grains, helping endosperm softening and cover harshening, which improves the separation process.

product

is influenced by the physical and mechanical properties of seeds and of the intermediate prod‐ ucts (size distribution, seeds hardness, moisture content) and by the design and functional pa‐ rameters of the roller mill (mutual arrangement of the rollers, differential speed, distance between the rollers, flutes profile, mutual position of the flutes), [3,4]. Effects of these factors are manifested in the size distribution of material particles, compositional distribution of the

Grinding Characteristics of Wheat in Industrial Mills

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Fang, Campbell et al. (2002) showed that if the distance between rollers increases from 0.3 mm to 0.7 mm, wheat seeds breakage in the gristing phase has a lower intensity, resulting in more particles of large sizes and less particles of smaller sizes. Distance between rollers indi‐ rectly influences the specific surface and energy consumption per mass unit and directly in‐ fluences the specific energy, [5]. Different flutes arrangements on the rollers lead to the obtaining of different size distributions. If the roller flutes are arranged in blade/blade posi‐ tion results in a relatively uniform size distribution, and back to back arrangement lead to a

Differential speed of milling rollers has a significant effect on the grinding of semolina, flour and wheat bran. With the increase of differential speed of rollers, it also increases the amount of semolina and decreases the amount of flour and wheat bran, [6]. This is due to the difference between shearing and compression forces which are applied on the particles. It is very important to know the size distribution of the material subjected to grinding, as well of the grist, so that appropriate adjustments can be made to roller mills, and also to choose the fabrics for the sieving frames of plansift compartments. Particles size distribution of the granular material can be determined using superposed sieve classifiers (sieve shak‐ ers), with different sizes of sieve holes. This can be assessed by various mathematical func‐

Experiments were performed on the material subjected to grinding (before and after grind‐ ing) and cumulative distribution curves were drawn for the sieved material, by computer aided regression analysis of the experimental data with Rosin-Rammler function. Based on the data obtained from particle size distribution were also determined other physical charac‐ teristics of the analyzed material: average particle size, grist modulus, specific surface of the granular material, surface increasement resulted from grinding within a passage (break),

Within this chapter are presented the flow diagrams for two wheat mills of different capaci‐ ties, one of 100 tons / 24 hours and one of 220 tons / 24 hours, from which it can be estimated

There are also presented the experimental results obtained from the particles size distribu‐ tion of the material subjected to grinding and of the resulted grist, in both technological phases, for the two mills, as well as particles size distribution of the material for various

Knowing of the mechanical characteristics of wheat seeds and of the grist particles, and also their size characteristics, volume and mass of the wheat seeds, is useful for estimating the

material, wear degree of the rollers, energy consumed for grinding, [4].

tions, from which, most used is the Rosin-Rammler function.

deep parabolic distribution, [1].

bulk density and specific mass.

the movement of products within the mill.

grinding machines of the analyzed mills.

energy required for crushing.

One of the fractions resulting from a plansifter compartment is composed of flour particles (with sizes under 160 µm), in a higher or lower percentage of the total flour that can be with‐ drawn in the industrial mill. To extract the full amount of flour from the wheat berries, mul‐ tiple passes (passages) are required. Some passages are part of coarse grinding phase (gristing), where the milling rollers have fluted surface, while other passages are part of milling phase (fine grinding), where the milling rollers have smooth surface.

Intermediate milling products are, mainly, grists (seed particles with various sizes), semoli‐ na (large, average and small) and dunsts (harsh and smooth). They all return in the grinding process for flour extraction, but the grists are grinded by mills with fluted rollers (gristing passages), while semolina and dunsts are grinded by mills with smooth rollers (milling pas‐ sages). Semolina and dunsts, as intermediate milling products, are particles of clean endo‐ sperm or with a small percentage of cohesive coat.

Particles obtained by grinding have sizes in a fairly wide range (1200-160 µm, within the mentioned fractions), average size of the particles of resulted fraction being determined by granulometric analysis using sieve classifier.

In roller mills, wheat seeds are grinded in the gristing phase by pairs of fluted rollers, thus be‐ ing obtained a wide range of particles with sizes from < 200 µm to > 2000 µm, [1], consisting in coat particles (of larger sizes) and endosperm particles (of smaller sizes), to be further separat‐ ed with plansifters. The milling process aims to grind the endosperm into finer particles of flour and semolina, while the coating and the seed particles must remain in large sizes to be separated by sifting, [2]. In gristing passages, milling rollers with fluted surface are used, and in milling passages, rollers with smooth surface are used. The quality of wheat milling process is influenced by the physical and mechanical properties of seeds and of the intermediate prod‐ ucts (size distribution, seeds hardness, moisture content) and by the design and functional pa‐ rameters of the roller mill (mutual arrangement of the rollers, differential speed, distance between the rollers, flutes profile, mutual position of the flutes), [3,4]. Effects of these factors are manifested in the size distribution of material particles, compositional distribution of the material, wear degree of the rollers, energy consumed for grinding, [4].

Intermediate product Intermediate product Intermediate product

Plansifter compartment, with 12 sifting frames

Roller mills

Intermediate product

Flour 5

Before the grinding process is started, grains must undergo the cleansing process. This is fol‐ lowed by a conditioning process that ensures a uniform moisture content for the entire lot of grains, helping endosperm softening and cover harshening, which improves the separation

One of the fractions resulting from a plansifter compartment is composed of flour particles (with sizes under 160 µm), in a higher or lower percentage of the total flour that can be with‐ drawn in the industrial mill. To extract the full amount of flour from the wheat berries, mul‐ tiple passes (passages) are required. Some passages are part of coarse grinding phase (gristing), where the milling rollers have fluted surface, while other passages are part of

Intermediate milling products are, mainly, grists (seed particles with various sizes), semoli‐ na (large, average and small) and dunsts (harsh and smooth). They all return in the grinding process for flour extraction, but the grists are grinded by mills with fluted rollers (gristing passages), while semolina and dunsts are grinded by mills with smooth rollers (milling pas‐ sages). Semolina and dunsts, as intermediate milling products, are particles of clean endo‐

Particles obtained by grinding have sizes in a fairly wide range (1200-160 µm, within the mentioned fractions), average size of the particles of resulted fraction being determined by

In roller mills, wheat seeds are grinded in the gristing phase by pairs of fluted rollers, thus be‐ ing obtained a wide range of particles with sizes from < 200 µm to > 2000 µm, [1], consisting in coat particles (of larger sizes) and endosperm particles (of smaller sizes), to be further separat‐ ed with plansifters. The milling process aims to grind the endosperm into finer particles of flour and semolina, while the coating and the seed particles must remain in large sizes to be separated by sifting, [2]. In gristing passages, milling rollers with fluted surface are used, and in milling passages, rollers with smooth surface are used. The quality of wheat milling process

milling phase (fine grinding), where the milling rollers have smooth surface.

Wheat seeds or other intermediate products

Intermediate product

**Figure 1.** Schematic diagram of a grinding passage

sperm or with a small percentage of cohesive coat.

granulometric analysis using sieve classifier.

process.

324 Food Industry

Fang, Campbell et al. (2002) showed that if the distance between rollers increases from 0.3 mm to 0.7 mm, wheat seeds breakage in the gristing phase has a lower intensity, resulting in more particles of large sizes and less particles of smaller sizes. Distance between rollers indi‐ rectly influences the specific surface and energy consumption per mass unit and directly in‐ fluences the specific energy, [5]. Different flutes arrangements on the rollers lead to the obtaining of different size distributions. If the roller flutes are arranged in blade/blade posi‐ tion results in a relatively uniform size distribution, and back to back arrangement lead to a deep parabolic distribution, [1].

Differential speed of milling rollers has a significant effect on the grinding of semolina, flour and wheat bran. With the increase of differential speed of rollers, it also increases the amount of semolina and decreases the amount of flour and wheat bran, [6]. This is due to the difference between shearing and compression forces which are applied on the particles.

It is very important to know the size distribution of the material subjected to grinding, as well of the grist, so that appropriate adjustments can be made to roller mills, and also to choose the fabrics for the sieving frames of plansift compartments. Particles size distribution of the granular material can be determined using superposed sieve classifiers (sieve shak‐ ers), with different sizes of sieve holes. This can be assessed by various mathematical func‐ tions, from which, most used is the Rosin-Rammler function.

Experiments were performed on the material subjected to grinding (before and after grind‐ ing) and cumulative distribution curves were drawn for the sieved material, by computer aided regression analysis of the experimental data with Rosin-Rammler function. Based on the data obtained from particle size distribution were also determined other physical charac‐ teristics of the analyzed material: average particle size, grist modulus, specific surface of the granular material, surface increasement resulted from grinding within a passage (break), bulk density and specific mass.

Within this chapter are presented the flow diagrams for two wheat mills of different capaci‐ ties, one of 100 tons / 24 hours and one of 220 tons / 24 hours, from which it can be estimated the movement of products within the mill.

There are also presented the experimental results obtained from the particles size distribu‐ tion of the material subjected to grinding and of the resulted grist, in both technological phases, for the two mills, as well as particles size distribution of the material for various grinding machines of the analyzed mills.

Knowing of the mechanical characteristics of wheat seeds and of the grist particles, and also their size characteristics, volume and mass of the wheat seeds, is useful for estimating the energy required for crushing.

For this purpose, in this paper are presented the results of some experimental research on the behaviour of wheat seeds in uniaxial compression tests between parallel plates. There are also presented the curves of variation for the crushing force and energy absorbed until the crushing point of seeds.

S m a l l < \$ % &

 

 

 

Grinding Characteristics of Wheat in Industrial Mills

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**Figure 2.** Technological diagram of gristing phase for a Bühler mill with capacity of 220 t / 24 h, [8]

 

semolina

**Figure 3.** Technological diagram for sorting of big semolina in Bühler mill, [7]

Soft dunst

Small semolina

for grinding Flour

Small Flour

 

 

 

> 

for onsumption Big semolina

The results presented and the obtained data are of real interest for the designers of roller mills, as well as for the manufacturers and users of such machines.

## **2. Technological diagrams for wheat grinding**

The technological passage consists of one or two pairs of milling rollers, both processing the same product, combined with one or more plansifter compartments for sieving.

Gristing is the technological phase aiming to fragment the wheat seed in particles of differ‐ ent sizes and to remove the endosperm from the coating. Particles resulted from first, sec‐ ond and third grinding phase vary in size, from breakages like half seeds to flour particles with very fine granulometry. As gristing is repeated, particles will get increasingly finer, the amount of white flour decreases, and seeds coating reaches the penultimate and last phase as fine dust, [7]. Thus, grist is the intermediate product obtained in the milling industry, by grinding grains by mean of roller mills with fluted surface.

Fig. 2 presents the technological diagram of gristing phase of the wheat in an industrial mill with the capacity of 220 t/24 h.

Milling unit consists of 9 double roller mills, of which the first processes, in both sections, the same material (whole seeds), two plansifters, together amounting 14 compartments, three double semolina machines and five brushes and bran finishers. The three phases of the process (gristing, milling, sorting) can be observed in fig.3 – fig.5.

Gristing phase consists of six simple mills with fluted rollers, four full and two half's of plansifter compartments and four bran finishers which process the coatings resulted from multiple grinding operations. The seeds are processed in a mill with double rollers placed in horizontal plane, noted by B1–B2.

The first grist is processed in passage B3, and the fractions obtained here will follow differ‐ ent routes, to the milling passages, or to the semolina machines or bran finishers, passages B4gr and B5f being responsible for the processing of material particles with high coating content, and passage B4f processes the second refuse from gristing passage B3, with frac‐ tions having the same characteristics processed in plansifter compartments. The develop‐ ment of gristing phase in directly connected to the type of meal and the degree of flour extraction. Products resulted from gristing are named intermediate products and they con‐ sist of: big grist, fine grist, big semolina, middle semolina, fine semolina, big dunst, soft dunst, flour and bran, [7].

**Figure 2.** Technological diagram of gristing phase for a Bühler mill with capacity of 220 t / 24 h, [8]

For this purpose, in this paper are presented the results of some experimental research on the behaviour of wheat seeds in uniaxial compression tests between parallel plates. There are also presented the curves of variation for the crushing force and energy absorbed until

The results presented and the obtained data are of real interest for the designers of roller

The technological passage consists of one or two pairs of milling rollers, both processing the

Gristing is the technological phase aiming to fragment the wheat seed in particles of differ‐ ent sizes and to remove the endosperm from the coating. Particles resulted from first, sec‐ ond and third grinding phase vary in size, from breakages like half seeds to flour particles with very fine granulometry. As gristing is repeated, particles will get increasingly finer, the amount of white flour decreases, and seeds coating reaches the penultimate and last phase as fine dust, [7]. Thus, grist is the intermediate product obtained in the milling industry, by

Fig. 2 presents the technological diagram of gristing phase of the wheat in an industrial mill

Milling unit consists of 9 double roller mills, of which the first processes, in both sections, the same material (whole seeds), two plansifters, together amounting 14 compartments, three double semolina machines and five brushes and bran finishers. The three phases of the

Gristing phase consists of six simple mills with fluted rollers, four full and two half's of plansifter compartments and four bran finishers which process the coatings resulted from multiple grinding operations. The seeds are processed in a mill with double rollers placed in

The first grist is processed in passage B3, and the fractions obtained here will follow differ‐ ent routes, to the milling passages, or to the semolina machines or bran finishers, passages B4gr and B5f being responsible for the processing of material particles with high coating content, and passage B4f processes the second refuse from gristing passage B3, with frac‐ tions having the same characteristics processed in plansifter compartments. The develop‐ ment of gristing phase in directly connected to the type of meal and the degree of flour extraction. Products resulted from gristing are named intermediate products and they con‐ sist of: big grist, fine grist, big semolina, middle semolina, fine semolina, big dunst, soft

same product, combined with one or more plansifter compartments for sieving.

mills, as well as for the manufacturers and users of such machines.

**2. Technological diagrams for wheat grinding**

grinding grains by mean of roller mills with fluted surface.

process (gristing, milling, sorting) can be observed in fig.3 – fig.5.

the crushing point of seeds.

326 Food Industry

with the capacity of 220 t/24 h.

horizontal plane, noted by B1–B2.

dunst, flour and bran, [7].

**Figure 3.** Technological diagram for sorting of big semolina in Bühler mill, [7]

Particles size of these components, resulted from sieving process, is determined by the size of the sieve holes used in sieving compartments. Depending on the particles size, semolina and dunsts can be classified as: big semolina with average size of 1200-630 µm; middle sem‐ olina 630–400 µm; fine semolina 400–310 µm; big dunsts 310–245 µm; soft dunsts 245–160 µm. Semolina sorting is dome in sorting phase (fig.2. ) A clear delineation between soft dunsts and flour can not be practically achieved, and therefore, are cases when soft dunsts (dm = 220 µm) are considered to be flour (flours granulosity is given by the sieves, with mean equivalent size of the particles below 160 µm).

**C1 C2 C5** 

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**C3 C4 C6** 

**Figure 4.** Technological flow of semolina grinding phase in wheat mill, capacity of 100 t/24 h, [11]

of frames in the first passage is then sent to the M2 grinding passage.

grinded products and the extraction of flour from these products.

as the other compartments of plane sieve.

and fig.5.

In breakage phase the technological diagram of mill contains five pairs of rolls, filled with one compartment of plane sieve, two semolina machines and three wheat bran finishers. The technological breakage phase is completed with one compartment of plane sieve with‐ out grinding machine, in which the material is sorted by fractions of different sizes as well

The first grist, obtained from seeds processing with the pair of fluted rollers Sr.1, is process‐ ed in passage Sr.2, and from here the fractions follow various routes, to grinding passages, to semolina machines to wheat bran finishers. The sifting material from the second and the last set of gristing passage Sr.1, is send to a plansifter compartment for division in fractions (Div.1), which next reach the MG1 and MG2 semolina machines. The refuse from the last set

The circulation of grist intermediate products in the technological diagram is shown in fig.4

In the grinding phase (fig.5), the technological diagram of milling unit consists of seven sim‐ ple roller mills, each fitted with one plansifter compartment for sorting in fractions of the

Particles of intermediate products can be highlited not only by their size, but also by shape, volume, specific mass, aerodynamic properties. Particles with rich coating have irregular shape in the form of foils with rolled or folded edges. Particles of clean endosperm have pol‐ yhedral shape with sharp edges and convex lateral surfaces.

Semolina is an intermediate product obtained in percentage of 25…30% in industrial wheat milling, is found as small granules and after cleaning is further milled to obtain flour or a food product known as "kitchen semolina". This is obtained in percentage of 2...3 % at wheat milling and it is cleaned in special semolina machines in order to remove coating par‐ ticles by the combined action of sieving and airflows. Dunst is a fine semolina obtained as intermediate product from the grinding of wheat or semolina.

After gristing phase it is important to sort the milling products using a wide range of sizes for sieve holes (1000...224 µm), followed by the cleaning of semolina and dunsts, the phase of semolina opening being no longer necessary, since most coating was already removed in the gristing phase (fig.3).

The unit is fully automated, all mill equipments starting and stopping from the computer, starting with the equipments from the final technological phases (bagging, flour homogeni‐ zation, sieving with plansifters, semolina cleaning, bran finishers, etc.) from the circuit of flour or intermediate products, while stopping begins with the first pair of rollers, i.e. re‐ verse of start up.

In fig.4 and fig.5 is presented the technological flow for a wheat mill with capacity of 100 t/24 h, in grinding phases (fig.4) and in the milling (breakage) phase of semolina (fig.5), [10].

The technological flow of wheat mill is ensured by 12 processing passages, with 12 pairs of milling rollers (6 double rollers of Buhler type) from which 5 gristing passages and 7 milling passages. In addition, the technological flow is fitted with a sorting passage (separate com‐ partment of plansifter).

Apart from the 12 technological passages, each consisting in a pair of roller mills and one plansifter compartment, the mill also has a double machine for semolina, three bran finish‐ ers and other auxiliary equipments (detachers, wheat brushes, filters and cleaning cyclones, etc.), as well as the proper elements for the pneumatic transport system from one equipment to another, according to the technological flow.

**Figure 4.** Technological flow of semolina grinding phase in wheat mill, capacity of 100 t/24 h, [11]

Particles size of these components, resulted from sieving process, is determined by the size of the sieve holes used in sieving compartments. Depending on the particles size, semolina and dunsts can be classified as: big semolina with average size of 1200-630 µm; middle sem‐ olina 630–400 µm; fine semolina 400–310 µm; big dunsts 310–245 µm; soft dunsts 245–160 µm. Semolina sorting is dome in sorting phase (fig.2. ) A clear delineation between soft dunsts and flour can not be practically achieved, and therefore, are cases when soft dunsts (dm = 220 µm) are considered to be flour (flours granulosity is given by the sieves, with

Particles of intermediate products can be highlited not only by their size, but also by shape, volume, specific mass, aerodynamic properties. Particles with rich coating have irregular shape in the form of foils with rolled or folded edges. Particles of clean endosperm have pol‐

Semolina is an intermediate product obtained in percentage of 25…30% in industrial wheat milling, is found as small granules and after cleaning is further milled to obtain flour or a food product known as "kitchen semolina". This is obtained in percentage of 2...3 % at wheat milling and it is cleaned in special semolina machines in order to remove coating par‐ ticles by the combined action of sieving and airflows. Dunst is a fine semolina obtained as

After gristing phase it is important to sort the milling products using a wide range of sizes for sieve holes (1000...224 µm), followed by the cleaning of semolina and dunsts, the phase of semolina opening being no longer necessary, since most coating was already removed in

The unit is fully automated, all mill equipments starting and stopping from the computer, starting with the equipments from the final technological phases (bagging, flour homogeni‐ zation, sieving with plansifters, semolina cleaning, bran finishers, etc.) from the circuit of flour or intermediate products, while stopping begins with the first pair of rollers, i.e. re‐

In fig.4 and fig.5 is presented the technological flow for a wheat mill with capacity of 100 t/24 h, in grinding phases (fig.4) and in the milling (breakage) phase of semolina (fig.5), [10].

The technological flow of wheat mill is ensured by 12 processing passages, with 12 pairs of milling rollers (6 double rollers of Buhler type) from which 5 gristing passages and 7 milling passages. In addition, the technological flow is fitted with a sorting passage (separate com‐

Apart from the 12 technological passages, each consisting in a pair of roller mills and one plansifter compartment, the mill also has a double machine for semolina, three bran finish‐ ers and other auxiliary equipments (detachers, wheat brushes, filters and cleaning cyclones, etc.), as well as the proper elements for the pneumatic transport system from one equipment

mean equivalent size of the particles below 160 µm).

yhedral shape with sharp edges and convex lateral surfaces.

intermediate product from the grinding of wheat or semolina.

the gristing phase (fig.3).

verse of start up.

328 Food Industry

partment of plansifter).

to another, according to the technological flow.

In breakage phase the technological diagram of mill contains five pairs of rolls, filled with one compartment of plane sieve, two semolina machines and three wheat bran finishers. The technological breakage phase is completed with one compartment of plane sieve with‐ out grinding machine, in which the material is sorted by fractions of different sizes as well as the other compartments of plane sieve.

The first grist, obtained from seeds processing with the pair of fluted rollers Sr.1, is process‐ ed in passage Sr.2, and from here the fractions follow various routes, to grinding passages, to semolina machines to wheat bran finishers. The sifting material from the second and the last set of gristing passage Sr.1, is send to a plansifter compartment for division in fractions (Div.1), which next reach the MG1 and MG2 semolina machines. The refuse from the last set of frames in the first passage is then sent to the M2 grinding passage.

The circulation of grist intermediate products in the technological diagram is shown in fig.4 and fig.5.

In the grinding phase (fig.5), the technological diagram of milling unit consists of seven sim‐ ple roller mills, each fitted with one plansifter compartment for sorting in fractions of the grinded products and the extraction of flour from these products.

**Figure 5.** Technological flow of the semolina grinding phase in a wheat mill with 100 t/24 h, [9]

All roller mills of both technological phases have the length of 1000 mm and diameter of 250 mm, with fluted surface, in the gristing phase, respectively smooth surface without flutes in the grinding phase. In the gristing phase, the ratio of the tangential speeds of fluted rollers is k=2.54, and in the grinding phase, for five pairs of rollers, k=2.54, and for two pairs of rollers k=1.5.

**Figure 6.** Arrangement of roller mills for the mill with capacity of 100 t /24 h

**P, kW n,**

**Passage**

**I (A)**

Characteristics of driving motors for mill rollers are given in table 1.

**rpm**

**No-load Load No-load Load**

Plansifters are driven by electric motors of 4 kW, cos φ = 0,81 and speed of 960 rot/min.

Double machine for semolina is driven by two moto-vibrators of 400 W and speed of 960 rpm.

**cos φ Passage**

**Table 1.** Characteristics of electric motors for the drive of mill rollers, for wheat mill with capacity of 100 t/24 h [9]

Sr. 1 19 45 30 960 0.83 M1 B 10 15 11 960 0.79 Sr. 2 21 37 22 960 0.83 M2 11,9 16 11 960 0.79 Sr. 3 23 32 18 975 0.82 M3 10,9 18 7,5 960 0.76 Sr. 4 13 30 15 970 0.81 M4 10,9 15 11 960 0.79 Sr. 5 13 17 11 960 0.79 M5 12 16 11 960 0.79 M1 A 10 15 11 960 0.79 M6 12 17 11 960 0.79

**I (A)**

**P, kW n,**

Grinding Characteristics of Wheat in Industrial Mills

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**rpm**

**cos φ**

As shown in fig.5, the products to be grinded into the grinding phase are products arriving from gristing phase (or breakage phase), inclusive from grists (Sr.1-6) or from semolina ma‐ chines and bran finishers. The siftings from MG1 and MG2 semolina machines, which are semolinas with sizes below 0.8-1.0 mm, are grinded in the first technological passages M1A and M1B, while the siftings from FT1 and FT3 bran finishers go to the last two grinders M4 and M5, which processed products with higher content of bran. In diagram, the first refusal from M1A and M1B grinders is led to M3 grinder, working with half compartment of plane sieve. It is noted that to grinders which grinded smaller particles of endosperm (about 0.40 mm), after the mill rollers in technological flow are placed detached of material, due to ag‐ glomerations arising from the compression of smaller particles of endosperm in the action zone of grinding rolls.

In fig.6 is shown the arrangement of rollers to a mill with 100 t /24 h capacity, where the samples for our determinations were collected.

**Figure 6.** Arrangement of roller mills for the mill with capacity of 100 t /24 h

**Figure 5.** Technological flow of the semolina grinding phase in a wheat mill with 100 t/24 h, [9]

k=1.5.

330 Food Industry

zone of grinding rolls.

samples for our determinations were collected.

