Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food Packaging Films

*Cecilia Rojas de Gante, Judith A. Rocha and Carlos P. Sáenz Collins*

## **Abstract**

The development of active food packaging is addressed using polyolefins such as LDPE and PVOH, as well as biopolymers from flour (sorghum and corn) and byproducts of the food industry. Bacteriocins (nisin, natamycin), plant extracts such as oregano and thyme, as well as native plants of the northeast region of Mexico (*Larrea tridentata*, *Schinus molle*, *Cordia boissieri*, *Leucophyllum frutescens*), and essential oils of oregano and thyme as antimicrobial agents have been studied. The effect exerted by the process of incorporation of the antimicrobial agent (casting, extrusion) on the barrier and mechanical properties of the package as well as the antimicrobial activity of the containers (broad spectrum or selective activity) has been observed and the establishment of methods for their traceability.

**Keywords:** sorghum, maize, flour, nisin, thyme, oregano, *Larrea tridentata*, *Schinus molle*, *Cordia boissieri*, *Leucophyllum frutescens*, *Listeria monocytogenes*, *Staphylococcus aureus*

#### **1. Introduction**

Since the last decade and a half (2000 to date), the main forces that have unleashed the greatest developments in the packaging of food are the great concern of society for the care of their integral health including its nutritional status through foods with less or no presence of additives but in convenient presentations that facilitate their preparation, heating, and intake as well as foods with therapeutic action. A consumer who is very concerned about the safety of food, where food packaging and storage systems do not represent or have physical, biological, or even toxicological risks, nor for the protection of the environment.

All of the previous demand constantly forces the change on the nature of the food packaging and consequently on the materials of which it is composed [1]. Therefore, new materials are being developed to comply with the above. First, packages that contain in their formulation substances that migrate from the container to the food exert a positive action avoiding deterioration reactions likewise increase the sensory quality through the positive migration of substances or have a therapeutic effect. In this category are the so-called active packaging [1]. Second, in relation

to the protection of the environment: the development of biodegradable packaging using, for example, biomaterials obtained from agri-food sources [1].

An active packaging is defined as the one that produces a change in the state of the packaged food to prolong its shelf life, improve its safety and quality, and provide a barrier between the food and its environment [1]. The mechanisms of action in active packages can be acting as emitting systems or as sequestering systems for substances. In the emitting systems, compounds or additives generally recognized as safe (GRAS), such as antioxidants and antimicrobial agents, are released into the food through the walls of the package. Sequestering systems remove undesirable compounds such as oxygen, H2O, ethylene, CO2, and impurities, among others [2]. Great diversity of active packaging is being developed in order to control the emission or absorption of substances and thus modify the environment of the product or directly the product. Thus, active packaging has substances or systems that absorb oxygen, ethylene, CO2 and humidity; others absorb or release desired aromas [2–4]. Other active packages contain active enzyme systems and antimicrobial substances or systems. All these active containers seek the elimination of microbial growth, the extension of the useful life, and/or the increase of organoleptic qualities of the product [5].

The proposal of our line of research is based on obtaining a series of products (active antimicrobial and active biodegradable packages according to a defined food or conservation need), as well as the processes for their elaboration. Most developments use materials obtained only from starches or proteins. With our development, a single product has a biopolymer matrix that includes both biopolymers (starches and proteins) in a single stage. By starting from a matrix that includes both biopolymers (starches and proteins and sometimes antimicrobials or antioxidants), unit operations are eliminated, which reduces costs of equipment and energy, consequently operating costs. The technological impact of the developments of the research line will be reflected in the conservation of food (fresh or dehydrated) through the use of antimicrobial/antioxidant active packaging that contribute to preserve the environment when they are discarded since they are potentially biodegradable.

From the scientific point of view, this solves a couple of problems at the same time, the first concerning the toxicological risk of the abuse of additives in the formulation and conservation of food and the second discarding the ecological and environmental problems generated by food packaging. Our developments will have, on the one hand, low environmental impact due to the development of biodegradable products from nature-friendly processes. On the other hand, they will have a high economic impact since currently in the country there are no companies dedicated to the development of biopolymer containers, creation of own technologies, and high added value to products of low commercial value.

#### **2. Antimicrobial active packaging developed at the Tecnológico de Monterrey**

#### **2.1 Biopolymer active packaging**

Our first works focused on the use of starches from several varieties of sorghum (high-production cereal in northeast of Mexico) whose different proportion in amylose and amylopectin plays an important role in the water vapor barrier of the containers reinforcing them with prolamines (kafirin and zein) to increase their impermeability [6] and use of antifungal agents such as the sorbates and benzoates of Na and K. The inclusion of broad-spectrum antimicrobial additives in plastic polymers and/ or biopolymers through the proprietary technology generated at the Tecnológico de Monterrey, for example, enabled active packaging to be obtained on a laboratory scale

**27**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

that reduced biological risks by manual or semi-manual packaging (risks of contamination with pathogens such as *Listeria monocytogenes* and *E. coli* O157: H7) [7–9]. On the other hand, there is great interest in the development and research of biopolymers obtained from agricultural sources. The matrices most commonly used to obtain this type of biopolymers are starch, proteins, and other polysaccharides. Some examples are corn zein, gluten and wheat gliadin, soy proteins, sorghum kafirin, cactus mucilage, and different types of starch (corn, potato, banana, tapioca, pea, waxy starch, and high amylose content, among others) [10–13]. In works carried out by our research group, it has been shown that it is possible to incorporate natural antimicrobial agents into films that could be used as active packaging. For example, Schause succeeded in establishing both the dry extraction conditions of starches and proteins from cereals such as sorghum (*Sorghum bicolor* Moench) as well as the casting process to obtain a film from sorghum flour and incorporate nisin as an antimicrobial active compound [8]. Nisin is a bacteriocin produced by some strains of *Lactococcus lactis* and *Streptococcus lactis* that has a broad antimicrobial power against Gram-positive bacteria. Nisin and lysozyme are used as a food preservative in dairy products as an inhibitor of *Clostridium tyrobutyricum*, *Clostridium butyricum*, *Clostridium saccharobutyricum* (causes swelling in cheese production), and pathogens like *Clostridium botulinum*, *Clostridium sporogenes* (which is used as a surrogate for *C. botulinum*), and *L. monocytogenes* [14]. The bactericidal action of nisin occurs in the cytoplasmic membrane, causing cell damage due to proton loss and damage to the integrity of the cell membrane [14]. Gram-negative bacteria have an outer membrane that protects the cytoplasmic membrane, so the bactericidal action of nisin is limited and the development of Gram-negative bacteria such as *E. coli* O157: H7

Subsequently, Ríos-Licea conducted a search of natural substances of broad spectrum, so he analyzed the antimicrobial activity of aqueous extracts of known plants. Ríos-Licea also succeeded in developing antimicrobial films by incorporating natural extracts of garlic and oregano into the same biopolymer matrix of sorghum flour using the method established by Schause [15]. However, it was necessary to incorporate high concentrations of natural extracts, due to the low

Tinoco-Pérez studied a variety of corn rich in anthocyanins (blue corn) by applying the process of dry milling and establishing the process to obtain active films in antioxidants (anthocyanins) from flour of this cereal [16]. Two biopolymers present in corn with a filmogenic capacity are starch and zein, the first being the most abundant in this grain [16]. There are a significant amount of reports published on films made from corn starch and zein; the effect of different additives, copolymers, and processes on the performance of films for different applications has been evaluated. In 2009, Mexico produced 29.4 million tons of corn using 38.5% of its total cultivated area. The production of this grain has shown an increase in its average annual growth rate of 2.1% in the period from 1994 to 2008. Of total corn production in 2008, 92% was white corn, 7% was blue corn, and 1% was of other varieties. Basically, white corn is destined for national consumption, yellow for export, and the rest of the varieties are commonly produced for self-consumption of rural populations. Among the 1% of the varieties not defined is the blue corn (*Zea mays* amylacea) [17, 18]. Blue corn (*Zea mays* amylacea) is a type of corn rich in anthocyanins (responsible for its pigmentation) and floury endosperm. It is cultivated in areas of dry climate and demands minimal care. Despite its nutraceutical potential, blue corn is only produced by rural communities for self-consumption due to its devalued commercial value, since the urbanized areas consume mainly white and yellow corn products. Among the few current uses of blue corn is the extraction of anthocyanins for use as natural food

potency of the antimicrobial activity of the commercial product tested.

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

and *Salmonella* would not be inhibited.

coloring and antioxidants [16, 19].

#### *Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

that reduced biological risks by manual or semi-manual packaging (risks of contamination with pathogens such as *Listeria monocytogenes* and *E. coli* O157: H7) [7–9]. On the other hand, there is great interest in the development and research of biopolymers obtained from agricultural sources. The matrices most commonly used to obtain this type of biopolymers are starch, proteins, and other polysaccharides. Some examples are corn zein, gluten and wheat gliadin, soy proteins, sorghum kafirin, cactus mucilage, and different types of starch (corn, potato, banana, tapioca, pea, waxy starch, and high amylose content, among others) [10–13]. In works carried out by our research group, it has been shown that it is possible to incorporate natural antimicrobial agents into films that could be used as active packaging. For example, Schause succeeded in establishing both the dry extraction conditions of starches and proteins from cereals such as sorghum (*Sorghum bicolor* Moench) as well as the casting process to obtain a film from sorghum flour and incorporate nisin as an antimicrobial active compound [8]. Nisin is a bacteriocin produced by some strains of *Lactococcus lactis* and *Streptococcus lactis* that has a broad antimicrobial power against Gram-positive bacteria. Nisin and lysozyme are used as a food preservative in dairy products as an inhibitor of *Clostridium tyrobutyricum*, *Clostridium butyricum*, *Clostridium saccharobutyricum* (causes swelling in cheese production), and pathogens like *Clostridium botulinum*, *Clostridium sporogenes* (which is used as a surrogate for *C. botulinum*), and *L. monocytogenes* [14]. The bactericidal action of nisin occurs in the cytoplasmic membrane, causing cell damage due to proton loss and damage to the integrity of the cell membrane [14]. Gram-negative bacteria have an outer membrane that protects the cytoplasmic membrane, so the bactericidal action of nisin is limited and the development of Gram-negative bacteria such as *E. coli* O157: H7 and *Salmonella* would not be inhibited.

Subsequently, Ríos-Licea conducted a search of natural substances of broad spectrum, so he analyzed the antimicrobial activity of aqueous extracts of known plants. Ríos-Licea also succeeded in developing antimicrobial films by incorporating natural extracts of garlic and oregano into the same biopolymer matrix of sorghum flour using the method established by Schause [15]. However, it was necessary to incorporate high concentrations of natural extracts, due to the low potency of the antimicrobial activity of the commercial product tested.

Tinoco-Pérez studied a variety of corn rich in anthocyanins (blue corn) by applying the process of dry milling and establishing the process to obtain active films in antioxidants (anthocyanins) from flour of this cereal [16]. Two biopolymers present in corn with a filmogenic capacity are starch and zein, the first being the most abundant in this grain [16]. There are a significant amount of reports published on films made from corn starch and zein; the effect of different additives, copolymers, and processes on the performance of films for different applications has been evaluated. In 2009, Mexico produced 29.4 million tons of corn using 38.5% of its total cultivated area. The production of this grain has shown an increase in its average annual growth rate of 2.1% in the period from 1994 to 2008. Of total corn production in 2008, 92% was white corn, 7% was blue corn, and 1% was of other varieties. Basically, white corn is destined for national consumption, yellow for export, and the rest of the varieties are commonly produced for self-consumption of rural populations. Among the 1% of the varieties not defined is the blue corn (*Zea mays* amylacea) [17, 18]. Blue corn (*Zea mays* amylacea) is a type of corn rich in anthocyanins (responsible for its pigmentation) and floury endosperm. It is cultivated in areas of dry climate and demands minimal care. Despite its nutraceutical potential, blue corn is only produced by rural communities for self-consumption due to its devalued commercial value, since the urbanized areas consume mainly white and yellow corn products. Among the few current uses of blue corn is the extraction of anthocyanins for use as natural food coloring and antioxidants [16, 19].

*Active Antimicrobial Food Packaging*

to the protection of the environment: the development of biodegradable packaging

An active packaging is defined as the one that produces a change in the state of the packaged food to prolong its shelf life, improve its safety and quality, and provide a barrier between the food and its environment [1]. The mechanisms of action in active packages can be acting as emitting systems or as sequestering systems for substances. In the emitting systems, compounds or additives generally recognized as safe (GRAS), such as antioxidants and antimicrobial agents, are released into the food through the walls of the package. Sequestering systems remove undesirable compounds such as oxygen, H2O, ethylene, CO2, and impurities, among others [2]. Great diversity of active packaging is being developed in order to control the emission or absorption of substances and thus modify the environment of the product or directly the product. Thus, active packaging has substances or systems that absorb oxygen, ethylene, CO2 and humidity; others absorb or release desired aromas [2–4]. Other active packages contain active enzyme systems and antimicrobial substances or systems. All these active containers seek the elimination of microbial growth, the extension of the useful life, and/or the

The proposal of our line of research is based on obtaining a series of products (active antimicrobial and active biodegradable packages according to a defined food or conservation need), as well as the processes for their elaboration. Most developments use materials obtained only from starches or proteins. With our development, a single product has a biopolymer matrix that includes both biopolymers (starches and proteins) in a single stage. By starting from a matrix that includes both biopolymers (starches and proteins and sometimes antimicrobials or antioxidants), unit operations are eliminated, which reduces costs of equipment and energy, consequently operating costs. The technological impact of the developments of the research line will be reflected in the conservation of food (fresh or dehydrated) through the use of antimicrobial/antioxidant active packaging that contribute to preserve the environ-

From the scientific point of view, this solves a couple of problems at the same time, the first concerning the toxicological risk of the abuse of additives in the formulation and conservation of food and the second discarding the ecological and environmental problems generated by food packaging. Our developments will have, on the one hand, low environmental impact due to the development of biodegradable products from nature-friendly processes. On the other hand, they will have a high economic impact since currently in the country there are no companies dedicated to the development of biopolymer containers, creation of own technolo-

ment when they are discarded since they are potentially biodegradable.

gies, and high added value to products of low commercial value.

**2. Antimicrobial active packaging developed at the Tecnológico de** 

Our first works focused on the use of starches from several varieties of sorghum (high-production cereal in northeast of Mexico) whose different proportion in amylose and amylopectin plays an important role in the water vapor barrier of the containers reinforcing them with prolamines (kafirin and zein) to increase their impermeability [6] and use of antifungal agents such as the sorbates and benzoates of Na and K. The inclusion of broad-spectrum antimicrobial additives in plastic polymers and/ or biopolymers through the proprietary technology generated at the Tecnológico de Monterrey, for example, enabled active packaging to be obtained on a laboratory scale

using, for example, biomaterials obtained from agri-food sources [1].

increase of organoleptic qualities of the product [5].

**26**

**Monterrey**

**2.1 Biopolymer active packaging**

Among the processes studied to obtain films from corn fractions are casting, different types of extrusion (double screw/flat die, single screw/flat die, and extrusion/calendering, among others), stretching of zein resins, and pressing by heat [10, 20–22]. The effects of various additives and chemical treatments, for example, plasticizers, hydrophobic agents, copolymers, and the use of chemically modified starches on the structural, molecular, thermal, mechanical, and barrier performance characteristics, have been studied extensively [22–25].

In the case of sorghum, the cultivation of this cereal is less demanding in agronomic terms than corn (water and nutrients) [26, 27]. Sorghum is the fifth most important grain in the world, being the United States the country with the highest production in the world, followed by India and Nigeria. For the year 2010, Mexico contributed with 10.5% of the total world production, equivalent to 6,250,000 metric tons [15, 16]. In Mexico, sorghum is the second most important grain in production after corn; during the period 1996–2006, sorghum production contributed with 22% of the total production of cereals [26].

In Mexico, this cereal is destined mainly for livestock feed and secondarily for human food and obtaining inputs such as starch, alcohol, glucose, acetone, and butanol. One of the great advantages of sorghum is that it has the capacity to adapt to arid and semiarid climatic conditions and to be resistant to drought for long periods [26]. In previous works, it was able to demonstrate that antimicrobial active films can be obtained from corn and sorghum flour [8, 15, 16].

The biopolymers obtained in this way through a technique and process patented by Tecnológico de Monterrey as PCT [28] have the advantage of being biodegradable because their chemical structure is primarily based on proteins and starches. Additionally, they have the possibility of forming films with plasticity (custom flexibility) and of being formulated also tailored to the requirements of the product to be packaged. Additionally, they can be heat sealed to form bags of different dimensions or not to be sealed and act as "active" pads or pads in combination with other packaging. In addition to the advantages in terms of sustainability, the interest in using these sources to produce biopolymers lies in adding value to agricultural products [8, 29].

It is important to note that for any application of the said technology, it will be necessary to make an adaptation of the formulations and the process to satisfy the specific protection requirements for each food to be packaged. For what it is proposed to demonstrate in this work, the film-like packages obtained by adapting the formulations and process of the said published patent work to preserve and keep refrigerated for 30–45 days a commercial presentation in slices of semi-matured cheese [30].

The biopolymeric antimicrobial films described in WO2010/024657 A1 from cereals are limited to the packaging of dry foods or as pads for adsorption of exudates and emission of antimicrobial agents for fresh meat and cheese products [28]. Because of its sensitivity to water and low mechanical resistance to contain products with intermediate moisture, the biopolymeric matrix was reformulated to improve both parameters [30]. The results of refrigerated shelf stability of the cheese in terms of the control capacity exercised by the antimicrobials used in this study (nisin and natamycin) through the active packaging against fungi and yeast were effective throughout storage compared to vacuum packaging (control). The results of the microbial kinetics throughout the refrigerated storage for the fungi and yeast count showed the effectiveness of the active packaging. The development of fungi and yeasts remained controlled, showing the effectiveness of this emerging food preservation technology [30].

The plasticizing effects of two different polyols (glycerol and sorbitol) on the mechanical, thermal, and microstructural properties of flour films were studied by Valderrama and Rojas, and the results showed that films plasticized with sorbitol had better mechanical properties and less affinity for water than those plasticized with

**29**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

glycerol. The attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectra of blue corn flour plasticizer with sorbitol showed the presence of the additional

linkages between sorbitol and a polymeric matrix. The effect of the plasticizer on the glass transition temperature (Tg) showed that Tg decreased as the plasticizer content increased. Plasticized glycerol films showed lower Tg values than those with sorbitol. Observations by scanning electron microscopy (SEM) showed that it was necessary to add plasticizer to maintain film integrity. The sorbitol-plasticized flour films revealed better adhesion between phases, and these films showed a compact structure [31]. Finally, bioplastics were produced through thermoplastic processing using different cereals derived raw materials, namely, blue maize flour (BM), white sorghum flour (WS), maize starch, and the maize prolamin (zein). The overall performance of the bioplastics was investigated emphasizing on the study of the effect of different process strategies on the compatibilization of the starch and prolamin using mixtures of urea and formamide (UF) and maleated starch (MS) as compatibilizing agents [32, 33]. Results suggest that two competing phenomena, thermoplasticization and degradation, occurred simultaneously during the thermoplastic process. Fourier-transform infrared (FTIR) spectroscopy analysis evidenced the chemical changes induced by these phenomena. Moreover, chemical modification had also a major effect on the properties of the produced materials. WS films made with chemically modified flour increased their tensile strength in 29%, as compared to their native counterparts. Thermogravimetric analysis and FTIR analysis showed that the chemical interaction between starch and zein occurred more extensively in

films made with formamide than those made with maleated starch [32, 33].

In Valderrama's work, natural aqueous extracts are exchanged for essential oils because they have a higher concentration of antimicrobial active substances. It analyzed essential oils of oregano, thyme, tea tree, and mint, which have greater antimicrobial activity than the natural extracts used by Ríos-Licea [15]. In particular, the effect of incorporating two essential oils such as oregano (*Origanum vulgare*) and thyme (*Thymus vulgaris*) on polyolefin materials such as low-density polyethyl-

The mechanical, barrier, and antimicrobial properties of the packaging were evaluated against *Salmonella typhimurium*, *Listeria monocytogenes*, and *Escherichia coli* O157:H7. The results demonstrate that films developed by extrusion incorporating 4% (w/w) of essential oils had a higher inhibitory effect than those obtained using the ionizing treatment. The packaging developed by extrusion containing 1% (w/w) showed a positive inhibitory effect, while those obtained by the ionizing treatment had no inhibitory effect against any of the test microorganisms. The incorporation of essential oils on the LDPE films generated a plasticizer effect, whereas the ones obtained by means of ionizing treatment did significantly affect

A simple and rapid Fourier-transform infrared (FTIR) spectroscopy method was developed by Valderrama and Rojas to determine the main essential oil components (carvacrol, thymol, and p-cymene) in the antimicrobial LDPE films incorporated with oregano (*Origanum vulgare*) and thyme (*Thymus vulgaris*) essential oils. The ATR-FTIR spectroscopy with chemometrics, using the PLS-first derivative spectra, could predict the active compounds content accurate to an r2 >0.99 and a standard error of prediction (SEP) of <0.7. The developed method was successfully applied to predict the concentration of active compounds: carvacrol, thymol, and p-cymene in oregano and thyme essential oils with results compared to those of the GC-MS

characteristic of the carbonyl peak, which confirms the chemical

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

band at 1745 cm<sup>−</sup><sup>1</sup>

**2.2 Plastic active packaging**

ene (LDPE) and polypropylene (PP) was studied.

the barrier properties of the films Valderrama and Rojas [9].

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

glycerol. The attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectra of blue corn flour plasticizer with sorbitol showed the presence of the additional band at 1745 cm<sup>−</sup><sup>1</sup> characteristic of the carbonyl peak, which confirms the chemical linkages between sorbitol and a polymeric matrix. The effect of the plasticizer on the glass transition temperature (Tg) showed that Tg decreased as the plasticizer content increased. Plasticized glycerol films showed lower Tg values than those with sorbitol. Observations by scanning electron microscopy (SEM) showed that it was necessary to add plasticizer to maintain film integrity. The sorbitol-plasticized flour films revealed better adhesion between phases, and these films showed a compact structure [31].

Finally, bioplastics were produced through thermoplastic processing using different cereals derived raw materials, namely, blue maize flour (BM), white sorghum flour (WS), maize starch, and the maize prolamin (zein). The overall performance of the bioplastics was investigated emphasizing on the study of the effect of different process strategies on the compatibilization of the starch and prolamin using mixtures of urea and formamide (UF) and maleated starch (MS) as compatibilizing agents [32, 33]. Results suggest that two competing phenomena, thermoplasticization and degradation, occurred simultaneously during the thermoplastic process. Fourier-transform infrared (FTIR) spectroscopy analysis evidenced the chemical changes induced by these phenomena. Moreover, chemical modification had also a major effect on the properties of the produced materials. WS films made with chemically modified flour increased their tensile strength in 29%, as compared to their native counterparts. Thermogravimetric analysis and FTIR analysis showed that the chemical interaction between starch and zein occurred more extensively in films made with formamide than those made with maleated starch [32, 33].

#### **2.2 Plastic active packaging**

*Active Antimicrobial Food Packaging*

Among the processes studied to obtain films from corn fractions are casting, different types of extrusion (double screw/flat die, single screw/flat die, and extrusion/calendering, among others), stretching of zein resins, and pressing by heat [10, 20–22]. The effects of various additives and chemical treatments, for example, plasticizers, hydrophobic agents, copolymers, and the use of chemically modified starches on the structural, molecular, thermal, mechanical, and barrier perfor-

In the case of sorghum, the cultivation of this cereal is less demanding in agronomic terms than corn (water and nutrients) [26, 27]. Sorghum is the fifth most important grain in the world, being the United States the country with the highest production in the world, followed by India and Nigeria. For the year 2010, Mexico contributed with 10.5% of the total world production, equivalent to 6,250,000 metric tons [15, 16]. In Mexico, sorghum is the second most important grain in production after corn; during the period 1996–2006, sorghum production contributed

In Mexico, this cereal is destined mainly for livestock feed and secondarily for human food and obtaining inputs such as starch, alcohol, glucose, acetone, and butanol. One of the great advantages of sorghum is that it has the capacity to adapt to arid and semiarid climatic conditions and to be resistant to drought for long periods [26]. In previous works, it was able to demonstrate that antimicrobial active

The biopolymers obtained in this way through a technique and process patented by Tecnológico de Monterrey as PCT [28] have the advantage of being biodegradable because their chemical structure is primarily based on proteins and starches. Additionally, they have the possibility of forming films with plasticity (custom flexibility) and of being formulated also tailored to the requirements of the product to be packaged. Additionally, they can be heat sealed to form bags of different dimensions or not to be sealed and act as "active" pads or pads in combination with other packaging. In addition to the advantages in terms of sustainability, the interest in using these sources to produce biopolymers lies in adding value to agricultural

It is important to note that for any application of the said technology, it will be necessary to make an adaptation of the formulations and the process to satisfy the specific protection requirements for each food to be packaged. For what it is proposed to demonstrate in this work, the film-like packages obtained by adapting the formulations and process of the said published patent work to preserve and keep refrigerated for 30–45 days a commercial presentation in slices of semi-matured cheese [30]. The biopolymeric antimicrobial films described in WO2010/024657 A1 from cereals are limited to the packaging of dry foods or as pads for adsorption of exudates and emission of antimicrobial agents for fresh meat and cheese products [28]. Because of its sensitivity to water and low mechanical resistance to contain products with intermediate moisture, the biopolymeric matrix was reformulated to improve both parameters [30]. The results of refrigerated shelf stability of the cheese in terms of the control capacity exercised by the antimicrobials used in this study (nisin and natamycin) through the active packaging against fungi and yeast were effective throughout storage compared to vacuum packaging (control). The results of the microbial kinetics throughout the refrigerated storage for the fungi and yeast count showed the effectiveness of the active packaging. The development of fungi and yeasts remained controlled, showing the effectiveness of this emerging food preservation technology [30]. The plasticizing effects of two different polyols (glycerol and sorbitol) on the mechanical, thermal, and microstructural properties of flour films were studied by Valderrama and Rojas, and the results showed that films plasticized with sorbitol had better mechanical properties and less affinity for water than those plasticized with

mance characteristics, have been studied extensively [22–25].

films can be obtained from corn and sorghum flour [8, 15, 16].

with 22% of the total production of cereals [26].

