*SAKE* **Alcoholic Beverage Production in Japanese Food Industry**

Makoto Kanauchi

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[47] Pengue WA (2005) Transgenic crops in Argentina: The ecological and social debt.

[48] Tomei J & Upham P (2009) Argentinean soy-based biodiesel: An introduction to pro‐

month-old children in Madagascar. The Journal of Nutrition 138, 2448-2452.

ian and Malawian children. Ecology of Food Nutrition 29, 219-234.

Bulletin of Science Technology Society 25(4), 314-322.

38 Food Industry

duction and impacts. Energy Policy 37(10), 3890-3898.

[49] FEWS NET (2012) Price watch: May food prices, June 29, 2012.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53153

## **1. Introduction**

*SAKE* brewing is an important sector of the Japanese food industry. It has maintained a strong relation with the culture in areas producing it, as have other alcoholic beverages such as wine, beer, and tequila in other countries. *SAKE* has a history extending back 1000 years into antiquity, and brewers' skills and techniques have been cultivated scientifically for lon‐ ger than the discipline of chemistry has even existed. Particularly, low-temperature steriliza‐ tion of *SAKE* was conducted in the 16th century, before Louis Pasteur invented pasteurization. The method is carefully described in old Japanese literature.

The significance of *SAKE* culture and its old techniques of brewing has been investigated us‐ ing modern scientific analysis and brewing research methods. Furthermore, in *SAKE* brewing, unique techniques have been examined, such as fermenting under low temperature, achiev‐ ing more than 18% high alcohol concentrations without distillation, open fermentation sys‐ tems without sterilization, and creation of a fruity aroma in *SAKE*. Furthermore, yeast, mold, and the raw material––rice––have bred to be suitable *SAKE* brewing. Preferences for *SAKE* among young (20–30s) consumers have been elucidated recently, and the potential for new *SAKE* development has been reported. This report describes the history of *SAKE*, propagation methods of *SAKE*, its production materials, and recent research related to it.

## **2. History of** *SAKE*

Cultivation of rice, the raw material for *SAKE* brewing, originated in China. Seed rice har‐ vested more than 10 millennia ago have been found in Kiangsi province and Hunan prov‐ ince in China. Probably, Japanese rice was introduced from China, where rice was cultivated

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

in dry fields using dry rice cultivation methods. Introduced from China early, rice was culti‐ vated in dry fields in Japan also. However, the method of rice cultivation in paddy fields boosted yields to higher levels than those achieved in dry fields. The wet method brought social changes: a reliable labor force is necessary for cultivation by planting rice in paddy fields, harvesting it, and maintaining paddy fields, equipment, and irrigation. The labor force resources from families were limited. More labor was required from settlements. The settlements formed communities. Later communities formed ancient Japan. At that time, rice was of particular value: it was a divine food. *SAKE*, made from that divine food of rice, was also revered as blessed. It was used as a sacrifice to the gods. Moreover, people believed in a divine spirit indwelt in rice. Extending that belief, people believed that intoxication by alcohol beverages as *SAKE* made from rice brought gods into the human body. Further‐ more, they solidified the community by sharing divine foods as rice and beverage as *SAKE* among members as they cooperated in rice cultivation [1].

Between the Asuka-Nara Era and Heian Era (5th – 8th century), imperial families and cour‐ tiers established huge craft factories of which wide areas were dedicated to crafts. Many technicians and workers were employed in them, monopolizing practical technologies of all areas. Brewery also continued in such factories, and *SAKE* was brewed using advanced