All roller mills of both technological phases have the length of 1000 mm and diameter of 250 mm, with fluted surface, in the gristing phase, respectively smooth surface without flutes in the grinding phase. In the gristing phase, the ratio of the tangential speeds of fluted rollers is k=2.54, and in the grinding phase, for five pairs of rollers, k=2.54, and for two pairs of rollers

As shown in fig.5, the products to be grinded into the grinding phase are products arriving from gristing phase (or breakage phase), inclusive from grists (Sr.1-6) or from semolina ma‐ chines and bran finishers. The siftings from MG1 and MG2 semolina machines, which are semolinas with sizes below 0.8-1.0 mm, are grinded in the first technological passages M1A and M1B, while the siftings from FT1 and FT3 bran finishers go to the last two grinders M4 and M5, which processed products with higher content of bran. In diagram, the first refusal from M1A and M1B grinders is led to M3 grinder, working with half compartment of plane sieve. It is noted that to grinders which grinded smaller particles of endosperm (about 0.40 mm), after the mill rollers in technological flow are placed detached of material, due to ag‐ glomerations arising from the compression of smaller particles of endosperm in the action

In fig.6 is shown the arrangement of rollers to a mill with 100 t /24 h capacity, where the

Plansifters are driven by electric motors of 4 kW, cos φ = 0,81 and speed of 960 rot/min. Double machine for semolina is driven by two moto-vibrators of 400 W and speed of 960 rpm. Characteristics of driving motors for mill rollers are given in table 1.


**Table 1.** Characteristics of electric motors for the drive of mill rollers, for wheat mill with capacity of 100 t/24 h [9]

According to relevant regulations, on the technological diagram (fig.4 or fig.5) should be written the characteristics of grinding rollers: length, diameter (ex.1000x250, in mm), num‐ ber of flutes and their inclination (ex.7/cm, I=8%), flute angles (ex.35/65), mutual arrange‐ ment of the flutes (ex.S/S), speed ratio (ex.k=2.5), and the characteristics of fabrics used in plansifter frames (ex.3x46 – 3 frames with 46 wires per inch or 3xX for flour frames), at sem‐ olina machines (ex.42, which represents the number of wires per inch or 1000-500, which is frame size) or at bran finishers (ex.0.5 – size of fabric hole).

**•** The Rosin-Rammler distribution, for material particles with larger sizes than sieve holes,

where: R(*x*) is the mass percentage weight of fraction with larger particles than x (which re‐ mained on the sieve with meshes with size x); x – is the sieves meshes size by which the

( ) 100 1{ ( ) }

**•** The logistics type distribution with two parameters is defined by the relation:

( ) 100

Of these characteristics are important: the bulk density, ρ<sup>v</sup> (kg/m3

); material porosity, ε (%) and others.

e

processed, the density of the material, ρ (kg/m3

preciated by natural slope angle, ψ (<sup>o</sup>

method was used (xylene 0.8254 kg/cm3

*<sup>e</sup> R x <sup>e</sup>*

1

*x x*

a b

where: R(*x*) and x have the significance from relationships (1-3) α and β are logistical con‐

entry and exit of the grinding machine, dm (mm); angle of internal friction of particles ap‐

Of particular importance is the equivalent size of seeds subjected to grinding in the first

*The density* is the ratio between the sample mass and the volume of the particle in it. To de‐ termine the densities of wheat seeds, respectively the grinding products, the pycnometrical

*The porosity* is the property of granular materials, respectively of the grains, to not occupy the entire volume of storage, with an intergranular space. Knowing the values of bulk densi‐

).

ty and material density, the porosity was evaluated using the following relation, [15]:

(%) 1 / 100 % ( ) ( ) *<sup>v</sup>*

 rr +

a b

where: R(*x*) and x have the significance from to relationship (1), k - the module product par‐ ticles size (the size of sieve mesh through which, theoretical, pass all the sample particles

particles rest; b and n are the own coefficients of grinding material.

**•** The Schuhman distribution is defined by the relation:

( ) 100 *<sup>n</sup> b x Rx e*- = (1)

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333

*<sup>a</sup> R x* = - *x k* (2)

<sup>+</sup> = <sup>+</sup> (3)

); angle of material friction with the surfaces working

=- (4)

); the equivalent sizes of material particle at

), of the material to be

is expressed by the relation:

(100%)), a - the distribution module.

stants.

components, φ (<sup>o</sup>

technological passage.

Sieve frames from top of compartments are fitted with metal mesh as they separate seed brokens of relatively large sizes (which would wear quite quickly the textile fabrics), while flour frames from the lower set are fitted with frames with plastic or textile fabrics.

Lately, textile fabrics have been replaced with sieve frames with meshes of plastic fabric. Ac‐ cording to literature, fot the technological diagram of the analyzed mill, the equivalence be‐ tween the sieve number and the size of its holes, as they are specified in the diagram, is shown in table 2.


**Table 2.** Equivalence between sieve number and hole sizes

## **3. Physical and granulometric characteristics of seeds and grinding products**

In the grinding process is necessary to know the physico-mechanical characteristics of the material at the entry and exit from a processing machine, in this case, roller mills.

Main factors influencing the process of grain grinding are the physico-mechanical properties of seeds and of the grinding products, the constructive and functional characteristics of the grinding machines as well as the technological regime, most of those factors having a ran‐ dom character.

As a result of grinding it is obtained a mass of particles with various smaller sizes and dif‐ ferent geometrical shapes (grist).

Granulometric distribution of the grinded material and of the material leaving the grinding process can be assessed by the cumulative weight (%) of material passing through the sieve holes of classifier T(x) or which are refused by its sieves R(x), calculated on base of mass weight (%) of the fractions from the sieve. (R(x)+T(x)=100). The mathematical expression of granulometric distribution in case of grinded biological materials, is based on laws of math‐ ematical statistical method of small particles, [11-14].

There will be defined three usual types of laws of cumulative granulometric distribution.

**•** The Rosin-Rammler distribution, for material particles with larger sizes than sieve holes, is expressed by the relation:

$$R(x) = 100 \cdot e^{-b \cdot x^a} \tag{1}$$

where: R(*x*) is the mass percentage weight of fraction with larger particles than x (which re‐ mained on the sieve with meshes with size x); x – is the sieves meshes size by which the particles rest; b and n are the own coefficients of grinding material.

**•** The Schuhman distribution is defined by the relation:

According to relevant regulations, on the technological diagram (fig.4 or fig.5) should be written the characteristics of grinding rollers: length, diameter (ex.1000x250, in mm), num‐ ber of flutes and their inclination (ex.7/cm, I=8%), flute angles (ex.35/65), mutual arrange‐ ment of the flutes (ex.S/S), speed ratio (ex.k=2.5), and the characteristics of fabrics used in plansifter frames (ex.3x46 – 3 frames with 46 wires per inch or 3xX for flour frames), at sem‐ olina machines (ex.42, which represents the number of wires per inch or 1000-500, which is

Sieve frames from top of compartments are fitted with metal mesh as they separate seed brokens of relatively large sizes (which would wear quite quickly the textile fabrics), while

Lately, textile fabrics have been replaced with sieve frames with meshes of plastic fabric. Ac‐ cording to literature, fot the technological diagram of the analyzed mill, the equivalence be‐ tween the sieve number and the size of its holes, as they are specified in the diagram, is

**Sieve no.** 18 20 26 36 40 46 48 50 54 56 60 VIII IX X XI **Hole size (µm)** 1170 1050 780 520 470 390 370 350 320 310 280 180 170 150 130

**3. Physical and granulometric characteristics of seeds and grinding**

material at the entry and exit from a processing machine, in this case, roller mills.

In the grinding process is necessary to know the physico-mechanical characteristics of the

Main factors influencing the process of grain grinding are the physico-mechanical properties of seeds and of the grinding products, the constructive and functional characteristics of the grinding machines as well as the technological regime, most of those factors having a ran‐

As a result of grinding it is obtained a mass of particles with various smaller sizes and dif‐

Granulometric distribution of the grinded material and of the material leaving the grinding process can be assessed by the cumulative weight (%) of material passing through the sieve holes of classifier T(x) or which are refused by its sieves R(x), calculated on base of mass weight (%) of the fractions from the sieve. (R(x)+T(x)=100). The mathematical expression of granulometric distribution in case of grinded biological materials, is based on laws of math‐

There will be defined three usual types of laws of cumulative granulometric distribution.

flour frames from the lower set are fitted with frames with plastic or textile fabrics.

frame size) or at bran finishers (ex.0.5 – size of fabric hole).

**Table 2.** Equivalence between sieve number and hole sizes

shown in table 2.

332 Food Industry

**products**

dom character.

ferent geometrical shapes (grist).

ematical statistical method of small particles, [11-14].

$$R(\mathbf{x}) = 100 \cdot \left\{ 1 - \left( \mathbf{x} / k \right)^{\mathbf{a}} \right\} \tag{2}$$

where: R(*x*) and x have the significance from to relationship (1), k - the module product par‐ ticles size (the size of sieve mesh through which, theoretical, pass all the sample particles (100%)), a - the distribution module.

**•** The logistics type distribution with two parameters is defined by the relation:

$$R(\mathbf{x}) = 100 \cdot \frac{e^{\alpha + \beta \cdot \mathbf{x}}}{1 + e^{\alpha + \beta \cdot \mathbf{x}}} \tag{3}$$

where: R(*x*) and x have the significance from relationships (1-3) α and β are logistical con‐ stants.

Of these characteristics are important: the bulk density, ρ<sup>v</sup> (kg/m3 ), of the material to be processed, the density of the material, ρ (kg/m3 ); the equivalent sizes of material particle at entry and exit of the grinding machine, dm (mm); angle of internal friction of particles ap‐ preciated by natural slope angle, ψ (<sup>o</sup> ); angle of material friction with the surfaces working components, φ (<sup>o</sup> ); material porosity, ε (%) and others.

Of particular importance is the equivalent size of seeds subjected to grinding in the first technological passage.

*The density* is the ratio between the sample mass and the volume of the particle in it. To de‐ termine the densities of wheat seeds, respectively the grinding products, the pycnometrical method was used (xylene 0.8254 kg/cm3 ).

*The porosity* is the property of granular materials, respectively of the grains, to not occupy the entire volume of storage, with an intergranular space. Knowing the values of bulk densi‐ ty and material density, the porosity was evaluated using the following relation, [15]:

$$\mathfrak{a}(\%) = \left(1 - \rho\_v \mid \rho\right) \cdot 100 \left(\%\right) \tag{4}$$

*The static friction coefficient.* The most common method for determining the coefficient of stat‐ ic friction is inclined plane method which was used in this paper. It was used a device with adjustable incline plane, [15]. Two sets of determinations were realized on three types of surfaces: glossy fiberglass, steel sheet and cotton canvas.

Assessing parameters of the grinding process are: grinding degree, grinding finesse and spe‐ cific energy consumption at grinding.

*Grinding degree and grinding finesse* are determined by granulometric analysis, using a sieve overlay classifier with oscillatory movement.

*Grinding degree* is defined by the λ index and represents the ratio between equivalent sizes of particles before and after grinding, *De,* respectively *dm*, or the ratio between the outer surface of the particles resulted in the grinding process and the initial surface of the particle subject‐ ed to grinding, *S*<sup>f</sup> , respectively *S*<sup>i</sup> :

$$\mathcal{A} = \mathcal{D}\_e \int d\_m = \mathcal{S}\_f \int \mathcal{S}\_i \tag{5}$$

The material tested in the experiments was taken from the entry, respectively from the exit

The experimental data characterizing the physical properties of the grist obtained are shown in table 3. Also, in table 4 and table 5 are presented the results of size distribution analysis on mixtures of material entering and leaving the rolls placed in the technological grinding

**Static friction coefficient µ**

From table 3 it is noted that the static coefficient values, on the glossy fiberglass and metal are within the limits set in various specialized papers, while the values obtained in the ex‐ periments on cotton canvas fall in broad limits, probably due to material fractions moisture, but also because of its granularity, this phenomenon is observed, especially, to flours and

**(mm)**

) of the fractions from the sieving machine classifier sieves and of the cumulative

(%) for the collected gritting, at entrance "I" and exit "E" from the mentioned rolls (only M1A, and M1B), [9]

 **(%) pi**

0,00 0,90 0,00 34,40 0,00 0,00 0,40 0,00 7,70 0,00 0,18 1,00 0,90 16,30 34,40 0,13 0,60 0,40 8,00 7,70 0,25 3,70 1,90 10,30 50,70 0,18 3,90 1,00 23,80 15,70 0,32 33,30 5,60 15,80 61,00 0,25 22,10 4,90 35,60 39,50 0,50 49,10 38,90 12,50 76,80 0,32 48,90 27,00 21,90 75,10 0,71 12,00 88,00 10,70 89,30 0,40 24,10 75,90 3,00 97,00 dM1AI = 0,55 dM1AE = 0,33 dM1BI = 0,36 dM1BE = 0,26

**Cotton canvas Glossy fiberglass Steel sheet** M1A E 1.74 - >1.76 68,7 0.61 – 0.85 39,43 M1B I 1.24 – 1.82 56,4 0.45 – 0.67 30,19 M2 I >1.76 61 0.6 – 0.86 43,97 M3 E >1.76 72,4 0.58 – 0.82 38,35 M4 E >1.76 67,5 0.58 – 0.73 41,96 M5 I >1.76 64,3 0.60 – 0.88 46,62 M6 E >1.76 69,5 0.54 – 0.71 40,17

**Natural slope angle ψ**

Grinding Characteristics of Wheat in Industrial Mills

http://dx.doi.org/10.5772/53160

335

**M1B – I M1B - E**

 **(%) pi**

 **(%) Ti**

 **(%)**

 **(%) Ti**

of each pair of milling rollers (from the five pairs of the phase).

**Table 3.** The values of static friction coefficient and natural slope angles [15]

**M1A - I M1A – E li**

 **(%) Ti**

relatively small particle fractions of endosperm.

 **(%) pi**

phase.

**Break**

**li (mm)**

weights Ti

**pi**

**Table 4.** The ponder values (pi

 **(%) Ti**

Absolute value of the increase of particles outer surface in the grinding process Δ*S*, is given by:

$$\mathbf{AS} = \mathbf{S}\_f \mathbf{\cdot} \cdot \mathbf{S}\_l = \mathbf{S}\_l(\mathbf{\lambda} \cdot \mathbf{1}) \tag{6}$$

*The grinding finesse* has been appreciated by the geometric mean diameter dm of the grinding particles which was determined by the size distribution analysis, using the relation of weighted average:

$$d\_m = (1/100) \cdot \sum\_{i=0}^{n} p\_i d\_{i'} \tag{7}$$

where: pi is mass weight of fraction remaining on the sieve *i* of the classifier, di, is diameter (average value) of fractions particle on the sieve *i*, considered the arithmetic average of the sieves holes size that contain fraction *i*.

*The surface areaand the surface increase.* Knowing the mean diameter of particles of a granular mixture, their specific surface *S*e.m is determined with the relation, [10,15]:

$$S\_{e.m.} = \text{6/} \,\rho \cdot d\_m \,\, (m^2 / \,\text{kg}),\tag{8}$$

where: ρ is the density of the particles.

There are presented the results of some experimental research on the physical characteristics of grinding products on the technological flow of gristing phase of wheat from a mill with capacity of 100 t / 24 h (SC Spicul Rosiori de Vede, Teleorman, Romania).

The material tested in the experiments was taken from the entry, respectively from the exit of each pair of milling rollers (from the five pairs of the phase).

The experimental data characterizing the physical properties of the grist obtained are shown in table 3. Also, in table 4 and table 5 are presented the results of size distribution analysis on mixtures of material entering and leaving the rolls placed in the technological grinding phase.


**Table 3.** The values of static friction coefficient and natural slope angles [15]

*The static friction coefficient.* The most common method for determining the coefficient of stat‐ ic friction is inclined plane method which was used in this paper. It was used a device with adjustable incline plane, [15]. Two sets of determinations were realized on three types of

Assessing parameters of the grinding process are: grinding degree, grinding finesse and spe‐

*Grinding degree and grinding finesse* are determined by granulometric analysis, using a sieve

*Grinding degree* is defined by the λ index and represents the ratio between equivalent sizes of particles before and after grinding, *De,* respectively *dm*, or the ratio between the outer surface of the particles resulted in the grinding process and the initial surface of the particle subject‐

*Dd S S em f i*

D*S = - = ( - 1) SSS fi i*

Absolute value of the increase of particles outer surface in the grinding process Δ*S*, is given

l

*The grinding finesse* has been appreciated by the geometric mean diameter dm of the grinding particles which was determined by the size distribution analysis, using the relation of

<sup>0</sup> (1 / 100) . , *<sup>n</sup>*

(average value) of fractions particle on the sieve *i*, considered the arithmetic average of the

*The surface areaand the surface increase.* Knowing the mean diameter of particles of a granular

2

There are presented the results of some experimental research on the physical characteristics of grinding products on the technological flow of gristing phase of wheat from a mill with

. . 6 / ( / ), *e m <sup>m</sup> S d m kg* = r

mixture, their specific surface *S*e.m is determined with the relation, [10,15]:

capacity of 100 t / 24 h (SC Spicul Rosiori de Vede, Teleorman, Romania).

is mass weight of fraction remaining on the sieve *i* of the classifier, di, is diameter

= = (5)

*<sup>m</sup> i i <sup>i</sup> <sup>d</sup> p d* <sup>=</sup> <sup>=</sup> å (7)

(8)

(6)

surfaces: glossy fiberglass, steel sheet and cotton canvas.

cific energy consumption at grinding.

ed to grinding, *S*<sup>f</sup>

weighted average:

sieves holes size that contain fraction *i*.

where: ρ is the density of the particles.

where: pi

by:

334 Food Industry

overlay classifier with oscillatory movement.

, respectively *S*<sup>i</sup>

:

l

From table 3 it is noted that the static coefficient values, on the glossy fiberglass and metal are within the limits set in various specialized papers, while the values obtained in the ex‐ periments on cotton canvas fall in broad limits, probably due to material fractions moisture, but also because of its granularity, this phenomenon is observed, especially, to flours and relatively small particle fractions of endosperm.


**Table 4.** The ponder values (pi ) of the fractions from the sieving machine classifier sieves and of the cumulative weights Ti (%) for the collected gritting, at entrance "I" and exit "E" from the mentioned rolls (only M1A, and M1B), [9]


 **(%) Ri (%) pi**

 **(%) Ri (%) pi**

0.00 26.00 0.00 42.60 0.00 0.00 5.20 0.00 5.80 0.00 0.71 25.20 26.00 27.50 42.60 0.25 3.90 5.20 3.90 5.80 1.00 28.30 51.20 20.70 70.10 0.32 21.10 9.10 22.60 9.70 1.40 11.90 79.50 3.60 90.80 0.50 28.20 30.20 29.70 32.30 2.00 7.80 91.40 5.00 94.40 0.71 35.30 58.40 32.70 62.00 2.80 0.80 99.20 0.60 99.40 1.00 6.30 93.70 5.30 94.70 d4I = 1.06 mm d4E = 0.84 mm d5I = 0.65 mm d5E = 0.63 mm

for the fractions on the shaker sieves of the sifter machine and of the cumulative

(%) for the collected grinded products, at entry "I" and exit "E" from pairs of mentioned rollers (Sr.1…

Bulk density [kg/c.m.]

**Figure 7.** Variation of mean diameter and bulk density of grinding intermediate products on the grinding technologi‐

Sieves used in granulometric analysis with sieve classifier and the results obtained by analy‐

sis are given in table 7, for each of the five technological passages, at the entry end exit from

0 0.5 1 1.5 2 2.5 3 3.5 4

cal flow with grinding rollers [15]

the respective mill rollers.

 **(%) Ri (%) pi**

**li**

**Table 7.** Values of weights (%) pi

percentages Ri

Sr.5), [10]

**pi**

 **(mm) Sr.1 - E li**

**pi**

**li**

Equivalent size [mm]

Sr. 1 Sr. 2 Sr. 3 Sr. 4 Sr.5 M 1A M1B M2 M3 M4 M5 M6

Break

 **(mm) Sr.2 - I Sr.2 - E li**

 **(mm**) **Sr.4 - I Sr.4 - E li**

 **(%) Ri (%) pi**

0.00 24.20 0.00 0.00 2.00 0.00 34.70 0.00 0.00 13.40 0.00 43.10 0.00 1.00 8.40 24.20 0.71 6.00 2.00 11.50 34.70 0.71 22.50 13.40 20.60 43.10 1.40 15.10 32.60 1.00 19.20 8.00 22.20 46.20 1.00 22.80 35.90 23.00 63.70 2.00 20.10 47.70 1.40 13.90 27.20 11.50 68.40 1.40 12.00 58.70 5.10 86.70 2.80 27.00 67.80 2.00 29.50 41.10 14.90 79.90 2.00 20.10 70.70 7.20 91.80 4.00 5.20 94.80 2.80 29.40 70.60 5.20 94.80 2.80 9.20 90.80 1.00 99.00 d1E = 2.13 mm d2I = 2.23 mm d2E = 1.22 mm d3I = 1.51 mm d2E = 0.90 mm

 **(%) Ri (%) pi**

Entry Exit

Sr. 1 Sr. 2 Sr. 3 Sr. 4 Sr.5 M 1A M1B M2 M3 M4 M5 M6

 **(mm) Sr.3 - I Sr.3 - E**

 **(mm) Sr.5 - I Sr.5 - E**

 **(%) Ri (%) pi**

 **(%) Ri (%) pi**

 **(%**) **Ri (%)**

 **(%) Ri (%)**

Break

Entry Exit

http://dx.doi.org/10.5772/53160

337

Grinding Characteristics of Wheat in Industrial Mills

The sign \* in table 3, for negative values of specific surface increases, means that at the passage through milling rollers with smooth surface, agglomeration of gritting particles occurs.

**Table 5.** The values of grinding degree, specific surface, surface increase and porosity

Based on the data obtained from the experiments and presented in table 6, were mapped graphics, using MS Excel version 7.0 program (fig.6), the variations of mean diameter and bulk density to technological breakage passage of milling unit.


**Table 6.** Physico-mechanical characteristics of grinding products at gristing passages of wheat, from the mill with capacity of 100 t / 24 h, [10]

Correlation between individual volume of the seeds, calculated with the relation: *V* =(1 / 6) *π l w t*(where: l, w, t represent the measured length, width, and thickness of each seed, and the seeds are assimilated with ellipsoid geometrical bodies) and their weight is presented in fig.7.

**Break**

336 Food Industry

**Equivalent size(I/E)**

capacity of 100 t / 24 h, [10]

presented in fig.7.

**Grinding**

with smooth surface, agglomeration of gritting particles occurs.

**Table 5.** The values of grinding degree, specific surface, surface increase and porosity

bulk density to technological breakage passage of milling unit.

**degree Bulk density True density Specific surface**

M1A 0,55-0,33 1,68 560,0-389,5 1344,9-1247,1 8,13-14,72 6,595 58,3-68,7 M1B 0,36-0,26 1,385 583,5-499,0 1338,7-1372,0 12,34-16,72 4,38 56,4-63,6 M2 0,19-0,17 1,113 480,5-437,5 1233,3-1313,4 26,04-27,55 1,513 61-66,7 M3 0,35-0,45 0,788 363,5-308,5 1252,9-1119,8 13,55-11,95 -1,605\* 71-72,4 M4 0,22-0,24 0,940 452,5-419,5 1290,6-1290,6 21,02-19,76 -1,252\* 64,9-67,5 M5 0,22-0,24 0,924 430,5-419,5 1205,4-1210,2 22,74-20,89 -1,854\* 64,3-65,3 M6 0,24-0,27 0,903 416,0-373,0 1274,4-1224,5 19,40-18,23 -1,164\* 67,4-69,5

The sign \* in table 3, for negative values of specific surface increases, means that at the passage through milling rollers

Based on the data obtained from the experiments and presented in table 6, were mapped graphics, using MS Excel version 7.0 program (fig.6), the variations of mean diameter and

**Physical characteristic Sr.1-I Sr.1-E Sr.2-I Sr.2-E Sr.3-I Sr.3-E Sr.4-I Sr.4-E Sr.5-I Sr.5-E Bulk density, ρv (kg/m3)** 713.0 381.5 482.0 346.5 267.8 292.0 255.0 257.0 269.0 266.0 **Density, ρ (kg/m3)** 1239 1250 1219 1200 1100 1063 1016 1130 1100 1191 **Equivalent size, (mm)** 3.76 2.13 2.23 1.22 1.51 0.90 1.06 0.84 0.65 0.63

**Grinding degree, λ** 1.76 1.83 1.67 1.26 1.03

**Specific surface, (m2/kg)** 1.29 2.25 2.21 4.10 3.61 6.27 5.57 6.32 8.39 8.00\* **Surface increase, ΔS (m2/kg)** 0.96 1.89 2.66 0.75 –0.39\* **Natural slope angle, ψ (gr.)** 21.8 37.8 37.1 37.5 44.6 39.0 41.1 39.2 42.6 44.4 **Porosity, ε (%)** 42.5 69.5 60.5 71.1 75.7 72.5 74.9 77.3 75.5 77.7

**Table 6.** Physico-mechanical characteristics of grinding products at gristing passages of wheat, from the mill with

Correlation between individual volume of the seeds, calculated with the relation: *V* =(1 / 6) *π l w t*(where: l, w, t represent the measured length, width, and thickness of each seed, and the seeds are assimilated with ellipsoid geometrical bodies) and their weight is

**mm λ g/dm3 g/dm3 x103 m2/kg x103 m2/kg %**

**Surface**

**increase Porosity**

**Figure 7.** Variation of mean diameter and bulk density of grinding intermediate products on the grinding technologi‐ cal flow with grinding rollers [15]

Sieves used in granulometric analysis with sieve classifier and the results obtained by analy‐ sis are given in table 7, for each of the five technological passages, at the entry end exit from the respective mill rollers.