**28**

products [8, 29].

In Valderrama's work, natural aqueous extracts are exchanged for essential oils because they have a higher concentration of antimicrobial active substances. It analyzed essential oils of oregano, thyme, tea tree, and mint, which have greater antimicrobial activity than the natural extracts used by Ríos-Licea [15]. In particular, the effect of incorporating two essential oils such as oregano (*Origanum vulgare*) and thyme (*Thymus vulgaris*) on polyolefin materials such as low-density polyethylene (LDPE) and polypropylene (PP) was studied.

The mechanical, barrier, and antimicrobial properties of the packaging were evaluated against *Salmonella typhimurium*, *Listeria monocytogenes*, and *Escherichia coli* O157:H7. The results demonstrate that films developed by extrusion incorporating 4% (w/w) of essential oils had a higher inhibitory effect than those obtained using the ionizing treatment. The packaging developed by extrusion containing 1% (w/w) showed a positive inhibitory effect, while those obtained by the ionizing treatment had no inhibitory effect against any of the test microorganisms. The incorporation of essential oils on the LDPE films generated a plasticizer effect, whereas the ones obtained by means of ionizing treatment did significantly affect the barrier properties of the films Valderrama and Rojas [9].

A simple and rapid Fourier-transform infrared (FTIR) spectroscopy method was developed by Valderrama and Rojas to determine the main essential oil components (carvacrol, thymol, and p-cymene) in the antimicrobial LDPE films incorporated with oregano (*Origanum vulgare*) and thyme (*Thymus vulgaris*) essential oils. The ATR-FTIR spectroscopy with chemometrics, using the PLS-first derivative spectra, could predict the active compounds content accurate to an r2 >0.99 and a standard error of prediction (SEP) of <0.7. The developed method was successfully applied to predict the concentration of active compounds: carvacrol, thymol, and p-cymene in oregano and thyme essential oils with results compared to those of the GC-MS

method. The described nondestructive method can be applied to make the traceability of active compounds of essential oils in antimicrobial food packaging [34].

The work of Rocha is described below, who worked with the same essential oils of oregano and thyme that Valderrama used to obtain active plastic containers. This was due to the fact that they presented greater antimicrobial activity than the aqueous extracts of oregano and garlic from previous studies in our research group [9, 15]. It also proposed the use of a polymeric film for the preparation of the active container with essential oils, in order to present an alternative to vacuum cheese packaging. For this project, polyvinyl alcohol (PVOH) has been chosen for the preparation of the packaging due to its unique characteristics: permeability, biodegradability, and its facility to form films by the casting method. The purpose of this work is to propose an alternative, a packaging that is not dependent on complex plastic structures that requires vacuum packaging for provide the high barrier. The main challenge of the present project is the incorporation of essential oils that are lipophilic to a hydrophilic PVOH matrix, which is why it was suggested encapsulating them in cyclodextrins.

#### **3. Control of the development of** *Listeria monocytogenes* **in fresh cheese during shelf life at refrigeration by means of an antimicrobial PVOH film (pad) with microcapsules of active compounds of oregano and thyme**

The presence of *Listeria* spp. has been found in cheeses from developed countries such as the United States, Sweden, France, Germany, Italy, Brazil, and Japan. Hence, there is an urgent need to find alternative conservation systems, which allow to contribute to the inhibition of this type of pathogen. Listeriosis infections represent only 0.02% of cases of diseases in the USA; however, this bacterium is responsible for 25% of deaths in outbreaks related to food [35, 36]. Especially worrisome is the fact that it can survive pasteurization and be able to develop even in refrigeration temperatures. Hence, the importance of finding new technologies for their control and/ or elimination, in particular, considering that the use of antimicrobial compounds in dairy products is restricted, that there is great interest in using natural compounds for this purpose but, above all, that these substances are added to the containers and not to the food, which can be achieved through the active packaging, is the objective of the present study. The development of *L. monocytogenes* has a particular health interest, because it is responsible for a fifth of the deaths related to foodborne diseases, especially considering that it survives the pasteurization process and develops even in refrigeration. It is important to highlight the tendency to decrease and, in some cases, eliminate preservatives in food. In dairy products, the direct use of antimicrobial agents is specifically forbidden, so the use of natural additives is becoming an alternative. If these are combined with the primary function of packaging to maintain sanitary safety and minimize the toxicological impacts of food, we are getting an active packaging. An active container that inhibits its development in fresh cheese during its storage in refrigeration can help to reduce the incidence of outbreaks and deaths due to this bacterium. The main goal was to develop an active packaging system that allows to control the development of pathogenic bacteria, in particular *Listeria monocytogenes* in refrigerated fresh cheeses, using natural antibacterial agents. As specific objectives: select and establish the conditions of incorporation of essential oils in a hydrophilic polymer, polyvinyl alcohol (PVOH), studying three methods of incorporation and formation of films; determine the antimicrobial activity of the films obtained against *Salmonella typhimurium*, *Listeria monocytogenes*, and *Escherichia coli* O157: H7 as target microorganisms; and, finally, check the effectiveness of the film and packaging system in the inhibition of the development of *L. monocytogenes* in fresh goat cheese, during 29 days of refrigerated storage.

**31**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

The selected essential oils were oregano (OEO) and thyme (TEO) (Primavera Life) for their potent antimicrobial activity and their availability in the national market. The antimicrobial activity of these inclusion complexes such as films using *Salmonella typhimurium*, *Listeria monocytogenes*, and *Escherichia coli* O157:H7 as target microorganisms was determined. Polyvinyl alcohol (PVOH) soluble in hot water, 87–90% hydrolyzed, with a molecular weight of 30,000–70,000 (Sigma-Aldrich), and glycerol (DEQ ) were used for the production of active film. To obtain the inclusion complexes of the essential oils, crystalline α-cyclodextrin and β-cyclodextrin (Sigma-Aldrich) were used. The plastic films for the cheese packaging were LDPE film made by extrusion by Valderrama [9] and multilayer film Zublon® 5CR (Zubex Industrial S.A. de C.V.). For the activation of the microorganisms, the following selective broths were used: UVM-modified Listeria Enrichment Broth for *L. monocytogenes* (Becton Dickinson, DIFCO, México), Brilliant green bile lactose broth (BRILA broth) for *E.coli* O157: H7 (Merck KGaA, Germany), and Brain Heart Infusion broth (BHI broth) for *S. typhimurium* (Merck KGaA, Germany). For the plate count and antimicrobial activity tests, the following were used: Oxford Agar for *L. monocytogenes* (Becton Dickinson, DIFCO, México), SS agar for *Salmonella* and *Shigella* (Merck KGaA, Germany), Modified EC Broth and Bacto Agar for *E. coli* (Becton Dickinson, DIFCO, México). Oxford Agar with Oxford selective supplement (Becton Dickinson, DIFCO, México) was used for the counting of *L. monocytogenes* in the fresh cheese packaged experiment. For the goat cheese, fresh goat cheese, CAPRICO brand Cabrero cheese, was obtained in 400 g presentations directly with the company CAPRICO (manufacturing lot JL09210PN) located in Linares, N.L.

*3.1.2 Process for developing PVOH films with active microcapsules of essential oils*

*3.1.3 Thermal stability of essential oils and confirmation of inclusion complex* 

if these would be affected during the film making process, the thermal stability of the same and their active compounds were evaluated. Firstly, a thermal evaluation of the essential oils of oregano and thyme was carried out, as well as its main active components with carvacrol, thymol, and p-cymene standards. Next, the β-CD and the inclusion complexes of OEO and TEO to confirm the formation of such complexes and not only a physical mixture. Finally, a thermal evaluation of the PVOH films with the inclusion complexes of CD:EO of oregano and thyme was made to determine the optimal storage temperature of the active films. The thermal evaluation was performed with a home DTA validated by Martínez and collaborators [37]. For each substance, at least two runs of food matrix were performed to verify the repeatability of the analysis.

*formation by differential thermal analysis (DTA)*

The PVOH films were elaborated adapting the method used by Schause [8] and Ríos-Licea [15], previously studying three methods of incorporation of the essential oils in it: dispersion, emulsification, and formation of inclusion complexes with α and β cyclodextrins (CD) and the following variables: amount of PVOH, type and amount of cyclodextrin, coprecipitation strategy, solvent (water, ethanol), and concentration of EO. The encapsulation with β-CD is being the one selected for the incorporation of EO in the PVOH film. A PVOH film without inclusion complex was made as a control. With the resulting films, pads of 8 × 4 cm were made to be

In order to determine the degradation temperature of the essential oils and establish

*3.1.1 Selection of substances, materials, microorganisms, and cheese*

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

used in the active packaging system.

**3.1 Methods**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

#### **3.1 Methods**

*Active Antimicrobial Food Packaging*

**thyme**

method. The described nondestructive method can be applied to make the traceability of active compounds of essential oils in antimicrobial food packaging [34]. The work of Rocha is described below, who worked with the same essential oils of oregano and thyme that Valderrama used to obtain active plastic containers. This was due to the fact that they presented greater antimicrobial activity than the aqueous extracts of oregano and garlic from previous studies in our research group [9, 15]. It also proposed the use of a polymeric film for the preparation of the active container with essential oils, in order to present an alternative to vacuum cheese packaging. For this project, polyvinyl alcohol (PVOH) has been chosen for the preparation of the packaging due to its unique characteristics: permeability, biodegradability, and its facility to form films by the casting method. The purpose of this work is to propose an alternative, a packaging that is not dependent on complex plastic structures that requires vacuum packaging for provide the high barrier. The main challenge of the present project is the incorporation of essential oils that are lipophilic to a hydrophilic PVOH matrix, which is why it was suggested encapsulating them in cyclodextrins.

**3. Control of the development of** *Listeria monocytogenes* **in fresh cheese during shelf life at refrigeration by means of an antimicrobial PVOH film (pad) with microcapsules of active compounds of oregano and** 

The presence of *Listeria* spp. has been found in cheeses from developed countries such as the United States, Sweden, France, Germany, Italy, Brazil, and Japan. Hence, there is an urgent need to find alternative conservation systems, which allow to contribute to the inhibition of this type of pathogen. Listeriosis infections represent only 0.02% of cases of diseases in the USA; however, this bacterium is responsible for 25% of deaths in outbreaks related to food [35, 36]. Especially worrisome is the fact that it can survive pasteurization and be able to develop even in refrigeration temperatures. Hence, the importance of finding new technologies for their control and/ or elimination, in particular, considering that the use of antimicrobial compounds in dairy products is restricted, that there is great interest in using natural compounds for this purpose but, above all, that these substances are added to the containers and not to the food, which can be achieved through the active packaging, is the objective of the present study. The development of *L. monocytogenes* has a particular health interest, because it is responsible for a fifth of the deaths related to foodborne diseases, especially considering that it survives the pasteurization process and develops even in refrigeration. It is important to highlight the tendency to decrease and, in some cases, eliminate preservatives in food. In dairy products, the direct use of antimicrobial agents is specifically forbidden, so the use of natural additives is becoming an alternative. If these are combined with the primary function of packaging to maintain sanitary safety and minimize the toxicological impacts of food, we are getting an active packaging. An active container that inhibits its development in fresh cheese during its storage in refrigeration can help to reduce the incidence of outbreaks and deaths due to this bacterium. The main goal was to develop an active packaging system that allows to control the development of pathogenic bacteria, in particular *Listeria monocytogenes* in refrigerated fresh cheeses, using natural antibacterial agents. As specific objectives: select and establish the conditions of incorporation of essential oils in a hydrophilic polymer, polyvinyl alcohol (PVOH), studying three methods of incorporation and formation of films; determine the antimicrobial activity of the films obtained against *Salmonella typhimurium*, *Listeria monocytogenes*, and *Escherichia coli* O157: H7 as target microorganisms; and, finally, check the effectiveness of the film and packaging system in the inhibition of the development of *L. monocytogenes* in

**30**

fresh goat cheese, during 29 days of refrigerated storage.

#### *3.1.1 Selection of substances, materials, microorganisms, and cheese*

The selected essential oils were oregano (OEO) and thyme (TEO) (Primavera Life) for their potent antimicrobial activity and their availability in the national market. The antimicrobial activity of these inclusion complexes such as films using *Salmonella typhimurium*, *Listeria monocytogenes*, and *Escherichia coli* O157:H7 as target microorganisms was determined. Polyvinyl alcohol (PVOH) soluble in hot water, 87–90% hydrolyzed, with a molecular weight of 30,000–70,000 (Sigma-Aldrich), and glycerol (DEQ ) were used for the production of active film. To obtain the inclusion complexes of the essential oils, crystalline α-cyclodextrin and β-cyclodextrin (Sigma-Aldrich) were used. The plastic films for the cheese packaging were LDPE film made by extrusion by Valderrama [9] and multilayer film Zublon® 5CR (Zubex Industrial S.A. de C.V.). For the activation of the microorganisms, the following selective broths were used: UVM-modified Listeria Enrichment Broth for *L. monocytogenes* (Becton Dickinson, DIFCO, México), Brilliant green bile lactose broth (BRILA broth) for *E.coli* O157: H7 (Merck KGaA, Germany), and Brain Heart Infusion broth (BHI broth) for *S. typhimurium* (Merck KGaA, Germany). For the plate count and antimicrobial activity tests, the following were used: Oxford Agar for *L. monocytogenes* (Becton Dickinson, DIFCO, México), SS agar for *Salmonella* and *Shigella* (Merck KGaA, Germany), Modified EC Broth and Bacto Agar for *E. coli* (Becton Dickinson, DIFCO, México). Oxford Agar with Oxford selective supplement (Becton Dickinson, DIFCO, México) was used for the counting of *L. monocytogenes* in the fresh cheese packaged experiment. For the goat cheese, fresh goat cheese, CAPRICO brand Cabrero cheese, was obtained in 400 g presentations directly with the company CAPRICO (manufacturing lot JL09210PN) located in Linares, N.L.

#### *3.1.2 Process for developing PVOH films with active microcapsules of essential oils*

The PVOH films were elaborated adapting the method used by Schause [8] and Ríos-Licea [15], previously studying three methods of incorporation of the essential oils in it: dispersion, emulsification, and formation of inclusion complexes with α and β cyclodextrins (CD) and the following variables: amount of PVOH, type and amount of cyclodextrin, coprecipitation strategy, solvent (water, ethanol), and concentration of EO. The encapsulation with β-CD is being the one selected for the incorporation of EO in the PVOH film. A PVOH film without inclusion complex was made as a control. With the resulting films, pads of 8 × 4 cm were made to be used in the active packaging system.

#### *3.1.3 Thermal stability of essential oils and confirmation of inclusion complex formation by differential thermal analysis (DTA)*

In order to determine the degradation temperature of the essential oils and establish if these would be affected during the film making process, the thermal stability of the same and their active compounds were evaluated. Firstly, a thermal evaluation of the essential oils of oregano and thyme was carried out, as well as its main active components with carvacrol, thymol, and p-cymene standards. Next, the β-CD and the inclusion complexes of OEO and TEO to confirm the formation of such complexes and not only a physical mixture. Finally, a thermal evaluation of the PVOH films with the inclusion complexes of CD:EO of oregano and thyme was made to determine the optimal storage temperature of the active films. The thermal evaluation was performed with a home DTA validated by Martínez and collaborators [37]. For each substance, at least two runs of food matrix were performed to verify the repeatability of the analysis.

#### *3.1.4 Microbiological evaluation and disk diffusion method*

To determine the antimicrobial activity of PVOH films, the disk diffusion method was applied (Kirby-Bauer method). After preparing and inoculating the agar with 106 CFU of each microorganism, samples of the films were cut in the form of 6 mm diameter disks and deposited on the agar, evaluating both the rough and smooth side of the films [7, 38, 39]. After 24 hours of incubation at 37 ± 1°C in inverted position, the inhibition halo was measured with a digital micrometer (Mitutoyo Digimatic 2,931,051 m, 0.001 mm sensitivity).

#### *3.1.5 Control study of L. monocytogenes in fresh goat cheese using an active packaging system*

The packaging system consisted of a pad of PVOH with EO microcapsules of oregano and thyme in a LDPE bag. First, 7 × 7 cm bags with LDPE film of 0.023 ± 0.003 mm thickness obtained by extrusion by Valderrama [9] were made, which were obtained by sealing two films on three sides with a vacuum packing machine Torrey brand. In the same way, bags were obtained with the multilayer film (Zublon® 5CR from Zubex Industrial). Second, in aseptic conditions, portions of cheese of 3 cm × 3 cm and 10 ± 0.5 g of weight were cut and exposed to UV treatment for 15 min on each side, a methodology adapted from Suppakul [40] for the purpose to reduce the interference of microorganisms typical of cheese in the study. Then, the samples were packed in the bags of the four treatments to be analyzed and inoculated with 100 mL of *Listeria monocytogenes* at a concentration of 5 × 103 CFU/mL. Finally, the bags were heat sealed in a packaging machine (TORREY) and stored in a refrigerator (Torrey Model VRD42) at 4 ± 1°C for up to 29 days.

The four treatments evaluated were (1) multilayer bag for vacuum packaging as control, (2) LDPE bag with PVOH "pad" without essential oils, (3) LDPE bag with PVOH "pad" with inclusion complex of β-CD:OEO, essential oil at a concentration of 25% in the film, and (4) LDPE bag with PVOH "pad" with inclusion complex of β-CD:TEO, essential oil at a concentration of 25% in the film.

The inhibition kinetics of *L. monocytogenes* was determined in the cheese samples packaged at 0, 1, 3, 5, 7, 15, 22, 26, and 29 days at refrigerated conditions. The analysis of the microbial count proceeded according to NOM110-SSA1-1994, making decimal dilutions and plate count [41]. The LabVIEW software for differential thermal analysis was used.

#### **3.2 Results**

#### *3.2.1 PVOH films process with microcapsules of essential oils of oregano and thyme*

It was possible to produce PVOH films with the inclusion complexes of oregano and thyme in all the experimental conditions. Films with 1, 4, and 15% EO were prepared with molar ratio 1:10, which they are shown in **Figure 1**. Films made with 1% EO were those most similar to the PVOH control film. Continuous, elastic, and transparent films were obtained. The higher the concentration of the essential oil in the film, the more presence of the inclusion complex affects the transparency of the film, with the PVOH matrix being observed as white, as can be seen in the film at 15% EO (**Figure 1**). It should be noted, however, that although the inclusion complex is observed in the film, no migration of this or the essential oil to the touch is perceived, which is why it has been well incorporated into the PVOH matrix. The films also presented less transparency when approaching the β-CD:EO ratio at 1:1

**33**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

molar proportions; this is because a greater amount of inclusion complex tends to saturate the film. The films whose inclusion complex was dissolved in 30% ethanol also showed greater transparency than those in which it was prepared in water; this is because the inclusion complex in the 30% ethanol solution was better solubilized.

Firstly, the thermal stability of β-cyclodextrin and the essential oils of oregano and thyme, as well as its components (carvacrol, thymol, and p-cymene), was evaluated to confirm the formation of inclusion complexes. The thermograms of the carvacrol, thymol, and p-cymene standards are shown in **Figure 2** and **3**. Here it is shown that these three components are stable up to a temperature of 182°C, which refers to the boiling temperature of the p-cymene. The melting point of thymol shown in **Figure 2** is in agreement with that obtained by Ponce, which reports the melting point of thymol at 50°C [42]. The boiling point of p-cymene matches with the one reported by the supplier (178–180°C Sigma-Aldrich). Carvacrol was analyzed by broadening the study temperatures, as shown in **Figure 3**. This compound has an interesting behavior, since it has a crystallization temperature of −20°C followed by a melting point of 2°C and a point of boiling of 240° C. Sigma-Aldrich reports its melting point at 3–4°C and its boiling point at 236–237°C, which also coincides with that reported by Dahmane, which reports the boiling point of carvacrol at 237.7°C [43]. The closeness of the crystallization and fusion transitions does not allow the existence of a solid state of this intermediate substance at the reported temperatures. A similar behavior is reported by Ponce for cinnamaldehyde [42]. According to the obtained in **Figure 5**, it was successful to form an inclusion complex, since otherwise the signal of the boiling point would have been shown at 214°C

The thermogram obtained by DTA of the β-CD:EO inclusion complexes of oregano and thyme (**Figure 5**) shows that they are stable at temperatures below 115°C, so there is no risk of degradation of the active compounds if the inclusion complex dissolves PVOH in situ in a solution with inclusion complex at 8°C (process B of PVOH film making with inclusion complexes). **Figure 6** shows the thermogram of the PVOH films with the inclusion complexes of oregano and thyme. The film made with essential oil of thyme had a little moisture on its surface, so we can see a couple of peaks at 0 and 100°C corresponding to the melting and boiling point of water, respectively. PVOH control films and those with inclusion complexes of essential oil of oregano and thyme are stable up to a temperature of 110°C, which indicates that they can be stored in shelves at room temperature without problem.

*3.2.2 Thermal stability of essential oils and confirmation of inclusion complex* 

*Appearance of PVOH films with different concentrations of inclusion complex. Films elaborated at* 

*concentrations of 1, 4, and 15% of EO by process A (molar ratio β-CD: AO 1:10).*

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

*formation by DTA*

**Figure 1.**

of the essential oils (**Figure 4**).

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

**Figure 1.**

*Active Antimicrobial Food Packaging*

*packaging system*

ential thermal analysis was used.

**3.2 Results**

agar with 106

*3.1.4 Microbiological evaluation and disk diffusion method*

(Mitutoyo Digimatic 2,931,051 m, 0.001 mm sensitivity).

*3.1.5 Control study of L. monocytogenes in fresh goat cheese using an active* 

stored in a refrigerator (Torrey Model VRD42) at 4 ± 1°C for up to 29 days.

The inhibition kinetics of *L. monocytogenes* was determined in the cheese samples packaged at 0, 1, 3, 5, 7, 15, 22, 26, and 29 days at refrigerated conditions. The analysis of the microbial count proceeded according to NOM110-SSA1-1994, making decimal dilutions and plate count [41]. The LabVIEW software for differ-

*3.2.1 PVOH films process with microcapsules of essential oils of oregano and thyme*

It was possible to produce PVOH films with the inclusion complexes of oregano and thyme in all the experimental conditions. Films with 1, 4, and 15% EO were prepared with molar ratio 1:10, which they are shown in **Figure 1**. Films made with 1% EO were those most similar to the PVOH control film. Continuous, elastic, and transparent films were obtained. The higher the concentration of the essential oil in the film, the more presence of the inclusion complex affects the transparency of the film, with the PVOH matrix being observed as white, as can be seen in the film at 15% EO (**Figure 1**). It should be noted, however, that although the inclusion complex is observed in the film, no migration of this or the essential oil to the touch is perceived, which is why it has been well incorporated into the PVOH matrix. The films also presented less transparency when approaching the β-CD:EO ratio at 1:1

β-CD:TEO, essential oil at a concentration of 25% in the film.

The four treatments evaluated were (1) multilayer bag for vacuum packaging as control, (2) LDPE bag with PVOH "pad" without essential oils, (3) LDPE bag with PVOH "pad" with inclusion complex of β-CD:OEO, essential oil at a concentration of 25% in the film, and (4) LDPE bag with PVOH "pad" with inclusion complex of

To determine the antimicrobial activity of PVOH films, the disk diffusion method was applied (Kirby-Bauer method). After preparing and inoculating the

form of 6 mm diameter disks and deposited on the agar, evaluating both the rough and smooth side of the films [7, 38, 39]. After 24 hours of incubation at 37 ± 1°C in inverted position, the inhibition halo was measured with a digital micrometer

The packaging system consisted of a pad of PVOH with EO microcapsules of oregano and thyme in a LDPE bag. First, 7 × 7 cm bags with LDPE film of 0.023 ± 0.003 mm thickness obtained by extrusion by Valderrama [9] were made, which were obtained by sealing two films on three sides with a vacuum packing machine Torrey brand. In the same way, bags were obtained with the multilayer film (Zublon® 5CR from Zubex Industrial). Second, in aseptic conditions, portions of cheese of 3 cm × 3 cm and 10 ± 0.5 g of weight were cut and exposed to UV treatment for 15 min on each side, a methodology adapted from Suppakul [40] for the purpose to reduce the interference of microorganisms typical of cheese in the study. Then, the samples were packed in the bags of the four treatments to be analyzed and inoculated with 100 mL of *Listeria monocytogenes* at a concentration of 5 × 103 CFU/mL. Finally, the bags were heat sealed in a packaging machine (TORREY) and

CFU of each microorganism, samples of the films were cut in the

**32**

*Appearance of PVOH films with different concentrations of inclusion complex. Films elaborated at concentrations of 1, 4, and 15% of EO by process A (molar ratio β-CD: AO 1:10).*

molar proportions; this is because a greater amount of inclusion complex tends to saturate the film. The films whose inclusion complex was dissolved in 30% ethanol also showed greater transparency than those in which it was prepared in water; this is because the inclusion complex in the 30% ethanol solution was better solubilized.

#### *3.2.2 Thermal stability of essential oils and confirmation of inclusion complex formation by DTA*

Firstly, the thermal stability of β-cyclodextrin and the essential oils of oregano and thyme, as well as its components (carvacrol, thymol, and p-cymene), was evaluated to confirm the formation of inclusion complexes. The thermograms of the carvacrol, thymol, and p-cymene standards are shown in **Figure 2** and **3**. Here it is shown that these three components are stable up to a temperature of 182°C, which refers to the boiling temperature of the p-cymene. The melting point of thymol shown in **Figure 2** is in agreement with that obtained by Ponce, which reports the melting point of thymol at 50°C [42]. The boiling point of p-cymene matches with the one reported by the supplier (178–180°C Sigma-Aldrich). Carvacrol was analyzed by broadening the study temperatures, as shown in **Figure 3**. This compound has an interesting behavior, since it has a crystallization temperature of −20°C followed by a melting point of 2°C and a point of boiling of 240° C. Sigma-Aldrich reports its melting point at 3–4°C and its boiling point at 236–237°C, which also coincides with that reported by Dahmane, which reports the boiling point of carvacrol at 237.7°C [43]. The closeness of the crystallization and fusion transitions does not allow the existence of a solid state of this intermediate substance at the reported temperatures. A similar behavior is reported by Ponce for cinnamaldehyde [42]. According to the obtained in **Figure 5**, it was successful to form an inclusion complex, since otherwise the signal of the boiling point would have been shown at 214°C of the essential oils (**Figure 4**).