*SAKE* Alcoholic Beverage Production in Japanese Food Industry

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41

During the Heian period (8th century and thereafter), *SAKE* rose to importance for use in regional ceremonies or banquets. *SAKE* brewing by SAKE-NO-TSUKASA was both a *SAKE* brewery and a supervisory office of the imperial court. Some kinds of *SAKE* were brewed for emperor, imperial family, and the aristocracy for use at ceremonies or banquets. Brewing methods were described in *ENGISHIKI*, an ancient book of codes and procedures related to national rites and prayers. For example, *GOSYU* was specially brewed for the Emperor us‐ ing steamed rice, *KOJI*, and mother water. The mash was fermented using wild fermentative yeast for ten days; then the mash was filtrated. The resultant *SAKE* was used for subsequent brewing as mother water. The *SAKE* brewed steamed rice, *KOJI*, and strained *SAKE* brewing were repeated four times to produce *SAKE* with a very sweet taste [1]. The literature in this period described *SAKE* of more than two kinds. The minor aristocracy and many people

Between the later Heian Era and Muromachi Era via the Kamakura Era (12th–16th century) *SAKE* was produced and sold at Buddhist temples and private breweries. During that peri‐ od, it was a popular alcoholic beverage. In the 12th century, the feudal government issued alcohol prohibition laws many times to maintain security. Officers destroyed *SAKE* contain‐

At the beginning of the Muromachi Era, according to the '*GOSHU-NO-NIKKI*', *SAKE* was brewed already using a modern process in which rice-*KOJI* and steamed rice and water were mashed successively step-by-step. Moreover, the techniques applied lactic-acid fer‐ mentation, which demonstrates protection of the mash from bacterial contamination and

During the 16th century, the *TAMON-IN* Diary was written for 100 years. *TAMON-IN* were small temples belonging to the *KOFUKUJI* temple in Nara. The diary described heating methods used to kill contaminated germs already in this century. In Europe, Louis Pasteur announced low-temperature pasteurization of wine and milk in 1865. However, Japanese

During the Edo Era, the brewing season extended from the autumnal equinox to the vernal. However, results show good tasting *SAKE* brewing conducted in midwinter using a method called '*KANZUKURI*'. The brewing techniques of those brewers in the Ikeda, Itami, and Na‐ da districts (Osaka City and Hyogo Prefecture) held the leadership in *SAKE* brewing at that time. During the Genroku period (end of the 17th Century), the total number of breweries

After the Meiji Era, *SAKE* brewing methods changed drastically based on European science. Many improvements of *SAKE* brewing were accomplished by applying beer-brewing meth‐ ods directly. However, many special techniques are used in *SAKE* production. For example,

brewers had acquired experimentally pasteurized *SAKE* during the 16th century [5].

were not able to drink *SAKE* because it was extremely expensive.

dominant growth of yeast during *SAKE* production [1, 5].

was reported as greater than 27,000 [5].

technologies during those eras.

ers throughout cities.

Alcoholic beverages can be made from cereals as beer, *SAKE*, or whisky. Saccharification processing is extremely important. Generally, it is an important feature of Asian alcoholic beverage production that mold cultivate in cereal, so-called '*KOJI*', is used for production. However, according to ancient literature, Osumi-no-Kuni-Fudoki, which recorded the cul‐ ture and geography of Kagoshima in ancient times before production of *SAKE* using *KOJI*, alcoholic beverages were made with saliva as a saccharifying agent with a method of chew‐ ing rice in the mouth. It was produced by that method until the eighth century [1].