**Table 7.** Values of weights (%) pi for the fractions on the shaker sieves of the sifter machine and of the cumulative percentages Ri (%) for the collected grinded products, at entry "I" and exit "E" from pairs of mentioned rollers (Sr.1… Sr.5), [10]

Based on the results obtained by granulometric analysis with the sieve classifier were tested by nonlinear regression analysis, the three laws of cumulative distribution for the refuse of the sieves R(x) (Rosin-Rammler function, Schuhman function and two parameters logistical function), for products entering the process, and for the products leaving the pairs of rollers, in the gristing phase of the grinding process. Experimental points and the curves of cumula‐ tive distribution for the refuse of the sieves (R(x)), using the three functions (eq.1, eq.2, eq.3), for some grinding products are presented in fig.8.

**•** The total grinding degree of the wheat breakage phase at the analyzed mill is approxi‐

**Proof Corrections Form – Gheorghe VOICU**

**PROOF CORRECTIONS FORM** 

**No. Delete Replace with** 

 

Sr.2-E

0

0

0

**Figure 9.** The curves described by the cumulative distribution laws (1), (2), (3) towards the experimental points R(%) for the gritting product from the five roll pairs (Sr.2…Sr.5) [10]; (I-entrance; E-exit); Rosin-Rammler; - - - - Schu‐

20

40

60

80

T,R (%)

100

 

20

40

60

80

100 T,R (%)

Sr.3-I

l, (mm)

� for T(%); for R(%)

20

40

60

80

100 T,R (%)

**Author(s) Name(s): Voicu Gheorghe, Biris Sorin-Stefan, Stefan Elena-Madalina, Constantin** 

**Chapter Title: GRINDING CHARACTERISTICS OF WHEAT IN INDUSTRIAL MILLS** 

**•** It is appreciated that, in all cases, at seeds wheat grinding in the complex roller mills, we

other methods, Schuhman and two parameter logistic, also can be used with satisfactory

≥0,982), but the

l, (mm)

l, (mm)

l, (mm)

special (Picture) in the final paper

http://dx.doi.org/10.5772/53160

be replaced

Grinding Characteristics of Wheat in Industrial Mills

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

 

Sr.3-E

Sr.4-E

Will replace with that of the initial work. Group in the initial paper, Copy and Paste

339

1

can consider that the best law of distribution is the Rosin-Rammler (1), (R<sup>2</sup>

paper submitted. In addition there are two

mately λ = 7, correspondent to a coarse gritting (crushing);

**23** Fig.12 Figure 12 should be replaced with that of the

**17** Fig.9 Symbols of experimental points were modified

from submitted paper.

overlapping figures.

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

 

l, (mm)

Sr.4-I

l, (mm)

**Gabriel-Alexandru, Ungureanu Nicoleta** 

results.

Fig.9

 

100 Sr.2-I

 

0

0

man; ― ‧ ― ‧ logistical function)

20

40

60

80

100 T,R (%)

20

40

60

80

100 T,R (%)

0

20

40

60

80

T,R (%)

**Page No.** 

**Line** 

**Figure 8.** Correlation between volume and the mass of wheat seeds in an technological mixture (before grinding) [16]

The coefficient values k, a, b, n, α and β, from the cumulative distribution relations Rosin– Rammler, Schuhman and the two parameters logistical function, as well as the R2 correlation coefficient values (which verifies the distribution adequacy degree expressed through the (1), (2), (3) relations), correspondent for the nine analyzed probes (from the five roll pairs) are presented in table 8.

From the analysis and interpretation of the obtained data for the 9 probes, which come from the mill rolls with rifles (for the coarse gritting in the breaking passages) (fig.9), following conclusions were found:


special (Picture) in the final paper

1

**•** The total grinding degree of the wheat breakage phase at the analyzed mill is approxi‐ mately λ = 7, correspondent to a coarse gritting (crushing); **23** Fig.12 Figure 12 should be replaced with that of the paper submitted. In addition there are two be replaced

**Gabriel-Alexandru, Ungureanu Nicoleta** 

from submitted paper.

**Page No.** 

**Line** 

**Proof Corrections Form – Gheorghe VOICU**

**PROOF CORRECTIONS FORM** 

**No. Delete Replace with** 

**Author(s) Name(s): Voicu Gheorghe, Biris Sorin-Stefan, Stefan Elena-Madalina, Constantin** 

**Chapter Title: GRINDING CHARACTERISTICS OF WHEAT IN INDUSTRIAL MILLS** 

Based on the results obtained by granulometric analysis with the sieve classifier were tested by nonlinear regression analysis, the three laws of cumulative distribution for the refuse of the sieves R(x) (Rosin-Rammler function, Schuhman function and two parameters logistical function), for products entering the process, and for the products leaving the pairs of rollers, in the gristing phase of the grinding process. Experimental points and the curves of cumula‐ tive distribution for the refuse of the sieves (R(x)), using the three functions (eq.1, eq.2, eq.3),

y = 666.64x + 3.2023

= 0.9132

0.01 0.02 0.03 0.04 0.05 0.06 Seeds weight, g

**Figure 8.** Correlation between volume and the mass of wheat seeds in an technological mixture (before grinding) [16]

The coefficient values k, a, b, n, α and β, from the cumulative distribution relations Rosin–

coefficient values (which verifies the distribution adequacy degree expressed through the (1), (2), (3) relations), correspondent for the nine analyzed probes (from the five roll pairs)

From the analysis and interpretation of the obtained data for the 9 probes, which come from the mill rolls with rifles (for the coarse gritting in the breaking passages) (fig.9), following

**•** For the vast analyzed material probes, from the mills flux, the best law of cumulative dis‐

**•** For the two parameter distribution law, the R2 correlation coefficient presents close values

correlation

≥0.982, time in which

≥0,963, at half the probes

≥0.933 (usually

Rammler, Schuhman and the two parameters logistical function, as well as the R2

tribution is the Rosin-Rammler (1) with a correlation coefficient R2

≥0.956) can be used with satisfactory results, in these cases;

from the ones obtained through the Rosin-Rammler function, R2

the Schuhman type distribution law with a correlation coefficient R2

R2

for some grinding products are presented in fig.8.

Seeds calculated volume, mm3

are presented in table 8.

conclusions were found:

being very close;

R2

338 Food Industry

**•** It is appreciated that, in all cases, at seeds wheat grinding in the complex roller mills, we can consider that the best law of distribution is the Rosin-Rammler (1), (R<sup>2</sup> ≥0,982), but the other methods, Schuhman and two parameter logistic, also can be used with satisfactory results. overlapping figures. 

**Figure 9.** The curves described by the cumulative distribution laws (1), (2), (3) towards the experimental points R(%) for the gritting product from the five roll pairs (Sr.2…Sr.5) [10]; (I-entrance; E-exit); Rosin-Rammler; - - - - Schu‐ man; ― ‧ ― ‧ logistical function)


ing of particles that passes through sieve no. 40 (with holes opening 0.47 mm) was extracted flour F (mean size of particles 0.08 mm). This fraction with fraction C1-DIV1' and with the two fractions C2-DIV1 of the second plansifter compartment are directed to the sorting-di‐ viding compartment DIV1 (compartment C5). Mean particle sizes of fraction DIV1", from compartment C1, are 0.31 mm (equal to the opening of sieve holes which refused them,

**C1 DIV1' li**

**Ti (%)**

**C2 DIV1' li**

**Ti (%)**

0.000 34.40 0.00 0.000 23.50 0.00 0.000 5.60 0.00 0.000 19.10 0.00 0,000 17.70 0.00 0.0000.30 0.00 0.710 11.50 34.40 1.000 30.10 23.50 0.180 4.00 5.60 0.045 34.90 19.10 0,090 9.40 17.70 0.1251.60 0.30 1.000 22.20 45.90 1.400 13.50 53.60 0.250 7.70 9.60 0.063 21.60 54.00 0,125 22.30 27.10 0.1805.60 1.90 1.400 11.60 68.10 2.000 17.60 67.10 0.400 26.10 17.30 0.090 16.10 75.60 0,180 9.60 49.40 0.25019.20 7.50 2.000 15.00 79.70 2.500 12.40 84.70 0.500 49.20 43.70 0.125 6.70 91.70 0,200 25.10 59.00 0.31544.50 26.70 2.800 5.30 94.70 3.150 2.90 97.10 0.710 7.40 92.60 0.160 1.60 98.40 0,250 15.90 84.10 0.40028.80 71.20 d2E = 1.22 mm d2Break3 = 1.56 mm d2DIV1' = 0.52 mm d2F = 0.07 mm d2M2 = 0.17 mm d2DIV1" = 0.37 mm

(%) of sieved fractions and of the cumulative weights Ti

The last components of plansifter compartments in gristing passage shave higher content of coating particles which are found in the upper layers of material on the frames, thus being recommended that they do not separate through the holes, even if their sizes are about the size of endosperm particles, to be further removed in semolina machines (sieving motion leads to the layering of mixture components by density). Flour particles have mean sizes un‐ der 0.18 mm in all plansifter compartments, while particles of last refuse from the five pas‐ sages fitted with pairs of rollers have mean sizes over 0.37 mm (see Table 9). Values of

**(mm)**

**pi (%)**

0.000 24.20 0.00 0.000 10.20 0.00 0.000 1.10 0.00 0.000 4.20 0.00 0.000 6.00 0.00 0.0000.60 0.00 1.000 8.20 24.20 1.000 21.30 10.20 0.180 2.30 1.10 0.045 45.10 4.20 0.125 8.00 6.00 0.0901.90 0.60 1.400 15.10 32.40 1.400 14.60 31.50 0.250 5.00 3.40 0.063 24.30 49.30 0.180 12.80 14.000.12541.50 2.50 2.000 20.20 47.50 2.000 21.60 46.10 0.400 51.70 8.40 0.090 18.80 73.60 0.250 24.50 26.800.18015.00 44.00 2.800 27.10 67.70 2.500 20.70 67.70 0.630 28.60 60.10 0.125 6.30 92.40 0.315 32.40 51.300.20030.10 59.00 4.000 5.20 94.80 4.000 11.60 88.40 0.710 11.30 88.70 0.160 1.30 98.70 0.400 16.30 83.700.25010.90 89.10 d1E = 2.13 mm d1Break2 = 2.27 mm d1DIV1' = 0.58 mm d1F = 0.08 mm d1DIV1" = 0.31 mm d1M2 = 0.19 mm

**(mm)**

**pi (%)**

**C1 F li**

**Ti (%)**

**C2 F li**

**Ti (%)** **(mm)**

**pi (%)**

**(mm)**

**pi (%)**

**C1 DIV1" li**

Grinding Characteristics of Wheat in Industrial Mills

http://dx.doi.org/10.5772/53160

**Ti (%)**

**C2 M2 li**

**Ti (%)** **(mm)**

(%) for products collected at the

**pi (%)**

**C2 DIV1"**

**Ti (%)**

**(mm )**

**C1 M2**

**Ti (%)** 341

**pi (%)**

proving that here also the sieving is incomplete).

**pi (%)**

**C1 Break 2 li**

**Ti (%)**

**C2 Break 3 li**

**Ti (%)** **(mm)**

**pi (%)**

**(mm)**

**pi (%)**

**(mm)**

**(mm)**

**pi (%)**

entrance, respectively exit of plansifter compartments, C1 and C2

**li (mm)**

**li (mm)** **C1 Entrance li**

**Ti (%)**

**C2 Entrance li**

**Table 9.** Values of weights pi

**Ti (%)**

**pi (%)**

**pi (%)**

**Table 8.** The coefficient values a, k, b, n, α and β and of the R<sup>2</sup> correlation coefficients, for the three size distribution laws tested, for the gritted products from the "I" entry to the "E" exit between the mentioned roll pairs (Sr.1...Sr.5), [10]

In plansifter compartments, material fractions are separated and sorted, as any granular ma‐ terial is made of particles with sizes between a minimum and a maximum value, in the inte‐ rior of the mixture the size distribution being characterised by various distribution laws.

It must be mentioned that material particles, being extracted from various areas of the seed (from exterior to interior) have different mechanical characteristics and composition. This, and the different sizes of particles gives a different behaviour of the particles during grinding.

Thus is important to study and to know the size distribution of the particles of each fraction obtained in each frame set of the six plansifter compartments.

Size of sieve holes used for the experiments and the amount of material fractions on each sieve (individual and cumulative) for the separated material are presented in table 9.

In every fraction there is a percentage of material with sizes smaller than the size of the sieve hole, which means that sieving is incomplete, even if the number of frames is quite high. However, the average particle size of fraction C1–Break 2 is 2.27 mm, much larger than the opening of sieve holes of the package (1.05 mm). This shows that here are obtained the parts of seed with quite large sizes, which must be reintroduced in the grinding process at the passage Break 2.

At the second set of sieving frames of plansifter compartment C1, the opening of fabric holes is 470 µm (no. 40), but mean size of particles of fraction C1-DIV1' is 0.58 mm, slightly larger than the opening of the holes. It is noticed (Table 9) that there are particles with sizes smaller than the size of holes which remain unseparated (at least 8.4%). This phenomenon is valid for all sets of sieves in the plansifter with six compartments, as can be seen from the analysis of the results presented in table 9.

Composition of fraction C1-DIV1" of plansifter compartment C1 consists of the refuse of sieve frames no. 56 (with holes opening 0.31 mm), after the sieved of the second set, consist‐ ing of particles that passes through sieve no. 40 (with holes opening 0.47 mm) was extracted flour F (mean size of particles 0.08 mm). This fraction with fraction C1-DIV1' and with the two fractions C2-DIV1 of the second plansifter compartment are directed to the sorting-di‐ viding compartment DIV1 (compartment C5). Mean particle sizes of fraction DIV1", from compartment C1, are 0.31 mm (equal to the opening of sieve holes which refused them, proving that here also the sieving is incomplete).

**Law type Coeff. Sr.1-E Sr.2-I Sr.2-E Sr.3-I Sr.3-E Sr.4-I Sr.4-E Sr.5-I Sr.5-E Rosin-Rammler (eq.1)** b 0.224 0.114 0.665 0.411 1.025 0.701 1.169 2.472 2.652

**Schuhman (eq.2)** k 4.201 3.398 2.893 2.966 2.531 2.532 2.431 1.025 1.016

**Table 8.** The coefficient values a, k, b, n, α and β and of the R<sup>2</sup> correlation coefficients, for the three size distribution laws tested, for the gritted products from the "I" entry to the "E" exit between the mentioned roll pairs (Sr.1...Sr.5),

In plansifter compartments, material fractions are separated and sorted, as any granular ma‐ terial is made of particles with sizes between a minimum and a maximum value, in the inte‐ rior of the mixture the size distribution being characterised by various distribution laws.

It must be mentioned that material particles, being extracted from various areas of the seed (from exterior to interior) have different mechanical characteristics and composition. This, and the different sizes of particles gives a different behaviour of the particles during grinding.

Thus is important to study and to know the size distribution of the particles of each fraction

Size of sieve holes used for the experiments and the amount of material fractions on each

In every fraction there is a percentage of material with sizes smaller than the size of the sieve hole, which means that sieving is incomplete, even if the number of frames is quite high. However, the average particle size of fraction C1–Break 2 is 2.27 mm, much larger than the opening of sieve holes of the package (1.05 mm). This shows that here are obtained the parts of seed with quite large sizes, which must be reintroduced in the grinding process at the

At the second set of sieving frames of plansifter compartment C1, the opening of fabric holes is 470 µm (no. 40), but mean size of particles of fraction C1-DIV1' is 0.58 mm, slightly larger than the opening of the holes. It is noticed (Table 9) that there are particles with sizes smaller than the size of holes which remain unseparated (at least 8.4%). This phenomenon is valid for all sets of sieves in the plansifter with six compartments, as can be seen from the analysis

Composition of fraction C1-DIV1" of plansifter compartment C1 consists of the refuse of sieve frames no. 56 (with holes opening 0.31 mm), after the sieved of the second set, consist‐

sieve (individual and cumulative) for the separated material are presented in table 9.

obtained in each frame set of the six plansifter compartments.

**Logistic with two parameters (eq.3)**

340 Food Industry

passage Break 2.

of the results presented in table 9.

[10]

n 1.659 2.302 1.382 1.747 1.682 2.220 2.093 2.852 2.817 R2 0,988 0,987 0,996 0,982 0,996 0,996 0,996 0,998 0,999

a 0.996 1723 0.674 0.960 0.495 0.711 0.464 1.710 1.639 R2 0,999 0,981 0,985 0,956 0,958 0,933 0.940 0,991 0.987

α 2.573 3.701 2.243 2.744 2.760 3.397 3.216 4.347 4.303 β -1.245 -1.666 -2.053 -1.981 -3.345 -3.380 -4.056 -6.739 -6.878 R2 0.984 0,974 0.972 0.963 0.988 0.995 0.994 0.997 0997


**Table 9.** Values of weights pi (%) of sieved fractions and of the cumulative weights Ti (%) for products collected at the entrance, respectively exit of plansifter compartments, C1 and C2

The last components of plansifter compartments in gristing passage shave higher content of coating particles which are found in the upper layers of material on the frames, thus being recommended that they do not separate through the holes, even if their sizes are about the size of endosperm particles, to be further removed in semolina machines (sieving motion leads to the layering of mixture components by density). Flour particles have mean sizes un‐ der 0.18 mm in all plansifter compartments, while particles of last refuse from the five pas‐ sages fitted with pairs of rollers have mean sizes over 0.37 mm (see Table 9). Values of coefficients b and n in the equation of relationship Rosin–Rammler cumulative distribution law (eq.1), for the material which passed through sieve holes in granulometric analysis, and the correlation coefficients R2 and χ<sup>2</sup> have high values which show the adequacy degree of the given function with the experimental data. In all cases, for all fractions obtained during gristing phase of wheat in the studied mill, the correlation is very good, appreciated by val‐ ues of coefficient R2 ≥ 0.926.

As it can be noticed from fig.10, there are fractions having most particles of sizes close to the minim value of sieve classifier holes, but there are also components with particles with sizes from the mean size to the maximum size of the sieve holes used for granulometric analysis.


0,0 0,5 1,0 1,5 2,0 2,5 3,0

Oriffice aperture li, mm

)


Oriffice aperture li, mm

Sifted material (cumulative), %

**Figure 10.** Curves of granulometric distribution given by eq. (1) in correlation with experimental data for grinding

Main stress to which seeds are subjected, while passing through mill rollers, is given by the type of rollers surface, namely smooth or fluted. Regardless the surface type, one of the main stress during grinding is compression (or crushing), especially if the mill rollers have smooth surface. To estimate the behaviour of seeds while passing through the rollers, exper‐ imental research is required on the compression stress of seeds from various wheat varieties, knowing that not all varieties have similar mechanical characteristics. Even seeds from the same variety have different behaviour, due to the irregular development stage in the ear,

The compression of wheat seeds is performed in three different stages: the first stage is elas‐ tically deformation, characterized by the proportionality between the compression force and the deformation; the second stage is plastic deformation, characterized by large increases of seed deformation at small increases of compression force; the last stage consists in cracking or rupture, being characterized by seed crushing when reaching a certain value of compres‐

fractions in plansifter compartments during gristing phase of wheat in a mill with capacity of 100 t /24 h

**4. Some mechanical characteristics of wheat seeds in uniaxial**

 C3 FT1 T(x)=100(1-e-b\*xn

b=0.206 n=2.654 R2 =0.976

 C6 M5 T(x)=100(1-e-b\*xn

b=188,250 n=3,148 R2 =0.999

)

Grinding Characteristics of Wheat in Industrial Mills

http://dx.doi.org/10.5772/53160

343

Sifted material (cumulative), %

Oriffice aperture li, mm

)


Oriffice aperture li, mm

**compression tests**

and also from one ear to another.

sion force, [17-20].

Sifted material (cumulative), %

 C2 Break 3 T(x)=100(1-e-b\*xn

b=0.327 n=1.925 R2 =0.986

 C4 FT2 T(x)=100(1-e-b\*xn

b=0.026 n=4,556 R2 =0.995 )

Sifted material (cumulative), %

However, most components show mean profile (with central inflection point) of the separa‐ tion curves which demonstrates the correct choosing of sieve classifier sizes (made from a set of 30 sieves by trying to take into consideration the arrangement in geometric distribu‐ tion with holes ratio of 2).

From the analysis of coefficients b and n from Rosin–Rammler law (eq.1) it is noticed that values of coefficient b are 0.2–1.5 10<sup>3</sup> for most analyzed fractions, generally with high values, for the small size components of the particles (flour or dunsts), 1 106 – 5 10<sup>7</sup> , giving the size characteristics of such particles (Table 10).


**Table 10.** Values of coefficients b and n and correlation coefficient R2 for Rosin – Rammler granulometric distribution, for the granulometric distribution law for fractions of the two plansifter compartments

Values of exponent n indicate the uniformity or the irregularity degree of particles from the analyzed fractions.

The analysis of this exponent values for the fractions of each plansifter compartment (Table 9) shows that they have a wide range of values, even for the same type of grinding product (for example flour – F), which shows the irregularity of particles, both for a given fraction and between fractions.

coefficients b and n in the equation of relationship Rosin–Rammler cumulative distribution law (eq.1), for the material which passed through sieve holes in granulometric analysis, and

the given function with the experimental data. In all cases, for all fractions obtained during gristing phase of wheat in the studied mill, the correlation is very good, appreciated by val‐

As it can be noticed from fig.10, there are fractions having most particles of sizes close to the minim value of sieve classifier holes, but there are also components with particles with sizes from the mean size to the maximum size of the sieve holes used for granulometric analysis.

However, most components show mean profile (with central inflection point) of the separa‐ tion curves which demonstrates the correct choosing of sieve classifier sizes (made from a set of 30 sieves by trying to take into consideration the arrangement in geometric distribu‐

From the analysis of coefficients b and n from Rosin–Rammler law (eq.1) it is noticed that values of coefficient b are 0.2–1.5 10<sup>3</sup> for most analyzed fractions, generally with high values,

**compartmen**t

C4

**Table 10.** Values of coefficients b and n and correlation coefficient R2 for Rosin – Rammler granulometric distribution,

Values of exponent n indicate the uniformity or the irregularity degree of particles from the

The analysis of this exponent values for the fractions of each plansifter compartment (Table 9) shows that they have a wide range of values, even for the same type of grinding product (for example flour – F), which shows the irregularity of particles, both for a given fraction

for the granulometric distribution law for fractions of the two plansifter compartments

C1 Break 2 0.169 1.964 0.987 18.890 C4 F 2.06·103 5.665 0.997 1.144 C1 DIV1" 37.812 3.412 0.993 9.147 C4 M4 1.15⋅10<sup>3</sup> 3.967 0.976 36.414 C1 DIV1' 15.782 6.033 0.997 6.245 C4 FT2 0.027 4.557 0.996 9.071 C1 F 2.2·103 3.051 0.938 142.827 C4 Break 5 2.590 2.983 0.999 1.091 C1 M2 2.86·103 5.028 0.988 20.440 C4 M5 1.6·103 6.054 0.999 3.370

for the small size components of the particles (flour or dunsts), 1 106 – 5 10<sup>7</sup>

**<sup>b</sup> <sup>n</sup> R² χ² Plansifter**

have high values which show the adequacy degree of

, giving the size

**b n R² χ²**

C4 Entrance 0.621 1.958 0.988 21.914

and χ<sup>2</sup>

the correlation coefficients R2

tion with holes ratio of 2).

**Plansifter compartment**

analyzed fractions.

and between fractions.

C1

characteristics of such particles (Table 10).