The thermogram obtained by DTA of the β-CD:EO inclusion complexes of oregano and thyme (**Figure 5**) shows that they are stable at temperatures below 115°C, so there is no risk of degradation of the active compounds if the inclusion complex dissolves PVOH in situ in a solution with inclusion complex at 8°C (process B of PVOH film making with inclusion complexes). **Figure 6** shows the thermogram of the PVOH films with the inclusion complexes of oregano and thyme. The film made with essential oil of thyme had a little moisture on its surface, so we can see a couple of peaks at 0 and 100°C corresponding to the melting and boiling point of water, respectively. PVOH control films and those with inclusion complexes of essential oil of oregano and thyme are stable up to a temperature of 110°C, which indicates that they can be stored in shelves at room temperature without problem.

The PVOH control film has a peak at 150°C, which refers to the point of fusion of unplasticized PVOH. The last peak corresponds to the degradation of PVOH at 230°C, which is supported by Holland and Hay [44].

**Figure 2.** *Thermogram of thymol and p-cymene.*

**35**

the microcapsule [45].

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

*3.2.3 Antimicrobial activity of the active films in vitro against L. monocytogenes,* 

The films made with a concentration of 25% essential oil of oregano and thyme presented broad-spectrum antimicrobial activity by inhibiting the growth against the Gram-positive and Gram-negative microorganisms evaluated. The antimicrobial activity and the inhibition halo against *E. coli* O157: H7, *L. monocytogenes*, and *S. typhimurium* is shown in **Figure 7**. In this test the antimicrobial activity of both films was very similar against *E. coli* O157: H7 and *S. typhimurium*. The opposite occurred against *L. monocytogenes*, where the films with 25% of TEO showed higher antimicrobial activity than the films with 25% of OEO. The halos of inhibition against *E. coli* O157: H7 and *S. typhimurium* shown in **Figure 7(A)** and **(C)** suggest that the rough side of the film shows a slightly higher antimicrobial activity against microorganisms, a capacity that was confirmed in the analysis against *L. monocytogenes* (**Figure 7(B)**). The treatment of the film with the highest antimicrobial activity was that elaborated at 25% of essential oil, with the inclusion complex in molar ratio β-CD:EO 1:10 and evaluated by its rough side. In experimental designs not reported in this work, we could verify that both, the concentration of the essential oil (1, 4, 8, 15%) and the molar ratio of the inclusion complex β-CD:EO (1:2 and 1:5), are factors that influence the antimicrobial activity, as well as the speed of diffusion of the antimicrobial through the walls of

The antimicrobial activity of the films is mainly due to the phenol group of carvacrol and p-cymene. The concentration of these compounds in the essential oils of oregano and thyme used for the production of films is shown in **Table 1**. The

*S. typhimurium, and E. coli O157: H7*

*Thermogram of PVOH films with inclusion complexes of OEO and TEO.*

*Thermogram of the inclusion complexes of OEO and TEO with β–CD.*

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

**Figure 5.**

**Figure 6.**

**Figure 4.** *Thermogram of thyme and oregano essential oils.*

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

**Figure 5.** *Thermogram of the inclusion complexes of OEO and TEO with β–CD.*

**Figure 6.** *Thermogram of PVOH films with inclusion complexes of OEO and TEO.*

#### *3.2.3 Antimicrobial activity of the active films in vitro against L. monocytogenes, S. typhimurium, and E. coli O157: H7*

The films made with a concentration of 25% essential oil of oregano and thyme presented broad-spectrum antimicrobial activity by inhibiting the growth against the Gram-positive and Gram-negative microorganisms evaluated. The antimicrobial activity and the inhibition halo against *E. coli* O157: H7, *L. monocytogenes*, and *S. typhimurium* is shown in **Figure 7**. In this test the antimicrobial activity of both films was very similar against *E. coli* O157: H7 and *S. typhimurium*. The opposite occurred against *L. monocytogenes*, where the films with 25% of TEO showed higher antimicrobial activity than the films with 25% of OEO. The halos of inhibition against *E. coli* O157: H7 and *S. typhimurium* shown in **Figure 7(A)** and **(C)** suggest that the rough side of the film shows a slightly higher antimicrobial activity against microorganisms, a capacity that was confirmed in the analysis against *L. monocytogenes* (**Figure 7(B)**). The treatment of the film with the highest antimicrobial activity was that elaborated at 25% of essential oil, with the inclusion complex in molar ratio β-CD:EO 1:10 and evaluated by its rough side. In experimental designs not reported in this work, we could verify that both, the concentration of the essential oil (1, 4, 8, 15%) and the molar ratio of the inclusion complex β-CD:EO (1:2 and 1:5), are factors that influence the antimicrobial activity, as well as the speed of diffusion of the antimicrobial through the walls of the microcapsule [45].

The antimicrobial activity of the films is mainly due to the phenol group of carvacrol and p-cymene. The concentration of these compounds in the essential oils of oregano and thyme used for the production of films is shown in **Table 1**. The

*Active Antimicrobial Food Packaging*

230°C, which is supported by Holland and Hay [44].

The PVOH control film has a peak at 150°C, which refers to the point of fusion of unplasticized PVOH. The last peak corresponds to the degradation of PVOH at

**34**

**Figure 4.**

**Figure 3.**

*Carvacrol thermogram.*

**Figure 2.**

*Thermogram of thymol and p-cymene.*

*Thermogram of thyme and oregano essential oils.*

**Figure 7.**

*Antimicrobial activity of PVOH fims with inclusion complexes against pathogenic bacteria. 7a against E. coli, 7b against L. monocytogenes, and 7c against S. typhimurium.*

phenol group is essential for bacterial inhibition, since it destabilizes the cytoplasmic membrane and also functions as a proton exchanger which reduces the pH gradient in the membrane and causes cell collapse and death [46–48]. The destabilization of the membrane occurs because carvacrol and thymol have affinity for lipids and accumulates in the bilayer between fatty acid chains, which causes changes in the conformation of the membrane. This mechanism of action does not present p-cymene; however, it has been found to have a synergy with phenols, expanding the membrane and destabilizing it [46]. The position of the hydroxyl group in the phenolic compounds does not seem to influence the degree of antimicrobial activity so that the activity of carvacrol and thymol is similar.

**37**

**Figure 8.**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

*3.2.4 Analysis of the control of L. monocytogenes in fresh cheese packed during* 

The antimicrobial activity of the PVOH films with inclusion complexes of oregano and thyme in a model with fresh cheese inoculated with *L. monocytogenes* and stored in a rack refrigerated at 4°C was evaluated. As seen in **Figure 8**, the pathogenic microorganism shows inhibition when it is packed with the films developed with the antimicrobial agents, since the fresh cheese develops fewer colonies than that packaged with PVOH control or with the multilayer film under vacuum. The concentration of *L. monocytogenes* gradually decreased in the cheeses packaged with the active pads as shown in **Figure 8**. After 15 days of storage, the cheese packed with the films with oregano and thyme no longer had a microbial count; this performance was better than in the cheeses packaged with the vacuum multilayer film (red line) that did present a microbial account. This fact would have been interpreted as that the films developed with inclusion complexes of oregano and thyme presented bactericidal activity against *L. monocytogenes* in cheese, in refrigerated shelf after 15 days of exposure. Unfortunately, the films do not have bactericidal activity since colonies were detected on days 22 and 26 of storage. For this reason, it is concluded that the active films have a bacteriostatic control against *L. monocytogenes*. It is also observed in **Figure 8**, that the vacuum multilayer film showed greater control of the microbial load of the inoculated cheese, than the package with PVOH film without essential oils. This is due to the fact that in vacuum packaging with multilayer film, the oxygen available in the head space is reduced, together with the gas impermeability of the film, preventing microorganisms from developing. *L. monocytogenes* in particular is

*Antimicrobial activity of PVOH films with inclusion complexes against fresh cheese inoculated with L. monocytogenes in refrigerated storage. PVOH films with 25% of OEO and TEO incorporated as inclusion* 

*complex. PVOH and multilayer vacuum packages were used as controls.*

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

*storage in a refrigerated rack*

*Concentration of phenolic compounds in the OEO and TEO.*

**Table 1.**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*


**Table 1.**

*Active Antimicrobial Food Packaging*

**36**

**Figure 7.**

phenol group is essential for bacterial inhibition, since it destabilizes the cytoplasmic membrane and also functions as a proton exchanger which reduces the pH gradient in the membrane and causes cell collapse and death [46–48]. The destabilization of the membrane occurs because carvacrol and thymol have affinity for lipids and accumulates in the bilayer between fatty acid chains, which causes changes in the conformation of the membrane. This mechanism of action does not present p-cymene; however, it has been found to have a synergy with phenols, expanding the membrane and destabilizing it [46]. The position of the hydroxyl group in the phenolic compounds does not seem to influence the degree of antimicrobial activity

*Antimicrobial activity of PVOH fims with inclusion complexes against pathogenic bacteria. 7a against E. coli,* 

so that the activity of carvacrol and thymol is similar.

*7b against L. monocytogenes, and 7c against S. typhimurium.*

*Concentration of phenolic compounds in the OEO and TEO.*

#### *3.2.4 Analysis of the control of L. monocytogenes in fresh cheese packed during storage in a refrigerated rack*

The antimicrobial activity of the PVOH films with inclusion complexes of oregano and thyme in a model with fresh cheese inoculated with *L. monocytogenes* and stored in a rack refrigerated at 4°C was evaluated. As seen in **Figure 8**, the pathogenic microorganism shows inhibition when it is packed with the films developed with the antimicrobial agents, since the fresh cheese develops fewer colonies than that packaged with PVOH control or with the multilayer film under vacuum. The concentration of *L. monocytogenes* gradually decreased in the cheeses packaged with the active pads as shown in **Figure 8**. After 15 days of storage, the cheese packed with the films with oregano and thyme no longer had a microbial count; this performance was better than in the cheeses packaged with the vacuum multilayer film (red line) that did present a microbial account. This fact would have been interpreted as that the films developed with inclusion complexes of oregano and thyme presented bactericidal activity against *L. monocytogenes* in cheese, in refrigerated shelf after 15 days of exposure. Unfortunately, the films do not have bactericidal activity since colonies were detected on days 22 and 26 of storage. For this reason, it is concluded that the active films have a bacteriostatic control against *L. monocytogenes*. It is also observed in **Figure 8**, that the vacuum multilayer film showed greater control of the microbial load of the inoculated cheese, than the package with PVOH film without essential oils. This is due to the fact that in vacuum packaging with multilayer film, the oxygen available in the head space is reduced, together with the gas impermeability of the film, preventing microorganisms from developing. *L. monocytogenes* in particular is

#### **Figure 8.**

*Antimicrobial activity of PVOH films with inclusion complexes against fresh cheese inoculated with L. monocytogenes in refrigerated storage. PVOH films with 25% of OEO and TEO incorporated as inclusion complex. PVOH and multilayer vacuum packages were used as controls.*

an aerobic bacterium, which is why the vacuum-packed product has control over its development during the time of storage.

#### **3.3 Conclusions**

Active PVOH films were obtained with essential oils of oregano and thyme, which showed broad-spectrum antibacterial activity by inhibiting pathogenic Gram-positive and Gram-negative bacteria specifically against *L. monocytogenes*, *E. coli* O157: H7, and *S. typhimurium*. The best conditions for the production of active films were 25% essential oil and elaboration of inclusion complex with a relation of 1:10 β-CD:EO. The active pad elaborated in the aforementioned conditions presented bacteriostatic activity against *L. monocytogenes* in cheese inoculated, packaged, and stored at 4°C for 29 days. The proposed packaging system ("pad" of the developed active film and a low-density polyethylene bag) can be an alternative to vacuum packaging using a multilayer film for cheeses. The experimental results showed that they provide a shelf life equivalent to vacuum packaging. In addition to the control of microbial activity, the proposed system is more accessible to small cheese producers as no special packaging technology is required other than a heat sealer machine. Likewise, the proposed packaging system can help reduce the incidence of outbreaks of diseases transmitted by foods contaminated with *Listeria monocytogenes*.

#### **4. Antimicrobial agents from plants of the northeast of Mexico**

As Mexico is a country that stands out for its floristic richness and taking into account the extensive knowledge of medicinal plants that since the pre-Columbian Era conserve Mexicans, mainly those of rural communities, it was natural that we were interested in the study of incorporation of some of them as active substances to be included in polymer matrices for food packaging.

#### **4.1** *Larrea tridentata* **plant as an alternative source for obtaining antimicrobial extracts**

The flora of arid zones represents a great potential for the wealth based on its biological specialization, since it is the product of thousands of years of physiological adaptation for its survival [50]. The governor plant (*L. tridentata*) typically develops under conditions of these zones. The *Larrea* genus includes five species of evergreen shrubs distributed throughout the Americas; its name was given in honor of the Spanish cleric Juan Antonio Hernández Larrea who was dean of the Zaragoza Chapter and bishop of Valladolid. This plant is commonly known as the governor, due to its dominance in the large areas of the arid zones of northern Mexico, but it is also known as guamis, sonora, tasajo, jarilla, creosote, and hediondilla due to its characteristic smell, mainly after the rain. In the Seri language, it is called "haaxat," and in the English language it has the common names of "creosote bush" and "greasewood" [49, 50].

The governor plant has a wide range of adaptation in elevation since it is located in the Valley of Death in California located 86 m below sea level, to more than 2500 m in the sierras of northern Mexico. Its growth is good in dry plains and plateaus, also around hills and slopes, and in several types of soils except clayey, saline, or granitic [50]. The lifetime of this plant is negatively correlated with disturbance and soil compaction, being intolerant to soils with high phosphorus content [49]. In Mexico, the distribution of the governor plant is in part of the Sonoran Desert,

**39**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

which includes the states of Baja California, Baja California Sur and Sonora, and in the Chihuahuan Desert, which includes the states of Chihuahua, Durango, Coahuila, Nuevo León, Zacatecas, and San Luis Potosí [50]. The infusion of the whole *L. tridentata* plant especially of the branches is used in urinary tract disorders to undo the kidney stones. The firing of branches, root, and bark is used to treat discomforts such as kidney pain and bladder inflammation. The decoction of the leaves is suggested in vaginal washes in gynecological problems such as female sterility. The infusion of branches, root, and bark is used in baths to treat hemorrhoids, fever, malaria, pimples, bumps, good healing, and rheumatism. And the infusion of the leaves is used as a remedy for gallstones, rheumatism, dermatitis, hepatitis, antiseptic, gastric discomforts, venereal diseases, and tuberculosis, in addition to

Among the uses that the governor plant has traditionally had, it stands out in its use as an antioxidant that was given to it in the United States since 1943; although in the decade of the 1990s, it was suspended by the US Food and Drug Administration (FDA) due to the strong interaction of nordihydroguaiaretic acid (NDGA) comment on results that were not found in the extracts studied, with several enzymatic processes. NDGA inhibits enzymatic activity, in addition to inhibiting the signaling pathway of lipoxygenase in which arachidonic acid generates leukotrienes and other

**4.2** *Cordia boissieri* **plant as an alternative source for obtaining antimicrobial** 

The anacahuita plant (*Cordia boissieri*) is the official flower of the state of Nuevo León, México. The genus *Cordia* gets its name in honor of the sixteenthcentury German botanist Valerius Cordus and the *boissieri* species gets its name in honor of the nineteenth-century French botanist Boissier [56]. This plant is commonly known by the names of anacahuita, Mexican olive, Texas olive, wild olive, trompillo, and rasca viejo [56]. *C. boissieri* is a shrub or small tree up to 5 m high, with ovate leaves, 15–20 cm long and velvety surface. The flowers are white, grouped from 5 to 8, with the yellow center, up to 45 mm in length. The fruit is ovoid from 25 to 30 mm, brownish-green to purple, fleshy, sweet, and contains 1–4 seeds [56]. This plant species is native to North America. It is mainly distributed in Mexico, in the states of Nuevo León, Coahuila, Tamaulipas, San Luis Potosí, and Veracruz, and in the State of Texas in the United States. There are reports that the fruits of the *C. boissieri* plant are used as a remedy for coughs and colds. Traditionally the leaves of the plant are used to treat rheumatism and bronchial problems. Also in traditional medicine, the flowers are used in the treatment of

**4.3** *Leucophyllum frutescens* **plant as an alternative source for obtaining** 

The ash plant (*Leucophyllum frutescens*) is an evergreen shrub. This plant is commonly known, in Mexico, with the name of ash and in the United States with the names of Texas ranger, Texas sage, silverleaf, and barometer bush, because the flowering is triggered by moisture [58]. It is a grey bush of 1.5–2 m in height. The silver-grey and green leaves are covered with silver hair. The violet to purple flowers are bell-shaped or funnel with five lobes and two lips and reach to measure 2.5 cm in length, which appear intermittently from spring to autumn. The fruit has the shape of a small capsule [58]. This plant is native to northern Mexico and the Southwestern United States. It is a species that is part of the medium and high bushes that develop

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

having antiamoebic activity [49, 51, 52].

oxygenated products [53–55].

diseases of bacterial origin [51, 56, 57].

**antimicrobial extracts**

**extracts**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

which includes the states of Baja California, Baja California Sur and Sonora, and in the Chihuahuan Desert, which includes the states of Chihuahua, Durango, Coahuila, Nuevo León, Zacatecas, and San Luis Potosí [50]. The infusion of the whole *L. tridentata* plant especially of the branches is used in urinary tract disorders to undo the kidney stones. The firing of branches, root, and bark is used to treat discomforts such as kidney pain and bladder inflammation. The decoction of the leaves is suggested in vaginal washes in gynecological problems such as female sterility. The infusion of branches, root, and bark is used in baths to treat hemorrhoids, fever, malaria, pimples, bumps, good healing, and rheumatism. And the infusion of the leaves is used as a remedy for gallstones, rheumatism, dermatitis, hepatitis, antiseptic, gastric discomforts, venereal diseases, and tuberculosis, in addition to having antiamoebic activity [49, 51, 52].

Among the uses that the governor plant has traditionally had, it stands out in its use as an antioxidant that was given to it in the United States since 1943; although in the decade of the 1990s, it was suspended by the US Food and Drug Administration (FDA) due to the strong interaction of nordihydroguaiaretic acid (NDGA) comment on results that were not found in the extracts studied, with several enzymatic processes. NDGA inhibits enzymatic activity, in addition to inhibiting the signaling pathway of lipoxygenase in which arachidonic acid generates leukotrienes and other oxygenated products [53–55].

#### **4.2** *Cordia boissieri* **plant as an alternative source for obtaining antimicrobial extracts**

The anacahuita plant (*Cordia boissieri*) is the official flower of the state of Nuevo León, México. The genus *Cordia* gets its name in honor of the sixteenthcentury German botanist Valerius Cordus and the *boissieri* species gets its name in honor of the nineteenth-century French botanist Boissier [56]. This plant is commonly known by the names of anacahuita, Mexican olive, Texas olive, wild olive, trompillo, and rasca viejo [56]. *C. boissieri* is a shrub or small tree up to 5 m high, with ovate leaves, 15–20 cm long and velvety surface. The flowers are white, grouped from 5 to 8, with the yellow center, up to 45 mm in length. The fruit is ovoid from 25 to 30 mm, brownish-green to purple, fleshy, sweet, and contains 1–4 seeds [56]. This plant species is native to North America. It is mainly distributed in Mexico, in the states of Nuevo León, Coahuila, Tamaulipas, San Luis Potosí, and Veracruz, and in the State of Texas in the United States. There are reports that the fruits of the *C. boissieri* plant are used as a remedy for coughs and colds. Traditionally the leaves of the plant are used to treat rheumatism and bronchial problems. Also in traditional medicine, the flowers are used in the treatment of diseases of bacterial origin [51, 56, 57].

#### **4.3** *Leucophyllum frutescens* **plant as an alternative source for obtaining antimicrobial extracts**

The ash plant (*Leucophyllum frutescens*) is an evergreen shrub. This plant is commonly known, in Mexico, with the name of ash and in the United States with the names of Texas ranger, Texas sage, silverleaf, and barometer bush, because the flowering is triggered by moisture [58]. It is a grey bush of 1.5–2 m in height. The silver-grey and green leaves are covered with silver hair. The violet to purple flowers are bell-shaped or funnel with five lobes and two lips and reach to measure 2.5 cm in length, which appear intermittently from spring to autumn. The fruit has the shape of a small capsule [58]. This plant is native to northern Mexico and the Southwestern United States. It is a species that is part of the medium and high bushes that develop

*Active Antimicrobial Food Packaging*

**3.3 Conclusions**

**extracts**

"greasewood" [49, 50].

development during the time of storage.

foods contaminated with *Listeria monocytogenes*.

to be included in polymer matrices for food packaging.

**4. Antimicrobial agents from plants of the northeast of Mexico**

As Mexico is a country that stands out for its floristic richness and taking into account the extensive knowledge of medicinal plants that since the pre-Columbian Era conserve Mexicans, mainly those of rural communities, it was natural that we were interested in the study of incorporation of some of them as active substances

**4.1** *Larrea tridentata* **plant as an alternative source for obtaining antimicrobial** 

The flora of arid zones represents a great potential for the wealth based on its biological specialization, since it is the product of thousands of years of physiological adaptation for its survival [50]. The governor plant (*L. tridentata*) typically develops under conditions of these zones. The *Larrea* genus includes five species of evergreen shrubs distributed throughout the Americas; its name was given in honor of the Spanish cleric Juan Antonio Hernández Larrea who was dean of the Zaragoza Chapter and bishop of Valladolid. This plant is commonly known as the governor, due to its dominance in the large areas of the arid zones of northern Mexico, but it is also known as guamis, sonora, tasajo, jarilla, creosote, and hediondilla due to its characteristic smell, mainly after the rain. In the Seri language, it is called "haaxat," and in the English language it has the common names of "creosote bush" and

The governor plant has a wide range of adaptation in elevation since it is located

in the Valley of Death in California located 86 m below sea level, to more than 2500 m in the sierras of northern Mexico. Its growth is good in dry plains and plateaus, also around hills and slopes, and in several types of soils except clayey, saline, or granitic [50]. The lifetime of this plant is negatively correlated with disturbance and soil compaction, being intolerant to soils with high phosphorus content [49]. In Mexico, the distribution of the governor plant is in part of the Sonoran Desert,

an aerobic bacterium, which is why the vacuum-packed product has control over its

Active PVOH films were obtained with essential oils of oregano and thyme, which showed broad-spectrum antibacterial activity by inhibiting pathogenic Gram-positive and Gram-negative bacteria specifically against *L. monocytogenes*, *E. coli* O157: H7, and *S. typhimurium*. The best conditions for the production of active films were 25% essential oil and elaboration of inclusion complex with a relation of 1:10 β-CD:EO. The active pad elaborated in the aforementioned conditions presented bacteriostatic activity against *L. monocytogenes* in cheese inoculated, packaged, and stored at 4°C for 29 days. The proposed packaging system ("pad" of the developed active film and a low-density polyethylene bag) can be an alternative to vacuum packaging using a multilayer film for cheeses. The experimental results showed that they provide a shelf life equivalent to vacuum packaging. In addition to the control of microbial activity, the proposed system is more accessible to small cheese producers as no special packaging technology is required other than a heat sealer machine. Likewise, the proposed packaging system can help reduce the incidence of outbreaks of diseases transmitted by

**38**

preferably in *lomeríos* of capricious soils [59]. Reports were found that in traditional medicine the leaves of *Lecophyllum frutescens* are used in the treatment of diseases caused by bacteria [51]. No work has been found on the compounds present in *Leucophyllum frutescens* to which an antibacterial action against *S. aureus* can be attributed.

#### **4.4** *Schinus molle* **plant as an alternative source for obtaining antimicrobial extracts**

The plant pirul (*Schinus molle*) is a perennial tree native to South America and naturalized in Mexico by Viceroy Antonio de Mendoza in the sixteenth century [60]. This plant is commonly known by the names of pirul in Mexico, aguaribay in Argentina, anacahuita in Uruguay, molle in Peru and, false pepper in Colombia [60]. It is a tree from 4 to 15 m high. The leaves are compound, alternate, 15–30 cm long, hung, with milky sap, and yellowish green. Its flowers are axillary panicles in the terminal leaves, 10–15 cm long, yellowish in color. The fruits are drupes in hanging clusters, each fruit 5–9 mm in diameter, pink or red [60]. *S. molle* is distributed, in Mexico, by the states of Aguascalientes, Chiapas, Coahuila, Federal District, Durango, Guanajuato, Guerrero, Hidalgo, State of Mexico, Jalisco, Michoacán, Morelos, Nuevo León, Oaxaca, Puebla, Querétaro, San Luis Potosí, Sinaloa, Tlaxcala, Veracruz, and Zacatecas. It is also naturalized in California, the Canary Islands, and China [61, 62]. It has been reported that the leaves of the *Schinus molle* plant serve to remedy respiratory diseases and for the treatment of skin wounds. For its part, the resin is also used to treat oral conditions [51, 60]. No works have been found on the compounds present in the leaves of the *Schinus molle* plant to which an antibacterial action against *S. aureus* can be attributed in an alcoholic extract.