C1 Entrance 0.222 1.663 0.988 17.039

≥ 0.926.

ues of coefficient R2

342 Food Industry

**Figure 10.** Curves of granulometric distribution given by eq. (1) in correlation with experimental data for grinding fractions in plansifter compartments during gristing phase of wheat in a mill with capacity of 100 t /24 h

## **4. Some mechanical characteristics of wheat seeds in uniaxial compression tests**

Main stress to which seeds are subjected, while passing through mill rollers, is given by the type of rollers surface, namely smooth or fluted. Regardless the surface type, one of the main stress during grinding is compression (or crushing), especially if the mill rollers have smooth surface. To estimate the behaviour of seeds while passing through the rollers, exper‐ imental research is required on the compression stress of seeds from various wheat varieties, knowing that not all varieties have similar mechanical characteristics. Even seeds from the same variety have different behaviour, due to the irregular development stage in the ear, and also from one ear to another.

The compression of wheat seeds is performed in three different stages: the first stage is elas‐ tically deformation, characterized by the proportionality between the compression force and the deformation; the second stage is plastic deformation, characterized by large increases of seed deformation at small increases of compression force; the last stage consists in cracking or rupture, being characterized by seed crushing when reaching a certain value of compres‐ sion force, [17-20].

Compression test is an objective method for determining the mechanical properties of cereal seeds and also one of the best techniques for determining the modulus of elasticity by the study of their behaviour at compression stress, using force-deformation curve, [21,22].

By performing uniaxial compression tests on wheat seeds, force-deformation curve is ob‐ tained, giving the possibility to determine hardness, apparent modulus of elasticity, crush‐ ing resistance, force and deformation and energy consumption in various specific points of the curve (i.e. rupture point) and maximum stress in the material, [21,23].

Cereal seeds have a different behaviour under the action of compression forces, depending on their moisture content, [17,20], variety, development stage, geometric sizes, individual mass, glassiness, (soft cereals and hard cereals) etc.

In fig.1 is presented a typical force-deformation curve for compressed Flamura wheat seed.

The bioyield point is the point on the force – deformation curve at which the force decreases or remains constant with increasing deformation. Force in the rupture point (rupture force) is the minimum required force for the wheat seed to break (rupture). Deformation at bio‐ yield and rupture points is the deformation at loading direction, [24,25]. Values of force and deformation to bioyield and rupture points are directly read from force-deformation curve and recorded by machine used for compression test, [21].

Energy absorbed in bioyield and rupture points could be determined from the area under the force-deformation curve between the initial point and the bioyield and rupture point, re‐ spectively, using equation [24,25]:

$$\mathcal{W} = \frac{FD}{2} \text{(mJ)}\tag{9}$$

Based on a standard method (ASAE 2008, [21]), for a seed placed between two parallel plates, the modulus of elasticity could be calculated with following equation, [20,21,21]:

> ( ) 3 2 <sup>2</sup> 1 2 3 2 <sup>1</sup>

*D R R* mé ù = + ê ú ë û

where: E – modulus of elasticity for cereal seeds, (MPa); ku – coefficient which depends on the geometrical properties of wheat seeds (ku = 1,303 - adapted from calculus tables of Koz‐ ma and Cunningham, 1962); *F* – compression force, (N); *D* – seed deformation (m); µ - Pois‐ son ratio, (µ = 0,3 for wheat seeds); *R*' and *R*'*1* – small and large radius of the curvature of

**Figure 12.** Estimation of curvature radius and force-deformation curve of wheat seed, (adapted from [25,26]) PL –

According to the standard method (ASAE 2008, [21]), also presented by Mohsenin in [25,26],

2

4 '

*<sup>1</sup>* (fig.12) can be calculated using relations (11)

*<sup>H</sup> <sup>R</sup>* @ (11)

<sup>+</sup> @ (12)

and *R'*

' 2

1

where: *H* is seed thickness, (m), and *L* is seed length, (m), in undistorted state.

 2 *H L <sup>R</sup> H* ' '

(10)

345

Grinding Characteristics of Wheat in Industrial Mills

http://dx.doi.org/10.5772/53160

2

0.338 1 1 1

*uk F*

convex surface seed in contact with the flat surface, (m), (see fig.12, left).

*E*

proportional limit; PI – point of inflection; Pc – point of calculation

curvature radius of convex surface, *R'*

and (12):

where: *W* is energy absorbed (mJ), *F* is force in bioyield or rupture point (N), *D* is deforma‐ tion in bioyield or rupture point (mm), (see fig.11).

**Figure 11.** A typical force –deformation curve of wheat grain (type Flamura), [20]

Based on a standard method (ASAE 2008, [21]), for a seed placed between two parallel plates, the modulus of elasticity could be calculated with following equation, [20,21,21]:

Compression test is an objective method for determining the mechanical properties of cereal seeds and also one of the best techniques for determining the modulus of elasticity by the study of their behaviour at compression stress, using force-deformation curve, [21,22].

By performing uniaxial compression tests on wheat seeds, force-deformation curve is ob‐ tained, giving the possibility to determine hardness, apparent modulus of elasticity, crush‐ ing resistance, force and deformation and energy consumption in various specific points of

Cereal seeds have a different behaviour under the action of compression forces, depending on their moisture content, [17,20], variety, development stage, geometric sizes, individual

In fig.1 is presented a typical force-deformation curve for compressed Flamura wheat seed.

The bioyield point is the point on the force – deformation curve at which the force decreases or remains constant with increasing deformation. Force in the rupture point (rupture force) is the minimum required force for the wheat seed to break (rupture). Deformation at bio‐ yield and rupture points is the deformation at loading direction, [24,25]. Values of force and deformation to bioyield and rupture points are directly read from force-deformation curve

Energy absorbed in bioyield and rupture points could be determined from the area under the force-deformation curve between the initial point and the bioyield and rupture point, re‐

(mJ) <sup>2</sup>

where: *W* is energy absorbed (mJ), *F* is force in bioyield or rupture point (N), *D* is deforma‐

0 0,5 1 1,5 2 2,5

Deformation, mm

Crushing (breaking) domain

*F D <sup>W</sup>* <sup>=</sup> (9)

the curve (i.e. rupture point) and maximum stress in the material, [21,23].

mass, glassiness, (soft cereals and hard cereals) etc.

and recorded by machine used for compression test, [21].

tion in bioyield or rupture point (mm), (see fig.11).

**Figure 11.** A typical force –deformation curve of wheat grain (type Flamura), [20]

Elastic deformation

> Bioyield point

Rupture point

Plastic deformation

Force, N

spectively, using equation [24,25]:

344 Food Industry

$$E = \frac{0.338 \, k\_{\mu}^{3/2} \, F \left(1 - \mu^2\right)}{D^{3/2}} \left[\frac{1}{R} + \frac{1}{R\_1}\right]^{3/2} \tag{10}$$

where: E – modulus of elasticity for cereal seeds, (MPa); ku – coefficient which depends on the geometrical properties of wheat seeds (ku = 1,303 - adapted from calculus tables of Koz‐ ma and Cunningham, 1962); *F* – compression force, (N); *D* – seed deformation (m); µ - Pois‐ son ratio, (µ = 0,3 for wheat seeds); *R*' and *R*'*1* – small and large radius of the curvature of convex surface seed in contact with the flat surface, (m), (see fig.12, left).

**Figure 12.** Estimation of curvature radius and force-deformation curve of wheat seed, (adapted from [25,26]) PL – proportional limit; PI – point of inflection; Pc – point of calculation

According to the standard method (ASAE 2008, [21]), also presented by Mohsenin in [25,26], curvature radius of convex surface, *R'* and *R' <sup>1</sup>* (fig.12) can be calculated using relations (11) and (12):

$$R' \equiv \frac{H}{2} \tag{11}$$

$$R\_1 \stackrel{\cdot}{=} \frac{H + L^2 \not\!\!/ 4}{2H} \tag{12}$$

2

where: *H* is seed thickness, (m), and *L* is seed length, (m), in undistorted state.

H

H

R'

H

R'

R'

R'

H

L

R'

H

H

L

R'

L

This method was used by many researchers to determine the modulus of elasticity for differ‐ ent agricultural products, [27-30].

On the histograms were traced the variation curves for the analyzed parameters by regres‐ sion analysis of the values given by the histogram, using the normal function presented in

where: px(%) is the percentage weight of each class interval (number of seeds with values in the considered class interval); a – class interval for each analyzed parameter; b and c are re‐ gression coefficients of the analyzed function (*b* is the standard deviation, *c* is the values

Values of coefficients for the regression function (eq.13) used in statistical analysis and val‐

Length l, (mm) 0.20 0.404 6.443 0.989 0.20 0.501 6.186 0.971 Width w, (mm) 0.10 0.202 3.429 0.974 0.20 0.284 2.994 0.981 Thickness t, (mm) 0.20 0.248 3.058 0.975 0.20 0.315 2.664 0.983 Mass m, (g) 0.01 0.008 0.051 0.988 0.01 0.009 0.037 0.981 Volume V, (mm3) 5.00 5.870 35.57 0.968 5.00 6.207 26.23 0.985 Bioyield force F1, (N) 20.0 41.36 122.64 0.921 20.0 39.48 104.70 0.888 Bioyield energy W1, (J) 0.01 0.024 0.036 0.923 0.01 0.017 0.026 0.884

**Table 11.** Values of coefficients for regression equation (eq.13) and its correlation with experimental data [20]

Analysis of histograms and variation curves, as well as of data in table 11, shows that all analyzed parameters have almost normal distribution, assessed by values of correlation co‐

Using standard method (ASAE 2008, [21]) and equations (10), (11) and (12) were determined the values of modululs of elasticity for wheat seeds of Flamura, Trivale and Glosa varieties,

Fig.15 shows the machine used for uniaxial compression tests between parallel plates of

From the sample of 100 determinations for each variety of wheat, were selected the 50 most representative determinations, being kept the values found for force and absolute deforma‐

in this paper being presented their mean values, (table 12).

<sup>1</sup> <sup>2</sup> <sup>100</sup> 2

*<sup>x</sup> pae* p*b*

<sup>2</sup> <sup>1</sup>

*x c b*

æ -ö - ç ÷ è ø <sup>=</sup> (13)

Grinding Characteristics of Wheat in Industrial Mills

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347

for data given by histograms are presented in Table 11.

**Flamura wheat variety Trivale wheat variety a b c R2 a b c R2**

equation (13), [23]:

ues of correlation coefficient R2

**Measured parameters of wheat seeds**

mean).

efficient R2

tion of the seed.

.

weat seeds and their position.

According to the standard method (ASAE 2008), values of force *F* and deformation *D*, from equation (2) are calculated for the proportionality area of force-deformation curve in the point of calculation *Pc* (fig.12). The position of this point is estimated visually, as the point is located halfway between curve origin and proportionality limit PL (fig.12, right). It was found that the point of calculation *Pc* is located lower than the point of inflection, also estab‐ lished visually, [21].

To determine the variation of mechanical resistance characteristics of wheat seeds from the same variety, compression tests were performed for sets of 100 seeds of three varieties of Ro‐ manian wheat (Flamura, Glosa and Trivale – soft wheat), using Hounsfield mechanical test‐ ing machine, at a constant speed of the crushing device of 5 mm min-1, using a force cell of 1000 N. Were graphically plotted the force-deformation curves for each seed, and from each diagram were collected data about: force, deformation and energy absorbed in the bioyield point (F1, ε1, W1), respectively in the final point (rupture), (F2, ε2, W2).

The analysis of measured data showed that the seeds of Flamura variety were larger than Trivale variety, for all three main sizes, and for their volume. The same goes for seeds mass. Flamura variety seeds were more uniform as size and mass. Regarding the mechanical char‐ acteristics of wheat seeds, it was found that compression forces, for bioyield point and for final seeds crushing, were smaller for Trivale variety than Flamura. The same goes for ener‐ gy absorbed to the bioyield point, respectively to crushing. Since the sizes of Trivale seeds were smaller than Flamura seeds, the deformations carried to the bioyield point, respective‐ ly to crushing, were smaller for Trivale than Flamura, but the standard deviation of the val‐ ues was smaller for Flamura for deformations, showing that Flamura seeds were more regular in terms of deformations (until crushing).

In fig.13 are presented two examples of force-deformation curves for two varieties of wheat, and in fig.14 are presented the histograms of bioyield force and energy absorbed for seed crushing.

**Figure 13.** Examples of force-deformation curves for the two wheat varieties, [23]

On the histograms were traced the variation curves for the analyzed parameters by regres‐ sion analysis of the values given by the histogram, using the normal function presented in equation (13), [23]:

This method was used by many researchers to determine the modulus of elasticity for differ‐

According to the standard method (ASAE 2008), values of force *F* and deformation *D*, from equation (2) are calculated for the proportionality area of force-deformation curve in the point of calculation *Pc* (fig.12). The position of this point is estimated visually, as the point is located halfway between curve origin and proportionality limit PL (fig.12, right). It was found that the point of calculation *Pc* is located lower than the point of inflection, also estab‐

To determine the variation of mechanical resistance characteristics of wheat seeds from the same variety, compression tests were performed for sets of 100 seeds of three varieties of Ro‐ manian wheat (Flamura, Glosa and Trivale – soft wheat), using Hounsfield mechanical test‐ ing machine, at a constant speed of the crushing device of 5 mm min-1, using a force cell of 1000 N. Were graphically plotted the force-deformation curves for each seed, and from each diagram were collected data about: force, deformation and energy absorbed in the bioyield

The analysis of measured data showed that the seeds of Flamura variety were larger than Trivale variety, for all three main sizes, and for their volume. The same goes for seeds mass. Flamura variety seeds were more uniform as size and mass. Regarding the mechanical char‐ acteristics of wheat seeds, it was found that compression forces, for bioyield point and for final seeds crushing, were smaller for Trivale variety than Flamura. The same goes for ener‐ gy absorbed to the bioyield point, respectively to crushing. Since the sizes of Trivale seeds were smaller than Flamura seeds, the deformations carried to the bioyield point, respective‐ ly to crushing, were smaller for Trivale than Flamura, but the standard deviation of the val‐ ues was smaller for Flamura for deformations, showing that Flamura seeds were more

In fig.13 are presented two examples of force-deformation curves for two varieties of wheat, and in fig.14 are presented the histograms of bioyield force and energy absorbed for seed

Force, N

w = 12% l = 6,78 m m b = 2,85 m m c = 2,7 m m

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4 1 .6 1 .8 2 .0 2 .2 2 .4 D e fo rm a ti o n , m m

**Trivale**

point (F1, ε1, W1), respectively in the final point (rupture), (F2, ε2, W2).

regular in terms of deformations (until crushing).

0.0 0.5 1.0 1.5 2.0 2.5 3 .0 D e fo rm a tio n , m m

**Figure 13.** Examples of force-deformation curves for the two wheat varieties, [23]

**Flamura**

w = 12% l = 6,15 m m b = 3,41 m m c = 2,87 m m

Force, N

ent agricultural products, [27-30].

lished visually, [21].

346 Food Industry

crushing.

H

H

R'

H

R'

R'

R'

H

L

R'

H

H

L

R'

L

$$p\_x = 100a \frac{1}{\sqrt{2\pi}} e^{-\frac{1}{2}\left(\frac{x-c}{b}\right)^2} \tag{13}$$

where: px(%) is the percentage weight of each class interval (number of seeds with values in the considered class interval); a – class interval for each analyzed parameter; b and c are re‐ gression coefficients of the analyzed function (*b* is the standard deviation, *c* is the values mean).

Values of coefficients for the regression function (eq.13) used in statistical analysis and val‐ ues of correlation coefficient R2 for data given by histograms are presented in Table 11.


**Table 11.** Values of coefficients for regression equation (eq.13) and its correlation with experimental data [20]

Analysis of histograms and variation curves, as well as of data in table 11, shows that all analyzed parameters have almost normal distribution, assessed by values of correlation co‐ efficient R2 .

Using standard method (ASAE 2008, [21]) and equations (10), (11) and (12) were determined the values of modululs of elasticity for wheat seeds of Flamura, Trivale and Glosa varieties, in this paper being presented their mean values, (table 12).

Fig.15 shows the machine used for uniaxial compression tests between parallel plates of weat seeds and their position.

From the sample of 100 determinations for each variety of wheat, were selected the 50 most representative determinations, being kept the values found for force and absolute deforma‐ tion of the seed.

**Figure 14.** Histograms and variation curves for the force in bioyeld point and the energy consumption in rupture point for wheat seeds [23]

Force-deformation curves, for each of the 50 determinations (of a variety) were processed so that each has the same origin (same starting point), and the intervals of reading (recorded) to be the same. Values for the parameter on the ordinate (forces in the mentioned points) were averaged (arithmetic average for the 50 determinations was calculated) for the same value of deformation (parameter on the abscissa), and these values were used to retrace the force-deformation curve, which respresents the curve of mean values of compression force (fig.16). Using the approximately normal distribution, were statistically estimated the limits within which the mean force-deformation curve is found, for a confidence interval of 95%. For normal distribution, the confidence interval corresponding to 95% confidence level ranges between +/- 1,96, considered standard deviations. Thus, the confidence interval of mean curve was calculated using the following equation:

$$
\Delta \mu = m \pm 1.96 \frac{\sigma}{\sqrt{n}} \tag{14}
$$

On the curve of mean values (fig.16), were determined the values of mechanical characteris‐ tics mentioned before (forces and deformations in the characteristic points) and it was calcu‐ lated the value of modulus of elasticity using the standard method (ASAE 2008, [21]), for

Knowing the forces and deformations in the points of bioyield and rupture, from the area under the force-deformation curve between the initial point and the bioyield and rupture point, respectively, using equation (1), energy absorbed in bioyield and rupture point was

> Bioyield force Fb, (N) 93.2 83.1 98.0 98.4 81.1 94.0 Bioyield energy Wb, (J) - - - 0.028 0.018 0.016 Rupture force Fr, (N) 107.8 90.5 103.6 104.2 83.2 94.7 Rupture energy Wr, (J) - - - 0.038 0.018 0.016

Relative deformation, δ<sup>b</sup> 0.138 0.092 0.077 - - -

Relative deformation, δ<sup>r</sup> 0.099 0.109 0.086 - - -

Modulus of elasticity, (MPa) 313 364 486 298 369 468

**Table 12.** Values of measured and determined parameters in uniaxial compression test [20]

**Compression table**

**the mean curve**

**Compression head**

**Loading**

Grinding Characteristics of Wheat in Industrial Mills

http://dx.doi.org/10.5772/53160

349

**Mean of parameters values Values of parameters read from**

**Flamura Trivale Glosa Flamura Trivale Glosa**

0.304 0.267 0.260 0.464 0.348 0.292

0.419 0.320 0.290 0.576 0.400 0.360

**Wheat seed**

mean curve (for the three varieties of wheat).

**Figure 15.** Hounsfield - Mechanical testing machine used in compression test [20]

determined.

Bioyield deformation

Rupture deformation

**Measured parameters of wheat seeds**

Absolute deformation, Db (mm)

Absolute deformation, Dr (mm)

where: *μ* is the confidence interval, and *m* is the mean value of the analyzed parameter (in this case, the compression force) and *σ* / *n* =*Sm* is the standard error of the mean, *σ* – stand‐ ard deviation, and *n* –number of seeds from each variety of wheat (in this paper, n = 50).

On the curve of mean values (fig.16), were determined the values of mechanical characteris‐ tics mentioned before (forces and deformations in the characteristic points) and it was calcu‐ lated the value of modulus of elasticity using the standard method (ASAE 2008, [21]), for mean curve (for the three varieties of wheat).

**Figure 15.** Hounsfield - Mechanical testing machine used in compression test [20]

**Figure 14.** Histograms and variation curves for the force in bioyeld point and the energy consumption in rupture

Force-deformation curves, for each of the 50 determinations (of a variety) were processed so that each has the same origin (same starting point), and the intervals of reading (recorded) to be the same. Values for the parameter on the ordinate (forces in the mentioned points) were averaged (arithmetic average for the 50 determinations was calculated) for the same value of deformation (parameter on the abscissa), and these values were used to retrace the force-deformation curve, which respresents the curve of mean values of compression force (fig.16). Using the approximately normal distribution, were statistically estimated the limits within which the mean force-deformation curve is found, for a confidence interval of 95%. For normal distribution, the confidence interval corresponding to 95% confidence level ranges between +/- 1,96, considered standard deviations. Thus, the confidence interval of

> *<sup>m</sup>* 1.96 *<sup>n</sup>* s

where: *μ* is the confidence interval, and *m* is the mean value of the analyzed parameter (in this case, the compression force) and *σ* / *n* =*Sm* is the standard error of the mean, *σ* – stand‐ ard deviation, and *n* –number of seeds from each variety of wheat (in this paper, n = 50).

= ± (14)

m

mean curve was calculated using the following equation:

point for wheat seeds [23]

348 Food Industry

Knowing the forces and deformations in the points of bioyield and rupture, from the area under the force-deformation curve between the initial point and the bioyield and rupture point, respectively, using equation (1), energy absorbed in bioyield and rupture point was determined.


**Table 12.** Values of measured and determined parameters in uniaxial compression test [20]

Analysis of data presented in table 12 showed that the values of bioyield force, respectively values of the force in the point of rupture of wheat seeds, determined from the mean curve are very close to the values of these forces obtained from the force-deformation curves for each particular seed.

known laws, from which most used is Rosin-Rammler distribution function, with high cor‐

Grinding Characteristics of Wheat in Industrial Mills

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351

However, it is shown that there can also be used with good results the Schuhman and logis‐ tical two parameters distribution laws, the finding suggest that the type of granulometric distribution law which best describes the size of grinded biological materials depends on material nature and the place and role of roller mill used for grinding in the general techno‐ logical flow. Knowledge of adequate mathematical models describing the size distribution of grinded materials is useful in all engineering activities related to the processes on the flow

Values of mechanical characteristics of wheat seeds (regardless the variety) are necessary to estimate the energy consumed for their grinding in grain mills. A great influence on the grinding energy is given by the crushing force and their relative and absolute deformation,

For some wheat varieties presented in this chapter, compression force in the rupture point, determined from force-deformation curves has values of 100-110 N, for seed moisture con‐

Crushing energy has values of 0.02-0.04 J, for each wheat seed, but it is influenced by the moisture of seeds and by seed arrangement during compression: on width "sideways" or on

Regarding the modulus of elasticity, its values are between 313-487 MPa, being greater as moisture is lower. It was found that lower moisture content resulted in higher values of modulus of elasticity and to lower values of rupture energy, which confirm that wetter seeds have greater plasticity than dry seeds, so they have higher energy consumption.

[1] Fang Ch., Campbell G.M. Stress-Strain Analysis and Visual Observation of Wheat Kernel Breakage During Roller Milling Using Fluted Rolls. Cereal Chemistry 2002;

[2] Mazlina S., Kamal M. Evaluation of the potential role recycle within the flour milling

relation coefficient R2

tent of about 12%.

**Author details**

**References**

79(4) 511–517.

thickness "laying flat".

.

of complex roller mills of last generation.

determined by experimental research of uniaxial compression.

Gheorghe Voicu, Sorin-Stefan Biris, Elena-Madalina Stefan, Gabriel-Alexandru Constantin and Nicoleta Ungureanu

break system. Universiti Putra Malaysia, TLN 2006; 42.

"Politehnica" University of Bucharest, Romania

**Figure 16.** Mean curves force-deformation for three wheat varieties and 95 % confidence interval, [20]

Analysis of curves presented in figure 15 shows that they have similar shapes for the three varieties of wheat, and also within each of them and the force-deformation curves for each individual seed analyzed from each variety of wheat.

As absolute values of the force in the bioyield point, respectively in the rupture point, they are found in between 83.1 N for Trivale variety and 98.0 N for Glosa variety regarding the bioyield force, respectively 90.5 N for Trivale and 107.8 N for Flamura (values calculated with arithmetic average of the 50 determinations). These values are very close to the values presented in literature [31], where is stated that crushing force (rupture) of wheat seeds is of approximately 100 N.

On the relative deformation of seeds, during the compression tests, for the force in the bio‐ yield point (bioyield force), respectively rupture, data in table 12 also show relatively close values for the wheat seeds of the three varieties.

## **5. Conclusions**

Development of technological gristing process of the wheat in a mill is very important for the entire technological flow of the mill, having a great influence on the degree of flour ex‐ traction, without excessive grinding of seed coating.

Based on material samples taken from the entrance and exit of each pair of milling rollers it can be determined, by laboratory analysis, the equivalent average sizes of the material, grinding degree in the passage, and the specific surface of material particles.

Granulometric analysis of the material to be grinded or of the grinded material at mill roll‐ ers, and of the sorted fractions in plansifter compartments show a distribution after multiple known laws, from which most used is Rosin-Rammler distribution function, with high cor‐ relation coefficient R2 .

However, it is shown that there can also be used with good results the Schuhman and logis‐ tical two parameters distribution laws, the finding suggest that the type of granulometric distribution law which best describes the size of grinded biological materials depends on material nature and the place and role of roller mill used for grinding in the general techno‐ logical flow. Knowledge of adequate mathematical models describing the size distribution of grinded materials is useful in all engineering activities related to the processes on the flow of complex roller mills of last generation.

Values of mechanical characteristics of wheat seeds (regardless the variety) are necessary to estimate the energy consumed for their grinding in grain mills. A great influence on the grinding energy is given by the crushing force and their relative and absolute deformation, determined by experimental research of uniaxial compression.

For some wheat varieties presented in this chapter, compression force in the rupture point, determined from force-deformation curves has values of 100-110 N, for seed moisture con‐ tent of about 12%.

Crushing energy has values of 0.02-0.04 J, for each wheat seed, but it is influenced by the moisture of seeds and by seed arrangement during compression: on width "sideways" or on thickness "laying flat".

Regarding the modulus of elasticity, its values are between 313-487 MPa, being greater as moisture is lower. It was found that lower moisture content resulted in higher values of modulus of elasticity and to lower values of rupture energy, which confirm that wetter seeds have greater plasticity than dry seeds, so they have higher energy consumption.

## **Author details**

Analysis of data presented in table 12 showed that the values of bioyield force, respectively values of the force in the point of rupture of wheat seeds, determined from the mean curve are very close to the values of these forces obtained from the force-deformation curves for

**Figure 16.** Mean curves force-deformation for three wheat varieties and 95 % confidence interval, [20]

individual seed analyzed from each variety of wheat.

values for the wheat seeds of the three varieties.

traction, without excessive grinding of seed coating.

Analysis of curves presented in figure 15 shows that they have similar shapes for the three varieties of wheat, and also within each of them and the force-deformation curves for each

As absolute values of the force in the bioyield point, respectively in the rupture point, they are found in between 83.1 N for Trivale variety and 98.0 N for Glosa variety regarding the bioyield force, respectively 90.5 N for Trivale and 107.8 N for Flamura (values calculated with arithmetic average of the 50 determinations). These values are very close to the values presented in literature [31], where is stated that crushing force (rupture) of wheat seeds is of

On the relative deformation of seeds, during the compression tests, for the force in the bio‐ yield point (bioyield force), respectively rupture, data in table 12 also show relatively close

Development of technological gristing process of the wheat in a mill is very important for the entire technological flow of the mill, having a great influence on the degree of flour ex‐

Based on material samples taken from the entrance and exit of each pair of milling rollers it can be determined, by laboratory analysis, the equivalent average sizes of the material,

Granulometric analysis of the material to be grinded or of the grinded material at mill roll‐ ers, and of the sorted fractions in plansifter compartments show a distribution after multiple

grinding degree in the passage, and the specific surface of material particles.

each particular seed.

350 Food Industry

approximately 100 N.

**5. Conclusions**

Gheorghe Voicu, Sorin-Stefan Biris, Elena-Madalina Stefan, Gabriel-Alexandru Constantin and Nicoleta Ungureanu

"Politehnica" University of Bucharest, Romania

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**Chapter 16**

**Technological Options of**

Amalia Conte, Luisa Angiolillo, Marcella Mastromatteo and Matteo Alessandro Del Nobile

http://dx.doi.org/10.5772/53151

**1. Introduction**

**Packaging to Control Food Quality**

Additional information is available at the end of the chapter

The shelf life of perishable foods as meat, poultry, fish, fruit, vegetables and fresh cerealbased products is limited by various factors that generally bring to changes in odor, flavor, color and texture until to their complete unacceptability. Packaging is the main tool to pre‐ vent product deterioration and prolong its shelf life. The package protects the food against physical, chemical and biological damage. It also acts as a physical barrier to oxygen, mois‐ ture, volatile chemical compounds and microorganisms that are detrimental to food. The package has to be considered as an integral part of the preservation system because it pro‐ vides a barrier between the food and the external environment. It is usually a composite item meeting several different needs [1]. What we call the preservation role is a fundamental requirement of food packaging, since it is directly related to the safety of the consumer. Package performance depends on numerous variables, such as the initial food quality, the processing operations, the size, the shape and appearance of package, the distribution meth‐ od and the disposal of packages. Generally specking, the properties which determine their adequacy to meet performance requirements can be grouped into the following categories: mechanical, thermal, optical and mass transport properties. Mass transport phenomena are of great importance to food packaging with plastics, since a polymeric matrix is permeable to moisture, oxygen, carbon dioxide, nitrogen and other low molecular weight compounds. Glass and metal packaging materials are not permeable to low molecular weight com‐ pounds, whereas paper-based materials are too permeable. Hence, these last types of materi‐ als do not provide an opportunity for the designer to optimize the barrier properties for various applications. The polymers can provide a wide range (by three or four orders of

> © 2013 Conte et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.


**Chapter 16**

## **Technological Options of Packaging to Control Food Quality**

Amalia Conte, Luisa Angiolillo, Marcella Mastromatteo and Matteo Alessandro Del Nobile

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53151

## **1. Introduction**

[28] Baslar M., Kalkan F., Mazhar Kara M., Ertugay M.F., Correlation between the protein content and mechanical properties of wheat, Turkish Journal of Agriculture and For‐

[29] Moya M., Ayuga F., Guaita M., Aguado P.J. Mechanical properties of granular agri‐ cultural materials considered in silos designe. 15th ASCE Engineering Mechanics Conference, Columbia University, New York, (2002), http://www.civil.columbia.edu/

[30] Abbaspour-Fard M.H., Khodabakhshian R., Emadi B., Sadrnia H. Evaluation the Ef‐ fects of Some Relevant Parameters Elastic Modulus of Pumpkin Seed and its Kernel. International Journal of Biomaterials 2012; ID 271650, http://www.sciencedirect.com

[31] Emadi B., Khodabakhshian R., Abbaspour Fard M.H., Sadrnia H. Experimental com‐ parison of applying different theories in elasticity for determination of the elasticity modulus of agricultural produce. Pumpkin seed as a case study. Journal of Agricul‐

estry 2012; 36, http://mistug.tubitak.gov.tr (accessed May 2012).

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(accessed May 2012).

354 Food Industry

tural Technology 2011; 7(6) 1495-1508.

The shelf life of perishable foods as meat, poultry, fish, fruit, vegetables and fresh cerealbased products is limited by various factors that generally bring to changes in odor, flavor, color and texture until to their complete unacceptability. Packaging is the main tool to pre‐ vent product deterioration and prolong its shelf life. The package protects the food against physical, chemical and biological damage. It also acts as a physical barrier to oxygen, mois‐ ture, volatile chemical compounds and microorganisms that are detrimental to food. The package has to be considered as an integral part of the preservation system because it pro‐ vides a barrier between the food and the external environment. It is usually a composite item meeting several different needs [1]. What we call the preservation role is a fundamental requirement of food packaging, since it is directly related to the safety of the consumer. Package performance depends on numerous variables, such as the initial food quality, the processing operations, the size, the shape and appearance of package, the distribution meth‐ od and the disposal of packages. Generally specking, the properties which determine their adequacy to meet performance requirements can be grouped into the following categories: mechanical, thermal, optical and mass transport properties. Mass transport phenomena are of great importance to food packaging with plastics, since a polymeric matrix is permeable to moisture, oxygen, carbon dioxide, nitrogen and other low molecular weight compounds. Glass and metal packaging materials are not permeable to low molecular weight com‐ pounds, whereas paper-based materials are too permeable. Hence, these last types of materi‐ als do not provide an opportunity for the designer to optimize the barrier properties for various applications. The polymers can provide a wide range (by three or four orders of

© 2013 Conte et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

magnitude) of permeability for different applications, thus justifying studies aimed to en‐ sure adequate barrier protection. Therefore, in situations where food deteriorations are driv‐ en by either gas or moisture permeation to the ambient environment, an accurate choice of packaging mass transport properties may bring about an increase to product shelf life. Each category food has its specificity in quality attributes, storage conditions, expected shelf life and packaging tools applied. Together with transport properties of film, another valid pack‐ aging option to maintain product quality is represented by the proper selection of head‐ space conditions. Vacuum packaging and modified atmosphere packaging (MAP) are two widely used strategies for food preservation [2]. The first strategy means a complete lack of gas in the package whereas, under MAP, headspace environment may change during stor‐ age but there is no additional manipulation of the internal environment. Packaging under these conditions can protect products against deteriorative effects, which may include dis‐ coloration, off-flavor and off-odor development, nutrient loss, texture changes, pathogenici‐ ty, and other measurable factors. With the increasing demand for fresh and natural products without addition of dangerous chemicals, MAP or vacuum seem to be ideal methods of preservation for many foods, being simple and cheap to be applied. The few disadvantages are related to need of equipments and proper packaging materials and, in the specific case of MAP, to the limitation on retail for the increased pack volume of bags.

tamination or degradation. Active packaging developments are now focusing on incorpo‐ rating the agents into the polymeric matrices which constitute the package walls; the resulting materials act by releasing substances which have a positive effect on the food or by retaining undesired substances from the food or the internal atmosphere of the pack‐ age. The migration of a substance may be achieved by direct contact between food and packaging material or through gas phase diffusion from packaging layer to food surface. Although the former is the packaging situation usually meets, the latter solution has exert‐ ed interesting effects due to simple and wide applications. Among the migratory agent categories, a further division would be made between controlled and uncontrolled release systems. Even though uncontrolled delivery packages intended for food applications are more abundant, controlled release systems are of industrial relevance due to their apti‐ tude to prevent sensorial or toxicological problems or inefficiency of the system, caused by a too high or a too low concentration of delivered substance [8]. The active packaging technology provides several advantages compared to direct addition of active compounds, such as lower amounts of active substances required, localisation of the activity to the sur‐ face, migration from film to food matrix and elimination of additional steps within a standard process intended to introduce the active compounds at the industrial processing level such as mixing, immersion, or spraying. New regulations, the Commission Regula‐ tion (EC) No 450/2009 (EC, May 2009), and the Question Number EFSA-Q-2005-041 (EF‐ SA, July 2009), together with Regulation 1935/2004 (EC, October 2004) make active

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The current work aims to overview the main technological options of packaging to control food quality. In particular, the attention will be focused on the use of proper headspace con‐ ditions and applied active packaging systems. Case studies are given for main food catego‐

Vacuum, gas flushing or controlled permeability of the pack are valid techniques to control biochemical, enzymatic and microbial degradations so as to avoid or decrease the main deg‐ radations that might occur in food. This allows the preservation of fresh state of the food product without temperature or chemical treatments used by competitive preservation tech‐ niques, such as canning, freezing, dehydration and other processes. MAP is the replacement of air in a pack with a single gas or mixtures of gases; the proportion of each component is fixed when the mixture is introduced. No control is exerted over the initial composition, and the gas composition is likely to change with time owing to the diffusion of gases into and out of the product, the permeation of gases into and out of the pack, and the effects of the product and microbial metabolism [10]. MAP was first recorded in 1927 as an extension of shelf life of apples by storing them in atmosphere with reduced oxygen and increased car‐ bon dioxide concentrations. In 1930s it was used to transport fruit in the holds of ships. In‐ creasing the carbon dioxide concentration surrounding beef carcasses transported long distances an increase in shelf life by up to 100% was shown [11]. Marks and Spenser intro‐

packaging possible within the European Union [9].

**2. Headspace conditions**

ries, such as dairy products, meat, fish, fruit and vegetables.

Today the efforts to improve the performances of packaging with clear effects on food quali‐ ty can be directed towards two many working areas, green polymers and active packaging. The performance expected from bio-plastic materials used in food packaging application is containing the food and protecting it from the environment while maintaining food quality. It is obvious that to perform these functions is important to control and modify their me‐ chanical and barrier properties, that consequently depend on the structure of the polymeric packaging material. In addition, it is important to study the change that can occur on the characteristics of the bioplastics during the time of interaction with the food. Studies of the literature show up that only a limited amount of biopolymers are used for food packaging application [3, 4]. Unlike the usual wrap, films, labels and laminates came from fossil fuel resources, the use of biodegradable polymers represents a real step in the right direction to preserve us from environmental pollution. This kind of packaging materials needs more re‐ search, more added value like the introduction of smart and intelligent molecules able to give information about the properties of the food inside the package and nutritional values. It is necessary to make researches on this kind of material to enhance barrier properties, to ensure food properties integrity, to incorporate intelligent labeling, to give to the consumer the possibility to have more detailed product information than the current system [5, 6].

Active packaging is the most relevant innovative idea applied for consumer satisfaction. It has been defined as a system in which the product, the package and the environment in‐ teract in a positive way to extend shelf life of product or to achieve some characteristics that cannot be obtained otherwise [7]. In many present-day active packaging technologies the active agent is placed in the package with the food, in a small sachet, pad or device manufactured from a permeable material which allows the active compound to achieve its purpose but prevents direct contact with the food product, protecting the food from con‐ tamination or degradation. Active packaging developments are now focusing on incorpo‐ rating the agents into the polymeric matrices which constitute the package walls; the resulting materials act by releasing substances which have a positive effect on the food or by retaining undesired substances from the food or the internal atmosphere of the pack‐ age. The migration of a substance may be achieved by direct contact between food and packaging material or through gas phase diffusion from packaging layer to food surface. Although the former is the packaging situation usually meets, the latter solution has exert‐ ed interesting effects due to simple and wide applications. Among the migratory agent categories, a further division would be made between controlled and uncontrolled release systems. Even though uncontrolled delivery packages intended for food applications are more abundant, controlled release systems are of industrial relevance due to their apti‐ tude to prevent sensorial or toxicological problems or inefficiency of the system, caused by a too high or a too low concentration of delivered substance [8]. The active packaging technology provides several advantages compared to direct addition of active compounds, such as lower amounts of active substances required, localisation of the activity to the sur‐ face, migration from film to food matrix and elimination of additional steps within a standard process intended to introduce the active compounds at the industrial processing level such as mixing, immersion, or spraying. New regulations, the Commission Regula‐ tion (EC) No 450/2009 (EC, May 2009), and the Question Number EFSA-Q-2005-041 (EF‐ SA, July 2009), together with Regulation 1935/2004 (EC, October 2004) make active packaging possible within the European Union [9].

The current work aims to overview the main technological options of packaging to control food quality. In particular, the attention will be focused on the use of proper headspace con‐ ditions and applied active packaging systems. Case studies are given for main food catego‐ ries, such as dairy products, meat, fish, fruit and vegetables.

## **2. Headspace conditions**

magnitude) of permeability for different applications, thus justifying studies aimed to en‐ sure adequate barrier protection. Therefore, in situations where food deteriorations are driv‐ en by either gas or moisture permeation to the ambient environment, an accurate choice of packaging mass transport properties may bring about an increase to product shelf life. Each category food has its specificity in quality attributes, storage conditions, expected shelf life and packaging tools applied. Together with transport properties of film, another valid pack‐ aging option to maintain product quality is represented by the proper selection of head‐ space conditions. Vacuum packaging and modified atmosphere packaging (MAP) are two widely used strategies for food preservation [2]. The first strategy means a complete lack of gas in the package whereas, under MAP, headspace environment may change during stor‐ age but there is no additional manipulation of the internal environment. Packaging under these conditions can protect products against deteriorative effects, which may include dis‐ coloration, off-flavor and off-odor development, nutrient loss, texture changes, pathogenici‐ ty, and other measurable factors. With the increasing demand for fresh and natural products without addition of dangerous chemicals, MAP or vacuum seem to be ideal methods of preservation for many foods, being simple and cheap to be applied. The few disadvantages are related to need of equipments and proper packaging materials and, in the specific case

356 Food Industry

of MAP, to the limitation on retail for the increased pack volume of bags.

Today the efforts to improve the performances of packaging with clear effects on food quali‐ ty can be directed towards two many working areas, green polymers and active packaging. The performance expected from bio-plastic materials used in food packaging application is containing the food and protecting it from the environment while maintaining food quality. It is obvious that to perform these functions is important to control and modify their me‐ chanical and barrier properties, that consequently depend on the structure of the polymeric packaging material. In addition, it is important to study the change that can occur on the characteristics of the bioplastics during the time of interaction with the food. Studies of the literature show up that only a limited amount of biopolymers are used for food packaging application [3, 4]. Unlike the usual wrap, films, labels and laminates came from fossil fuel resources, the use of biodegradable polymers represents a real step in the right direction to preserve us from environmental pollution. This kind of packaging materials needs more re‐ search, more added value like the introduction of smart and intelligent molecules able to give information about the properties of the food inside the package and nutritional values. It is necessary to make researches on this kind of material to enhance barrier properties, to ensure food properties integrity, to incorporate intelligent labeling, to give to the consumer the possibility to have more detailed product information than the current system [5, 6].

Active packaging is the most relevant innovative idea applied for consumer satisfaction. It has been defined as a system in which the product, the package and the environment in‐ teract in a positive way to extend shelf life of product or to achieve some characteristics that cannot be obtained otherwise [7]. In many present-day active packaging technologies the active agent is placed in the package with the food, in a small sachet, pad or device manufactured from a permeable material which allows the active compound to achieve its purpose but prevents direct contact with the food product, protecting the food from con‐ Vacuum, gas flushing or controlled permeability of the pack are valid techniques to control biochemical, enzymatic and microbial degradations so as to avoid or decrease the main deg‐ radations that might occur in food. This allows the preservation of fresh state of the food product without temperature or chemical treatments used by competitive preservation tech‐ niques, such as canning, freezing, dehydration and other processes. MAP is the replacement of air in a pack with a single gas or mixtures of gases; the proportion of each component is fixed when the mixture is introduced. No control is exerted over the initial composition, and the gas composition is likely to change with time owing to the diffusion of gases into and out of the product, the permeation of gases into and out of the pack, and the effects of the product and microbial metabolism [10]. MAP was first recorded in 1927 as an extension of shelf life of apples by storing them in atmosphere with reduced oxygen and increased car‐ bon dioxide concentrations. In 1930s it was used to transport fruit in the holds of ships. In‐ creasing the carbon dioxide concentration surrounding beef carcasses transported long distances an increase in shelf life by up to 100% was shown [11]. Marks and Spenser intro‐ duced MAP for meat in 1979; the success of this product led, two years later, to the introduc‐ tion of MAP for bacon, fish, sliced cooked meats and cooked shellfish. MAP techniques are now used on a wide range of fresh or chilled foods, including raw and cooked meats and poultry, fish, fresh pasta, fruit and vegetables and more recently coffee, tea and bakery products. The advantage of MAP for the consumer are:

age while if the film is fully permeable, the atmosphere inside the pack becomes the same as the air outside. In semi-permeable films, the atmosphere in the pack rise to a gas equilibri‐ um. Packs for MAP are made from one or more polymers: polyvinylchloride (PVC), poly‐ ethylene terephthalate (PET), polyethylene (PE), and polypropylene (PP), depending on the characteristics desired for the final use. There are many factors which must be taken into ac‐

**•** barrier properties: permeability to various gases and water vapor transmission rate;

**•** sealing reliability: ability to seal to itself and to container;

change in smell, taste, texture, appearance, toxicity

products

CO2

**•** anti-fog properties: good product visibility;

Type of products vegetables, bakery cooked

**Table 1.** Main degradation factors and roles of MAP

Role of MAP gas mixture mainly constituted by

ing and easy-peel seals for convenient opening.

**•** machine capability: capacity of trouble-free operation (resistance to tearing, possibility to

**•** special characteristics: possibility of heating without removing product from the packag‐

The main gases used in MAP are: oxygen, nitrogen and carbon dioxide. These three gases are used in different combination according to the product and the needs of manufacturer and consumer. The choice for a particular combination is influenced by the microbiologi‐ cal flora and the sensitivity of the product to gases and color stability requirements. The basic concept of MAP of fresh foods is the replacement of the air surrounding the food in the package with a mixture of atmospheric gases different in proportion from that of air. Oxygen is the most important gas being used by both aerobic spoilage microorganisms and plant tissues and taking part in some enzymatic reactions responsible for food deteri‐ oration. For these reasons, under MAP, oxygen is either excluded or set as low as possi‐ ble. This gas is generally set at low levels to reduce oxidative deterioration of foods, particularly in high fat product. Oxygen generally stimulates growth of aerobic bacteria, inhibiting growth of anaerobic bacteria, although there is a wide variation in the sensitivi‐ ty of anaerobes to this gas. The exceptions occur when oxygen is needed for fruit and

Spoilage Rancidity Enzymatic browning

less nutritional value, rancid

products containing vitamins

N2 or other neutral gas replacement of air

taste browning of vegetables

N2 or other neutral gas replacement of air

and fats vegetables

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count:

be heat-formed)

Effects on products


The disadvantages of MAP are:


MAP does not increase significantly the shelf life of every type of food since some products that undergo processes such as smoking, curing, etc., already have extended shelf lives be‐ cause of pre-packaging treatments. In these cases MAP may improve other quality aspects such as color stability or slice separation. The safety and the stability of foods depend on mi‐ croorganisms initially present, being unable to overcome various adverse factors, both ex‐ trinsic and intrinsic to the food. Modification of the atmosphere surrounding the food may provide one condition to inhibit microbial growth. In table 1 there is a description of the principal degradations that take part in a common food product and the role of MAP in con‐ trasting these factors. The combination of chilled temperatures and MAP generally results in a more effective and safer storage regime and longer shelf life [12]. Atmospheres within the product are influenced by the type of material used in the package and the initial gas mix‐ ture used. Some materials allow diffusion of gases in and/or out of the package during stor‐ age while if the film is fully permeable, the atmosphere inside the pack becomes the same as the air outside. In semi-permeable films, the atmosphere in the pack rise to a gas equilibri‐ um. Packs for MAP are made from one or more polymers: polyvinylchloride (PVC), poly‐ ethylene terephthalate (PET), polyethylene (PE), and polypropylene (PP), depending on the characteristics desired for the final use. There are many factors which must be taken into ac‐ count:


duced MAP for meat in 1979; the success of this product led, two years later, to the introduc‐ tion of MAP for bacon, fish, sliced cooked meats and cooked shellfish. MAP techniques are now used on a wide range of fresh or chilled foods, including raw and cooked meats and poultry, fish, fresh pasta, fruit and vegetables and more recently coffee, tea and bakery

**•** increased distribution area and reduced transport costs due to less frequent deliveries;

**•** reduction in production and storage costs due to better utilization of labor, space and

**•** increase of pack volume which will adversely affect transport costs and retail display

MAP does not increase significantly the shelf life of every type of food since some products that undergo processes such as smoking, curing, etc., already have extended shelf lives be‐ cause of pre-packaging treatments. In these cases MAP may improve other quality aspects such as color stability or slice separation. The safety and the stability of foods depend on mi‐ croorganisms initially present, being unable to overcome various adverse factors, both ex‐ trinsic and intrinsic to the food. Modification of the atmosphere surrounding the food may provide one condition to inhibit microbial growth. In table 1 there is a description of the principal degradations that take part in a common food product and the role of MAP in con‐ trasting these factors. The combination of chilled temperatures and MAP generally results in a more effective and safer storage regime and longer shelf life [12]. Atmospheres within the product are influenced by the type of material used in the package and the initial gas mix‐ ture used. Some materials allow diffusion of gases in and/or out of the package during stor‐

**•** increased shelf life allowing less frequent loading of retail display shelves;

**•** improved presentation-clear view of product and all round visibility;

**•** hygienic stackable pack, sealed and free from product drip and odor;

**•** cost of analytical equipment to ensure that correct gas mixtures are used;

**•** cost of quality assurance systems to prevent distribution of leakers;

**•** benefits of MAP are lost once the pack is opened or leaks.

products. The advantage of MAP for the consumer are:

**•** reduction in retail waste;

358 Food Industry

equipment.

space;

**•** easy separation of sliced products;

The disadvantages of MAP are:

**•** little or no need of chemical preservatives;

**•** centralized packaging and portion control;

**•** capital cost of gas packaging machinery;

**•** cost of gases and packaging materials;

**•** special characteristics: possibility of heating without removing product from the packag‐ ing and easy-peel seals for convenient opening.