#### **4.5 Inhibition of** *Staphylococcus aureus* **with extracts of anacahuita (***Cordia boissieri***), governor (***Larrea tridentata***), ash (***Leucophyllum frutescens***), and pirul (***Schinus molle***) with potential application in active packaging**

#### *4.5.1 Introduction*

*Staphylococcus aureus* is recognized as one of the main pathogenic agents for humans [63]. This microorganism is a natural inhabitant of the man's skin without causing damage to it, but when the skin's defenses diminish, it can cause a disease [64]. *S. aureus* produces abscesses and superficial lesions of the skin and causes impetigo, septicemia, and fevers, besides producing infections in the nervous system, endocarditis, and osteomyelitis [63]. It also has an extraordinary ability to develop resistance to antimicrobials and has the potential to cause viable infections to be fatal. It is responsible for 32–47% of infections in the skin and subcutaneous tissue [65]. Annually *S. aureus* causes around 100,000 deaths in hospitalized patients in the USA [66]. Several research groups have focused their studies on the antimicrobial activity of various natural extracts. Medicinal plants are considered a potential source of new drugs because of their phytochemical content and their little toxic effect [67]. Molina-Salinas et al. reported that the methanolic extracts of *Cordia boissieri* and *Leucophyllum frutescens* show inhibitory activity against *Streptococcus pneumoniae* and *Mycobacterium tuberculosis*, respectively, and that the hexanic extract of *Schinus molle* exhibits inhibitory activity against *S. aureus* [68]. For its part, Tello-Baca reported that the aqueous extract of *Larrea tridentata* has inhibitory activity against *Escherichia coli* and *S. aureus* [69].

The objective of the present investigation was to evaluate if the alcoholic extracts of the plants of anacahuita (*Cordia boissieri*), governor (*Larrea tridentata*), ash

**41**

*S. aureus* (108

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

(*Leucophyllum frutescens*), and pirul (*Schinus molle*) exhibit antimicrobial activity against *S. aureus* and select those with potential use in the formulation of active food packaging.

*Vegetal material*: The first variable of this research was the origin of the plant material. The plants were obtained in two ways: collection and purchase. The collection was carried out in the municipality of García, Nuevo León, and Mexico, and the purchase was made at the San Judas Hierbería in Monterrey, Nuevo León, Mexico. *Preparation of the extracts:* The flowers of *C. boissieri* and the leaves of *L. frutescens*, *L. tridentata*, and *S. molle* were used to prepare the extracts. The plants were subjected to a fine grind in a porcelain mortar. The samples were passed through a sieve with 1 mm mesh. The second variable is the nature of the solvent (ethanol and methanol, at 70% v/v). The extracts were prepared at 6% (w/v). The extraction of the active compounds was carried out by soaking for 15 min at 35°C on a stirring and heating plate (PMC). The solutions were left to stand for 48 h at room temperature in hermetically sealed containers protected from light. The extracts were filtered on Panama flax cloth to remove large particles. Subsequently, the samples were centrifuged at 7000 rpm for 10 min. Finally, a filtration in a Kitasato flask with Whatman paper No. 4 was carried out. The obtained extracts were stored at 4°C in

*Evaluation of inhibitory activity*: The inhibitory activity was evaluated by the disk diffusion method in Trypticase Soy Agar (Becton Dickinson) (NCCLS, 2003). The tested extracts were purchased *C. boissieri* extracted with ethanol (ACE), purchased *C. boissieri* extracted with methanol (ACM), collected *C. boissieri* extracted with ethanol (ARE), collected *C. boissieri* extracted with methanol (ARM), purchased *L. frutescens* extracted with ethanol (CCE), purchased *L. frutescens* extracted with methanol (CCM), *L. frutescens* collected extracted with ethanol (CRE), *L. frutescens* collected extracted with methanol (CRM), purchased *L. tridentata* extracted with ethanol (GCE), purchased *L. tridentata* extracted with methanol (GCM), *L. tridentata* harvested extracted with ethanol (GRE), *L. tridentata* harvested extracted with methanol (GRM), purchased *S. molle* extracted with ethanol (PCE), purchased *S. molle* extracted with methanol (PCM), *S. molle* collected extracted with ethanol (PRE), and collected *S. molle* extracted with methanol

incubated at 37°C for 24 and 48 h. Negative controls were used for ethanol and methanol, as the extraction solvent, and as positive controls, kanamycin (50 mg/ml) and chloramphenicol (34 mg/ ml), because they are broad-spectrum antibiotics. The tests were done in triplicate. The statistical analysis, to select the best extract of each of the plants, was

*Minimum inhibitory concentration:* The tube dilution method was used to determine the minimum inhibitory concentration of the selected extracts (NCCLS, 2000). Five concentrations of each extract (100, 200, 300, 400, and 500 μl) were placed in tubes with 5 ml of Trypticase Soy liquid medium (Becton Dickinson) with 500 μl of

formed in triplicate. To determine if the inhibitory activity of the extracts is bacteri-

medium of Trypticase Soybean (Becton Dickinson) was placed in 50/50 ratio with each one of the extracts was reseeded on Trypticase Soy Agar (Becton Dickinson) by the swab technique and incubated 24 h at 37°C and (b) a sample of bacterial cells from the inhibition halo formed was reseeded on Trypticase Soy Agar with striatum (Becton Dickinson) by each of the extracts and incubated at 37°C for 24 h. The tests were performed in triplicate. To select the two plants with the greatest inhibition, a

CFU/ml). The tubes were incubated at 37°C for 24 h. The tests were per-

CFU/ml. Plates were

CFU/ml) in liquid

glass containers, hermetically sealed and covered against light.

(PRM). *S. aureus* (ATCC 6538) was used at a concentration of 108

cidal or bacteriostatic, two tests were performed: (a) *S. aureus* (108

statistical analysis was performed using the Kruskal-Wallis test.

carried out using the Kruskal-Wallis test.

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

*4.5.2 Materials and methods*

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

(*Leucophyllum frutescens*), and pirul (*Schinus molle*) exhibit antimicrobial activity against *S. aureus* and select those with potential use in the formulation of active food packaging.

#### *4.5.2 Materials and methods*

*Active Antimicrobial Food Packaging*

attributed.

**extracts**

in an alcoholic extract.

*4.5.1 Introduction*

preferably in *lomeríos* of capricious soils [59]. Reports were found that in traditional medicine the leaves of *Lecophyllum frutescens* are used in the treatment of diseases caused by bacteria [51]. No work has been found on the compounds present in *Leucophyllum frutescens* to which an antibacterial action against *S. aureus* can be

**4.4** *Schinus molle* **plant as an alternative source for obtaining antimicrobial** 

**4.5 Inhibition of** *Staphylococcus aureus* **with extracts of anacahuita (***Cordia* 

**pirul (***Schinus molle***) with potential application in active packaging**

*Staphylococcus aureus* is recognized as one of the main pathogenic agents for humans [63]. This microorganism is a natural inhabitant of the man's skin without causing damage to it, but when the skin's defenses diminish, it can cause a disease [64]. *S. aureus* produces abscesses and superficial lesions of the skin and causes impetigo, septicemia, and fevers, besides producing infections in the nervous system, endocarditis, and osteomyelitis [63]. It also has an extraordinary ability to develop resistance to antimicrobials and has the potential to cause viable infections to be fatal. It is responsible for 32–47% of infections in the skin and subcutaneous tissue [65]. Annually *S. aureus* causes around 100,000 deaths in hospitalized patients in the USA [66]. Several research groups have focused their studies on the antimicrobial activity of various natural extracts. Medicinal plants are considered a potential source of new drugs because of their phytochemical content and their little toxic effect [67]. Molina-Salinas et al. reported that the methanolic extracts of *Cordia boissieri* and *Leucophyllum frutescens* show inhibitory activity against *Streptococcus pneumoniae* and *Mycobacterium tuberculosis*, respectively, and that the hexanic extract of *Schinus molle* exhibits inhibitory activity against *S. aureus* [68]. For its part, Tello-Baca reported that the aqueous extract of *Larrea tridentata* has

The objective of the present investigation was to evaluate if the alcoholic extracts

of the plants of anacahuita (*Cordia boissieri*), governor (*Larrea tridentata*), ash

inhibitory activity against *Escherichia coli* and *S. aureus* [69].

*boissieri***), governor (***Larrea tridentata***), ash (***Leucophyllum frutescens***), and** 

The plant pirul (*Schinus molle*) is a perennial tree native to South America and naturalized in Mexico by Viceroy Antonio de Mendoza in the sixteenth century [60]. This plant is commonly known by the names of pirul in Mexico, aguaribay in Argentina, anacahuita in Uruguay, molle in Peru and, false pepper in Colombia [60]. It is a tree from 4 to 15 m high. The leaves are compound, alternate, 15–30 cm long, hung, with milky sap, and yellowish green. Its flowers are axillary panicles in the terminal leaves, 10–15 cm long, yellowish in color. The fruits are drupes in hanging clusters, each fruit 5–9 mm in diameter, pink or red [60]. *S. molle* is distributed, in Mexico, by the states of Aguascalientes, Chiapas, Coahuila, Federal District, Durango, Guanajuato, Guerrero, Hidalgo, State of Mexico, Jalisco, Michoacán, Morelos, Nuevo León, Oaxaca, Puebla, Querétaro, San Luis Potosí, Sinaloa, Tlaxcala, Veracruz, and Zacatecas. It is also naturalized in California, the Canary Islands, and China [61, 62]. It has been reported that the leaves of the *Schinus molle* plant serve to remedy respiratory diseases and for the treatment of skin wounds. For its part, the resin is also used to treat oral conditions [51, 60]. No works have been found on the compounds present in the leaves of the *Schinus molle* plant to which an antibacterial action against *S. aureus* can be attributed

**40**

*Vegetal material*: The first variable of this research was the origin of the plant material. The plants were obtained in two ways: collection and purchase. The collection was carried out in the municipality of García, Nuevo León, and Mexico, and the purchase was made at the San Judas Hierbería in Monterrey, Nuevo León, Mexico.

*Preparation of the extracts:* The flowers of *C. boissieri* and the leaves of *L. frutescens*, *L. tridentata*, and *S. molle* were used to prepare the extracts. The plants were subjected to a fine grind in a porcelain mortar. The samples were passed through a sieve with 1 mm mesh. The second variable is the nature of the solvent (ethanol and methanol, at 70% v/v). The extracts were prepared at 6% (w/v). The extraction of the active compounds was carried out by soaking for 15 min at 35°C on a stirring and heating plate (PMC). The solutions were left to stand for 48 h at room temperature in hermetically sealed containers protected from light. The extracts were filtered on Panama flax cloth to remove large particles. Subsequently, the samples were centrifuged at 7000 rpm for 10 min. Finally, a filtration in a Kitasato flask with Whatman paper No. 4 was carried out. The obtained extracts were stored at 4°C in glass containers, hermetically sealed and covered against light.

*Evaluation of inhibitory activity*: The inhibitory activity was evaluated by the disk diffusion method in Trypticase Soy Agar (Becton Dickinson) (NCCLS, 2003). The tested extracts were purchased *C. boissieri* extracted with ethanol (ACE), purchased *C. boissieri* extracted with methanol (ACM), collected *C. boissieri* extracted with ethanol (ARE), collected *C. boissieri* extracted with methanol (ARM), purchased *L. frutescens* extracted with ethanol (CCE), purchased *L. frutescens* extracted with methanol (CCM), *L. frutescens* collected extracted with ethanol (CRE), *L. frutescens* collected extracted with methanol (CRM), purchased *L. tridentata* extracted with ethanol (GCE), purchased *L. tridentata* extracted with methanol (GCM), *L. tridentata* harvested extracted with ethanol (GRE), *L. tridentata* harvested extracted with methanol (GRM), purchased *S. molle* extracted with ethanol (PCE), purchased *S. molle* extracted with methanol (PCM), *S. molle* collected extracted with ethanol (PRE), and collected *S. molle* extracted with methanol (PRM). *S. aureus* (ATCC 6538) was used at a concentration of 108 CFU/ml. Plates were incubated at 37°C for 24 and 48 h. Negative controls were used for ethanol and methanol, as the extraction solvent, and as positive controls, kanamycin (50 mg/ml) and chloramphenicol (34 mg/ ml), because they are broad-spectrum antibiotics. The tests were done in triplicate. The statistical analysis, to select the best extract of each of the plants, was carried out using the Kruskal-Wallis test.

*Minimum inhibitory concentration:* The tube dilution method was used to determine the minimum inhibitory concentration of the selected extracts (NCCLS, 2000). Five concentrations of each extract (100, 200, 300, 400, and 500 μl) were placed in tubes with 5 ml of Trypticase Soy liquid medium (Becton Dickinson) with 500 μl of *S. aureus* (108 CFU/ml). The tubes were incubated at 37°C for 24 h. The tests were performed in triplicate. To determine if the inhibitory activity of the extracts is bactericidal or bacteriostatic, two tests were performed: (a) *S. aureus* (108 CFU/ml) in liquid medium of Trypticase Soybean (Becton Dickinson) was placed in 50/50 ratio with each one of the extracts was reseeded on Trypticase Soy Agar (Becton Dickinson) by the swab technique and incubated 24 h at 37°C and (b) a sample of bacterial cells from the inhibition halo formed was reseeded on Trypticase Soy Agar with striatum (Becton Dickinson) by each of the extracts and incubated at 37°C for 24 h. The tests were performed in triplicate. To select the two plants with the greatest inhibition, a statistical analysis was performed using the Kruskal-Wallis test.


#### **Table 2.**

*Diameters of the inhibition halo against S. aureus of the alcoholic extracts of C. boissieri, L. frutescens, L. tridentata, and S. molle plants.*

*Characterization of the extracts:* The two selected extracts were analyzed by gas chromatography (Agilent 6890)/mass spectrometry (Agilent 5973) (GC/MS). An HP-5MS column (30 m × 0.25 mm × 0.25 mm) was used for the separation of the components. Helium as a carrier gas has a flow rate of 15 ml/min. The injection temperature was 270°C in "split" mode. The temperature of the column, after a period of 1 min at 80°C, was increased to 320°C at a rate of 15°C min<sup>−</sup><sup>1</sup> , and maintained at

**43**

**Table 3.**

*plants with the highest inhibition against S. aureus.*

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

this temperature for 20 min. The mass spectrum had an ionization energy of 70 eV, a temperature of the ionization source of 230°C, and a quadrupole temperature of 150°C. The compounds were characterized with respect to the "Wiley7n.1" database.

According to the obtained results in **Table 2**, we can observe that all alcoholic extracts of the plants *C. boissieri*, *L. frutescens*, *L. tridentata*, and *S. molle* have inhibitory activity against *S. aureus*. The tested extracts showed an increase in the diameter of the inhibition halo after 24–48 h (except for ACM and CCM extracts); this may be due to the fact that increasing the contact time increases the diffusion of the active compounds toward the middle. From the *C. boissieri* plant extracts, ACE (48 h), MCA (24 h), ARE (24 and 48 h), and MRA (24 and 48 h) showed a significantly higher inhibition than controls (p < 0.05), being the ARM extract (24 and 48 h) the one that showed the highest inhibitory activity against *S. aureus*. Of the extracts of *L. frutescens*, CCM (48 h), CRE (24 and 48 h), and CRM (48 h) showed a significantly higher inhibitory activity than the controls (p < 0.05). CRE (24 and 48 h) was the extract with the highest inhibition against *S. aureus*. All evaluated extracts of *L. tridentata* showed a significantly higher inhibition than that of the controls (p < 0.05), presenting GRE (24 and 48 h) as the extract with greater inhibition against *S. aureus* and with a lower variance. In extracts of *S. molle*, PCM (24 and 48 h), PRE (24 and 48 h), and PRM (24 and 48 h) showed a significantly higher inhibition than that of the controls (p < 0.05), finding that PRE (24 and

48 h) is the extract with greater inhibitory activity against *S. aureus*.

**Table 3** shows the minimum inhibitory concentration of the extract with the highest inhibition of each of the plants studied. The extract of the plant *L. tridentata* had the lowest minimum inhibitory concentration on *S. aureus* with 20 μl/ml, followed by the extract of *S. molle* with 80 μl/ml. Extracts of *C. boissieri* and *L. frutescens* had the highest minimum inhibitory concentration with 100 μl/ml each. It was also determined that the extracts of *L. tridentata* and *S. molle* have inhibitory activity against *S. aureus* of the bactericidal type and that the extracts of *C. boissieri* and *L. frutescens* show inhibitory activity of bacteriostatic type. **Figure 9** shows the comparison of the inhibition diameters of the extracts with greater inhibition of each of the plants studied. The GRE (24 and 48 h) and PRE (24 and 48 h) extracts have a significantly higher inhibitory activity than the ARM extracts (24 and 48 h) and CRE (24 and 48 h) (p < 0.05). From GC/MS of *L. tridentata* extract (GRE), 12 compounds were identified, being 9,12-octadecanoic acid and 3,4′, 5,6,7-pentahydroxyflavone at 20.36 and 32.44 min, respectively, compounds they identified with greater certainty. Of the *S. molle* extract (PRE), 10 compounds were identified by GC/MS, with α-pinene compounds being

*Minimum inhibitory concentration of the extracts of C. boissieri, L. frutescens, L. tridentata, and S. molle* 

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

*4.5.3 Results*

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

this temperature for 20 min. The mass spectrum had an ionization energy of 70 eV, a temperature of the ionization source of 230°C, and a quadrupole temperature of 150°C. The compounds were characterized with respect to the "Wiley7n.1" database.

#### *4.5.3 Results*

*Active Antimicrobial Food Packaging*

**42**

**Table 2.**

*L. tridentata, and S. molle plants.*

*Diameters of the inhibition halo against S. aureus of the alcoholic extracts of C. boissieri, L. frutescens,*

of 1 min at 80°C, was increased to 320°C at a rate of 15°C min<sup>−</sup><sup>1</sup>

*Characterization of the extracts:* The two selected extracts were analyzed by gas chromatography (Agilent 6890)/mass spectrometry (Agilent 5973) (GC/MS). An HP-5MS column (30 m × 0.25 mm × 0.25 mm) was used for the separation of the components. Helium as a carrier gas has a flow rate of 15 ml/min. The injection temperature was 270°C in "split" mode. The temperature of the column, after a period

, and maintained at

According to the obtained results in **Table 2**, we can observe that all alcoholic extracts of the plants *C. boissieri*, *L. frutescens*, *L. tridentata*, and *S. molle* have inhibitory activity against *S. aureus*. The tested extracts showed an increase in the diameter of the inhibition halo after 24–48 h (except for ACM and CCM extracts); this may be due to the fact that increasing the contact time increases the diffusion of the active compounds toward the middle. From the *C. boissieri* plant extracts, ACE (48 h), MCA (24 h), ARE (24 and 48 h), and MRA (24 and 48 h) showed a significantly higher inhibition than controls (p < 0.05), being the ARM extract (24 and 48 h) the one that showed the highest inhibitory activity against *S. aureus*.

Of the extracts of *L. frutescens*, CCM (48 h), CRE (24 and 48 h), and CRM (48 h) showed a significantly higher inhibitory activity than the controls (p < 0.05). CRE (24 and 48 h) was the extract with the highest inhibition against *S. aureus*. All evaluated extracts of *L. tridentata* showed a significantly higher inhibition than that of the controls (p < 0.05), presenting GRE (24 and 48 h) as the extract with greater inhibition against *S. aureus* and with a lower variance. In extracts of *S. molle*, PCM (24 and 48 h), PRE (24 and 48 h), and PRM (24 and 48 h) showed a significantly higher inhibition than that of the controls (p < 0.05), finding that PRE (24 and 48 h) is the extract with greater inhibitory activity against *S. aureus*.

**Table 3** shows the minimum inhibitory concentration of the extract with the highest inhibition of each of the plants studied. The extract of the plant *L. tridentata* had the lowest minimum inhibitory concentration on *S. aureus* with 20 μl/ml, followed by the extract of *S. molle* with 80 μl/ml. Extracts of *C. boissieri* and *L. frutescens* had the highest minimum inhibitory concentration with 100 μl/ml each. It was also determined that the extracts of *L. tridentata* and *S. molle* have inhibitory activity against *S. aureus* of the bactericidal type and that the extracts of *C. boissieri* and *L. frutescens* show inhibitory activity of bacteriostatic type. **Figure 9** shows the comparison of the inhibition diameters of the extracts with greater inhibition of each of the plants studied. The GRE (24 and 48 h) and PRE (24 and 48 h) extracts have a significantly higher inhibitory activity than the ARM extracts (24 and 48 h) and CRE (24 and 48 h) (p < 0.05).

From GC/MS of *L. tridentata* extract (GRE), 12 compounds were identified, being 9,12-octadecanoic acid and 3,4′, 5,6,7-pentahydroxyflavone at 20.36 and 32.44 min, respectively, compounds they identified with greater certainty. Of the *S. molle* extract (PRE), 10 compounds were identified by GC/MS, with α-pinene compounds being


#### **Table 3.**

*Minimum inhibitory concentration of the extracts of C. boissieri, L. frutescens, L. tridentata, and S. molle plants with the highest inhibition against S. aureus.*

#### **Figure 9.**

*Diameter of the inhibition halo of the extracts with greater inhibition against S. aureus of each of the plants C. boissieri, L. frutescens, L. tridentata, and S. molle. ARM, C. boissieri collected extracted with methanol; CRE, L. frutescens harvested extracted with ethanol; GRE, L. tridentata harvested extracted with ethanol; and PRE, S. molle harvested extracted with ethanol.*

identified with greater certainty at 5.07 min, camphene at 5.30 min, p-mentha-1.5 comes at 6.12 min, p-mentha-1 (7), 2-diene at 6.51 min, 3 (15), 6-caryophylladiene at 12.15 min, 1 (10), 4 (15), 5-germacratriene a 12.92 min, 1 (10), 4-candinadiene at 13.39 min, 1.3-elemandien-11-ol at 13.71 min, and 4.9-cadinadiene at 16.27 min. The presence of 9, 12-octadecanoic acid and 3, 4 ', 5, 6, 7-pentahydroxyflavone was identified in the ethanolic extract of *L. tridentata* harvested and α-pinene; camphene; p-mentha-1, 5-diene; p-mentha-1 (7), 2-diene; 3 (15), 6-caryophyldiene; 1 (10), 4 (15), 5-germacratriene; 1 (10), 4-candinadiene; 1, 3-elemandien-11-ol; and 4, 9candinadiene in the ethanol extract of *S. molle* collected.

#### *4.5.4 Discussion*

For the four plants evaluated, there is greater inhibition in the extracts formulated with the harvested plants than with the purchased plants, since in the purchased plants, the storage time and the management that has been given are not known. In the extracts of the plants *L. frutescens*, *L. tridentata*, and *S. molle*, the ethanol was the solvent with which greater diameters were obtained in the inhibition zone. In the extracts of the *C. boissieri* plant, the solvent that allowed greater inhibition was methanol. The best inhibition results were obtained from the extracts of *L. tridentata* and *S. molle* plants. These plants represent a great antimicrobial potential; because as a plant that develops in arid conditions, it has a richness based on its biological specialization, since it is the product of thousands of years of physiological adaptation for its survival [50]. The inhibitory activity of *L. tridentata* can be attributed to the interaction of compounds present in the extracts, among which the 3,4′-5,6,7-pentahydroxyflavone exhibits an important role because the flavonoids are compounds with recognized antimicrobial activity [70]. The results obtained indicate that the four extracts exhibit antimicrobial activity, whether bactericidal or bacteriostatic. The activity observed was always greater than that of the controls (70% ethanol (E), kanamycin at a concentration of 50 mg/ml (K) and chloramphenicol at a concentration of 34 mg/ml (F)). Of the four extracts analyzed, that corresponding to *L. tridentata* showed the highest antimicrobial activity and the

**45**

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food…*

lowest minimum inhibitory concentration. The extract corresponding to *C. boissieri* showed the lowest antimicrobial activity and the highest minimum inhibitory

The presence of 9, 12-octadecanoic acid and 3, 4 ', 5, 6, 7-pentahydroxyflavone was identified in the ethanolic extract of *L. tridentata* harvested and α-pinene; camphene; p-mentha-1, 5-diene; p-mentha-1 (7), 2-diene; 3 (15), 6-caryophyldiene; 1 (10), 4 (15), 5-germacratriene; 1 (10), 4-candinadiene; 1, 3-elemandien-11-ol; and

In a previous work, Sáenz-Collins demonstrated that it was possible to obtain active antimicrobial PVOH biofilms against *S. aureus*, with potential use as dressings due to their biocompatibility. The extracts have no effect on the formation of biofilms. It was found that the higher the concentration of the extract in the biofilm, the greater the inhibition against *S. aureus*. Also, it was demonstrated that the alcoholic extracts had antimicrobial activity against Gram-negative bacteria as *Salmonella* and *E. coli* [71]. The drying temperature of the biofilm shows a diminishing effect on the antimicrobial activity; however, this remains present. It was demonstrated that the alcoholic extracts based on methanol and ethanol of *L. tridentata* show antimicrobial activity against *S. aureus* and that the ethanolic extract is more active. Sáenz-Collins also verified, through gas chromatography coupled to a mass spectrometer, that all the extracts of the governor plant possess an important amount of compounds with potential antimicrobial activity such as 4-vinylguaiacol, 4-hydroxybenzoic acid, and norisoguaiacin [71]. Although these plants can be purchased in some traditional local markets for medicinal use, their potential as a source of natural antimicrobial agents

In the present work, it was demonstrated that the alcoholic extracts (ethanolic and methanolic) of the plants *C. boissieri*, *L. tridentata*, *L. frutescens*, and *S. molle* have inhibitory activity against *S. aureus*. The ethanolic extracts of the harvested plants show a greater halo of inhibition, being *L. tridentata* and *S. molle* the plants that present the best results. The results obtained indicate that the four extracts exhibit antimicrobial activity, whether bactericidal or bacteriostatic. Of the four extracts analyzed, that corresponding to *L. tridentata* showed the highest antimicrobial activity and the lowest minimum inhibitory concentration. The extract corresponding to *C. boissieri* showed the lowest antimicrobial activity and the highest minimum inhibitory concentration in relation to the other extracts. It was demonstrated that the antimicrobial activity of *L. tridentata* and *S. molle* is bacteri-

*DOI: http://dx.doi.org/10.5772/intechopen.80779*

concentration in relation to the other extracts.

4, 9 candinadiene in the ethanol extract of *S. molle* collected.

for use in active food packaging must be further investigated.

cidal and have potential use in active food packaging.

*4.5.5 Conclusions*

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

lowest minimum inhibitory concentration. The extract corresponding to *C. boissieri* showed the lowest antimicrobial activity and the highest minimum inhibitory concentration in relation to the other extracts.

The presence of 9, 12-octadecanoic acid and 3, 4 ', 5, 6, 7-pentahydroxyflavone was identified in the ethanolic extract of *L. tridentata* harvested and α-pinene; camphene; p-mentha-1, 5-diene; p-mentha-1 (7), 2-diene; 3 (15), 6-caryophyldiene; 1 (10), 4 (15), 5-germacratriene; 1 (10), 4-candinadiene; 1, 3-elemandien-11-ol; and 4, 9 candinadiene in the ethanol extract of *S. molle* collected.

In a previous work, Sáenz-Collins demonstrated that it was possible to obtain active antimicrobial PVOH biofilms against *S. aureus*, with potential use as dressings due to their biocompatibility. The extracts have no effect on the formation of biofilms. It was found that the higher the concentration of the extract in the biofilm, the greater the inhibition against *S. aureus*. Also, it was demonstrated that the alcoholic extracts had antimicrobial activity against Gram-negative bacteria as *Salmonella* and *E. coli* [71]. The drying temperature of the biofilm shows a diminishing effect on the antimicrobial activity; however, this remains present. It was demonstrated that the alcoholic extracts based on methanol and ethanol of *L. tridentata* show antimicrobial activity against *S. aureus* and that the ethanolic extract is more active. Sáenz-Collins also verified, through gas chromatography coupled to a mass spectrometer, that all the extracts of the governor plant possess an important amount of compounds with potential antimicrobial activity such as 4-vinylguaiacol, 4-hydroxybenzoic acid, and norisoguaiacin [71]. Although these plants can be purchased in some traditional local markets for medicinal use, their potential as a source of natural antimicrobial agents for use in active food packaging must be further investigated.

#### *4.5.5 Conclusions*

*Active Antimicrobial Food Packaging*

*PRE, S. molle harvested extracted with ethanol.*

identified with greater certainty at 5.07 min, camphene at 5.30 min, p-mentha-1.5 comes at 6.12 min, p-mentha-1 (7), 2-diene at 6.51 min, 3 (15), 6-caryophylladiene at 12.15 min, 1 (10), 4 (15), 5-germacratriene a 12.92 min, 1 (10), 4-candinadiene at 13.39 min, 1.3-elemandien-11-ol at 13.71 min, and 4.9-cadinadiene at 16.27 min. The presence of 9, 12-octadecanoic acid and 3, 4 ', 5, 6, 7-pentahydroxyflavone was identified in the ethanolic extract of *L. tridentata* harvested and α-pinene; camphene; p-mentha-1, 5-diene; p-mentha-1 (7), 2-diene; 3 (15), 6-caryophyldiene; 1 (10), 4 (15), 5-germacratriene; 1 (10), 4-candinadiene; 1, 3-elemandien-11-ol; and 4,

*Diameter of the inhibition halo of the extracts with greater inhibition against S. aureus of each of the plants C. boissieri, L. frutescens, L. tridentata, and S. molle. ARM, C. boissieri collected extracted with methanol; CRE, L. frutescens harvested extracted with ethanol; GRE, L. tridentata harvested extracted with ethanol; and* 

For the four plants evaluated, there is greater inhibition in the extracts formulated with the harvested plants than with the purchased plants, since in the purchased plants, the storage time and the management that has been given are not known. In the extracts of the plants *L. frutescens*, *L. tridentata*, and *S. molle*, the ethanol was the solvent with which greater diameters were obtained in the inhibition zone. In the extracts of the *C. boissieri* plant, the solvent that allowed greater inhibition was methanol. The best inhibition results were obtained from the extracts of *L. tridentata* and *S. molle* plants. These plants represent a great antimicrobial potential; because as a plant that develops in arid conditions, it has a richness based on its biological specialization, since it is the product of thousands of years of physiological adaptation for its survival [50]. The inhibitory activity of *L. tridentata* can be attributed to the interaction of compounds present in the extracts, among which the 3,4′-5,6,7-pentahydroxyflavone exhibits an important role because the flavonoids are compounds with recognized antimicrobial activity [70]. The results obtained indicate that the four extracts exhibit antimicrobial activity, whether bactericidal or bacteriostatic. The activity observed was always greater than that of the controls (70% ethanol (E), kanamycin at a concentration of 50 mg/ml (K) and chloramphenicol at a concentration of 34 mg/ml (F)). Of the four extracts analyzed, that corresponding to *L. tridentata* showed the highest antimicrobial activity and the

9candinadiene in the ethanol extract of *S. molle* collected.

**44**

*4.5.4 Discussion*

**Figure 9.**

In the present work, it was demonstrated that the alcoholic extracts (ethanolic and methanolic) of the plants *C. boissieri*, *L. tridentata*, *L. frutescens*, and *S. molle* have inhibitory activity against *S. aureus*. The ethanolic extracts of the harvested plants show a greater halo of inhibition, being *L. tridentata* and *S. molle* the plants that present the best results. The results obtained indicate that the four extracts exhibit antimicrobial activity, whether bactericidal or bacteriostatic. Of the four extracts analyzed, that corresponding to *L. tridentata* showed the highest antimicrobial activity and the lowest minimum inhibitory concentration. The extract corresponding to *C. boissieri* showed the lowest antimicrobial activity and the highest minimum inhibitory concentration in relation to the other extracts. It was demonstrated that the antimicrobial activity of *L. tridentata* and *S. molle* is bactericidal and have potential use in active food packaging.

*Active Antimicrobial Food Packaging*

#### **Author details**

Cecilia Rojas de Gante\*, Judith A. Rocha and Carlos P. Sáenz Collins Departamento de Bioingeniería, Tecnológico de Monterrey, Campus Ciudad de México, Ciudad de México, Mexico

\*Address all correspondence to: crd@itesm.mx

© 2018 The Author(s). Licensee IntechOpen. 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.

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**46**

**Author details**

provided the original work is properly cited.

\*Address all correspondence to: crd@itesm.mx

México, Ciudad de México, Mexico

© 2018 The Author(s). Licensee IntechOpen. 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,

Departamento de Bioingeniería, Tecnológico de Monterrey, Campus Ciudad de

Cecilia Rojas de Gante\*, Judith A. Rocha and Carlos P. Sáenz Collins

[1] Brody AL. Active packaging becomes more active. Food Technology. 2005;**12**:82-84

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[16] Tinoco-Pérez B. Cuantificación de antocianinas en envases biopolimericos activos con potencial acción antioxidante a partir de harina de maíz azul (*Zea mais* amilacea) [Tesis de Maestría]. Monterrey, N.L.: Tecnológico de Monterrey; 2007

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[20] Dawson PL, Hirt DE, Rieck JR, Acton JC, Sotthibandhu A. Nisin release from films is affected by both protein type and film-forming method. Food Research International. 2003;**36**(9-10):959-968

[21] Pushpadass HA, Marx DB, Hanna AM. Effects of extrusion temperature and plasticizers on the physical and functional properties of starch films. Starch-Starke. 2008;**60**(10):527-538

[22] Wang Y, Rakotonirainy AM, Padua GW. Thermal behavior of zein-based biodegradable films. Starch-Starke. 2003;**55**(1):25-29

[23] López OV, Zaritzky NE, García MA. Physicochemical characterization of chemically modified corn starches related to rheological behavior, retrogradation and film forming capacity. Journal of Food Engineering. 2010;**100**(1):160-168

[24] Zeng M, Huang Y, Lu L, Fan L, Lourdin D. Effects of filler-matrix

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[28] Rojas de Gante C, Rios-Licea J, Tinoco-Pérez B. Method for producing biofilms from cereal grains, biofilms obtained said method and use thereof for producing receptacles for containing and preserving foods. Patent No. WO2010/024657 A1. 2010

[29] Parra D, Tadini C, Ponce P, Lugao A. Mechanical properties and water vapor transmission in some blends of cassava starch edible films. Carbohydrate Polymers. 2004;**58**(4):475478

[30] Cecilia Rojas-de G, Grissel Trujillo-de S, Carlos Patricio Sáenz C, Andrea Valderrama S. Desempeño de una película de maíz azul en el envasado de un queso de humedad intermedia. Revista de Biotecnologia en el Sector Agropecuario y Agroindustrial Ed. Especial No. 2. 2013:49-58

[31] Valderrama Solano AC, Rojas de Gante C. Development of biodegradable films based on blue corn flour with potential applications in food packaging.

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del Sur. 2008

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AJ. Evaluación de dos metodologías para determinar la actividad antimicrobiana de plantas medicinales. BLACMA.

[39] Pasterán F, Galas M. Manual de procedimientos, Sensibilidad a los antimicrobianos en Salmonella, Shigella y *E. coli*. Centro Regional de Referencia WHO- Global Salm. Surv. Para América

[40] Suppakul P, Sonneveld K, Bigger SW, Miltz J. Efficacy of polyethylenebased antimicrobial films containing principal consituents of basil. LWT Food Science and Technology.

[41] Norma Oficial Mexicana NOM-110-SSA1-1994 – Bienes y servicios. Preparación y dilución demuestras de alimentos para su análisis

[42] Ponce-Cevallos P, Buera M, Elizalde B. Encapsulation of cinnamon and thyme essential oils components (cinnamaldehyde and thymol) in β-cyclodextrin: Effect of interactions with water on complex stability. Journal of Food Engineering. 2010;**99**(1):70-75

[43] Dahmane M, Athman F, Sebih S, Guermouche MH, Bayle JP, Boudah S. Effect of the chain length on the thermal and analytical properties of laterally biforked nematogens. Journal of Chromatography A. 2010;**1217**(42):6562-6568

[44] Holland BJ, Hay JN. The thermal degradation of poly (vinyl alcohol).

[45] Rocha J. Desarrollo de películas de alcohol polivinílico (PVOH) con aceites esenciales naturales, para ser usados como "pads" (almohadillas) activos antimicrobianos en el envasado de quesos de cabra [Tesis de Maestría]. Monterrey N.L., México: Centro de

Polymer. 2001;**42**:67756783

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Effects of plasticizers on mechanical, thermal, and microstructural properties of flour films. Journal of Cereal Science.

[32] Trujillo-de Santiago G, Rojas-de Gante C, García-Lara S, Verdolotti L, Di Maio E, Iannace S. Strategies to produce thermoplastic starch–zein blends: Effect on phase compatibilization. Journal of Polymers and the Environment.

[33] Trujillo-de Santiago G, Rojas-de Gante C, García-Lara S, Verdolotti L, Di Maio E, Iannace S. Thermoplastic processing of blue maize and white sorghum flours to produce Bioplastics.

Journal of Polymers and the Environment. 2015;**23**(1):72-82

[34] Valderrama ACS, Rojas de

GC. Traceability of active compounds of essential oils in antimicrobial food packaging using a chemometric method by ATR-FTIR. American Journal of Analytical Chemistry. 2017;(8):726-741

[35] MacDonald PDM, Whitwam RE, Boggs JD, MacCormack JN, Anderson KL, Reardon JW, et al. Outbreak of listeriosis among Mexican immigrants as a result of consumption of illicitly produces Mexican-style cheese. Clinical Infectious Diseases.

[36] Indu MN, Hatha AAM, Abirosh

[37] Martínez LM, Videa M, Mederos F, Mesquita J. Constructing a highsensitivity, computerinterfaced, differential thermal analysis device for teaching and research. Journal of Chemical Education.

C, Harsha U, Vivekanandan G. Antimicrobial activity of some of the South-Indian spices against serotypes of *Escherichia coli*, *Salmonella*, *Listeria monocytogenes* and *Aeromonas hydrophila*. Brazilian Journal of Microbiology. 2006;**37**:153-158

2007;**84**(7):1222-1223

2005;**40**:677-682

2014;**60**:60-66

2015;**22**(4):508-524

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

Effects of plasticizers on mechanical, thermal, and microstructural properties of flour films. Journal of Cereal Science. 2014;**60**:60-66

*Active Antimicrobial Food Packaging*

activos con potencial acción

de Monterrey; 2007

[16] Tinoco-Pérez B. Cuantificación de antocianinas en envases biopolimericos morphology on mechanical properties of corn starch-zein thermomoulded films. Carbohydrate Polymers.

[25] Yang Q, Lue A, Qi H, Sun Y, Zhang X, Zhang L. Properties and bioapplications of blended cellulose and corn protein films. Macromolecular

Bioscience. 2009;**9**(9):849-856

[26] Financiera Rural. Monografía del Sorgo [Internet]. Available from: http://www.financierarural. gob.mx/informacionsectorrural/ Documents/Monografias/

MonografiaSorgo%28jun11%29.pdf

[27] U.S. Grains Council. Sorghum [Internet]. 2010. Available from: http://www.grains. org/index. php?option=com\_ content&view=article&id=

[28] Rojas de Gante C, Rios-Licea J, Tinoco-Pérez B. Method for producing biofilms from cereal grains, biofilms obtained said method and use thereof for producing receptacles for containing and preserving foods. Patent No. WO2010/024657 A1. 2010

[29] Parra D, Tadini C, Ponce P, Lugao A. Mechanical properties and water vapor transmission in some blends of cassava starch edible films. Carbohydrate Polymers.

[30] Cecilia Rojas-de G, Grissel Trujillo-de S, Carlos Patricio Sáenz C, Andrea Valderrama S. Desempeño de una película de maíz azul en el envasado de un queso de humedad intermedia. Revista de Biotecnologia en el Sector Agropecuario y Agroindustrial Ed.

Especial No. 2. 2013:49-58

[31] Valderrama Solano AC, Rojas de Gante C. Development of biodegradable films based on blue corn flour with potential applications in food packaging.

2004;**58**(4):475478

74&Itemid=120

2011;**84**(1):323-328

antioxidante a partir de harina de maíz azul (*Zea mais* amilacea) [Tesis de Maestría]. Monterrey, N.L.: Tecnológico

[17] SAGARPA. Producción de Maíz en México [Internet]. 2009. Available from: http://www.sagarpa.gob.mx/agricultura

[18] SIAP. Cierre de la producción agrícola por cultivo [Internet]. 2009. Available from: http://www.siap.gob. mx/index.php?option=com\_wrapper&v

iew=wrapper&Itemid=350

[19] De la Rosa-Millán J. Análisis fisicoquímico, estructural y molecular de almidones de diferentes variedades de maíz azul [Tesis Maestría en Ciencias en desarrollo de productos bióticos]. Yautepec (México): Instituto Politécnico Nacional, Dependencia; 2009. pp. 1-12

[20] Dawson PL, Hirt DE, Rieck JR, Acton JC, Sotthibandhu A. Nisin release from films is affected by both protein type and film-forming method. Food Research International.

[21] Pushpadass HA, Marx DB, Hanna AM. Effects of extrusion temperature and plasticizers on the physical and functional properties of starch films. Starch-Starke. 2008;**60**(10):527-538

[22] Wang Y, Rakotonirainy AM, Padua GW. Thermal behavior of zein-based biodegradable films. Starch-Starke.

[23] López OV, Zaritzky NE, García MA. Physicochemical characterization of chemically modified corn starches related to rheological behavior, retrogradation and film forming capacity. Journal of Food Engineering. 2010;**100**(1):160-168

[24] Zeng M, Huang Y, Lu L, Fan L, Lourdin D. Effects of filler-matrix

2003;**36**(9-10):959-968

2003;**55**(1):25-29

**48**

[32] Trujillo-de Santiago G, Rojas-de Gante C, García-Lara S, Verdolotti L, Di Maio E, Iannace S. Strategies to produce thermoplastic starch–zein blends: Effect on phase compatibilization. Journal of Polymers and the Environment. 2015;**22**(4):508-524

[33] Trujillo-de Santiago G, Rojas-de Gante C, García-Lara S, Verdolotti L, Di Maio E, Iannace S. Thermoplastic processing of blue maize and white sorghum flours to produce Bioplastics. Journal of Polymers and the Environment. 2015;**23**(1):72-82

[34] Valderrama ACS, Rojas de GC. Traceability of active compounds of essential oils in antimicrobial food packaging using a chemometric method by ATR-FTIR. American Journal of Analytical Chemistry. 2017;(8):726-741

[35] MacDonald PDM, Whitwam RE, Boggs JD, MacCormack JN, Anderson KL, Reardon JW, et al. Outbreak of listeriosis among Mexican immigrants as a result of consumption of illicitly produces Mexican-style cheese. Clinical Infectious Diseases. 2005;**40**:677-682

[36] Indu MN, Hatha AAM, Abirosh C, Harsha U, Vivekanandan G. Antimicrobial activity of some of the South-Indian spices against serotypes of *Escherichia coli*, *Salmonella*, *Listeria monocytogenes* and *Aeromonas hydrophila*. Brazilian Journal of Microbiology. 2006;**37**:153-158

[37] Martínez LM, Videa M, Mederos F, Mesquita J. Constructing a highsensitivity, computerinterfaced, differential thermal analysis device for teaching and research. Journal of Chemical Education. 2007;**84**(7):1222-1223

[38] Rojas JJ, García AM, López AJ. Evaluación de dos metodologías para determinar la actividad antimicrobiana de plantas medicinales. BLACMA. 2005;**4**(2):28

[39] Pasterán F, Galas M. Manual de procedimientos, Sensibilidad a los antimicrobianos en Salmonella, Shigella y *E. coli*. Centro Regional de Referencia WHO- Global Salm. Surv. Para América del Sur. 2008

[40] Suppakul P, Sonneveld K, Bigger SW, Miltz J. Efficacy of polyethylenebased antimicrobial films containing principal consituents of basil. LWT Food Science and Technology. 2008;**41**:779-788

[41] Norma Oficial Mexicana NOM-110-SSA1-1994 – Bienes y servicios. Preparación y dilución demuestras de alimentos para su análisis microbiológico

[42] Ponce-Cevallos P, Buera M, Elizalde B. Encapsulation of cinnamon and thyme essential oils components (cinnamaldehyde and thymol) in β-cyclodextrin: Effect of interactions with water on complex stability. Journal of Food Engineering. 2010;**99**(1):70-75

[43] Dahmane M, Athman F, Sebih S, Guermouche MH, Bayle JP, Boudah S. Effect of the chain length on the thermal and analytical properties of laterally biforked nematogens. Journal of Chromatography A. 2010;**1217**(42):6562-6568

[44] Holland BJ, Hay JN. The thermal degradation of poly (vinyl alcohol). Polymer. 2001;**42**:67756783

[45] Rocha J. Desarrollo de películas de alcohol polivinílico (PVOH) con aceites esenciales naturales, para ser usados como "pads" (almohadillas) activos antimicrobianos en el envasado de quesos de cabra [Tesis de Maestría]. Monterrey N.L., México: Centro de Biotecnología ITESM; 2011

[46] Ultee A, Bennik MHJ, Moezelaar R. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen *Bacillus cereus*. Applied and Environmental Microbiology. 2002;**68**(4):1561-1568

[47] Burt S. Essential oils: Their antibacterial properties and potential applications in foods, a review. International Journal of Food Microbiology. 2004;**94**:223-253

[48] Lambert RJW, Skandamis PN, Coote PJ, Nychas GJE. Study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of Applied Microbiology. 2001;**91**:453-462

[49] Vázquez-Yanes C, Batis-Muñoz AI, Alcocer-Silva MI, Gual-Díaz M, Sánchez-Dirzo C. Árboles y arbustos potencialmente valiosos para la restauración ecológica y la reforestación. Reporte técnico del proyecto J084. CONABIO - Instituto de Ecología, UNAM; 1999

[50] Lira-Saldivar RH. Estado actual del conocimiento sobre las propiedades biocidas de la Gobernadora [*Larrea tridentata* (D.C.) Coville]. Revista Mexicana de Fitopatología. 2003;**21**(2):214-222

[51] Adame J, Adame H. Plantas curativas del noreste mexicano. Monterrey: Ediciones Castillo; 2000

[52] García-Llamas F. La gran enciclopedia de las plantas curativas. México: Editorial Diana; 2005

[53] Bronfman M. Nuevos Factores de Transcripción Nuclear. Santiago: Pontificia Universidad Católica de Chile; 2002

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[57] Hernández-Magaña R, Gally-Jorda M. Plantas medicinales. México: Árbol Editorial; 1981

[58] Irish M. Trees and Shrubs for the Southwest. China: Congrees; 2008

[59] Villegas-Durán G, Bolaños-Medina A, Miranda-Sánchez JA, García-Aldape J, Galván-García OM. Flora nectarífera y polinífera en el estado de Tamaulipas. México: SAGARPA; 2003

[60] Hernández-Vázquez J, Sánchez-González S, González-Pacheco C, Luna-Peñaloza SR, Durán-Carmona V, Toledo-Ocampo A, Rodríguez-Velasco A, González-Lorenzo JR. Programa ambiental de la Universidad Autónoma de la Ciudad de México. 2011. Recuperado de: http://desarrollo.uacm.edu.mx.html

[61] Rzedowski GC, Calderón J. Flora del Bajío y regiones adyacentes. Pátzcuaro: Instituto de Ecología-Centro regional del Bajío; 1999

[62] Villaseñor JL, Espinosa FJ. Catálogo de malezas de México. México: UNAM; 1998

[63] Ruocco E, Donnarumma G, Baroni A, Tufano MA. Bacterial and viral skin diseases. Dermatologic Clinics. 2007;**25**(4):663-676

[64] Richardson AR, Libby SJ, Fang FC. A nitric oxide-inducible lactate dehydrogenase enables *Staphylococcus aureus* to resist innate immunity. Science. 2008;**319**(5870):1672-1676

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en Ciencias). 2009

Aprovechamiento de la planta gobernadora (*Larrea tridentata*) para la obtención de un agente activo antimicrobiano frente a *Staphylococcus aureus* con aplicación en biopelículas de alcohol polivinílico para uso médico en forma de apósitos [Tesis] (Maestría

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[66] Demain A, Sánchez S. Microbial drug discovery: 80 years of progress. The Journal of Antibiotics. 2009;**62**:5-16

[68] Molina-Salinas GM, Pérez-López A, Becerril-Montes P, Salazar-Aranda R, Said-Fernández S, Waksman-de Torres N. Evaluation of the flora of Northern Mexico for in vitro antimicrobial and antituberculosis activity. Journal of Ethnopharmacology.

[67] Beg AZ, Ahmad I. Effect of *Plumbago zeylanica* extract and certain curing agents on multidrug resistant bacteria of clinical origin. World Journal of Microbiology and Biotechnology.

2001;**32**(2):S114-S132

2000;**16**(8-9):841-844

2007;**109**(3):435-441

[69] Tello-Baca R. Actividad

antibacteriana de plants con actividad hemaglutinante [QFB dissertation]. Morelia, México: Universidad

Michoacana de San Nicolás de Hidalgo;

[70] Cushnie T, Lamb AJ. Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents.

*Using Native Plants of the Northeast of Mexico for Developing Active Antimicrobial Food… DOI: http://dx.doi.org/10.5772/intechopen.80779*

[65] Diekema DJ, Pfaller MA, Schmitz FJ, Smayevsky J, Bell J, Jones RN. Survey of infections due to Staphylococcus species: Frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY antimicrobial surveillance program, 1999. Clinical Infectious Diseases. 2001;**32**(2):S114-S132

*Active Antimicrobial Food Packaging*

[47] Burt S. Essential oils: Their antibacterial properties and potential applications in foods, a review. International Journal of Food Microbiology. 2004;**94**:223-253

[48] Lambert RJW, Skandamis PN, Coote PJ, Nychas GJE. Study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of Applied Microbiology. 2001;**91**:453-462

[49] Vázquez-Yanes C, Batis-Muñoz AI, Alcocer-Silva MI, Gual-Díaz M, Sánchez-Dirzo C. Árboles y arbustos potencialmente valiosos para la

restauración ecológica y la reforestación. Reporte técnico del proyecto J084. CONABIO - Instituto de Ecología,

[50] Lira-Saldivar RH. Estado actual del conocimiento sobre las propiedades biocidas de la Gobernadora [*Larrea tridentata* (D.C.) Coville]. Revista Mexicana de Fitopatología.

[51] Adame J, Adame H. Plantas curativas del noreste mexicano. Monterrey: Ediciones Castillo; 2000

[52] García-Llamas F. La gran

enciclopedia de las plantas curativas. México: Editorial Diana; 2005

[53] Bronfman M. Nuevos Factores de Transcripción Nuclear. Santiago: Pontificia Universidad Católica de Chile; 2002

[54] Bruneton J. Farmacognosia: fitoquímica, plantas medicinales.

[55] FDA/CFSAN. Illnesses and Injuries Associated With the Use of Selected

Zaragoza: Acribia; 2001

UNAM; 1999

2003;**21**(2):214-222

[46] Ultee A, Bennik MHJ, Moezelaar R. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen *Bacillus cereus*. Applied and Environmental Microbiology. 2002;**68**(4):1561-1568

Dietary Supplements. Miami: Dockets Management Branch Food and Drug

[56] Alvarado MA, Foroughbakhch R, Jurado E, Rocha A. Caracterización morfológica y nutricional del fruto de anacahuita (*Cordia boissieri*) en dos localidades del Noreste de México. Phyton, International Journal of Experimental Botany. 2004;**73**:85-90

[57] Hernández-Magaña R, Gally-Jorda M. Plantas medicinales. México: Árbol

[58] Irish M. Trees and Shrubs for the Southwest. China: Congrees; 2008

[59] Villegas-Durán G, Bolaños-Medina A, Miranda-Sánchez JA, García-Aldape J, Galván-García OM. Flora nectarífera y polinífera en el estado de Tamaulipas.

[60] Hernández-Vázquez J, Sánchez-González S, González-Pacheco C, Luna-Peñaloza SR, Durán-Carmona V, Toledo-Ocampo A, Rodríguez-Velasco A, González-Lorenzo JR. Programa ambiental de la Universidad Autónoma de la Ciudad de México. 2011. Recuperado de: http://desarrollo.uacm.edu.mx.html

[61] Rzedowski GC, Calderón J. Flora del Bajío y regiones adyacentes. Pátzcuaro: Instituto de Ecología-Centro regional

[62] Villaseñor JL, Espinosa FJ. Catálogo de malezas de México. México: UNAM;

[63] Ruocco E, Donnarumma G, Baroni A, Tufano MA. Bacterial and viral skin diseases. Dermatologic Clinics.