#### **Table 1.** Main degradation factors and roles of MAP

The main gases used in MAP are: oxygen, nitrogen and carbon dioxide. These three gases are used in different combination according to the product and the needs of manufacturer and consumer. The choice for a particular combination is influenced by the microbiologi‐ cal flora and the sensitivity of the product to gases and color stability requirements. The basic concept of MAP of fresh foods is the replacement of the air surrounding the food in the package with a mixture of atmospheric gases different in proportion from that of air. Oxygen is the most important gas being used by both aerobic spoilage microorganisms and plant tissues and taking part in some enzymatic reactions responsible for food deteri‐ oration. For these reasons, under MAP, oxygen is either excluded or set as low as possi‐ ble. This gas is generally set at low levels to reduce oxidative deterioration of foods, particularly in high fat product. Oxygen generally stimulates growth of aerobic bacteria, inhibiting growth of anaerobic bacteria, although there is a wide variation in the sensitivi‐ ty of anaerobes to this gas. The exceptions occur when oxygen is needed for fruit and vegetable respiration, color retention as in the case of red meat or to avoid anaerobic con‐ ditions in white fish [13]. One of the main function of oxygen is the maintenance of myo‐ globin in its oxygenated form, oxymyoglobin because this is the form responsible for the bright red color, which most consumers associate to fresh meat. Carbon dioxide is both water and lipid soluble and although is not bactericide or fungicide, it has a bacteriostatic and fungistatic properties. The effect on microorganisms consists in the extension of the lag phase and a decrease of growth rate. The effectiveness of this gas is influenced by its original and final concentrations, the storage temperature, the partial pressure of carbon dioxide, the initial bacterial population, the microbial growth phase, the growth medium used, the acidity, the water activity and the type of product being packaged [10, 14-16]. Yeasts which produce carbon dioxide during growth are stimulated by high levels of car‐ bon dioxide and thus for some products where they are potentially a major cause of spoil‐ age, MAP may not be an advisable option. Also *Clostridium perfrigens* and *botulinum* are not affected by the presence of carbon dioxide and their growth is encouraged by anaero‐ bic conditions. In general, carbon dioxide is most effective in foods where the normal spoilage microorganisms consist of aerobic and gram negative psychrotropic bacteria [17]. For maximum antimicrobial effect, the storage temperature of the product should be kept as low as possible, because the solubility of carbon dioxide decreases dramatically with in‐ creasing temperature, thus improper temperature could eliminate the beneficial effects of carbon dioxide. The absorption of carbon dioxide is dependent on the moisture and fat content of the product. If product absorbs excess carbon dioxide the total volume inside the package will be reduced, giving a vacuum package look known as pack collapse. Ex‐ cess carbon dioxide absorption in combination with package collapse can also reduce wa‐ ter holding capacity of meats, resulting in unsightly drip. Genigeorgis [18] suggested that the antimicrobial activity of carbon dioxide was a result of the gas being absorbed onto the surface of the product forming carbonic acid, subsequent ionization of the carbonic acid and a reduction in pH. Other theories have been summarized:

**PRODUCT Temperature Shelf life under MAP Shelf life in AIR**

a

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Technological Options of Packaging to Control Food Quality

Toast bread Room 2-3 months 10 days

Croissant, milk bread Room 6 weeks Several days Pizza 4-5°C 30 days Several days Hamburger, hot dog rolls 4-5°C 30 days 1 week Cakes with cream Room 25-30 days -

Emmenthal 2-4°C 4-5 weeks A few days Bovine mozzarella 2-4°C 6-8 days 3 days Robiola, Crescenza 2-4°C 3-4 weeks 1 week Cheese slices 2-4°C 2-3 months 2-3 months Gorgonzola 2-4°C 30 days 10 days

The properties of meat that are important in determining shelf life include water binding ca‐ pacity, color, microbial quality, lipid stability and palatability. The variables that influence the shelf life of packaged fresh meat are: product, package and headspace, packaging equip‐ ment, storage temperature, and additives. Plastic film properties, shrinkage, strength, oxy‐ gen transmission, moisture transmission, and anti-fog agents are important for meat package materials [19]. Fresh meat packaging is only minimally permeable to moisture to prevent desiccation, while gas permeability varies with the applications. MAP (commonly 70-80% O2 and 20-30% CO2 ) and vacuum packaging are widely used methods for packaging meat. Packaging under high oxygen concentration, however, may cause an increase in the lipid and protein oxidation. These reactions affect the functional, sensory and nutritional quality of meat products. Lipid oxidation leads to discoloration, increase drip-loss, off-odors and production of toxic compounds. In addition, these modifications can negatively affect the sensory quality of meat products in terms of texture, tenderness and color [20]. Protein oxidation can also result in the loss of enzyme activity and protein solubility, as well as in the formation of protein complexes and non enzymatic browning products. In a recent study [21], the effects of MAP (70% O2 and 30% CO2) and vacuum skin packaging on protein oxidation and texture of pork were investigated. Packaging under MAP containing high lev‐ el of O2 resulted in protein cross-linking, which reduced tenderness and juiciness of pork. Rowe et al. [20] proposed that the oxidation of muscle proteins may have a negative effect on beef tenderness that was attributed to an inactivation of µ-calpain with a subsequent de‐ crease in proteolysis. Zakrys et al. [22]compared the effects of high levels of oxygen (80% O2 20% CO2) with vacuum packaging and showed that high O2 levels lead to high myosin inter‐

Cake Room 40-60 days -

Parmesan in pieces 2-4°C 40-60 days -


**Table 2.** Examples of food shelf life under MAP and air

a


Nitrogen is an inert gas which has been used as a packaging filler for many years to prevent pack collapse because of its low solubility in water and lipid. In MAP products, especially fresh meat packed in high concentrations of carbon dioxide, pack collapse oc‐ curs because of the solubility of carbon dioxide in meat tissue. Nitrogen is also used to replace oxygen in MAP products, to prevent rancidity and inhibit growth of aerobic or‐ ganisms. The gas combination depends on product characteristics. Table 2 reports a com‐ parison between storage in MAP and in air for different products. Some relevant casestudies that highlight the benefits and limits of MAP for main food categories are reported hereinafter.


a - means that air was never used to store product.

vegetable respiration, color retention as in the case of red meat or to avoid anaerobic con‐ ditions in white fish [13]. One of the main function of oxygen is the maintenance of myo‐ globin in its oxygenated form, oxymyoglobin because this is the form responsible for the bright red color, which most consumers associate to fresh meat. Carbon dioxide is both water and lipid soluble and although is not bactericide or fungicide, it has a bacteriostatic and fungistatic properties. The effect on microorganisms consists in the extension of the lag phase and a decrease of growth rate. The effectiveness of this gas is influenced by its original and final concentrations, the storage temperature, the partial pressure of carbon dioxide, the initial bacterial population, the microbial growth phase, the growth medium used, the acidity, the water activity and the type of product being packaged [10, 14-16]. Yeasts which produce carbon dioxide during growth are stimulated by high levels of car‐ bon dioxide and thus for some products where they are potentially a major cause of spoil‐ age, MAP may not be an advisable option. Also *Clostridium perfrigens* and *botulinum* are not affected by the presence of carbon dioxide and their growth is encouraged by anaero‐ bic conditions. In general, carbon dioxide is most effective in foods where the normal spoilage microorganisms consist of aerobic and gram negative psychrotropic bacteria [17]. For maximum antimicrobial effect, the storage temperature of the product should be kept as low as possible, because the solubility of carbon dioxide decreases dramatically with in‐ creasing temperature, thus improper temperature could eliminate the beneficial effects of carbon dioxide. The absorption of carbon dioxide is dependent on the moisture and fat content of the product. If product absorbs excess carbon dioxide the total volume inside the package will be reduced, giving a vacuum package look known as pack collapse. Ex‐ cess carbon dioxide absorption in combination with package collapse can also reduce wa‐ ter holding capacity of meats, resulting in unsightly drip. Genigeorgis [18] suggested that the antimicrobial activity of carbon dioxide was a result of the gas being absorbed onto the surface of the product forming carbonic acid, subsequent ionization of the carbonic

acid and a reduction in pH. Other theories have been summarized:

**•** penetration of membranes resulting in changes of intracellular pH;

**•** direct changes to physic-chemical properties of proteins.

reported hereinafter.

360 Food Industry

**•** direct inhibition of enzyme systems or decreases in rate of enzyme reactions;

**•** alteration of cell membrane function including effects on nutrient uptake and absorption;

Nitrogen is an inert gas which has been used as a packaging filler for many years to prevent pack collapse because of its low solubility in water and lipid. In MAP products, especially fresh meat packed in high concentrations of carbon dioxide, pack collapse oc‐ curs because of the solubility of carbon dioxide in meat tissue. Nitrogen is also used to replace oxygen in MAP products, to prevent rancidity and inhibit growth of aerobic or‐ ganisms. The gas combination depends on product characteristics. Table 2 reports a com‐ parison between storage in MAP and in air for different products. Some relevant casestudies that highlight the benefits and limits of MAP for main food categories are

#### **Table 2.** Examples of food shelf life under MAP and air

The properties of meat that are important in determining shelf life include water binding ca‐ pacity, color, microbial quality, lipid stability and palatability. The variables that influence the shelf life of packaged fresh meat are: product, package and headspace, packaging equip‐ ment, storage temperature, and additives. Plastic film properties, shrinkage, strength, oxy‐ gen transmission, moisture transmission, and anti-fog agents are important for meat package materials [19]. Fresh meat packaging is only minimally permeable to moisture to prevent desiccation, while gas permeability varies with the applications. MAP (commonly 70-80% O2 and 20-30% CO2 ) and vacuum packaging are widely used methods for packaging meat. Packaging under high oxygen concentration, however, may cause an increase in the lipid and protein oxidation. These reactions affect the functional, sensory and nutritional quality of meat products. Lipid oxidation leads to discoloration, increase drip-loss, off-odors and production of toxic compounds. In addition, these modifications can negatively affect the sensory quality of meat products in terms of texture, tenderness and color [20]. Protein oxidation can also result in the loss of enzyme activity and protein solubility, as well as in the formation of protein complexes and non enzymatic browning products. In a recent study [21], the effects of MAP (70% O2 and 30% CO2) and vacuum skin packaging on protein oxidation and texture of pork were investigated. Packaging under MAP containing high lev‐ el of O2 resulted in protein cross-linking, which reduced tenderness and juiciness of pork. Rowe et al. [20] proposed that the oxidation of muscle proteins may have a negative effect on beef tenderness that was attributed to an inactivation of µ-calpain with a subsequent de‐ crease in proteolysis. Zakrys et al. [22]compared the effects of high levels of oxygen (80% O2 20% CO2) with vacuum packaging and showed that high O2 levels lead to high myosin inter‐ molecular cross-links, low free thiol groups and high carbonyl content, demonstrating that a significant level of protein oxidation occurred. This protein oxidation was found to have a negative effect on meat tenderness. Results from this study suggested that high oxygen in‐ duced changes in myosin and intermolecular cross-linking, increased disulphide bond for‐ mation, protein oxidation and drip loss compared to vacuum packaged. Color of meat is a very important quality attribute that influences consumer acceptance of meat. The surface color of meat depends on the quantity of myoglobin present, on its chemical state and also on the chemical and physical conditions of other components. Meat showing a bright red color is assumed to be fresh, while oxidation of heme iron to form methamyoglobin produ‐ ces the brown color which consumers find undesirable. An interesting study conducted by Mastromatteo et al. [23] evaluated the combination of different MAPs (from 20% to 40% of CO2 ; from 5% to 20% of O2 and from 75% to 40% of N2 ) with natural essential oils on shelf life of reduced pork back fat content sausages. They found that lemon and thymol recorded the highest sensory score while all the investigated MAPs showed an antimicrobial effect; moreover, low carbon dioxide concentrations caused low color variations during storage. The combination of MAP and thymol was able to further improve the shelf life of meat, in fact the microbial threshold was never reached. A shelf life of more than 5 days for thymol-MAP samples was obtained, respect to the other investigated samples (2 days). To sum up, integration of meat characteristics with available packaging materials, equipment into cur‐ rent cold chain logistical and information systems have resulted in a sufficiently high state of complexity that has caused uncertainty and confusion among industry, regulatory agen‐ cy, and consumer segments [13]. Meat and packaging industry must continue to work on systems that will ensure safe and palatable products. The review of Belcher [24] well sum‐ marized packaging developments that are resulting from numerous trends taking place in the meat industry and in the retail sector. Moreover, alternative non-thermal preservation technologies such as high hydrostatic pressure, super chilling, natural biopreservatives and active packaging have been proposed because they are also effective against spores. To in‐ crease their efficacy, a combination of several preservation technologies under the so-called hurdle concept has to be investigated [25].

being quickly established if the produce is sealed in an impermeable film with low initial O2 concentration. Subsequently anaerobic respiration of the produce will be initiated at very low O2 concentrations, resulting in the accumulation of ethanol, acetaldehyde and organic acids and deterioration of organoleptic properties. Rate of respiration is influenced by the initial gas concentration so that, for example, reducing the oxygen to 2% and increasing the carbon dioxide concentration to 5%, results in more than 10-fold reduction of respiration rate in vegetables such as broccoli [32]. The maintenance of color is important and in red peppers, MAP has been shown to increase carotenoid retention and reduce browning [33]. The combination of storage time and temperature has been shown to be important in ex‐ tending the shelf life of fruit in terms of texture, weight loss, pH and other nutritional changes. Temperature is also a factor in determining respiration rates of fruits. In freshly harvested beansprouts for example, which not only have a high respiration rate but also are characterized by high initial microbial populations, Varoquaux et al. [34], observed a 10-fold increase in respiration rate at 16.5 °C. In the case of fruit and vegetables with high respira‐ tion rates, there is an optimum initial atmosphere concentration to ensure minimal growth of aerobic spoilage bacteria together with an optimal film permeability to delay the develop‐ ment of anaerobic respiration and necrosis of vegetable. One of the obvious ways in which produce may be assessed for freshness is in terms of wilting and shriveling which are due to loss of moisture. Fruits and vegetables lose moisture when the relative humidity in the pack‐ aging is less than 80-95% of saturation and reduction in quality occurs if 3-6% of the produce moisture is lost [35]. Most films used for MAP of fruit and vegetables are relatively good water vapor barriers and are able to maintain a high relative humidity inside the pack. The relative humidity within a pack is influenced by the rate at which the product loses water vapor and by vapor transmission rate of the packaging film. Successful applications include broccoli florets, cauliflower florets, carrots, peeled garlic [36]. LDPE was found a good alter‐ native to PVC for wrapping these vegetables. Comparative evaluation of the effect of stor‐ age temperature fluctuation on MAP of selected fruit and vegetables like mushrooms and mature green tomatoes was also studied by Tano et al. [37]. The quality of the products stor‐ ed under temperature fluctuating regime was severely affected as indicated by extensive browning, loss of firmness, weight loss increase, ethanol level in plant tissue and infection, due to physiological damage and excessive condensation, compared to products stored at constant temperature. It was clear that temperature fluctuation can seriously compromise the benefits of MAP and safety of the product. Temperature is the most effective environ‐ mental factor in the prevention of fruit ripening. Both ripening and ethylene production rates increase with increase in temperature. To delay fruit ripening, temperature should be held as close to 0 °C as possible. The use of MAP as a supplement to proper temperature maintenance in the effort to delay ripening is effective for all fruits. Reducing oxygen con‐ centration below 8% and/or elevating Carbon dioxide concentration above 1% retards fruit ripening. Successful applications include broccoli slaw, coleslaw, dry slaw, casserole mix, and mixed salads. Degradation of cut vegetables in terms of appearance was delayed by N2 gas packaging and vegetables remained acceptable at temperature below 5 °C after 5 days. MAP may have the effect of increasing shelf life of some vegetables in terms of sensory properties but does not reduce growth of some microorganisms such as *L. monocytogenes*

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As regard fresh-cut fruits and vegetables, process increases respiration rate and causes ma‐ jor tissue disruption as enzymes and substrates normally sequestered within the vacuole. Processing also increases wound-induced ethylene, water activity and surface area per unit volume, which, may accelerate water loss and enhance microbial growth since sugars also become readily available. These physiological changes may be accompanied by flavor loss, cut surface discoloration, color loss, increased rate of vitamin loss, shrinkage and shorter shelf life. MAP is largely used for minimally processed fruit and vegetables. It relies on the modification of the atmosphere inside the package in order to extend the food shelf life by reducing the respiration of the product and consequently its degradation rate. The effect of MAP on quality of many fresh-cut products has been studied and successful applications in‐ clude mushroom [26], apples [27], tomato [28], butterhead lettuce [29], potato [30], or kiwi‐ fruit [31]. These products are metabolically active for long periods after harvesting due to both endogenous activity, such as respiration, and external factors such as physical injury, microbial flora, water loss and storage temperature. Respiration may result in anaerobiosis, being quickly established if the produce is sealed in an impermeable film with low initial O2 concentration. Subsequently anaerobic respiration of the produce will be initiated at very low O2 concentrations, resulting in the accumulation of ethanol, acetaldehyde and organic acids and deterioration of organoleptic properties. Rate of respiration is influenced by the initial gas concentration so that, for example, reducing the oxygen to 2% and increasing the carbon dioxide concentration to 5%, results in more than 10-fold reduction of respiration rate in vegetables such as broccoli [32]. The maintenance of color is important and in red peppers, MAP has been shown to increase carotenoid retention and reduce browning [33]. The combination of storage time and temperature has been shown to be important in ex‐ tending the shelf life of fruit in terms of texture, weight loss, pH and other nutritional changes. Temperature is also a factor in determining respiration rates of fruits. In freshly harvested beansprouts for example, which not only have a high respiration rate but also are characterized by high initial microbial populations, Varoquaux et al. [34], observed a 10-fold increase in respiration rate at 16.5 °C. In the case of fruit and vegetables with high respira‐ tion rates, there is an optimum initial atmosphere concentration to ensure minimal growth of aerobic spoilage bacteria together with an optimal film permeability to delay the develop‐ ment of anaerobic respiration and necrosis of vegetable. One of the obvious ways in which produce may be assessed for freshness is in terms of wilting and shriveling which are due to loss of moisture. Fruits and vegetables lose moisture when the relative humidity in the pack‐ aging is less than 80-95% of saturation and reduction in quality occurs if 3-6% of the produce moisture is lost [35]. Most films used for MAP of fruit and vegetables are relatively good water vapor barriers and are able to maintain a high relative humidity inside the pack. The relative humidity within a pack is influenced by the rate at which the product loses water vapor and by vapor transmission rate of the packaging film. Successful applications include broccoli florets, cauliflower florets, carrots, peeled garlic [36]. LDPE was found a good alter‐ native to PVC for wrapping these vegetables. Comparative evaluation of the effect of stor‐ age temperature fluctuation on MAP of selected fruit and vegetables like mushrooms and mature green tomatoes was also studied by Tano et al. [37]. The quality of the products stor‐ ed under temperature fluctuating regime was severely affected as indicated by extensive browning, loss of firmness, weight loss increase, ethanol level in plant tissue and infection, due to physiological damage and excessive condensation, compared to products stored at constant temperature. It was clear that temperature fluctuation can seriously compromise the benefits of MAP and safety of the product. Temperature is the most effective environ‐ mental factor in the prevention of fruit ripening. Both ripening and ethylene production rates increase with increase in temperature. To delay fruit ripening, temperature should be held as close to 0 °C as possible. The use of MAP as a supplement to proper temperature maintenance in the effort to delay ripening is effective for all fruits. Reducing oxygen con‐ centration below 8% and/or elevating Carbon dioxide concentration above 1% retards fruit ripening. Successful applications include broccoli slaw, coleslaw, dry slaw, casserole mix, and mixed salads. Degradation of cut vegetables in terms of appearance was delayed by N2 gas packaging and vegetables remained acceptable at temperature below 5 °C after 5 days. MAP may have the effect of increasing shelf life of some vegetables in terms of sensory properties but does not reduce growth of some microorganisms such as *L. monocytogenes*

molecular cross-links, low free thiol groups and high carbonyl content, demonstrating that a significant level of protein oxidation occurred. This protein oxidation was found to have a negative effect on meat tenderness. Results from this study suggested that high oxygen in‐ duced changes in myosin and intermolecular cross-linking, increased disulphide bond for‐ mation, protein oxidation and drip loss compared to vacuum packaged. Color of meat is a very important quality attribute that influences consumer acceptance of meat. The surface color of meat depends on the quantity of myoglobin present, on its chemical state and also on the chemical and physical conditions of other components. Meat showing a bright red color is assumed to be fresh, while oxidation of heme iron to form methamyoglobin produ‐ ces the brown color which consumers find undesirable. An interesting study conducted by Mastromatteo et al. [23] evaluated the combination of different MAPs (from 20% to 40% of CO2 ; from 5% to 20% of O2 and from 75% to 40% of N2 ) with natural essential oils on shelf life of reduced pork back fat content sausages. They found that lemon and thymol recorded the highest sensory score while all the investigated MAPs showed an antimicrobial effect; moreover, low carbon dioxide concentrations caused low color variations during storage. The combination of MAP and thymol was able to further improve the shelf life of meat, in fact the microbial threshold was never reached. A shelf life of more than 5 days for thymol-MAP samples was obtained, respect to the other investigated samples (2 days). To sum up, integration of meat characteristics with available packaging materials, equipment into cur‐ rent cold chain logistical and information systems have resulted in a sufficiently high state of complexity that has caused uncertainty and confusion among industry, regulatory agen‐ cy, and consumer segments [13]. Meat and packaging industry must continue to work on systems that will ensure safe and palatable products. The review of Belcher [24] well sum‐ marized packaging developments that are resulting from numerous trends taking place in the meat industry and in the retail sector. Moreover, alternative non-thermal preservation technologies such as high hydrostatic pressure, super chilling, natural biopreservatives and active packaging have been proposed because they are also effective against spores. To in‐ crease their efficacy, a combination of several preservation technologies under the so-called

As regard fresh-cut fruits and vegetables, process increases respiration rate and causes ma‐ jor tissue disruption as enzymes and substrates normally sequestered within the vacuole. Processing also increases wound-induced ethylene, water activity and surface area per unit volume, which, may accelerate water loss and enhance microbial growth since sugars also become readily available. These physiological changes may be accompanied by flavor loss, cut surface discoloration, color loss, increased rate of vitamin loss, shrinkage and shorter shelf life. MAP is largely used for minimally processed fruit and vegetables. It relies on the modification of the atmosphere inside the package in order to extend the food shelf life by reducing the respiration of the product and consequently its degradation rate. The effect of MAP on quality of many fresh-cut products has been studied and successful applications in‐ clude mushroom [26], apples [27], tomato [28], butterhead lettuce [29], potato [30], or kiwi‐ fruit [31]. These products are metabolically active for long periods after harvesting due to both endogenous activity, such as respiration, and external factors such as physical injury, microbial flora, water loss and storage temperature. Respiration may result in anaerobiosis,

hurdle concept has to be investigated [25].

362 Food Industry

and *Salmonella* Enterica. Therefore, the use of appropriate pre-harvest and postharvest sani‐ tation practices to prevent contamination remains the most important measure for ensuring the microbiological safety of ready-to-eat fresh-cut products.

tory quality. Gammariello et al. [51] evaluated the shelf life of Stracciatella cheese packaged in four different gas mixtures at 8 °C and showed that MAP 50:50 and 95:5 (O2:CO2) pro‐ longed the sensorial acceptability limit by delayed growth of spoilage bacteria, without af‐ fecting the dairy microflora. Del Nobile et al. [52] suggest that MAP of Ricotta with 95% carbon dioxide inhibits microbial growth without effects on lactic acid bacteria, probably due to their facultative anaerobic nature, and also maintains the natural color of Ricotta.

Technological Options of Packaging to Control Food Quality

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MAP found wide application also for fresh fish. Fish such as herring and haddock bene‐ fits from being packaged under MAP since this reduces the production of peroxides which affect fish sensory characteristics and hence shelf-life [53]. However, high levels of carbon dioxide may result in carbon dioxide dissolution into the fish flesh, causing de‐ formation or collapse of the packaging and also affecting the product color. The result‐ ing drop in pH of the tissue may cause a decrease in the flesh's water holding capacity and drip may occur, reducing shelf-life. Fresh hake stored in up to 60% carbon dioxide exerted a shelf life significantly longer than those stored in air. MAP inhibits bacterial growth, reduces the formation of total volatile bases and trimethylamine and delays al‐ terations in protein functionality [54]. In cooked fish such as smoked blue cod and smoked atlantic and silver salmon, a high concentration of carbon dioxide increases shelf life without showing drip or muscle exudate observed in fresh fish. carbon dioxide ex‐ tents fish shelf life due to the inhibition of Gram negative and lactic acid bacteria. car‐ bon dioxide concentrations in all MAP fishery products should be carefully monitored, especially when stored for long periods of time, because carbon dioxide does not inhibit *C. botulinum* and the effect of temperature abuse may increase the risk of botulism in

those products which contain spores of non-proteolytic *C. botulinum* [55, 56].