[64] Richardson AR, Libby SJ, Fang FC. A nitric oxide-inducible lactate dehydrogenase enables *Staphylococcus aureus* to resist innate immunity. Science. 2008;**319**(5870):1672-1676

México: SAGARPA; 2003

Administration; 1993

Editorial; 1981

del Bajío; 1999

2007;**25**(4):663-676

1998

**50**

[66] Demain A, Sánchez S. Microbial drug discovery: 80 years of progress. The Journal of Antibiotics. 2009;**62**:5-16

[67] Beg AZ, Ahmad I. Effect of *Plumbago zeylanica* extract and certain curing agents on multidrug resistant bacteria of clinical origin. World Journal of Microbiology and Biotechnology. 2000;**16**(8-9):841-844

[68] Molina-Salinas GM, Pérez-López A, Becerril-Montes P, Salazar-Aranda R, Said-Fernández S, Waksman-de Torres N. Evaluation of the flora of Northern Mexico for in vitro antimicrobial and antituberculosis activity. Journal of Ethnopharmacology. 2007;**109**(3):435-441

[69] Tello-Baca R. Actividad antibacteriana de plants con actividad hemaglutinante [QFB dissertation]. Morelia, México: Universidad Michoacana de San Nicolás de Hidalgo; 2009

[70] Cushnie T, Lamb AJ. Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents. 2005;**26**:343-356

[71] Sáenz-Collins CP.

Aprovechamiento de la planta gobernadora (*Larrea tridentata*) para la obtención de un agente activo antimicrobiano frente a *Staphylococcus aureus* con aplicación en biopelículas de alcohol polivinílico para uso médico en forma de apósitos [Tesis] (Maestría en Ciencias). 2009

**53**

**Chapter 4**

Review

**Abstract**

product.

**1. Food packaging**

Protein-Based Active Film as

*Nurul Saadah Said and Norizah Mhd Sarbon*

Antimicrobial Food Packaging: A

This review discusses the protein-based active film as antimicrobial food packaging derived from various sources such as gelatin, casein, whey and zein-based protein. The films properties that exhibit antimicrobial activity are being reviewed along with their application in food packaging industry. This paper also studies the inhibition activity by antimicrobial agents from organic and metallic sources which were incorporated into the protein-based film. Nowadays, protein-based film has emerged as one of the most extensively studied in food packaging sector as it exhibits good mechanical, optical, and oxygen barrier properties. In addition, protein-based film also showed good compatibility to polar surfaces while having effective control on the release of additives and bioactive compounds in food packaging system. This paper also detailed out information on antimicrobial food packaging in order to increase consumer awareness regarding food safety and healthy lifestyle while maintaining the quality and prolonged the shelf life of food

**Keywords:** protein, biopolymer, edible film, active packaging, antimicrobial agents

of products by decreasing the risk of tampering and adulteration [3].

Food packaging is defined as a way of preparing food for transport, distribution, storage, and retailing till the end use while ensuring safe delivery to the ultimate customer [1]. Packaging systems are characterized into three groups which are primary, secondary, and tertiary packaging according to their layers or functions. Primary packaging is the first level of packaging which involves direct contact with the products. While secondary packaging contains a number of primary packages that protect the primary packages from damage during shipment and storage and are also designed to be displayed onto the retail shelves. As tertiary packaging, it acts as distribution carrier which consists of a number of secondary and primary packages [2]. Food packaging is designed to protect and maintain the quality and safety of foods from chemical, biological, and physical deterioration while helping in extending the product's shelf life [3]. In addition, its basic functions are commonly served as containment, protection or preservation, and communication and convenience purpose [1]. Packaging is also helpful in reducing municipal solid waste disposal and the cost of many food products by facilitating large-scale production and efficiency in bulk distribution. Food packaging also ensures the safety

#### **Chapter 4**

## Protein-Based Active Film as Antimicrobial Food Packaging: A Review

*Nurul Saadah Said and Norizah Mhd Sarbon*

#### **Abstract**

This review discusses the protein-based active film as antimicrobial food packaging derived from various sources such as gelatin, casein, whey and zein-based protein. The films properties that exhibit antimicrobial activity are being reviewed along with their application in food packaging industry. This paper also studies the inhibition activity by antimicrobial agents from organic and metallic sources which were incorporated into the protein-based film. Nowadays, protein-based film has emerged as one of the most extensively studied in food packaging sector as it exhibits good mechanical, optical, and oxygen barrier properties. In addition, protein-based film also showed good compatibility to polar surfaces while having effective control on the release of additives and bioactive compounds in food packaging system. This paper also detailed out information on antimicrobial food packaging in order to increase consumer awareness regarding food safety and healthy lifestyle while maintaining the quality and prolonged the shelf life of food product.

**Keywords:** protein, biopolymer, edible film, active packaging, antimicrobial agents

#### **1. Food packaging**

Food packaging is defined as a way of preparing food for transport, distribution, storage, and retailing till the end use while ensuring safe delivery to the ultimate customer [1]. Packaging systems are characterized into three groups which are primary, secondary, and tertiary packaging according to their layers or functions. Primary packaging is the first level of packaging which involves direct contact with the products. While secondary packaging contains a number of primary packages that protect the primary packages from damage during shipment and storage and are also designed to be displayed onto the retail shelves. As tertiary packaging, it acts as distribution carrier which consists of a number of secondary and primary packages [2]. Food packaging is designed to protect and maintain the quality and safety of foods from chemical, biological, and physical deterioration while helping in extending the product's shelf life [3]. In addition, its basic functions are commonly served as containment, protection or preservation, and communication and convenience purpose [1]. Packaging is also helpful in reducing municipal solid waste disposal and the cost of many food products by facilitating large-scale production and efficiency in bulk distribution. Food packaging also ensures the safety of products by decreasing the risk of tampering and adulteration [3].

However, there are major drawbacks regarding non-biodegradable food packaging which caused environmental problems that include changes to the carbon dioxide cycle, composting problems, and increasing level of toxic emissions [4]. Stimulated by the environmental and growing interest of health safety concerns from consumers, many researchers have now been concentrating on ways to develop biodegradable packaging. Food packaging from biodegradable polymers have raised attention due to their renewable and environmental-friendly characteristics. Studies from renewable of natural biopolymers sources were included from polysaccharides (starch and chitin), lipids (waxes and paraffin), proteins (collagen and gelatin), or the combination of these components [5–7]. Among those, protein possessed greater characteristics and potential in food packaging due to its ability in film-forming process with high mechanical and barrier properties [8].

#### **2. Protein-based food packaging**

Proteins are composed of amino acid chains linked by peptide bonds to form a primary structure [9]. It can be characterized according to its amino acid composition, geometrical conformation, solubility, molecular weight, sedimentation behavior, surface polarity, and native molecular configuration shape [10]. Protein generally existed in two main classes that are known as fibrous or globular proteins. Fibrous and globular proteins have different sizes, shapes, solubility, appearances, and functions. Fibrous protein served as the main structural materials of animal tissues, while the globular proteins have multiple functions such as formation of enzymes, cellular messengers, and amino acids. Fibrous proteins consist of repetition of a single unit to form chains that act as connective tissues and associated closely with each other in parallel structures to provide strength and joint mobility, while globular proteins consist of long chains with numerous branches and folded into complicated spherical structures held together by a combination of hydrogen, ionic, hydrophobic, and covalent (disulfide) bonds. The example of fibrous protein that has gained great attention in studies of food packaging material is collagen. As for the globular protein, many research have been conducted on the use of casein, wheat gluten, corn zein, soy protein, whey protein, and mung bean protein as a great potential to be utilized as edible food packaging [11]. In food packaging, protein is mainly used as it exhibits good mechanical, optical, and oxygen barrier properties. Furthermore, protein is able to promote good compatibility to polar surfaces and control the release of additives and bioactive compounds in food packaging system [8, 10]. Therefore, many research have been keen to produce protein-based packaging that emerge in the form of edible films and coatings from various protein sources such as gelatin, casein, whey, corn zein, pea, wheat gluten, amaranth, soy, mung bean, and peanut.

#### **2.1 Protein-based edible film**

Nowadays, biodegradable film for food packaging has drawn attention from many researchers as an alternative approach to solve the problem arise from petroleum-based polymeric material that possesses non-biodegradability properties which leads to a critical environmental issue and causes exhaustion of natural resources. Natural biopolymers such as protein are eco-friendly and exhibit nontoxic properties which also have comparable physicochemical characteristics with the synthetic polymeric film [12]. In general, edible film is defined as a standalone thin layer sheet formed from a biopolymer matrix and possessed structural integrity. It served as a moisture barrier or as a solute/gas barrier while being able

**55**

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review*

pea, wheat gluten, amaranth, soy, mung bean, and peanut.

to improve the mechanical and rheological characteristics of the intended products [13]. These films also help in improving product quality by imparting certain functions such as antioxidant, antimicrobials, or any other specific functions while providing physical protection and extending the shelf life of the food products. Protein-based films have been extensively utilized due to their relative abundance and good film-forming ability, contain high nutritional value, as well as provide desirable mechanical, gas barrier, and transparency properties [14]. Other than that, protein-based films also showed better mechanical properties than polysaccharide- and lipid-based films due to their unique structure that provides wider range of functional properties especially exhibiting a high intermolecular binding potential that is able to form a bond at a different position [14]. As widely known, protein-based edible film is being utilized from various kinds of protein sources which are mainly classified into two categories: animal and agro-based protein polymers. Several studies that have been conducted on animal-derived protein polymer include collagen, gelatin, fish myofibrillar protein, and whey protein, while the studies on agro-based protein polymer include the sources derived from corn zein,

The most preferred method to form edible protein-based film is by using solvent

casting. The films are formed from solutions or dispersions of the protein as the solvent/carrier evaporates. The solvent/carrier is generally limited to water, ethanol, or ethanol-water mixtures [11]. The method was technically done by spreading dilute film solution and plasticizer into Petri dish or plates and drying them under ambient conditions or controlled relative humidity. Commonly, large-scale production uses more sophisticated equipment that is able to generate larger protein films by mechanically spreading the solution to a fixed thickness. There are several parameters that need to be determined for continuous film production such as air temperature, surface properties of the substrate upon which the films are formed, flow rate, and drying time. The films can be dried under ambient conditions by using several methods that are hot air, infrared energy, or microwave energy. The physical properties of the final film regarding film morphology, appearance, and barrier and mechanical properties can be significantly affected by the drying method used [15]. The other alternative method for protein-based film forming is by using extrusion. Extrusion process used elevated temperature and shear in order to soften and melt the polymer and thus allow the cohesive film matrix to form. The use of extrusion has certain advantages over solvent casting method as it is able to reduce more time and energy inputs as well as raise the cost of biopolymer film formation into a competitive range

that is able to match and compete with the synthetic film production [15].

or gloss which seem to be more appealing to consumers [13, 16].

Meanwhile, edible coating is a more thinly edible film which is being formed directly onto the food or materials surface [16]. Edible coating can improve the physical and chemical integrity of the product by creating a modified overhead atmosphere and prevent the migration of moisture, oxygen, carbon dioxide, or any other solutes. It also acts as a carrier in terms of food additives (antioxidants, antimicrobials, and specific nutrients) while increasing the shelf life of the product. Furthermore, edible coatings can improve the product's appearance by adding color

Protein coating is often being processed by using two common methods that are known as wet and dry (mainly extrusion) processes that depend on the target structure either mono- or multilayer structure. Wet coatings from polymer solutions or suspensions are commonly done by using lacquering or spraying techniques. Rheological properties of the coating formulation are greatly influenced by

**2.2 Protein-based edible coating**

*DOI: http://dx.doi.org/10.5772/intechopen.80774*

#### *Protein-Based Active Film as Antimicrobial Food Packaging: A Review DOI: http://dx.doi.org/10.5772/intechopen.80774*

*Active Antimicrobial Food Packaging*

**2. Protein-based food packaging**

amaranth, soy, mung bean, and peanut.

**2.1 Protein-based edible film**

However, there are major drawbacks regarding non-biodegradable food packaging which caused environmental problems that include changes to the carbon dioxide cycle, composting problems, and increasing level of toxic emissions [4]. Stimulated by the environmental and growing interest of health safety concerns from consumers, many researchers have now been concentrating on ways to develop biodegradable packaging. Food packaging from biodegradable polymers have raised attention due to their renewable and environmental-friendly characteristics. Studies from renewable of natural biopolymers sources were included from polysaccharides (starch and chitin), lipids (waxes and paraffin), proteins (collagen and gelatin), or the combination of these components [5–7]. Among those, protein possessed greater characteristics and potential in food packaging due to its ability in

Proteins are composed of amino acid chains linked by peptide bonds to form a primary structure [9]. It can be characterized according to its amino acid composition, geometrical conformation, solubility, molecular weight, sedimentation behavior, surface polarity, and native molecular configuration shape [10]. Protein generally existed in two main classes that are known as fibrous or globular proteins. Fibrous and globular proteins have different sizes, shapes, solubility, appearances, and functions. Fibrous protein served as the main structural materials of animal tissues, while the globular proteins have multiple functions such as formation of enzymes, cellular messengers, and amino acids. Fibrous proteins consist of repetition of a single unit to form chains that act as connective tissues and associated closely with each other in parallel structures to provide strength and joint mobility, while globular proteins consist of long chains with numerous branches and folded into complicated spherical structures held together by a combination of hydrogen, ionic, hydrophobic, and covalent (disulfide) bonds. The example of fibrous protein that has gained great attention in studies of food packaging material is collagen. As for the globular protein, many research have been conducted on the use of casein, wheat gluten, corn zein, soy protein, whey protein, and mung bean protein as a great potential to be utilized as edible food packaging [11]. In food packaging, protein is mainly used as it exhibits good mechanical, optical, and oxygen barrier properties. Furthermore, protein is able to promote good compatibility to polar surfaces and control the release of additives and bioactive compounds in food packaging system [8, 10]. Therefore, many research have been keen to produce protein-based packaging that emerge in the form of edible films and coatings from various protein sources such as gelatin, casein, whey, corn zein, pea, wheat gluten,

Nowadays, biodegradable film for food packaging has drawn attention from many researchers as an alternative approach to solve the problem arise from petroleum-based polymeric material that possesses non-biodegradability properties which leads to a critical environmental issue and causes exhaustion of natural resources. Natural biopolymers such as protein are eco-friendly and exhibit nontoxic properties which also have comparable physicochemical characteristics with the synthetic polymeric film [12]. In general, edible film is defined as a standalone thin layer sheet formed from a biopolymer matrix and possessed structural integrity. It served as a moisture barrier or as a solute/gas barrier while being able

film-forming process with high mechanical and barrier properties [8].

**54**

to improve the mechanical and rheological characteristics of the intended products [13]. These films also help in improving product quality by imparting certain functions such as antioxidant, antimicrobials, or any other specific functions while providing physical protection and extending the shelf life of the food products.

Protein-based films have been extensively utilized due to their relative abundance and good film-forming ability, contain high nutritional value, as well as provide desirable mechanical, gas barrier, and transparency properties [14]. Other than that, protein-based films also showed better mechanical properties than polysaccharide- and lipid-based films due to their unique structure that provides wider range of functional properties especially exhibiting a high intermolecular binding potential that is able to form a bond at a different position [14]. As widely known, protein-based edible film is being utilized from various kinds of protein sources which are mainly classified into two categories: animal and agro-based protein polymers. Several studies that have been conducted on animal-derived protein polymer include collagen, gelatin, fish myofibrillar protein, and whey protein, while the studies on agro-based protein polymer include the sources derived from corn zein, pea, wheat gluten, amaranth, soy, mung bean, and peanut.

The most preferred method to form edible protein-based film is by using solvent casting. The films are formed from solutions or dispersions of the protein as the solvent/carrier evaporates. The solvent/carrier is generally limited to water, ethanol, or ethanol-water mixtures [11]. The method was technically done by spreading dilute film solution and plasticizer into Petri dish or plates and drying them under ambient conditions or controlled relative humidity. Commonly, large-scale production uses more sophisticated equipment that is able to generate larger protein films by mechanically spreading the solution to a fixed thickness. There are several parameters that need to be determined for continuous film production such as air temperature, surface properties of the substrate upon which the films are formed, flow rate, and drying time. The films can be dried under ambient conditions by using several methods that are hot air, infrared energy, or microwave energy. The physical properties of the final film regarding film morphology, appearance, and barrier and mechanical properties can be significantly affected by the drying method used [15]. The other alternative method for protein-based film forming is by using extrusion. Extrusion process used elevated temperature and shear in order to soften and melt the polymer and thus allow the cohesive film matrix to form. The use of extrusion has certain advantages over solvent casting method as it is able to reduce more time and energy inputs as well as raise the cost of biopolymer film formation into a competitive range that is able to match and compete with the synthetic film production [15].

#### **2.2 Protein-based edible coating**

Meanwhile, edible coating is a more thinly edible film which is being formed directly onto the food or materials surface [16]. Edible coating can improve the physical and chemical integrity of the product by creating a modified overhead atmosphere and prevent the migration of moisture, oxygen, carbon dioxide, or any other solutes. It also acts as a carrier in terms of food additives (antioxidants, antimicrobials, and specific nutrients) while increasing the shelf life of the product. Furthermore, edible coatings can improve the product's appearance by adding color or gloss which seem to be more appealing to consumers [13, 16].

Protein coating is often being processed by using two common methods that are known as wet and dry (mainly extrusion) processes that depend on the target structure either mono- or multilayer structure. Wet coatings from polymer solutions or suspensions are commonly done by using lacquering or spraying techniques. Rheological properties of the coating formulation are greatly influenced by the techniques that will be used for wet coating process. Different types of methods are applicable for the drying process such as drying under ambient conditions, hot air, infrared energy, or microwave energy. The protein coating properties that include morphology, appearance, and barrier and mechanical properties will be influenced by the drying method used. As for dry process, extrusion method is one of the most common techniques that is being applied in conventional industrial method for protein coating. An extruder works by allowing the polymer to melt at high temperature during a relatively short time. The mechanical action of the screw and temperature exerts the material to melt, convey, compress, shear, mix, undergo variation of its amorphous content, optionally react, and be finally shaped through a die of a desired shape [10].

The protein-based film and coating were commonly being tested on their mechanical (tensile strength, elongation at break, and Young's modulus), barrier, (water vapor permeability and oxygen permeability), and physical (color and transparency) properties. However, due to its hydrophilic nature, protein-based film and coating also have high sensitivity to moisture and poor water vapor barrier properties. Thus, many studies have been conducted in order to improve and modify the functionality of protein-based film and coating as food packaging which includes the addition of different substances or agents such as cross linkers, plasticizers, and additives with antioxidant and antimicrobial properties. The incorporation of certain additives into packaging systems that intended to maintain or extend the quality of product or shelf life is referred as active packaging [17].

#### **3. Active packaging**

Active packaging is a medium which allows the interaction between the packaging, product, and environment. These systems involved the chemical, physical, and biological activities which change conditions of the packed food and help in extending the product's sustainability and shelf life. Moreover, active packaging is also able to enhance the microbiological safety and the sensory properties while maintaining the quality of the intended product [18]. Commonly, active packaging systems are concerned with substances that absorb (scavengers) or release (emitters) gases or steam which actively modifies the atmosphere inside packaging. Scavengers are used to remove unwanted items that commonly involved with the absorption of oxygen, ethylene, moisture, carbon dioxide, and flavors/odors from the environment into the internal packaging, while emitters are designed to release desired items that have a positive impact on food into the packaging environment that are commonly associated with the emitter of carbon dioxide, antimicrobial agents, antioxidants, and flavors [18]. Among those, antimicrobial packaging has been considered as the most promising method which incorporated antimicrobial agents into food packaging system that help in controlling the undesirable growth of a microorganism while extending the product's safety and shelf life [19]. As protein structure is comprised of hydrophilic nature, it can allow the control release of additive and bioactive compounds which make the protein-based film as one of the most promising media to be used in designated active antimicrobial packaging application.

#### **3.1 Protein-based film as active packaging**

Protein-based edible films were usually made from protein solutions or dispersions as the solvent/carrier evaporates. The solvent/carrier is normally composed of either water, ethanol, or ethanol-water mixtures [11]. Even though they exhibit

**57**

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review*

sources such as gelatin, casein, whey, corn zein, and wheat gluten.

great inhibition toward growth of microorganism and pathogens.

agents against growth of microorganism and pathogens.

*3.1.2 Casein-based film as active packaging*

poor water resistance, however, they are better when compared to polysaccharides in film-forming ability with good mechanical and barrier properties [20]. Proteinbased film as active antimicrobial packaging is designed based on the diffusion of incorporated antimicrobial compounds to the product's surface while aiming to extend the shelf life period [21]. The antimicrobial activity depends on the rate of active compound diffusion by the antimicrobial agents which depends on several factors such as chemical compatibility with polymer matrix, headspace humidity, the physicochemical properties of the product which is being tested on, antimicrobial solubility in tested food, and also the released temperature [21]. Active antimicrobial packaging from protein-based edible films can be derived from various

Gelatin is a protein obtained by hydrolyzing the collagen contained in bones and skin of animals. Physical and chemical properties of the gelatin produced are greatly affected by the sources, age of animal, collagen type, and extraction method used [22]. The global gelatin production was 348.9 kilo tons in 2011 and is expected to reach 450.7 kilo tons in 2018, growing at a compound annual growth rate (CAGR) of 3.73% from 2012 to 2018 [23]. Among all protein sources, gelatin is being one of the most extensively studied due to its good filming properties while performing its duties to protect and extend the shelf life of food products. Many antimicrobial agents have been incorporated into a gelatin-based film such as metal ions, essential oils, natural extracts, polymers, organic acids, and bacteriocins which resulted in

For example, the gelatin-based active nanocomposite films containing silver nanoparticles (AgNPs) resulted in high antimicrobial activity against both Gramnegative (*Escherichia coli*) and Gram-positive (*Listeria monocytogenes*) bacteria. The bacterial inhibition might be due to the interaction of AgNPs with phosphorous and sulfur binding to microbial DNA and prevents bacterial replication which leads to cell death [24]. While, gelatin-based film with incorporation of oregano essential oil is found to be effective against Gram-negative (*Salmonella enteritidis* and *Escherichia coli*) and Gram-positive (*Staphylococcus aureus* and *Listeria monocytogenes*) bacteria. These antimicrobial properties might be attributed by the presence of two phenols (carvacrol and thymol) and monoterpene hydrocarbons (p-cymene and γ-terpinene) compounds which are present in oregano essential oils [25]. Based on another study, the incorporation of citric acid into the gelatin-based film also showed reduction of growth for Gram-negative bacteria (*Escherichia coli*) [26]. Citric acid has pKa 4.8 that makes cell membrane become permeable and allows the acid to enter the cell. Upon entering the cytoplasm, the acid will dissociate, thus lowering the internal pH of the cell which leads to disruption of cellular functions of a microorganism [27]. Based on the results of this study, it showed that gelatin with the incorporation of antimicrobial agents has resulted in excellent properties as active antimicrobial food packaging as they managed to inhibit microbial growth of the food product. Thus, it can be concluded that gelatin-based film has emerged as one of the most widely studied biopolymer in film processing sector as compared to other sources of protein-based film while showing great potential as a medium to release or emit active antimicrobial

Casein-based edible film production also has been numerously studied because they displayed high nutritional quality with good sensory properties. Casein is

*DOI: http://dx.doi.org/10.5772/intechopen.80774*

*3.1.1 Gelatin-based film as active packaging*

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review DOI: http://dx.doi.org/10.5772/intechopen.80774*

poor water resistance, however, they are better when compared to polysaccharides in film-forming ability with good mechanical and barrier properties [20]. Proteinbased film as active antimicrobial packaging is designed based on the diffusion of incorporated antimicrobial compounds to the product's surface while aiming to extend the shelf life period [21]. The antimicrobial activity depends on the rate of active compound diffusion by the antimicrobial agents which depends on several factors such as chemical compatibility with polymer matrix, headspace humidity, the physicochemical properties of the product which is being tested on, antimicrobial solubility in tested food, and also the released temperature [21]. Active antimicrobial packaging from protein-based edible films can be derived from various sources such as gelatin, casein, whey, corn zein, and wheat gluten.

#### *3.1.1 Gelatin-based film as active packaging*

*Active Antimicrobial Food Packaging*

a die of a desired shape [10].

**3. Active packaging**

the techniques that will be used for wet coating process. Different types of methods are applicable for the drying process such as drying under ambient conditions, hot air, infrared energy, or microwave energy. The protein coating properties that include morphology, appearance, and barrier and mechanical properties will be influenced by the drying method used. As for dry process, extrusion method is one of the most common techniques that is being applied in conventional industrial method for protein coating. An extruder works by allowing the polymer to melt at high temperature during a relatively short time. The mechanical action of the screw and temperature exerts the material to melt, convey, compress, shear, mix, undergo variation of its amorphous content, optionally react, and be finally shaped through

The protein-based film and coating were commonly being tested on their mechanical (tensile strength, elongation at break, and Young's modulus), barrier, (water vapor permeability and oxygen permeability), and physical (color and transparency) properties. However, due to its hydrophilic nature, protein-based film and coating also have high sensitivity to moisture and poor water vapor barrier properties. Thus, many studies have been conducted in order to improve and modify the functionality of protein-based film and coating as food packaging which includes the addition of different substances or agents such as cross linkers, plasticizers, and additives with antioxidant and antimicrobial properties. The incorporation of certain additives into packaging systems that intended to maintain or extend

the quality of product or shelf life is referred as active packaging [17].