Active packaging has been classified as a subset of smart packaging and referred to as the incorporation of certain additives into packaging film or within packaging containers with the aim of maintaining and extending product shelf life [57-59]. Another definition states that packaging may be termed active when it performs some desired role in food preservation other than providing an inert barrier to external conditions [60]. Hence, ac‐ tive packaging includes components of packaging systems that are capable of scavenging oxygen; absorbing carbon dioxide, moisture, ethylene and/or flavor/odor taints; releasing carbon dioxide, ethanol, antioxidants and/or other preservatives; and/or maintaining tem‐ perature control and/or compensating for temperature changes (Table 3). In the food and beverage market, growth of active packaging concepts is being driven by the growing use of packaged food, increasing demand for ready-prepared foods such as microwave meals, and increasing use of smaller package sizes. Although many active packaging technologies are still developmental, there are commercial successes, particularly in oxy‐ gen scavengers. Oxygen scavengers are easily oxidizable substances included in the pack‐ aging system to remove oxygen by means of a chemical reaction. The substance is

**3. Active packaging**

Shelf life of milk and milk-based products is limited because of their high water content and favorable pH for microbial growth [38-40]. The rapid spoilage adversely affects flavor and texture along with visual color changes of refrigerated raw and pasteurized milk, cottage cheese and other similar products. The responsible microorganisms include psychrotropic Gram negative bacteria, yeasts and moulds. These organisms produce extracellular protease and lipase activity, which reduce the functionality of milk proteins and fat and often pro‐ duce undesirable aromas. Gram positive bacteria particularly those producing lactic and acetic acids, can spoil dairy foods, but the number of organisms required are generally high‐ er than for Gram-negative bacteria, and the changes can be less noticeable. It has been re‐ ported that the product shelf life increases by low oxygen atmospheres because of the reduction in aerobic microorganisms. The antimicrobial effect of Carbon dioxide occurs near 10% level, and further increase in carbon dioxide affects growth of *Pseudomonas* and *Morax‐ ella*. The largest inhibition by carbon dioxide occurs with Gram negative psychrotrophs bac‐ teria [41]. The protective role of carbon dioxide is also important for mould proliferation; its function in creating an anaerobic environment with the displacement of existing molecular oxygen, its extra and intracellular pH decreasing effect and its destroying effect on the cell membrane make carbon dioxide an inhibitory substance towards microorganisms. The anti‐ microbial effect of carbon dioxide is dependent on many factors, including the partial pres‐ sure, application time, concentration of gas, temperature of the medium [42], volume of headspace, acidity, water activity of the medium and type of organism present [43]. The ap‐ plied composition for packaging of dairy products can vary from 10% to 100% carbon diox‐ ide, balanced with N2 as inert gas filler, to prevent package collapse as a result of carbon dioxide solubilization in the cheese. MAP has been applied to the packaging of cheese. The packaging of each type of cheese needs to be consider separately. Another fact to be consid‐ ered is that some cheeses are carbon dioxide producers, while other not. It is important that the levels of carbon dioxide are controlled because for certain cheese high levels of carbon dioxide have been found to impart off-flavor [44-46]. Cheese stored under carbon dioxide contained high concentrations of aldehydes and fatty acids and lower concentrations of alco‐ hols and esters than cheeses stored under nitrogen. Hard and semi-soft cheeses, such as cheddar, are commonly packed in 100% carbon dioxide or mixtures of carbon dioxide and nitrogen. Soft cheese have also a limited shelf life. An alternative to conventional packaging is to use MAP. Carbon dioxide acts both directly on moulds and indirectly by displacing oxygen. Vacuum packaging does not remove all of the oxygen and thus, moulds and yeasts can still occur [47]. The gas mixture typically used is 70% N2 and 30% CO2 to inhibit mould growth, to keep the package from collapsing and to prevent shred matting. Alves et al. [48] reported that atmospheres ≥ 50% carbon dioxide were more effective than air or 100% nitro‐ gen in improving shelf life of sliced mozzarella cheese. High carbon dioxide atmospheres have been shown to inhibit growth of lactic and mesophilic bacteria [49]. Piergiovanni et al. [50] compared Taleggio cheese packaged under four modified atmospheres and stored at 6 °C to conventional paper wrapping and found that samples packaged in MAP had satisfac‐ tory quality. Gammariello et al. [51] evaluated the shelf life of Stracciatella cheese packaged in four different gas mixtures at 8 °C and showed that MAP 50:50 and 95:5 (O2:CO2) pro‐ longed the sensorial acceptability limit by delayed growth of spoilage bacteria, without af‐ fecting the dairy microflora. Del Nobile et al. [52] suggest that MAP of Ricotta with 95% carbon dioxide inhibits microbial growth without effects on lactic acid bacteria, probably due to their facultative anaerobic nature, and also maintains the natural color of Ricotta.

MAP found wide application also for fresh fish. Fish such as herring and haddock bene‐ fits from being packaged under MAP since this reduces the production of peroxides which affect fish sensory characteristics and hence shelf-life [53]. However, high levels of carbon dioxide may result in carbon dioxide dissolution into the fish flesh, causing de‐ formation or collapse of the packaging and also affecting the product color. The result‐ ing drop in pH of the tissue may cause a decrease in the flesh's water holding capacity and drip may occur, reducing shelf-life. Fresh hake stored in up to 60% carbon dioxide exerted a shelf life significantly longer than those stored in air. MAP inhibits bacterial growth, reduces the formation of total volatile bases and trimethylamine and delays al‐ terations in protein functionality [54]. In cooked fish such as smoked blue cod and smoked atlantic and silver salmon, a high concentration of carbon dioxide increases shelf life without showing drip or muscle exudate observed in fresh fish. carbon dioxide ex‐ tents fish shelf life due to the inhibition of Gram negative and lactic acid bacteria. car‐ bon dioxide concentrations in all MAP fishery products should be carefully monitored, especially when stored for long periods of time, because carbon dioxide does not inhibit *C. botulinum* and the effect of temperature abuse may increase the risk of botulism in those products which contain spores of non-proteolytic *C. botulinum* [55, 56].

## **3. Active packaging**

and *Salmonella* Enterica. Therefore, the use of appropriate pre-harvest and postharvest sani‐ tation practices to prevent contamination remains the most important measure for ensuring

Shelf life of milk and milk-based products is limited because of their high water content and favorable pH for microbial growth [38-40]. The rapid spoilage adversely affects flavor and texture along with visual color changes of refrigerated raw and pasteurized milk, cottage cheese and other similar products. The responsible microorganisms include psychrotropic Gram negative bacteria, yeasts and moulds. These organisms produce extracellular protease and lipase activity, which reduce the functionality of milk proteins and fat and often pro‐ duce undesirable aromas. Gram positive bacteria particularly those producing lactic and acetic acids, can spoil dairy foods, but the number of organisms required are generally high‐ er than for Gram-negative bacteria, and the changes can be less noticeable. It has been re‐ ported that the product shelf life increases by low oxygen atmospheres because of the reduction in aerobic microorganisms. The antimicrobial effect of Carbon dioxide occurs near 10% level, and further increase in carbon dioxide affects growth of *Pseudomonas* and *Morax‐ ella*. The largest inhibition by carbon dioxide occurs with Gram negative psychrotrophs bac‐ teria [41]. The protective role of carbon dioxide is also important for mould proliferation; its function in creating an anaerobic environment with the displacement of existing molecular oxygen, its extra and intracellular pH decreasing effect and its destroying effect on the cell membrane make carbon dioxide an inhibitory substance towards microorganisms. The anti‐ microbial effect of carbon dioxide is dependent on many factors, including the partial pres‐ sure, application time, concentration of gas, temperature of the medium [42], volume of headspace, acidity, water activity of the medium and type of organism present [43]. The ap‐ plied composition for packaging of dairy products can vary from 10% to 100% carbon diox‐ ide, balanced with N2 as inert gas filler, to prevent package collapse as a result of carbon dioxide solubilization in the cheese. MAP has been applied to the packaging of cheese. The packaging of each type of cheese needs to be consider separately. Another fact to be consid‐ ered is that some cheeses are carbon dioxide producers, while other not. It is important that the levels of carbon dioxide are controlled because for certain cheese high levels of carbon dioxide have been found to impart off-flavor [44-46]. Cheese stored under carbon dioxide contained high concentrations of aldehydes and fatty acids and lower concentrations of alco‐

cheddar, are commonly packed in 100% carbon dioxide or mixtures of carbon dioxide and nitrogen. Soft cheese have also a limited shelf life. An alternative to conventional packaging is to use MAP. Carbon dioxide acts both directly on moulds and indirectly by displacing oxygen. Vacuum packaging does not remove all of the oxygen and thus, moulds and yeasts can still occur [47]. The gas mixture typically used is 70% N2 and 30% CO2 to inhibit mould growth, to keep the package from collapsing and to prevent shred matting. Alves et al. [48] reported that atmospheres ≥ 50% carbon dioxide were more effective than air or 100% nitro‐ gen in improving shelf life of sliced mozzarella cheese. High carbon dioxide atmospheres have been shown to inhibit growth of lactic and mesophilic bacteria [49]. Piergiovanni et al. [50] compared Taleggio cheese packaged under four modified atmospheres and stored at 6 °C to conventional paper wrapping and found that samples packaged in MAP had satisfac‐

Hard and semi-soft cheeses, such as

the microbiological safety of ready-to-eat fresh-cut products.

364 Food Industry

hols and esters than cheeses stored under nitrogen.

Active packaging has been classified as a subset of smart packaging and referred to as the incorporation of certain additives into packaging film or within packaging containers with the aim of maintaining and extending product shelf life [57-59]. Another definition states that packaging may be termed active when it performs some desired role in food preservation other than providing an inert barrier to external conditions [60]. Hence, ac‐ tive packaging includes components of packaging systems that are capable of scavenging oxygen; absorbing carbon dioxide, moisture, ethylene and/or flavor/odor taints; releasing carbon dioxide, ethanol, antioxidants and/or other preservatives; and/or maintaining tem‐ perature control and/or compensating for temperature changes (Table 3). In the food and beverage market, growth of active packaging concepts is being driven by the growing use of packaged food, increasing demand for ready-prepared foods such as microwave meals, and increasing use of smaller package sizes. Although many active packaging technologies are still developmental, there are commercial successes, particularly in oxy‐ gen scavengers. Oxygen scavengers are easily oxidizable substances included in the pack‐ aging system to remove oxygen by means of a chemical reaction. The substance is usually contained in sachets made of a material highly permeable to air but it can also be included in bottle closures or in the plastic film matrix. Different studies show that the use of scavengers led to faster reduction and to lower levels of residual oxygen, as compared to nitrogen flushing. The most common substances used are iron powder and ascorbic acid. Scavengers also differ in the reaction speed, from immediate action (0.5 to 1 day) to slow action (4 to 6 days), on the application, particularly the moisture content of the food, and on the function, i.e., oxygen scavenging only or dual function, such as absorbing or generating carbon dioxide, besides removing the oxygen. The scientific liter‐ ature contains a number of references which examine the influence of oxygen scavenger sachets on fresh beef discoloration. Gill and McGinnis [61] performed an oxygen absorp‐ tion kinetics study with a commercial oxygen scavenger (FreshPaxTM 200R) and report‐ ed that discoloration could be prevented in ground beef if large numbers of scavengers were used in each pack to bring residual oxygen to <10 ppm within 2 h at a storage temperature of -1.5 °C. The inclusion of oxygen scavengers (Ageless® SS200) in master packs flushed with 50% carbon dioxide and 50% nitrogen significantly improved color stability of M. longissimus dorsi and M. psoas major, relative to controls [62]. In addi‐ tion to fresh beef oxygen scavenging technology has also been applied to pork [63] and pork products, where, Martìnez, Djenane, Cilla, Beltràn, and Roncalès [64] reported that fresh pork sausages stored in 20% CO2 and 80% N2 plus an oxygen scavenger (Ageless® FX-40) for up to 20 days at 2 ± 1 °C reduced psychrotrophic aerobe counts and extended shelf life in terms of color and lipid stability. An alternative to sachets involves the incor‐ poration of the oxygen scavenger into the packaging structure itself. This UV light-acti‐ vated oxygen scavenging film, which structurally is composed of an oxygen scavenger layer extruded into a multilayer film, can reduce headspace oxygen levels from 1% to ppm levels in 4–10 days, compared to oxygen scavenging sachets. The OS2000TM scav‐ enging films found applications in a wide variety of food products including dried or smoked meat products and processed meats [65]. Berenzon and Saguy [66] evaluated the applicability of oxygen absorbers for extending shelf life of military ration crackers pack‐ aged in hermetically sealed tin cans and stored at 15, 25 and 35 °C for up to 52 weeks. Sensory evaluations suggested that crackers stored without oxygen absorbers developed oxidative rancid odors after 24 weeks at 25 and 35 °C. Independently of storage tempera‐ tures, no oxidative rancid odors were observed after 44 weeks with oxygen absorbers. Opposed to the currently available chemical oxygen scavengers, systems based upon nat‐ ural and biological components could have advantages towards consumer perception and sustainability [67]. A model system for a new oxygen scavenging poly(ethylene ter‐ ephthalate) (PET) bottle is proposed using an endospore-forming bacteria genus [68]. In‐ corporated spores could actively consume oxygen for minimum 15 days, after an activation period of 1–2 days at 30 °C under high humidity conditions. Although the sys‐ tem shows some clear opportunities, such as being a biological based system and its ca‐ pability of solving polymer compatibility and recyclability issues, towards the current chemical systems, further investigations are necessary to determine a possible interaction between spores and food product.

**Active packaging Action mechanisms Food applications**

Bread, cakes, cooked rice, biscuits, pizza, pasta, cheese, cured meats and fish, coffee, snack foods, dried foods

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other snack food products and

snack foods, fruit and vegetables.

Fish, meats, snack, cereals, dried foods, sandwiches, fruit and

cereals, poultry, dairy products and

Fruit, vegetables and other horticultural products.

Pizza crusts, cakes, bread, biscuits, fish

Fruit, vegetables and other horticultural products.

and bakery products.

vegetables.

beverages.

fruit.

and beverages.

Technological Options of Packaging to Control Food Quality

sponge cakes.

Slowed respiration rate, inhibited microbial growth. Coffee, fresh meats and fish, nuts and

Antimicrobial and antioxidant effect. Cereals, meats, fish, bread, cheese,

Malodorous constituents causing off-flavors are absorbed. Fruit juices, fried snack foods, fish,

Temperature control Able to maintain chilled temperature. Ready meals, meats, fish, poultry and

Another important example of scavenger is the packaging with ethylene scavenger proper‐ ty. Ethylene has long been recognized as a problem in post-harvest handling of horticultural products because it is responsible for a wide variety of undesirable effects: it accelerates the respiration of fruits and vegetables, as well as softening and ripening, and it is responsible for a number of specific post-harvest disorders. The removal of this gas from storage cham‐ bers and packages of fruits and vegetables is, therefore, of the utmost importance, and it is done as a regular practice in the case of chambers, although is only more recently done in the case of removal from a single package. Ethylene is a very reactive compound that can be altered in many ways, such as chemical cleavage and modification, absorption, adsorption, etc. This creates a diversity of opportunities for commercial applications for the removal of ethylene [69]. Most substances designed to remove ethylene from package are delivered ei‐ ther as sachets that go inside the package or are integrated into the packaging material, usu‐ ally a plastic polymer film. The most commonly used are based in potassium permanganate, activated carbon and activated earth. Meyer & Terry [70] studied the effect of 1-methylcyclo‐

Gas permeability responding to temperature changes to

inhibited undesirable oxidation of labile pigments and vitamins, controlled enzymatic discoloration, inhibited

Oxygen scavengers Slowed food metabolism, reduced oxidative rancidity,

growth of aerobic microorganisms.

Ethylene scavengers Slowed respiration rate, thus slowed softening and

Ethanol emitters Effective against mould, can also inhibit the growth of

Moisture absorbers Inhibited microbial growth and moisture related degradation of texture and flavor.

avoid anoxic conditions.

**Table 3.** Examples of active packaging systems

yeasts and bacteria.

ripening

Carbon dioxide scavengers/emitters

Preservative releasers

Flavor/odor absorbers

Temperature compensating


**Table 3.** Examples of active packaging systems

usually contained in sachets made of a material highly permeable to air but it can also be included in bottle closures or in the plastic film matrix. Different studies show that the use of scavengers led to faster reduction and to lower levels of residual oxygen, as compared to nitrogen flushing. The most common substances used are iron powder and ascorbic acid. Scavengers also differ in the reaction speed, from immediate action (0.5 to 1 day) to slow action (4 to 6 days), on the application, particularly the moisture content of the food, and on the function, i.e., oxygen scavenging only or dual function, such as absorbing or generating carbon dioxide, besides removing the oxygen. The scientific liter‐ ature contains a number of references which examine the influence of oxygen scavenger sachets on fresh beef discoloration. Gill and McGinnis [61] performed an oxygen absorp‐ tion kinetics study with a commercial oxygen scavenger (FreshPaxTM 200R) and report‐ ed that discoloration could be prevented in ground beef if large numbers of scavengers were used in each pack to bring residual oxygen to <10 ppm within 2 h at a storage temperature of -1.5 °C. The inclusion of oxygen scavengers (Ageless® SS200) in master packs flushed with 50% carbon dioxide and 50% nitrogen significantly improved color stability of M. longissimus dorsi and M. psoas major, relative to controls [62]. In addi‐ tion to fresh beef oxygen scavenging technology has also been applied to pork [63] and pork products, where, Martìnez, Djenane, Cilla, Beltràn, and Roncalès [64] reported that fresh pork sausages stored in 20% CO2 and 80% N2 plus an oxygen scavenger (Ageless® FX-40) for up to 20 days at 2 ± 1 °C reduced psychrotrophic aerobe counts and extended shelf life in terms of color and lipid stability. An alternative to sachets involves the incor‐ poration of the oxygen scavenger into the packaging structure itself. This UV light-acti‐ vated oxygen scavenging film, which structurally is composed of an oxygen scavenger layer extruded into a multilayer film, can reduce headspace oxygen levels from 1% to ppm levels in 4–10 days, compared to oxygen scavenging sachets. The OS2000TM scav‐ enging films found applications in a wide variety of food products including dried or smoked meat products and processed meats [65]. Berenzon and Saguy [66] evaluated the applicability of oxygen absorbers for extending shelf life of military ration crackers pack‐ aged in hermetically sealed tin cans and stored at 15, 25 and 35 °C for up to 52 weeks. Sensory evaluations suggested that crackers stored without oxygen absorbers developed oxidative rancid odors after 24 weeks at 25 and 35 °C. Independently of storage tempera‐ tures, no oxidative rancid odors were observed after 44 weeks with oxygen absorbers. Opposed to the currently available chemical oxygen scavengers, systems based upon nat‐ ural and biological components could have advantages towards consumer perception and sustainability [67]. A model system for a new oxygen scavenging poly(ethylene ter‐ ephthalate) (PET) bottle is proposed using an endospore-forming bacteria genus [68]. In‐ corporated spores could actively consume oxygen for minimum 15 days, after an activation period of 1–2 days at 30 °C under high humidity conditions. Although the sys‐ tem shows some clear opportunities, such as being a biological based system and its ca‐ pability of solving polymer compatibility and recyclability issues, towards the current chemical systems, further investigations are necessary to determine a possible interaction

between spores and food product.

366 Food Industry

Another important example of scavenger is the packaging with ethylene scavenger proper‐ ty. Ethylene has long been recognized as a problem in post-harvest handling of horticultural products because it is responsible for a wide variety of undesirable effects: it accelerates the respiration of fruits and vegetables, as well as softening and ripening, and it is responsible for a number of specific post-harvest disorders. The removal of this gas from storage cham‐ bers and packages of fruits and vegetables is, therefore, of the utmost importance, and it is done as a regular practice in the case of chambers, although is only more recently done in the case of removal from a single package. Ethylene is a very reactive compound that can be altered in many ways, such as chemical cleavage and modification, absorption, adsorption, etc. This creates a diversity of opportunities for commercial applications for the removal of ethylene [69]. Most substances designed to remove ethylene from package are delivered ei‐ ther as sachets that go inside the package or are integrated into the packaging material, usu‐ ally a plastic polymer film. The most commonly used are based in potassium permanganate, activated carbon and activated earth. Meyer & Terry [70] studied the effect of 1-methylcyclo‐

propene (1-MCP) and a newly developed palladium (Pd)-promoted ethylene scavenger (e + ®Ethylene Remover) on changes in firmness, color, fatty acids and sugar content of early and late season avocado (*Persea americana Mill.*), cv. Hass, during storage at 5 °C and subsequent ripening at 20 °C. Results have shown that the ®Ethylene Remover is effective at delaying ripening of avocado at low temperature, similarly to 1-MCP; however, subsequent ripening was not impaired. Similarly, but to a lesser extent and concomitant with trends in firmness retention and color changes, ®Ethylene Remover led to greater maintenance of mannoheptu‐ lose and perseitol than that of controls. Initial findings have demonstrated for the first time that the presence of a palladium-based scavenger was effective at removing ethylene to be‐ low physiologically active levels for preclimacteric green bananas and green avocado fruits. Reduced carbon dioxide production and control of color change from green to yellow was observed for the preclimacteric bananas. Results suggested that the normal and expected cli‐ macteric respiratory rise has been disrupted. Therefore, for the first time an ethylene scav‐ enger has been shown to be capable of extending shelf life even when the climacteric respiratory rise has already been initiated [71].

Antioxidant food packaging films were produced by incorporation of ascorbic acid, ferulic acid, quercetin, and green tea extract into an ethylene vinyl alcohol copolymer matrix. The efficiency of the films developed was determined by real packaging applications of brined sardines. The evolution of the peroxide index and the malondialdehyde content showed that, in general, the films improved sardine stability. Films with green tea extract offered the best protection against lipid oxidation [76]. A natural citrus extract was also sprayed onto the surface of polyethylene terephthalate trays to delay lipid oxidation of cooked tur‐ key meat slices, stored at 4 °C over 4 days [9]. The high surface roughness, demonstrated by optical profilometry, and the high level of solubility of the antioxidant in water al‐ lowed a good effectiveness of the citrus extract coating. Patties made of minced chicken breast and thigh packed in standard vacuum-packaging or in antioxidant active packag‐ ing containing rosemary extract were subjected to high pressure treatment (800 MPa, 10 min, 5 °C) and subsequently stored at 5 °C. The active packaging was able to delay sur‐ face lipid oxidation up to 25 days. The migration of α-tocopherol from a multilayer active packaging made up of high density polyethylene, ethylene vinyl alcohol and a layer of low density polyethylene containing the antioxidant, was studied. The antioxidant deliver‐ ing system delayed the lipid oxidation of whole milk powder and it was more effective at

Technological Options of Packaging to Control Food Quality

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369

The most investigated active systems are the packaging with antimicrobial properties, even if there has been little commercial activity in North America or Europe. Japan has historical‐ ly been a leader in antimicrobial use. To date the literature counts various research works and review papers dealing with advances in antimicrobial packaging. Generally specking most of them are focused on *in vitro* test and can be subdivided in various categories: (i) there are studies aimed to develop new active systems with recently exposed natural com‐ pounds; (ii) studies finalized to underline the release kinetic of the active agents from the matrix to the food, with the intent to realize controlled release systems; (iii) studies aimed to develop bio-based active systems and (iv) works that use the nanotechnology approach. Among the abundant list of research articles and reviews available in the scientific literature on this topic, some recent works have been selected. Kanatt et al. [78] studied active films of chitosan and polyvinyl alcohol containing aqueous mint extract/pomegranate peel extract. Ramos et al. [79] studied antimicrobial active films based on polypropylene (PP) and con‐ taining thymol and carvacrol at three different concentrations. Trans-2-hexenal was encap‐ sulated into β-cyclodextrins and incorporated into a poly(L-lactic acid) matrix by extrusion and casting [75]. A newly sinthetized polyester (poly-butylene adipate) containing covalent‐ ly bound quaternary phosphonium groups was developed by Anthierens et al. [80]. The re‐ sulting polyester showed great antimicrobial activity through direct contact without any migration of active groups. Among the bio-based active systems, various bio-active packag‐ ing were developed to control insect pest in granary weevils [81, 82]. The efficacy of edible films produced from whey protein isolate and glycerol, including incorporation of lactic acid, propionic acid, chitooligosaccharides and natamycin was assessed by Ramos et al. [83]. Suppakul et al. [84] studied the diffusion of linalool and methylchavicol from thin antimi‐ crobial low-density polyethylene-based films. Cellulose acetate-based mono and multilayer films including potassium sorbate were prepared using dry phase inversion technique [85].

temperatures higher than 20 °C [77].

In order to suppress spoilage and remove offensive odors in fresh products carbon dioxide absorbers are used. On the other hand, carbon dioxide emitters are useful in modified at‐ mosphere packaging, because carbon dioxide suppresses the bacteria that cause spoilage. Both carbon dioxide generators and absorbers are available in sachet format [72].