Active packaging is a medium which allows the interaction between the packaging, product, and environment. These systems involved the chemical, physical, and biological activities which change conditions of the packed food and help in extending the product's sustainability and shelf life. Moreover, active packaging is also able to enhance the microbiological safety and the sensory properties while maintaining the quality of the intended product [18]. Commonly, active packaging systems are concerned with substances that absorb (scavengers) or release (emitters) gases or steam which actively modifies the atmosphere inside packaging. Scavengers are used to remove unwanted items that commonly involved with the absorption of oxygen, ethylene, moisture, carbon dioxide, and flavors/odors from the environment into the internal packaging, while emitters are designed to release desired items that have a positive impact on food into the packaging environment that are commonly associated with the emitter of carbon dioxide, antimicrobial agents, antioxidants, and flavors [18]. Among those, antimicrobial packaging has been considered as the most promising method which incorporated antimicrobial agents into food packaging system that help in controlling the undesirable growth of a microorganism while extending the product's safety and shelf life [19]. As protein structure is comprised of hydrophilic nature, it can allow the control release of additive and bioactive compounds which make the protein-based film as one of the most promising media to be used in designated active antimicrobial packaging

Protein-based edible films were usually made from protein solutions or dispersions as the solvent/carrier evaporates. The solvent/carrier is normally composed of either water, ethanol, or ethanol-water mixtures [11]. Even though they exhibit

**56**

application.

**3.1 Protein-based film as active packaging**

Gelatin is a protein obtained by hydrolyzing the collagen contained in bones and skin of animals. Physical and chemical properties of the gelatin produced are greatly affected by the sources, age of animal, collagen type, and extraction method used [22]. The global gelatin production was 348.9 kilo tons in 2011 and is expected to reach 450.7 kilo tons in 2018, growing at a compound annual growth rate (CAGR) of 3.73% from 2012 to 2018 [23]. Among all protein sources, gelatin is being one of the most extensively studied due to its good filming properties while performing its duties to protect and extend the shelf life of food products. Many antimicrobial agents have been incorporated into a gelatin-based film such as metal ions, essential oils, natural extracts, polymers, organic acids, and bacteriocins which resulted in great inhibition toward growth of microorganism and pathogens.

For example, the gelatin-based active nanocomposite films containing silver nanoparticles (AgNPs) resulted in high antimicrobial activity against both Gramnegative (*Escherichia coli*) and Gram-positive (*Listeria monocytogenes*) bacteria. The bacterial inhibition might be due to the interaction of AgNPs with phosphorous and sulfur binding to microbial DNA and prevents bacterial replication which leads to cell death [24]. While, gelatin-based film with incorporation of oregano essential oil is found to be effective against Gram-negative (*Salmonella enteritidis* and *Escherichia coli*) and Gram-positive (*Staphylococcus aureus* and *Listeria monocytogenes*) bacteria. These antimicrobial properties might be attributed by the presence of two phenols (carvacrol and thymol) and monoterpene hydrocarbons (p-cymene and γ-terpinene) compounds which are present in oregano essential oils [25]. Based on another study, the incorporation of citric acid into the gelatin-based film also showed reduction of growth for Gram-negative bacteria (*Escherichia coli*) [26]. Citric acid has pKa 4.8 that makes cell membrane become permeable and allows the acid to enter the cell. Upon entering the cytoplasm, the acid will dissociate, thus lowering the internal pH of the cell which leads to disruption of cellular functions of a microorganism [27]. Based on the results of this study, it showed that gelatin with the incorporation of antimicrobial agents has resulted in excellent properties as active antimicrobial food packaging as they managed to inhibit microbial growth of the food product. Thus, it can be concluded that gelatin-based film has emerged as one of the most widely studied biopolymer in film processing sector as compared to other sources of protein-based film while showing great potential as a medium to release or emit active antimicrobial agents against growth of microorganism and pathogens.

#### *3.1.2 Casein-based film as active packaging*

Casein-based edible film production also has been numerously studied because they displayed high nutritional quality with good sensory properties. Casein is

commonly found in mammalian milk or in dairy products. Casein proteins comprise 80% of the total protein content in milk which precipitated from skim milk by acidifying the milk to produce acid casein to its isoelectric point of approximately 4.6 or the milk is treated with rennet to produce rennet casein. The casein is then being separated, washed, and dried [11, 28]. Casein is mainly comprised of three principal components, α, β, and κ, that formed colloidal micelles in milk which contains numerous amounts of casein molecules that are being stabilized by a calcium-phosphate bridge [11]. Due to excellent functional properties and natural abundant sources, caseins are used in numerous manufactured products such as in bakery applications, beverages, milk product, snack foods, edible films, etc. Casein or caseinates in the world market used in the food industry were reported in the range between 200,000 and 2,500,000 tons [28]. Caseins and caseinates can be prompted into edible films from aqueous solutions. Edible casein films are able to form a good barrier against oxygen and other nonpolar molecules because casein helps in supplying a great quantity of polar functional groups, such as hydroxyl and amino groups toward the film matrix. This property allows the casein film to be used as active packaging and can be combined with other packaging materials to protect products which are prone to oxidation or moisture [29].

In a study conducted by Arrieta et al. [30], sodium and calcium caseinate films with addition of carvacrol showed antibacterial effectiveness against both Gramnegative (*Escherichia coli*) and Gram-positive (*Staphylococcus aureus*) bacteria, while sodium caseinate-based edible film containing *Zataria multiflora Boiss*. essential oil exhibited a large inhibitory effect on Gram-positive (*Staphylococcus aureus*) followed by Gram-negative (*Salmonella Typhimurium* and *Escherichia coli*) bacteria [31]. Meanwhile, Oussalah et al. [32] mentioned that calcium caseinate and whey protein isolate edible films containing carboxymethyl cellulose with addition of 1% oregano essential oil showed inhibitory effect against Gram-negative bacteria that were *Escherichia coli* and *Pseudomonas* spp. on the surface of beefsteaks. The antimicrobial activity was mainly derived from phenolic compounds (carvacrol and thymol) which are present in the essential oil. The inhibition effect was done by interacting with the lipid bilayer of cytoplasmic membranes, causing them to be more permeable, which later induced and increased uptake of antibiotics by the bacterial cell [33]. From this study, it can be seen that caseinate film provides good matrices for the antimicrobial agent to release the active compounds that help to inhibit the growth of microorganisms.

#### *3.1.3 Whey protein-based film as active packaging*

Whey is a by-product derived from cheese-making process which is being defined as the remaining matter in the milk serum after coagulation of casein at pH 4.6 and temperature of 20°C. Whey protein is comprised of several individual proteins known as beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin, and immunoglobulins [15]. The global whey protein market is projected to reach the compound annual growth rate (CAGR) of 7.5% from 2018 to 2023, and the demand was estimated at a value of \$9.4 billion in 2017 [34]. The recovering process of whey solid component helps in reducing the organic pollution evolved from whey wastes while being able to optimally utilize the nutritional and functional properties provided by whey protein to be used in diverse sector [35]. The demand for whey protein among producers of food and beverages is increasing as they capitalize on the functional benefits of whey protein in various products such as sports nutrition, confectionery, bakery and ice cream products, infant formula, and health foods. Recent study has developed an alternative use of whey protein products to form edible film and coatings on surface of food products [36]. Whey protein-based films

**59**

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review*

are found to exhibit clear, odorless with good barrier properties to oxygen and lipids [37]. They also provide good matrices which allow the combination with other packaging materials to enhance the film's functionality as an active film against

A study reported that whey protein-based films incorporated with oregano and garlic essential oil resulted in larger inhibitory zones on Gram-negative (*Escherichia coli* and *Salmonella Enteritidis*) and Gram-positive bacteria (*Staphylococcus aureus*, *Lactobacillus plantarum*, and *Listeria monocytogenes*) [38], while another study of whey protein-based films incorporated with oregano essential oil showed antimicrobial activity against fungus species (*Penicillium commune*) [39]. The inhibition toward the microorganism was prompted by thymol and carvacrol compounds that are mainly present in essential oil. Based on another study of whey protein isolate (WPI) films supplemented with *Lactobacillus sakei*, the bacterial reductions were observed for Gram-negative (*Escherichia coli*) and Gram-positive bacteria (*Listeria monocytogenes*) after 36 hours and 120 hours of refrigerated storage on beef cube sample, respectively [40]. *Lactobacillus sakei* produced bacteriocin known as sakacin P which tends to exhibit antimicrobial properties [41]. Meanwhile, the incorporation of acetic, lactic, propionic, and benzoic acids (5%, v/v each) into whey protein-based edible film showed great inhibition zones against Gram-negative (*Escherichia coli* and *Salmonella* sp.) and Gram-positive bacteria (*Lactobacillus bulgaricus* and *Streptococcus thermophiles*) [42]. The use of acids causes acidification of growth media through acid dissociation into the cytoplasm which then induced the microbial inhibition [27]. Whey protein-based film also yields excellent results toward growth of microorganism as it provides good polymeric matrices for the antimicrobial agents to emit the active compounds into the packaging system.

Zein is a major protein in corn that is being classified as prolamin protein which dissolved in 70–80% ethanol. Zein is a relatively hydrophobic and thermoplastic material. The high content of nonpolar amino acids found in zein component might have an association with the hydrophobic nature of zein [11]. The corn is processed by using four different methods: wet-milling, dry milling, dry-grind processing, and alkaline treatment. After that, zein is being extracted from these products/ coproducts of corn which could result in different properties and end uses. Corn wet-milling process yields a protein-rich coproduct called corn gluten meal (CGM) from which zein has been commercially extracted. The other methods are drymilled corn (DMC) in which fibrous material is being separated from grits. As for the dry-grind ethanol process, the corn is ground along with the subsequent saccharification and fermentation of glucose to ethanol, leaving behind the coproduct distillers' dried grains with solubles (DDGS). Fractions such as cellulosic materials and protein are concentrated in DDGS due to conversion of starch to sugars and subsequently ethanol. While, alkaline treatment method has been mainly utilized for the use of human consumption and only has little basis for zein extraction. Most zein extractions have been based on aqueous alcohol extractions, but many other solvents were reported to be able to solubilize zein too [43]. A report by Informa Economics, Inc. [44] showed that zein was clarified as high-value product, and the cost for purified zein production had achieved \$9–30/lb. Zein proteins have been found to serve good materials for coating in pharmaceutical products and food ingredients as they exhibit tough and hydrophobic grease-proof coating properties that make them resistant against microbial attacks. Other potential applications of zein include its usage in fiber, adhesive, coating, ceramic, ink, cosmetic, textile, chewing gum, and biodegradable plastics [45]. In addition, numerous studies have

*DOI: http://dx.doi.org/10.5772/intechopen.80774*

*3.1.4 Zein protein-based film as active packaging*

microorganism or moisture.

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review DOI: http://dx.doi.org/10.5772/intechopen.80774*

*Active Antimicrobial Food Packaging*

commonly found in mammalian milk or in dairy products. Casein proteins comprise 80% of the total protein content in milk which precipitated from skim milk by acidifying the milk to produce acid casein to its isoelectric point of approximately 4.6 or the milk is treated with rennet to produce rennet casein. The casein is then being separated, washed, and dried [11, 28]. Casein is mainly comprised of three principal components, α, β, and κ, that formed colloidal micelles in milk which contains numerous amounts of casein molecules that are being stabilized by a calcium-phosphate bridge [11]. Due to excellent functional properties and natural abundant sources, caseins are used in numerous manufactured products such as in bakery applications, beverages, milk product, snack foods, edible films, etc. Casein or caseinates in the world market used in the food industry were reported in the range between 200,000 and 2,500,000 tons [28]. Caseins and caseinates can be prompted into edible films from aqueous solutions. Edible casein films are able to form a good barrier against oxygen and other nonpolar molecules because casein helps in supplying a great quantity of polar functional groups, such as hydroxyl and amino groups toward the film matrix. This property allows the casein film to be used as active packaging and can be combined with other packaging materials to

protect products which are prone to oxidation or moisture [29].

inhibit the growth of microorganisms.

*3.1.3 Whey protein-based film as active packaging*

In a study conducted by Arrieta et al. [30], sodium and calcium caseinate films with addition of carvacrol showed antibacterial effectiveness against both Gramnegative (*Escherichia coli*) and Gram-positive (*Staphylococcus aureus*) bacteria, while sodium caseinate-based edible film containing *Zataria multiflora Boiss*. essential oil exhibited a large inhibitory effect on Gram-positive (*Staphylococcus aureus*) followed by Gram-negative (*Salmonella Typhimurium* and *Escherichia coli*) bacteria [31]. Meanwhile, Oussalah et al. [32] mentioned that calcium caseinate and whey protein isolate edible films containing carboxymethyl cellulose with addition of 1% oregano essential oil showed inhibitory effect against Gram-negative bacteria that were *Escherichia coli* and *Pseudomonas* spp. on the surface of beefsteaks. The antimicrobial activity was mainly derived from phenolic compounds (carvacrol and thymol) which are present in the essential oil. The inhibition effect was done by interacting with the lipid bilayer of cytoplasmic membranes, causing them to be more permeable, which later induced and increased uptake of antibiotics by the bacterial cell [33]. From this study, it can be seen that caseinate film provides good matrices for the antimicrobial agent to release the active compounds that help to

Whey is a by-product derived from cheese-making process which is being defined as the remaining matter in the milk serum after coagulation of casein at pH 4.6 and temperature of 20°C. Whey protein is comprised of several individual proteins known as beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin, and immunoglobulins [15]. The global whey protein market is projected to reach the compound annual growth rate (CAGR) of 7.5% from 2018 to 2023, and the demand was estimated at a value of \$9.4 billion in 2017 [34]. The recovering process of whey solid component helps in reducing the organic pollution evolved from whey wastes while being able to optimally utilize the nutritional and functional properties provided by whey protein to be used in diverse sector [35]. The demand for whey protein among producers of food and beverages is increasing as they capitalize on the functional benefits of whey protein in various products such as sports nutrition, confectionery, bakery and ice cream products, infant formula, and health foods. Recent study has developed an alternative use of whey protein products to form edible film and coatings on surface of food products [36]. Whey protein-based films

**58**

are found to exhibit clear, odorless with good barrier properties to oxygen and lipids [37]. They also provide good matrices which allow the combination with other packaging materials to enhance the film's functionality as an active film against microorganism or moisture.

A study reported that whey protein-based films incorporated with oregano and garlic essential oil resulted in larger inhibitory zones on Gram-negative (*Escherichia coli* and *Salmonella Enteritidis*) and Gram-positive bacteria (*Staphylococcus aureus*, *Lactobacillus plantarum*, and *Listeria monocytogenes*) [38], while another study of whey protein-based films incorporated with oregano essential oil showed antimicrobial activity against fungus species (*Penicillium commune*) [39]. The inhibition toward the microorganism was prompted by thymol and carvacrol compounds that are mainly present in essential oil. Based on another study of whey protein isolate (WPI) films supplemented with *Lactobacillus sakei*, the bacterial reductions were observed for Gram-negative (*Escherichia coli*) and Gram-positive bacteria (*Listeria monocytogenes*) after 36 hours and 120 hours of refrigerated storage on beef cube sample, respectively [40]. *Lactobacillus sakei* produced bacteriocin known as sakacin P which tends to exhibit antimicrobial properties [41]. Meanwhile, the incorporation of acetic, lactic, propionic, and benzoic acids (5%, v/v each) into whey protein-based edible film showed great inhibition zones against Gram-negative (*Escherichia coli* and *Salmonella* sp.) and Gram-positive bacteria (*Lactobacillus bulgaricus* and *Streptococcus thermophiles*) [42]. The use of acids causes acidification of growth media through acid dissociation into the cytoplasm which then induced the microbial inhibition [27]. Whey protein-based film also yields excellent results toward growth of microorganism as it provides good polymeric matrices for the antimicrobial agents to emit the active compounds into the packaging system.

#### *3.1.4 Zein protein-based film as active packaging*

Zein is a major protein in corn that is being classified as prolamin protein which dissolved in 70–80% ethanol. Zein is a relatively hydrophobic and thermoplastic material. The high content of nonpolar amino acids found in zein component might have an association with the hydrophobic nature of zein [11]. The corn is processed by using four different methods: wet-milling, dry milling, dry-grind processing, and alkaline treatment. After that, zein is being extracted from these products/ coproducts of corn which could result in different properties and end uses. Corn wet-milling process yields a protein-rich coproduct called corn gluten meal (CGM) from which zein has been commercially extracted. The other methods are drymilled corn (DMC) in which fibrous material is being separated from grits. As for the dry-grind ethanol process, the corn is ground along with the subsequent saccharification and fermentation of glucose to ethanol, leaving behind the coproduct distillers' dried grains with solubles (DDGS). Fractions such as cellulosic materials and protein are concentrated in DDGS due to conversion of starch to sugars and subsequently ethanol. While, alkaline treatment method has been mainly utilized for the use of human consumption and only has little basis for zein extraction. Most zein extractions have been based on aqueous alcohol extractions, but many other solvents were reported to be able to solubilize zein too [43]. A report by Informa Economics, Inc. [44] showed that zein was clarified as high-value product, and the cost for purified zein production had achieved \$9–30/lb. Zein proteins have been found to serve good materials for coating in pharmaceutical products and food ingredients as they exhibit tough and hydrophobic grease-proof coating properties that make them resistant against microbial attacks. Other potential applications of zein include its usage in fiber, adhesive, coating, ceramic, ink, cosmetic, textile, chewing gum, and biodegradable plastics [45]. In addition, numerous studies have

been conducted on utilization of zein protein on development of biodegradable films as it exhibits good film-forming properties. Zein film production involves the development of hydrophobic, hydrogen, and limited disulfide bonds between zein chains in the film matrix [11]. Moreover, zein also showed good properties as carrier for antimicrobial agents such as lysozyme, lactoperoxidase, glucose oxidase, bacteriocins, plant phenolics, and essential oils [20].

A study conducted by Moradi et al. [46] has proved that zein-based film showed excellent antimicrobial properties through the release time of antibacterial agent from the film matrix into minced meat. In this literature, the zein and *Zataria multiflora Boiss.* essential oil-incorporated film has resulted in effective inhibition against both Gram-negative (*Escherichia coli*) and Gram-positive bacteria (*Listeria monocytogenes*) during 3 days of storage at 4°C. The result obtained corresponded with the finding by Kashiri et al. [21] which stated that the results showed that films containing *Zataria multiflora Boiss*. essential oil at 5% (g of essential oil/g of dry zein powder) achieved reductions of 1.18 log and 1.14 log against Gram-positive (*Listeria monocytogenes*) and Gram-negative (*Escherichia coli*) bacteria, respectively. While, as the concentration *Zataria multiflora Boiss*. essential oil being increased to 10%, the log reduction value increased to 2.16 log and 2.65 log for films against Gram-positive (*Listeria monocytogenes*) and Gram-negative (*Escherichia coli*) bacteria, respectively. From this study, the antimicrobial effect can be explained by the major compound (thymol and carvacrol) found in *Zataria multiflora Boiss.* essential oil which prevents the further growth of microorganism. The study also focused on addition of monolaurin into zein-based film which resulted in effective antimicrobial activity against Gram-positive bacteria (*Listeria monocytogenes*) during 3 days of storage at 4°C. The inhibition effect toward microorganism by monolaurin is caused by the interference with cytoplasmic membrane of microorganisms [46]. In another study by Mei et al. [47], the antimicrobial activity of silver nanocluster (AgNCs) and AgNO3- embedded zein film was tested on pathogenic *Escherichia coli*. The study showed that there were inhibition zones present with 1.95 and 2.05 mm at concentration of 10 μg Ag of AgNCs and AgNO3, respectively. Silver particles exhibit great antimicrobial activity as they can bind to the bacterial cell wall and cell membrane and inhibit the respiration process of microorganisms, while for the case of *Escherichia coli* inhibition, silver inhibits the uptake of phosphate while releasing succinate, proline, phosphate, mannitol, and glutamine from *Escherichia coli* cells [48]. Thus, based on all the results obtained by this study, it can be concluded that zein-based film does result in good compatibility with incorporation of antimicrobial agents. This is because the antimicrobial agents are able to release the antimicrobial compound agent into the packaging system and managed to inhibit the growth of microorganism.

#### **4. Antimicrobial agent in food packaging**

Active antimicrobial packaging involved the continuous interaction with the food product over specific shelf life by actively altering the internal environment [49]. In antimicrobial packaging system, the prevention and reducing growth rate of microorganism by extending the lag period will occur once the antimicrobial agents have been acquired [50]. An extensive study discussed the incorporation of antimicrobial agents in food packaging system from various organic and inorganic sources such as natural extracts (green tea), essential oils (clove, oregano, and thyme), enzyme (lysozyme), polymer (chitosan), organic acid (acetic acid, lactic acid, and benzoic acid), bacteriocins (nisin), and metal ions (zinc oxide and silver nanoparticles). However, due to stability of organic sources when exposed to extreme temperature, their use and application as antimicrobial agents might be

**61**

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review*

limited. Thus, inorganic metals have gained more interest as they are relatively more

Organic sources of antimicrobial agents can be comprised of animal and plant origin, microbial metabolites, and organic acids. As majority of organic antimicrobials derived from natural origin, they are thus able to inactivate microorganisms and enzymes without affecting the organoleptic or nutritional properties of the

Plant-derived antimicrobial agents possessed phenolic compounds that are able to alter the permeability of microbial cell and interfere with cell membrane functionality such as electron transport, protein synthesis, nutrient uptake, and enzyme activity, while the phenolic compounds also allow the loss of biomolecules such as ribose and sodium glutamate from inside the cell [52]. A wide range of study on antimicrobial agents that derived from plant origin mainly discussed on essential oils and natural extracts. The antimicrobial activity of essential oils is based on their molecular hydrophobicity which allows strong interaction with the lipids of cell membrane through their existence of phenolic compounds. This action will then increase the permeability of cell membrane and disturb the functionality and structure of the cell

which leads to leakage of ions and cytoplasmic content inside the cell [53].

In a study conducted by Yanwong and Threepopnatkul [54], the fish skin gelatin-edible films were incorporated with peppermint and citronella essential oils at different concentrations (10, 20, and 30%, w/w). The study showed that the incorporation of both essential oils exhibited excellent antibacterial properties against both Gram-negative (*Escherichia coli*) and Gram-positive (*Staphylococcus aureus*) bacteria. The inhibition activities were triggered by the presence of a major constituent in both essential oils that were identified as *p*-menthone and menthol and citronellal and citronellol for peppermint and citronella essential oils, respectively [55, 56]. While, Martucci et al. [57] observed the antimicrobial activity of oregano and lavender essential oils incorporated into gelatin film that were tested against *Escherichia coli* and *Staphylococcus aureus*. The study found that both essential oils exhibited good antimicrobial properties against the tested bacteria which were Gram-negative (*Escherichia coli*) and Gram-positive bacteria (*Staphylococcus aureus*) in concentrations above 2000 ppm. Oregano essential oil contained carvacrol and thymol, while lavender essential oil revealed a prevalence of linalool and camphor as their major compounds which exhibited good antimicrobial activity against tested microorganism. Another study mentioned that whey protein film with addition of 1–4% of cinnamon oil was tested against *Escherichia coli* and *Staphylococcus aureus*. However, the study showed antimicrobial activity only against *Staphylococcus aureus* with the highest inhibition zone at 4% addition of cinnamon oil into the whey protein film [58]. In addition, a major compound in cinnamon oil that exhibited the antimicrobial activity was identified as transcinnamaldehyde or cinnamaldehyde [59]. In the literature, it was mentioned that *Escherichia coli* exerted more resistance to cinnamon essential oil as compared to *Staphylococcus aureus*. This is due to the difference in bacteria's outer membrane structure. Gram-negative bacteria (*Escherichia coli*) possessed a thicker layer of the lipopolysaccharide outer membrane around the cell wall which is shown to be more resistant to hydrophobic substance of essential oil as compared with the Gram-positive (*Staphylococcus aureus*), which possesses single peptidoglycan layer

*DOI: http://dx.doi.org/10.5772/intechopen.80774*

**4.1 Organic sources as antimicrobial agents**

stable at higher temperatures [51].

*4.1.1 Plant-derived antimicrobial agent*

food products.

limited. Thus, inorganic metals have gained more interest as they are relatively more stable at higher temperatures [51].

#### **4.1 Organic sources as antimicrobial agents**

*Active Antimicrobial Food Packaging*

bacteriocins, plant phenolics, and essential oils [20].

been conducted on utilization of zein protein on development of biodegradable films as it exhibits good film-forming properties. Zein film production involves the development of hydrophobic, hydrogen, and limited disulfide bonds between zein chains in the film matrix [11]. Moreover, zein also showed good properties as carrier for antimicrobial agents such as lysozyme, lactoperoxidase, glucose oxidase,

A study conducted by Moradi et al. [46] has proved that zein-based film showed excellent antimicrobial properties through the release time of antibacterial agent from the film matrix into minced meat. In this literature, the zein and *Zataria multiflora Boiss.* essential oil-incorporated film has resulted in effective inhibition against both Gram-negative (*Escherichia coli*) and Gram-positive bacteria (*Listeria monocytogenes*) during 3 days of storage at 4°C. The result obtained corresponded with the finding by Kashiri et al. [21] which stated that the results showed that films containing *Zataria multiflora Boiss*. essential oil at 5% (g of essential oil/g of dry zein powder) achieved reductions of 1.18 log and 1.14 log against Gram-positive (*Listeria monocytogenes*) and Gram-negative (*Escherichia coli*) bacteria, respectively. While, as the concentration *Zataria multiflora Boiss*. essential oil being increased to 10%, the log reduction value increased to 2.16 log and 2.65 log for films against Gram-positive (*Listeria monocytogenes*) and Gram-negative (*Escherichia coli*) bacteria, respectively. From this study, the antimicrobial effect can be explained by the major compound (thymol and carvacrol) found in *Zataria multiflora Boiss.* essential oil which prevents the further growth of microorganism. The study also focused on addition of monolaurin into zein-based film which resulted in effective antimicrobial activity against Gram-positive bacteria (*Listeria monocytogenes*) during 3 days of storage at 4°C. The inhibition effect toward microorganism by monolaurin is caused by the interference with cytoplasmic membrane of microorganisms [46]. In another study by Mei et al. [47], the antimicrobial activity of silver nanocluster (AgNCs) and AgNO3- embedded zein film was tested on pathogenic *Escherichia coli*. The study showed that there were inhibition zones present with 1.95 and 2.05 mm at concentration of 10 μg Ag of AgNCs and AgNO3, respectively. Silver particles exhibit great antimicrobial activity as they can bind to the bacterial cell wall and cell membrane and inhibit the respiration process of microorganisms, while for the case of *Escherichia coli* inhibition, silver inhibits the uptake of phosphate while releasing succinate, proline, phosphate, mannitol, and glutamine from *Escherichia coli* cells [48]. Thus, based on all the results obtained by this study, it can be concluded that zein-based film does result in good compatibility with incorporation of antimicrobial agents. This is because the antimicrobial agents are able to release the antimicrobial compound agent into the

packaging system and managed to inhibit the growth of microorganism.