Ethanol emitters are particularly effective in extending the shelf life of high water activity baked products. The use of ethanol generating sachets or strips avoids the ethanol spraying directly onto the product surface prior to packaging [60]. The ethanol is absorbed or encap‐ sulated in a carrier material enclosed in sachets of selective permeability to ethanol to allow for ethanol accumulation in the headspace. The level of ethanol in the packaging headspace depends obviously on the sachet size and on product water activity.

Fragrances incorporated in packaging also found commercial use in food, personal care, pharmaceutical, and nutraceutical packaging. In food packaging, fragrances are being used as a marketing tool to create consumer awareness and to enhance brand image. Because cy‐ clodextrins are able to form inclusion complexes with various compounds, they present a potential interest as agents to retain or scavenge substances such as odors, bitter com‐ pounds, lactose, cholesterol, etc., or to add aromas, colors, or functional ingredients whose release could enhance the quality of the packaged product and extend its shelf life [73-75].

Control of moisture is also important for food preservation. In most cases, the packaging material itself is responsible for the control of moisture transfer between the internal and ex‐ ternal environment, providing an adequate barrier. There are situations however, where a greater control is needed to avoid the build-up of liquid water inside the package, therefore requiring liquid water control or humidity buffering as in the case of transpiration of fresh produce, melting of ice in fish transportation, temperature fluctuation in high water activity food packages and drip of tissue fluid from cut meats and produce [60]. To this aim, absorb‐ ent pads or sheets, anti-fog additives in the polymer film, humectant between two layers of a highly water vapor permeable film or sachets of inorganic desiccant salts, are generally used to accomplish liquid removal or humidity buffering.

Antioxidant food packaging films were produced by incorporation of ascorbic acid, ferulic acid, quercetin, and green tea extract into an ethylene vinyl alcohol copolymer matrix. The efficiency of the films developed was determined by real packaging applications of brined sardines. The evolution of the peroxide index and the malondialdehyde content showed that, in general, the films improved sardine stability. Films with green tea extract offered the best protection against lipid oxidation [76]. A natural citrus extract was also sprayed onto the surface of polyethylene terephthalate trays to delay lipid oxidation of cooked tur‐ key meat slices, stored at 4 °C over 4 days [9]. The high surface roughness, demonstrated by optical profilometry, and the high level of solubility of the antioxidant in water al‐ lowed a good effectiveness of the citrus extract coating. Patties made of minced chicken breast and thigh packed in standard vacuum-packaging or in antioxidant active packag‐ ing containing rosemary extract were subjected to high pressure treatment (800 MPa, 10 min, 5 °C) and subsequently stored at 5 °C. The active packaging was able to delay sur‐ face lipid oxidation up to 25 days. The migration of α-tocopherol from a multilayer active packaging made up of high density polyethylene, ethylene vinyl alcohol and a layer of low density polyethylene containing the antioxidant, was studied. The antioxidant deliver‐ ing system delayed the lipid oxidation of whole milk powder and it was more effective at temperatures higher than 20 °C [77].

propene (1-MCP) and a newly developed palladium (Pd)-promoted ethylene scavenger (e + ®Ethylene Remover) on changes in firmness, color, fatty acids and sugar content of early and late season avocado (*Persea americana Mill.*), cv. Hass, during storage at 5 °C and subsequent ripening at 20 °C. Results have shown that the ®Ethylene Remover is effective at delaying ripening of avocado at low temperature, similarly to 1-MCP; however, subsequent ripening was not impaired. Similarly, but to a lesser extent and concomitant with trends in firmness retention and color changes, ®Ethylene Remover led to greater maintenance of mannoheptu‐ lose and perseitol than that of controls. Initial findings have demonstrated for the first time that the presence of a palladium-based scavenger was effective at removing ethylene to be‐ low physiologically active levels for preclimacteric green bananas and green avocado fruits. Reduced carbon dioxide production and control of color change from green to yellow was observed for the preclimacteric bananas. Results suggested that the normal and expected cli‐ macteric respiratory rise has been disrupted. Therefore, for the first time an ethylene scav‐ enger has been shown to be capable of extending shelf life even when the climacteric

In order to suppress spoilage and remove offensive odors in fresh products carbon dioxide absorbers are used. On the other hand, carbon dioxide emitters are useful in modified at‐ mosphere packaging, because carbon dioxide suppresses the bacteria that cause spoilage.

Ethanol emitters are particularly effective in extending the shelf life of high water activity baked products. The use of ethanol generating sachets or strips avoids the ethanol spraying directly onto the product surface prior to packaging [60]. The ethanol is absorbed or encap‐ sulated in a carrier material enclosed in sachets of selective permeability to ethanol to allow for ethanol accumulation in the headspace. The level of ethanol in the packaging headspace

Fragrances incorporated in packaging also found commercial use in food, personal care, pharmaceutical, and nutraceutical packaging. In food packaging, fragrances are being used as a marketing tool to create consumer awareness and to enhance brand image. Because cy‐ clodextrins are able to form inclusion complexes with various compounds, they present a potential interest as agents to retain or scavenge substances such as odors, bitter com‐ pounds, lactose, cholesterol, etc., or to add aromas, colors, or functional ingredients whose release could enhance the quality of the packaged product and extend its shelf life [73-75]. Control of moisture is also important for food preservation. In most cases, the packaging material itself is responsible for the control of moisture transfer between the internal and ex‐ ternal environment, providing an adequate barrier. There are situations however, where a greater control is needed to avoid the build-up of liquid water inside the package, therefore requiring liquid water control or humidity buffering as in the case of transpiration of fresh produce, melting of ice in fish transportation, temperature fluctuation in high water activity food packages and drip of tissue fluid from cut meats and produce [60]. To this aim, absorb‐ ent pads or sheets, anti-fog additives in the polymer film, humectant between two layers of a highly water vapor permeable film or sachets of inorganic desiccant salts, are generally

Both carbon dioxide generators and absorbers are available in sachet format [72].

depends obviously on the sachet size and on product water activity.

used to accomplish liquid removal or humidity buffering.

respiratory rise has already been initiated [71].

368 Food Industry

The most investigated active systems are the packaging with antimicrobial properties, even if there has been little commercial activity in North America or Europe. Japan has historical‐ ly been a leader in antimicrobial use. To date the literature counts various research works and review papers dealing with advances in antimicrobial packaging. Generally specking most of them are focused on *in vitro* test and can be subdivided in various categories: (i) there are studies aimed to develop new active systems with recently exposed natural com‐ pounds; (ii) studies finalized to underline the release kinetic of the active agents from the matrix to the food, with the intent to realize controlled release systems; (iii) studies aimed to develop bio-based active systems and (iv) works that use the nanotechnology approach. Among the abundant list of research articles and reviews available in the scientific literature on this topic, some recent works have been selected. Kanatt et al. [78] studied active films of chitosan and polyvinyl alcohol containing aqueous mint extract/pomegranate peel extract. Ramos et al. [79] studied antimicrobial active films based on polypropylene (PP) and con‐ taining thymol and carvacrol at three different concentrations. Trans-2-hexenal was encap‐ sulated into β-cyclodextrins and incorporated into a poly(L-lactic acid) matrix by extrusion and casting [75]. A newly sinthetized polyester (poly-butylene adipate) containing covalent‐ ly bound quaternary phosphonium groups was developed by Anthierens et al. [80]. The re‐ sulting polyester showed great antimicrobial activity through direct contact without any migration of active groups. Among the bio-based active systems, various bio-active packag‐ ing were developed to control insect pest in granary weevils [81, 82]. The efficacy of edible films produced from whey protein isolate and glycerol, including incorporation of lactic acid, propionic acid, chitooligosaccharides and natamycin was assessed by Ramos et al. [83]. Suppakul et al. [84] studied the diffusion of linalool and methylchavicol from thin antimi‐ crobial low-density polyethylene-based films. Cellulose acetate-based mono and multilayer films including potassium sorbate were prepared using dry phase inversion technique [85]. Monolayer films, prepared using powdered cellulose and poly(vinyl) alcohol were coated with cellulose membrane to obtain multilayer films and sorbic acid was incorporated as an‐ timicrobial agent [86]. Cerisuelo et al. [87] studied by a mathematical model the release of carvacrol from an ethylene-vinyl alcohol coating on a polypropylene film while Del Nobile et al. [88] studied the release of thymol from zein-based films. Bierhalz et al. [89] studied re‐ lease behavior in water and diffusion coefficients of single and composite films based on al‐ ginate and pectin containing natamycin. Organic/inorganic compounds, essential oils, bacteria originated antibacterial proteins, enzymes and fruit extracts have shown great po‐ tential in inhibiting microbial growth in food stuff. However, the development of new resist‐ ant strains of bacteria to current antibiotics has led to the search for new bactericides that can effectively reduce the harmful effects of microorganisms. With the emergence of nano‐ technology, the search for effective biocidal agents has focused on the development of nano‐ structures of coinage metals like silver, copper, zinc and gold [90]. ZnO nanoparticles loaded starch-coated polyethylene film were developed by Tankhiwale and Bajpai [91]. Montmoril‐ lonite nanoclay and rosemary essential oil were incorporated into chitosan film to improve its physical and mechanical properties as well as antimicrobial and antioxidant behavior [92]. Silver nanoparticles (AgNPs) have been abundantly exploited for technological appli‐ cations as bactericidal agents. Recently, AgNPs were incorporated with success into biobased materials [93] and into a hydroxy-propyl methylcellulose matrix [94]. Although numerous antimicrobial systems continue to be investigated in food simulating models, real applications are limited by technical, aesthetic and regulatory barriers. To this regards, a few recent examples can be cited. Microencapsulated beta-cyclodextrin and trans-cinnamalde‐ hyde complex was incorporated into a multilayered edible coating made of chitosan and pectin to coat fresh-cut papaya that was then packaged in Ziploc trays with Ziploc lids for 15 days. The layer-by-layer assembly with incorporation of microencapsulated antimicrobial was effective in extending shelf life and quality of fruit [95]. The antimicrobial proteins lyso‐ zyme and lactoferrin were incorporated into paper containing carboxymethyl cellulose [96]. The antimicrobial activity on common food contaminants was also retained in the released protein, and a synergism between the two proteins was evident in tests carried out with pa‐ per containing both proteins. Lysozyme was most effective in preventing microbial growth when the system was applied to thin meat slices laid on paper sheets containing either or both antimicrobial proteins. Cellulose/silver nanocomposites were investigated to decrease the microbial loads in minimally processed foods and meat [97]. The active systems were synthesized by means of reduction by UV/heat of silver nitrate adsorbed on fluff pulp cellu‐ lose fibres. Minimally processed fruits and meat products were packaged in trays containing commercial absorbent pads or silver loaded absorbers and in contact with silver loaded ab‐ sorbers, spoilage counts were significantly reduced. Active packaging based on silver nano‐ particles, obtained by allowing silver ions from nitrate solutions to replace the Na+ of natural montmorillonite and then reduced by a thermal treatment, were applied to fruit sal‐ ad [98] and fresh dairy products [99, 100]. The striking feature of these works is the interest‐ ing antimicrobial effects, without compromising sensory properties. The antimicrobial effectiveness is usually complicated by several factors, including temperature, moisture lev‐ els, chemistry of the antimicrobial agent and release mechanism. Moreover, it is necessary to

consider odor or color change that an antimicrobial could provoke in the packaged product. The cost-benefit ratio of antimicrobials is also a limiting factor for commercial growth and rates of return in the food industry are small. All these considerations explain the limited diffusion of active systems, although several antimicrobials have been successful in the labo‐ ratory. Applications with good potential are value added products such as pre-sliced and

Technological Options of Packaging to Control Food Quality

http://dx.doi.org/10.5772/53151

371

Packaging design is clearly a fundamental part of a new launch product. Considering the importance of packaging in determining product shelf life, the correct approach allows considering on the same level of importance the product development and its packaging system. The key to successful packaging is selection of materials and designs that best bal‐ ance the competing needs of product characteristics, marketing considerations including distribution and consumer needs, environmental and waste management issues, and cost. Food packaging technologies also require integration with other processing and preserva‐ tion activities such as freezing, irradiation, pulsed electric fields, high pressure processing, and pulsed light. Globalization, packaging life cycles, and requirement for strict safety measures are increasing the pressure to produce new packaging systems able to transport food items and that also allow the traceability along the food distribution chain. Due to the diversity of product characteristics and basic food packaging demands and applica‐ tions, any packaging technologies offering to deliver more product and quality control in an economic and diverse manner would be favorably welcomed. To meet tomorrow's con‐ cerns, there continues to be a large amount of research to evaluate areas such as active packaging, traceability, sustainable resources and antimicrobial packaging. Advances in these areas will continue to give us a safe and sustainable food supply. The use of modi‐ fied atmosphere technique can extend shelf life. Its use does not eliminate the need for proper control of storage conditions, especially temperature, nor for the adequate training handlers at sensory characteristics and shelf life of many food products, inhibiting the growth of pathogenic bacteria. MAP will continue to be used in the future, most probably with several different MAP formats in use around the world. Mechanistic, logistical, and perception obstacles will require effort and ingenuity to overcome existing package and system difficulties and promote implementation of new processing and packaging tech‐ nologies. Moreover, the concept of combining antimicrobial/antioxidant agents within the package to control the deterioration and growth of microorganisms in food, will have a strong impact on both shelf life prolongation and food safety. Although the evidence sug‐ gests that active packaging is a promising technology, its potential cannot be fully realiz‐ ed unless major technical problems are overcome. More research related to the control of the migration of the active agents at rates suitable for different real food systems is still needed. Recognition of the benefits of active packaging technologies by the food industry, development of economically viable packaging systems and increased consumer accept‐

ance opens new frontiers for active packaging technology.

prepared foods.

**4. Final considerations**

consider odor or color change that an antimicrobial could provoke in the packaged product. The cost-benefit ratio of antimicrobials is also a limiting factor for commercial growth and rates of return in the food industry are small. All these considerations explain the limited diffusion of active systems, although several antimicrobials have been successful in the labo‐ ratory. Applications with good potential are value added products such as pre-sliced and prepared foods.

## **4. Final considerations**

Monolayer films, prepared using powdered cellulose and poly(vinyl) alcohol were coated with cellulose membrane to obtain multilayer films and sorbic acid was incorporated as an‐ timicrobial agent [86]. Cerisuelo et al. [87] studied by a mathematical model the release of carvacrol from an ethylene-vinyl alcohol coating on a polypropylene film while Del Nobile et al. [88] studied the release of thymol from zein-based films. Bierhalz et al. [89] studied re‐ lease behavior in water and diffusion coefficients of single and composite films based on al‐ ginate and pectin containing natamycin. Organic/inorganic compounds, essential oils, bacteria originated antibacterial proteins, enzymes and fruit extracts have shown great po‐ tential in inhibiting microbial growth in food stuff. However, the development of new resist‐ ant strains of bacteria to current antibiotics has led to the search for new bactericides that can effectively reduce the harmful effects of microorganisms. With the emergence of nano‐ technology, the search for effective biocidal agents has focused on the development of nano‐ structures of coinage metals like silver, copper, zinc and gold [90]. ZnO nanoparticles loaded starch-coated polyethylene film were developed by Tankhiwale and Bajpai [91]. Montmoril‐ lonite nanoclay and rosemary essential oil were incorporated into chitosan film to improve its physical and mechanical properties as well as antimicrobial and antioxidant behavior [92]. Silver nanoparticles (AgNPs) have been abundantly exploited for technological appli‐ cations as bactericidal agents. Recently, AgNPs were incorporated with success into biobased materials [93] and into a hydroxy-propyl methylcellulose matrix [94]. Although numerous antimicrobial systems continue to be investigated in food simulating models, real applications are limited by technical, aesthetic and regulatory barriers. To this regards, a few recent examples can be cited. Microencapsulated beta-cyclodextrin and trans-cinnamalde‐ hyde complex was incorporated into a multilayered edible coating made of chitosan and pectin to coat fresh-cut papaya that was then packaged in Ziploc trays with Ziploc lids for 15 days. The layer-by-layer assembly with incorporation of microencapsulated antimicrobial was effective in extending shelf life and quality of fruit [95]. The antimicrobial proteins lyso‐ zyme and lactoferrin were incorporated into paper containing carboxymethyl cellulose [96]. The antimicrobial activity on common food contaminants was also retained in the released protein, and a synergism between the two proteins was evident in tests carried out with pa‐ per containing both proteins. Lysozyme was most effective in preventing microbial growth when the system was applied to thin meat slices laid on paper sheets containing either or both antimicrobial proteins. Cellulose/silver nanocomposites were investigated to decrease the microbial loads in minimally processed foods and meat [97]. The active systems were synthesized by means of reduction by UV/heat of silver nitrate adsorbed on fluff pulp cellu‐ lose fibres. Minimally processed fruits and meat products were packaged in trays containing commercial absorbent pads or silver loaded absorbers and in contact with silver loaded ab‐ sorbers, spoilage counts were significantly reduced. Active packaging based on silver nano‐ particles, obtained by allowing silver ions from nitrate solutions to replace the Na+ of natural montmorillonite and then reduced by a thermal treatment, were applied to fruit sal‐ ad [98] and fresh dairy products [99, 100]. The striking feature of these works is the interest‐ ing antimicrobial effects, without compromising sensory properties. The antimicrobial effectiveness is usually complicated by several factors, including temperature, moisture lev‐ els, chemistry of the antimicrobial agent and release mechanism. Moreover, it is necessary to

370 Food Industry

Packaging design is clearly a fundamental part of a new launch product. Considering the importance of packaging in determining product shelf life, the correct approach allows considering on the same level of importance the product development and its packaging system. The key to successful packaging is selection of materials and designs that best bal‐ ance the competing needs of product characteristics, marketing considerations including distribution and consumer needs, environmental and waste management issues, and cost. Food packaging technologies also require integration with other processing and preserva‐ tion activities such as freezing, irradiation, pulsed electric fields, high pressure processing, and pulsed light. Globalization, packaging life cycles, and requirement for strict safety measures are increasing the pressure to produce new packaging systems able to transport food items and that also allow the traceability along the food distribution chain. Due to the diversity of product characteristics and basic food packaging demands and applica‐ tions, any packaging technologies offering to deliver more product and quality control in an economic and diverse manner would be favorably welcomed. To meet tomorrow's con‐ cerns, there continues to be a large amount of research to evaluate areas such as active packaging, traceability, sustainable resources and antimicrobial packaging. Advances in these areas will continue to give us a safe and sustainable food supply. The use of modi‐ fied atmosphere technique can extend shelf life. Its use does not eliminate the need for proper control of storage conditions, especially temperature, nor for the adequate training handlers at sensory characteristics and shelf life of many food products, inhibiting the growth of pathogenic bacteria. MAP will continue to be used in the future, most probably with several different MAP formats in use around the world. Mechanistic, logistical, and perception obstacles will require effort and ingenuity to overcome existing package and system difficulties and promote implementation of new processing and packaging tech‐ nologies. Moreover, the concept of combining antimicrobial/antioxidant agents within the package to control the deterioration and growth of microorganisms in food, will have a strong impact on both shelf life prolongation and food safety. Although the evidence sug‐ gests that active packaging is a promising technology, its potential cannot be fully realiz‐ ed unless major technical problems are overcome. More research related to the control of the migration of the active agents at rates suitable for different real food systems is still needed. Recognition of the benefits of active packaging technologies by the food industry, development of economically viable packaging systems and increased consumer accept‐ ance opens new frontiers for active packaging technology.

## **Author details**

Amalia Conte, Luisa Angiolillo, Marcella Mastromatteo and Matteo Alessandro Del Nobile\*

[12] Leistner L. Principles and applications of hurdle technology. In: New Methods of

Technological Options of Packaging to Control Food Quality

http://dx.doi.org/10.5772/53151

373

[13] McMillin KW. Where is MAP Going? A review and future potential of modified at‐

[14] Farber JM. Microbiological aspects of modified-atmosphere packaging technology-A

[15] Phillips CA. Review: Modified atmosphere packaging and its effects on the microbio‐ logical quality and safety of produce, International Journal of Food Science and Tech‐

[16] Church IJ, Parsons AL. Modified atmosphere packaging technology: A Review. Jour‐

[17] Hotchkiss JH. Microbiological Hazards of Controlled/Modified Atmosphere Food Packaging. Journal of the Science of Food and Agriculture 1989; 53(3) 41-49.

[18] Genigeorgis C. Microbial and safety implications of the use of modified atmospheres to extend the storage life of fresh meat and fish. International Journal of Food Micro‐

[19] Smith BS. The maturation of case-ready technology operational challenges. In Meat industry research conference pag117-118, 15-17 October 2001, Chicago, Illinois, USA.

[20] Rowe LJ, Maddock KR, Lonergan SM, Huff-Lonergan E. Influence of early post-mor‐ tem protein oxidation on beef quality. Journal of Animal Science 2004; 82 785-793.

[21] Lund MN, Lametsch R, Hviid MS, Jensen ON, Skibsted LH. High oxygen packaging atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi

[22] Zakrys-Waliwander PI, O'Sullivan MG, O'Neill EE, Kerry JP. The effects of high oxy‐ gen modified atmosphere packaging on protein oxidation of bovine M. longissimus

[23] Mastromatteo M, Incoronato AL, Conte A, Del Nobile MA. Shelf life of reduced pork back fat content sausages as affected by antimicrobial compounds and modified at‐ mosphere packaging. International Journal of Food Microbiology 2011; 150 1-7.

[24] Belcher J.N. Industrial packaging developments for the global meat market. Meat Sci‐

[25] Zhou GH, Xu XL, Liu Y. Preservation technologies for fresh meat – A review. Meat

[26] Simon A, Gonzales-Fandos E, Tobar V. The sensory and microbiological quality of fresh sliced mushroom (Agaricus bisporus L.) packaged in modified atmospheres.

International Journal of Food Science and Technology 2005; 40 943.

dorsi muscle during chilled storage. Food Chemistry 2012; 131 527-532.

food Preservation (ed.) G.W. Gould. 1995. p1-21. Glasgow, UK, Blackie.

mosphere packaging for meat. Meat Science 2008; 80 43–65.

nal of the Science of Food and Agriculture 1995; 67 143-152.

during chill storage. Meat Science 2007; 77 295-303.

Review. Journal of Food Protection 1993; 54(1) 58-70.

nology 1996; 31 463-479.

biology 1985; 1 237-251.

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Science 2010; 86 119–128.

\*Address all correspondence to: ma.delnobile@unifg.it

University of Foggia, Department of Agricultural Sciences, Food and Environment, Italy

## **References**


[12] Leistner L. Principles and applications of hurdle technology. In: New Methods of food Preservation (ed.) G.W. Gould. 1995. p1-21. Glasgow, UK, Blackie.

**Author details**

372 Food Industry

**References**

842

Amalia Conte, Luisa Angiolillo, Marcella Mastromatteo and Matteo Alessandro Del Nobile\*

University of Foggia, Department of Agricultural Sciences, Food and Environment, Italy

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**Chapter 17**

**Moisture-Dependent**

http://dx.doi.org/10.5772/53173

diets in order to improve human health.

**1. Introduction**

seed [9].

crop plant [7, 11].

**Engineering Properties**

**of Chia (***Salvia hispanica* **L.) Seeds**

Estefanía N. Guiotto, Vanesa Y. Ixtaina, Mabel C. Tomás and Susana M. Nolasco

Additional information is available at the end of the chapter

*Salvia hispanica* L., whose common name is chia, is an annual herbaceous plant belonging to the *Lamiaceae* or *Labiatae* family. This botanical species, native to southern Mexico and north‐ ern Guatemala, was an important crop in pre-Columbian Mesoamerica in conjunction with corn, beans and amaranth. Chia seeds were valuated not only for food, but also for medi‐ cines and paints [1]. Its cultivation was banned by Spanish conquerors and replaced by exot‐ ic crops (wheat and barley) [2]. Nowadays, chia seeds are being reintroduced to western

These seeds have been investigated and recommended due to their oil content with the highest proportion of α-linolenic acid (omega-3) compared to other natural source known to date [3, 4], and also because of their high levels of protein, antioxidant, dietary fiber, vita‐ mins and minerals [5, 6]. Chia seeds from Argentina exhibited 30.0 - 38.6 g oil/100 g, with 60.7 - 67.8 g/100 g of α-linolenic acid [7, 8]. Figure 1 shows the chemical composition of chia

Chia seed is traditionally consumed in Mexico, the southwestern U.S., and South America, but it is not widely known in Europe. However, in 2009, the European Union approved chia seeds as a novel food, allowing them to comprise up to 5% of a bread product's total matter [10]. Today, chia is mostly grown in Mexico, Bolivia, Argentina, Ecuador, Australia, and Guatemala, and it has been demonstrated that the species has great potential as a future

and reproduction in any medium, provided the original work is properly cited.

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© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