Active antimicrobial packaging involved the continuous interaction with the food product over specific shelf life by actively altering the internal environment [49]. In antimicrobial packaging system, the prevention and reducing growth rate of microorganism by extending the lag period will occur once the antimicrobial agents have been acquired [50]. An extensive study discussed the incorporation of antimicrobial agents in food packaging system from various organic and inorganic sources such as natural extracts (green tea), essential oils (clove, oregano, and thyme), enzyme (lysozyme), polymer (chitosan), organic acid (acetic acid, lactic acid, and benzoic acid), bacteriocins (nisin), and metal ions (zinc oxide and silver nanoparticles). However, due to stability of organic sources when exposed to extreme temperature, their use and application as antimicrobial agents might be

**4. Antimicrobial agent in food packaging**

**60**

Organic sources of antimicrobial agents can be comprised of animal and plant origin, microbial metabolites, and organic acids. As majority of organic antimicrobials derived from natural origin, they are thus able to inactivate microorganisms and enzymes without affecting the organoleptic or nutritional properties of the food products.

#### *4.1.1 Plant-derived antimicrobial agent*

Plant-derived antimicrobial agents possessed phenolic compounds that are able to alter the permeability of microbial cell and interfere with cell membrane functionality such as electron transport, protein synthesis, nutrient uptake, and enzyme activity, while the phenolic compounds also allow the loss of biomolecules such as ribose and sodium glutamate from inside the cell [52]. A wide range of study on antimicrobial agents that derived from plant origin mainly discussed on essential oils and natural extracts. The antimicrobial activity of essential oils is based on their molecular hydrophobicity which allows strong interaction with the lipids of cell membrane through their existence of phenolic compounds. This action will then increase the permeability of cell membrane and disturb the functionality and structure of the cell which leads to leakage of ions and cytoplasmic content inside the cell [53].

In a study conducted by Yanwong and Threepopnatkul [54], the fish skin gelatin-edible films were incorporated with peppermint and citronella essential oils at different concentrations (10, 20, and 30%, w/w). The study showed that the incorporation of both essential oils exhibited excellent antibacterial properties against both Gram-negative (*Escherichia coli*) and Gram-positive (*Staphylococcus aureus*) bacteria. The inhibition activities were triggered by the presence of a major constituent in both essential oils that were identified as *p*-menthone and menthol and citronellal and citronellol for peppermint and citronella essential oils, respectively [55, 56]. While, Martucci et al. [57] observed the antimicrobial activity of oregano and lavender essential oils incorporated into gelatin film that were tested against *Escherichia coli* and *Staphylococcus aureus*. The study found that both essential oils exhibited good antimicrobial properties against the tested bacteria which were Gram-negative (*Escherichia coli*) and Gram-positive bacteria (*Staphylococcus aureus*) in concentrations above 2000 ppm. Oregano essential oil contained carvacrol and thymol, while lavender essential oil revealed a prevalence of linalool and camphor as their major compounds which exhibited good antimicrobial activity against tested microorganism. Another study mentioned that whey protein film with addition of 1–4% of cinnamon oil was tested against *Escherichia coli* and *Staphylococcus aureus*. However, the study showed antimicrobial activity only against *Staphylococcus aureus* with the highest inhibition zone at 4% addition of cinnamon oil into the whey protein film [58]. In addition, a major compound in cinnamon oil that exhibited the antimicrobial activity was identified as transcinnamaldehyde or cinnamaldehyde [59]. In the literature, it was mentioned that *Escherichia coli* exerted more resistance to cinnamon essential oil as compared to *Staphylococcus aureus*. This is due to the difference in bacteria's outer membrane structure. Gram-negative bacteria (*Escherichia coli*) possessed a thicker layer of the lipopolysaccharide outer membrane around the cell wall which is shown to be more resistant to hydrophobic substance of essential oil as compared with the Gram-positive (*Staphylococcus aureus*), which possesses single peptidoglycan layer

structure [59]. Thus, from this study, it can be seen that all mentioned essential oils exhibited certain compounds that exerted good antimicrobial activity which could enhance the food product shelf life and stability.

#### *4.1.2 Animal-derived antimicrobial agent*

Antimicrobial agents originated from animal sources are commonly being used as they exhibited good resistance and inhibition toward growth of microorganism. They evolved as part of defense mechanisms in antimicrobial system. Most of the antimicrobial agents derived from animals emerged in the form of antimicrobial peptides such as pleurocidin, lactoferrin, defensins, and protamine [52]. These peptides were applicable as antibiotic resistant as they are able to destruct the cellular lipid bilayer membranes and can hinder even the fast-growing microorganism to mutate. Furthermore, they have good antimicrobial activity against both Gram-positive and Gram-negative bacteria while also showing antifungal and antiviral activities [60]. There are other effective antimicrobial enzymes which come from egg white, milk, and blood that are known as lysozyme. A study by Kaewprachu et al. [61] reported that minced pork wrapped with catechin-lysozyme which incorporated with gelatin film resulted in lower counts of total plate count, yeasts, and molds than minced pork that was wrapped with PVC film. The addition of lysozyme triggered the cleaving process of peptidoglycan in bacterial cell walls and resulted in lysis of bacterial cell. This result showed that catechin-lysozyme/gelatin film could inhibit the microbial growth, while there are also various studies on certain polysaccharides and lipids from animals that showed excellent antimicrobial activity. For example, Pisoschi et al. [52] stated that chitin derived from the exoskeletons of crustaceans, insects, mollusks, and the cell wall of microorganisms exhibited antimicrobial properties. Apart from that, chitosan which is obtained from the exoskeletons of crustaceans and arthropods and existed as a deacetylated form of chitin also showed effective antifungal and antibacterial activities [52]. In a literature studied by Malinowska-Pañczyk et al. [62], the incorporation of chitosan was observed on its ability as antimicrobial agent against Gram-negative (*Escherichia coli* and *Pseudomonas fluorescens*) and Gram-positive (*Staphylococcus aureus* and *Listeria innocua*) bacteria. The study then revealed all strains of bacteria were completely inactivated after 24 hours of incubation period for gelatin film incorporated with chitosan-90. As for gelatin films with addition of chitosan-73, the results showed that only *Pseudomonas fluorescens* and *Listeria innocua* were completely inactivated, while *Escherichia coli* and *Staphylococcus aureus* cells were partially inactivated after 24 hours of incubation. The inhibition factor was due to the cationic nature of chitosan which induced the electrostatic interaction between positively charged RN (CH3)3 + sites and negatively charged microbial cell membranes which led to cellular lysis [63]. Therefore, it can be seen that antimicrobial agents derived from animals resulted good inhibitory effect against microorganism and can be applied in food packaging for enhancing shelf life and quality of intended products.

#### **4.2 Metallic sources as antimicrobial agents**

Metals have been widely used as antimicrobial agents for a long time due to their ability to cause injuries to microbial cells by exerting oxidative stress, protein dysfunction, or membrane damage [64]. Metal ions such as copper, silver, zinc, palladium, and titanium have been studied as active antimicrobial agents against a wide spectrum of bacteria, yeast, and fungi [65]. Due to stability of organic sources at higher temperature, their application in food packaging may be limited, thus giving great advantage to metallic sources that are more stable at higher temperature [51].

**63**

pathogens.

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review*

Zinc particle especially zinc oxide (ZnO) is being widely proposed to be used as antimicrobial agents with broad range of other applications due to their specialty to survive under harsh environment. The antimicrobial activity by zinc oxide (ZnO) particles were proposed due to emission of zinc ions (Zn2+), which are able to penetrate into the bacteria's cell wall and affect the cytoplasmic content in the cell that leads to the death of bacteria. The incorporation of zinc oxide nanoparticles into gelatin was observed by Divya et al. [66] which revealed that the film showed higher inhibitory effect against Gram-negative bacteria (*Pseudomonas aeruginosa*) than Gram-positive (*Enterococcus faecalis*) bacteria. The results corresponded with the statement which suggested that ZnO induced photocatalytic mechanism related to the semiconductive properties of ZnO which lead to the formation of reactive oxygen species (ROS) and H2O2 which damaged the cell wall structure of bacteria [67, 68]. The literature by Pasquet et al. [67] also stated that the lipid bilayer membrane of Gram-negative bacteria was more sensitive toward reactive oxygen species (ROS) produced by ZnO particles than the thick membrane of Gram-positive bacteria that is coated by a peptidoglycan protective layer. Meanwhile, the study on gelatin/ZnO nanoparticles of nanocomposite films showed great antibacterial activity against both Gram-positive (*Listeria monocytogenes*) and Gram-negative (*Escherichia coli*) foodborne pathogenic bacteria. The study discussed that the antimicrobial activity toward both Gram-negative and Gram-positive bacteria was due to the easy penetration of nanoparticles in the cytoplasmic content of the cell which then leads to death of cell [69]. Thus, it can be concluded that incorporation of zinc particles into protein-based film helps in exerting antimicrobial activity against foodborne

The application of silver particles has received great attention from the researchers from all over the world due to their wide spectrum and application in antimicrobial packaging. Silver is often used in the size of nanoparticles as they give more potent effect against foodborne pathogen due to their enhanced catalytic reactivity owing to its large surface area to volume ratio. Based on a study conducted by Kanmani and Rhim [24], the incorporation of silver nanoparticles (AgNPs) into gelatin film was tested against Gram-positive (*Listeria monocytogenes*) and Gramnegative (*Escherichia coli*) bacteria. The results mentioned that the AgNP/gelatin biocomposite film showed high-inhibitory effect against both tested microorganisms. This might be due the interaction of AgNPs with compounds of protein and DNA in the cell that contain phosphorous and sulfur which prevent DNA replication and cause death of the cell. Furthermore, some study suggested that positively charged AgNPs are able to bind with negatively charged bacterial cell membranes that cause disruption of cell walls by shrinkage of the cytoplasm and membrane detachment that led to cell death [70]. In this literature, it also stated that AgNPs could penetrate the bacteria which inactivate the enzymes and induce the production of H2O2 and cause cell to die [24]. Meanwhile, another study by Mei et al. [47] observed on the antimicrobial activity of silver nanocluster (AgNC)-embedded zein film against pathogenic *Escherichia coli*. The study showed that adding 10 μg of AgNCs into zein film resulted in great antimicrobial effect as indicated by inhibition zones of 1.95. In this literature, it stated that the inhibitory effect was influenced by the release rate of Ag that was embedded in zein films. Since AgNCs had high surface to volume ratio due to its ultra-small size, it thus led to greater surface

*DOI: http://dx.doi.org/10.5772/intechopen.80774*

*4.2.1 Zinc particles as antimicrobial agents*

*4.2.2 Silver particles as antimicrobial agents*

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review DOI: http://dx.doi.org/10.5772/intechopen.80774*

#### *4.2.1 Zinc particles as antimicrobial agents*

*Active Antimicrobial Food Packaging*

enhance the food product shelf life and stability.

*4.1.2 Animal-derived antimicrobial agent*

structure [59]. Thus, from this study, it can be seen that all mentioned essential oils exhibited certain compounds that exerted good antimicrobial activity which could

Antimicrobial agents originated from animal sources are commonly being used as they exhibited good resistance and inhibition toward growth of microorganism. They evolved as part of defense mechanisms in antimicrobial system. Most of the antimicrobial agents derived from animals emerged in the form of antimicrobial peptides such as pleurocidin, lactoferrin, defensins, and protamine [52]. These peptides were applicable as antibiotic resistant as they are able to destruct the cellular lipid bilayer membranes and can hinder even the fast-growing microorganism to mutate. Furthermore, they have good antimicrobial activity against both Gram-positive and Gram-negative bacteria while also showing antifungal and antiviral activities [60]. There are other effective antimicrobial enzymes which come from egg white, milk, and blood that are known as lysozyme. A study by Kaewprachu et al. [61] reported that minced pork wrapped with catechin-lysozyme which incorporated with gelatin film resulted in lower counts of total plate count, yeasts, and molds than minced pork that was wrapped with PVC film. The addition of lysozyme triggered the cleaving process of peptidoglycan in bacterial cell walls and resulted in lysis of bacterial cell. This result showed that catechin-lysozyme/gelatin film could inhibit the microbial growth, while there are also various studies on certain polysaccharides and lipids from animals that showed excellent antimicrobial activity. For example, Pisoschi et al. [52] stated that chitin derived from the exoskeletons of crustaceans, insects, mollusks, and the cell wall of microorganisms exhibited antimicrobial properties. Apart from that, chitosan which is obtained from the exoskeletons of crustaceans and arthropods and existed as a deacetylated form of chitin also showed effective antifungal and antibacterial activities [52]. In a literature studied by Malinowska-Pañczyk et al. [62], the incorporation of chitosan was observed on its ability as antimicrobial agent against Gram-negative (*Escherichia coli* and *Pseudomonas fluorescens*) and Gram-positive (*Staphylococcus aureus* and *Listeria innocua*) bacteria. The study then revealed all strains of bacteria were completely inactivated after 24 hours of incubation period for gelatin film incorporated with chitosan-90. As for gelatin films with addition of chitosan-73, the results showed that only *Pseudomonas fluorescens* and *Listeria innocua* were completely inactivated, while *Escherichia coli* and *Staphylococcus aureus* cells were partially inactivated after 24 hours of incubation. The inhibition factor was due to the cationic nature of chitosan which induced the electrostatic interaction between positively charged RN (CH3)3

sites and negatively charged microbial cell membranes which led to cellular lysis [63]. Therefore, it can be seen that antimicrobial agents derived from animals resulted good inhibitory effect against microorganism and can be applied in food packaging for

Metals have been widely used as antimicrobial agents for a long time due to their ability to cause injuries to microbial cells by exerting oxidative stress, protein dysfunction, or membrane damage [64]. Metal ions such as copper, silver, zinc, palladium, and titanium have been studied as active antimicrobial agents against a wide spectrum of bacteria, yeast, and fungi [65]. Due to stability of organic sources at higher temperature, their application in food packaging may be limited, thus giving great advantage to metallic sources that are more stable at

enhancing shelf life and quality of intended products.

**4.2 Metallic sources as antimicrobial agents**

**62**

higher temperature [51].

Zinc particle especially zinc oxide (ZnO) is being widely proposed to be used as antimicrobial agents with broad range of other applications due to their specialty to survive under harsh environment. The antimicrobial activity by zinc oxide (ZnO) particles were proposed due to emission of zinc ions (Zn2+), which are able to penetrate into the bacteria's cell wall and affect the cytoplasmic content in the cell that leads to the death of bacteria. The incorporation of zinc oxide nanoparticles into gelatin was observed by Divya et al. [66] which revealed that the film showed higher inhibitory effect against Gram-negative bacteria (*Pseudomonas aeruginosa*) than Gram-positive (*Enterococcus faecalis*) bacteria. The results corresponded with the statement which suggested that ZnO induced photocatalytic mechanism related to the semiconductive properties of ZnO which lead to the formation of reactive oxygen species (ROS) and H2O2 which damaged the cell wall structure of bacteria [67, 68]. The literature by Pasquet et al. [67] also stated that the lipid bilayer membrane of Gram-negative bacteria was more sensitive toward reactive oxygen species (ROS) produced by ZnO particles than the thick membrane of Gram-positive bacteria that is coated by a peptidoglycan protective layer. Meanwhile, the study on gelatin/ZnO nanoparticles of nanocomposite films showed great antibacterial activity against both Gram-positive (*Listeria monocytogenes*) and Gram-negative (*Escherichia coli*) foodborne pathogenic bacteria. The study discussed that the antimicrobial activity toward both Gram-negative and Gram-positive bacteria was due to the easy penetration of nanoparticles in the cytoplasmic content of the cell which then leads to death of cell [69]. Thus, it can be concluded that incorporation of zinc particles into protein-based film helps in exerting antimicrobial activity against foodborne pathogens.

#### *4.2.2 Silver particles as antimicrobial agents*

The application of silver particles has received great attention from the researchers from all over the world due to their wide spectrum and application in antimicrobial packaging. Silver is often used in the size of nanoparticles as they give more potent effect against foodborne pathogen due to their enhanced catalytic reactivity owing to its large surface area to volume ratio. Based on a study conducted by Kanmani and Rhim [24], the incorporation of silver nanoparticles (AgNPs) into gelatin film was tested against Gram-positive (*Listeria monocytogenes*) and Gramnegative (*Escherichia coli*) bacteria. The results mentioned that the AgNP/gelatin biocomposite film showed high-inhibitory effect against both tested microorganisms. This might be due the interaction of AgNPs with compounds of protein and DNA in the cell that contain phosphorous and sulfur which prevent DNA replication and cause death of the cell. Furthermore, some study suggested that positively charged AgNPs are able to bind with negatively charged bacterial cell membranes that cause disruption of cell walls by shrinkage of the cytoplasm and membrane detachment that led to cell death [70]. In this literature, it also stated that AgNPs could penetrate the bacteria which inactivate the enzymes and induce the production of H2O2 and cause cell to die [24]. Meanwhile, another study by Mei et al. [47] observed on the antimicrobial activity of silver nanocluster (AgNC)-embedded zein film against pathogenic *Escherichia coli*. The study showed that adding 10 μg of AgNCs into zein film resulted in great antimicrobial effect as indicated by inhibition zones of 1.95. In this literature, it stated that the inhibitory effect was influenced by the release rate of Ag that was embedded in zein films. Since AgNCs had high surface to volume ratio due to its ultra-small size, it thus led to greater surface

+

contact with bacteria and consequently exhibited higher antimicrobial activity. Therefore, based on this study, it can be confirmed that addition of silver particles into protein-based film is able to exhibit mechanism of antibacterial action against microorganisms.

#### **5. Application of protein-based active film in food packaging**

Protein-based edible film has gained great interest due to its wide application as edible food packaging as compared to synthetic films. In addition, it is able to provide good matrix and acts as a medium for incorporation of antimicrobial and antioxidant agents into the film to release or emit their specific functions that help in enhancing the safety, stability, functionality, and shelf life of food products. Moreover, it also can be applied to control the diffusion rate of preservative substances from the product's surface to the internal environment of food. Meanwhile, protein-based packaging also is being extensively used as food wrapper and is being applied at the interfaces between different layers of heterogeneous food, while protein-based edible films could be used together with nonedible film as multilayer food packaging materials where it can be employed as internal layers that have direct contact with food materials [11]. Furthermore, due to good permeability against oxygen, carbon dioxide, and water vapor, protein-based film can be applied to the surfaces of fresh-cut food product in order to extend shelf life of the product by delaying color changes and ripening and prevent the effect of enzymatic browning and reducing moisture and aroma loss [71]. Thus, it can be concluded that protein-based edible film exhibits good characteristics through their mechanical and barrier properties as food packaging which is able to substitute the utilization synthetic film packaging.

#### **6. Conclusion**

Antimicrobial food packaging have gained great interest due to high inhibition of microbial activity that helps in prolonging the shelf life of packaged food and enhancing the food's safety while improving the functionality of the film. The incorporation of antimicrobial agents from various organic and inorganic sources into protein-based edible film has been discussed, and their effectiveness was mainly found depending on their activity against the target microorganism, the types of polymer used, the film's properties, and the factor based on the packaged food's composition, pH, water activity, as well as the storage and environmental condition. However, there are some challenges in creating good antimicrobial films that follow the regulatory and industry requirements which also aim in producing at low production cost and are able to meet with the consumer demands without altering the sensory characteristics of the intended packaged food. Therefore, more studies need to be done on biocomposite protein-based packaging films with incorporation of antimicrobial agents that might also require chemical, toxicological, and further test in securing more safe and approved products according to the standard food safety regulations while being able to deliver good means in protecting the safety and quality of packaged food.

**65**

**Author details**

provided the original work is properly cited.

Nurul Saadah Said and Norizah Mhd Sarbon\*

\*Address all correspondence to: norizah@umt.edu.my

Kuala Nerus, Terengganu, Malaysia

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review*

*DOI: http://dx.doi.org/10.5772/intechopen.80774*

© 2019 The Author(s). Licensee IntechOpen. 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,

School of Food Science and Technology, Universiti Malaysia Terengganu,

*Protein-Based Active Film as Antimicrobial Food Packaging: A Review DOI: http://dx.doi.org/10.5772/intechopen.80774*

*Active Antimicrobial Food Packaging*

microorganisms.

synthetic film packaging.

safety and quality of packaged food.

**6. Conclusion**

contact with bacteria and consequently exhibited higher antimicrobial activity. Therefore, based on this study, it can be confirmed that addition of silver particles into protein-based film is able to exhibit mechanism of antibacterial action against

Protein-based edible film has gained great interest due to its wide application as edible food packaging as compared to synthetic films. In addition, it is able to provide good matrix and acts as a medium for incorporation of antimicrobial and antioxidant agents into the film to release or emit their specific functions that help in enhancing the safety, stability, functionality, and shelf life of food products. Moreover, it also can be applied to control the diffusion rate of preservative substances from the product's surface to the internal environment of food. Meanwhile, protein-based packaging also is being extensively used as food wrapper and is being applied at the interfaces between different layers of heterogeneous food, while protein-based edible films could be used together with nonedible film as multilayer food packaging materials where it can be employed as internal layers that have direct contact with food materials [11]. Furthermore, due to good permeability against oxygen, carbon dioxide, and water vapor, protein-based film can be applied to the surfaces of fresh-cut food product in order to extend shelf life of the product by delaying color changes and ripening and prevent the effect of enzymatic browning and reducing moisture and aroma loss [71]. Thus, it can be concluded that protein-based edible film exhibits good characteristics through their mechanical and barrier properties as food packaging which is able to substitute the utilization

Antimicrobial food packaging have gained great interest due to high inhibition of microbial activity that helps in prolonging the shelf life of packaged food and enhancing the food's safety while improving the functionality of the film. The incorporation of antimicrobial agents from various organic and inorganic sources into protein-based edible film has been discussed, and their effectiveness was mainly found depending on their activity against the target microorganism, the types of polymer used, the film's properties, and the factor based on the packaged food's composition, pH, water activity, as well as the storage and environmental condition. However, there are some challenges in creating good antimicrobial films that follow the regulatory and industry requirements which also aim in producing at low production cost and are able to meet with the consumer demands without altering the sensory characteristics of the intended packaged food. Therefore, more studies need to be done on biocomposite protein-based packaging films with incorporation of antimicrobial agents that might also require chemical, toxicological, and further test in securing more safe and approved products according to the standard food safety regulations while being able to deliver good means in protecting the

**5. Application of protein-based active film in food packaging**

**64**

## **Author details**

Nurul Saadah Said and Norizah Mhd Sarbon\* School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia

\*Address all correspondence to: norizah@umt.edu.my

© 2019 The Author(s). Licensee IntechOpen. 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.

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Sciences. 2016;**5**(8):878-888

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**71**

**Chapter 5**

**Abstract**

life extension

**1. Introduction**

*Saroat Rawdkuen*

Edible Films Incorporated

Properties and Application

foods, and providing better quality with high safety.

with Active Compounds: Their

Antimicrobial compounds are food additives, which play a major role to reduce food spoilage. There are three main groups of antimicrobial compounds such as chemical agent, natural extract, and probiotics. The direct incorporations of the active compounds on the surface of food may have limited benefit because they are rapidly diffused from the food surface into the food product, resulting in the limited efficacy of these compounds. Thus, incorporation of antimicrobial compounds into packaging matrix, especially biopolymer film is a promising technique to reduce contaminations and inhibit, retard, and/or kill the microorganisms. Edible films are thin layer of natural polymers used to maintain the physicochemical quality of foods and extend their shelf life. A variety of biopolymeric-based materials including polysaccharides, proteins, and lipids have been extensively used for antimicrobial packaging and can be used as a carrier of active compounds. Incorporation of antimicrobial compounds may or may not enhance the mechanical properties and water vapor permeability of biopolymer films. The applications of active films can reduce contamination through the releasing of antimicrobial compound, thus reducing the risk from pathogen, extending shelf life of the packaged

**Keywords:** antimicrobial, edible film, bioactive compounds, natural extract, shelf

Antimicrobial compounds are functional additives, which play a major role to reduce food spoilage, maintain quality, and increase the shelf life of foodstuffs. There are three main groups of antimicrobial compounds such as chemical agent, natural extracts, and probiotics. Recently, consumers are increasingly seeking foods containing natural-occurring substances rather than synthetic additive because some synthetic additives can promote carcinogenic and toxicity, which is a clear concern to the health of consumers [1]. The use of antimicrobial compounds by directly adding to food products may cause the reduction of active compounds' activity and may change the organoleptic properties of the foods due to the complexity of food components and strong flavor of some agents. Thus, the incorporation of antimicrobial compounds into packaging matrix is a promising technique that can increase

antimicrobial efficiency and solve the limitation of directly adding.

#### **Chapter 5**

*Active Antimicrobial Food Packaging*

growth. Revista Brasileira de Farmacognosia. 2016;**26**(1):122-127

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[71] Kore VT, Tawade SS, Kabir J. Application of edible coatings on fruits and vegetables. Imperial Journal of Interdisciplinary Research.

2017;**3**(1):591-603

[65] Martucci JF, Ruseckaite RA. Antibacterial activity of gelatin/

films. Food Hydrocolloids.

Biology. 2018;**178**:211-218

2014;**460**:92-100

2017;**64**:70-77

copper (II)-exchanged montmorillonite

[66] Divya M, Vaseeharan B, Abinaya M, Vijayakumar S, Govindarajan M, Alharbi NS, et al. Biopolymer gelatincoated zinc oxide nanoparticles showed high antibacterial, antibiofilm and anti-angiogenic activity. Journal of Photochemistry and Photobiology, B:

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[70] Dakal TC, Kumar A, Majumdar RS, Yadav V. Mechanistic basis of antimicrobial actions of silver nanoparticles. Frontiers in Microbiology. 2016;**7**:1831

Letters. 2015;**7**(3):219-242

**70**