In China, ancient Chinese *KOJI* had been used from ancient times, as described in Chinese ancient texts such as the Chi-Min-Yao-Shu. Chinese *KOJI* is made from barley or wheat. It is kneaded cereal flour with water and hardened as a brick or cake. Modern *KOJI* is made from non-heated cereal flour or wheat as material. However, it is described that ancient *KOJI* made from a mixture of heat-treating material with mixed non-heated wheat flour, roasted wheat flour, and steamed wheat flour, and the mixture cultivated *KOJI* mold after kneading with water or extraction without inoculation of seed mold. Kanauchi and co-authors [2, 3] reported their features. Results show that *Aspergillus* spp. was grown on and within steamed cereal cake as the dominant *KOJI* mold, *Rhizopus* spp. was grown on and within a non-heat‐ ing cereal cake as the dominant *KOJI* mold. Furthermore, both *Aspergillus* spp. and *Rhizopus* spp. as dominant *KOJI* molds were grown on and within a roasted cereal cake and a cereal cake mixed with heat-treating of cereal materials of three kinds [2,3]. Mold strains were dominant selectively in cereal cake because denatured protein was impossible to decompose by *Rhizopus* spp., but *Aspergillus oryzae* was impossible to assimilate non-heated starch in wheat flour [2,3]. In modern China, non-heated cereals such as barley or peas are used, whereby *Rhizopus spp.* or similar physiological features have *Mucor* spp., which can grow on non-cereals, predominant in it. It is difficult to decompose denatured cereal protein to en‐ hance their uptake for nutrition of micro-organisms, *Rhizopus* spp. has a weak protease or peptidase to grow on steamed cereals [4].

It remains unclear whether Chinese type *KOJI* was used for *SAKE* production or not by an‐ cient Japanese. However, steamed rice is used for *SAKE* production where *Aspergillus* spp. has been used since ancient times.

Between the Asuka-Nara Era and Heian Era (5th – 8th century), imperial families and cour‐ tiers established huge craft factories of which wide areas were dedicated to crafts. Many technicians and workers were employed in them, monopolizing practical technologies of all areas. Brewery also continued in such factories, and *SAKE* was brewed using advanced technologies during those eras.

in dry fields using dry rice cultivation methods. Introduced from China early, rice was culti‐ vated in dry fields in Japan also. However, the method of rice cultivation in paddy fields boosted yields to higher levels than those achieved in dry fields. The wet method brought social changes: a reliable labor force is necessary for cultivation by planting rice in paddy fields, harvesting it, and maintaining paddy fields, equipment, and irrigation. The labor force resources from families were limited. More labor was required from settlements. The settlements formed communities. Later communities formed ancient Japan. At that time, rice was of particular value: it was a divine food. *SAKE*, made from that divine food of rice, was also revered as blessed. It was used as a sacrifice to the gods. Moreover, people believed in a divine spirit indwelt in rice. Extending that belief, people believed that intoxication by alcohol beverages as *SAKE* made from rice brought gods into the human body. Further‐ more, they solidified the community by sharing divine foods as rice and beverage as *SAKE*

Alcoholic beverages can be made from cereals as beer, *SAKE*, or whisky. Saccharification processing is extremely important. Generally, it is an important feature of Asian alcoholic beverage production that mold cultivate in cereal, so-called '*KOJI*', is used for production. However, according to ancient literature, Osumi-no-Kuni-Fudoki, which recorded the cul‐ ture and geography of Kagoshima in ancient times before production of *SAKE* using *KOJI*, alcoholic beverages were made with saliva as a saccharifying agent with a method of chew‐

In China, ancient Chinese *KOJI* had been used from ancient times, as described in Chinese ancient texts such as the Chi-Min-Yao-Shu. Chinese *KOJI* is made from barley or wheat. It is kneaded cereal flour with water and hardened as a brick or cake. Modern *KOJI* is made from non-heated cereal flour or wheat as material. However, it is described that ancient *KOJI* made from a mixture of heat-treating material with mixed non-heated wheat flour, roasted wheat flour, and steamed wheat flour, and the mixture cultivated *KOJI* mold after kneading with water or extraction without inoculation of seed mold. Kanauchi and co-authors [2, 3] reported their features. Results show that *Aspergillus* spp. was grown on and within steamed cereal cake as the dominant *KOJI* mold, *Rhizopus* spp. was grown on and within a non-heat‐ ing cereal cake as the dominant *KOJI* mold. Furthermore, both *Aspergillus* spp. and *Rhizopus* spp. as dominant *KOJI* molds were grown on and within a roasted cereal cake and a cereal cake mixed with heat-treating of cereal materials of three kinds [2,3]. Mold strains were dominant selectively in cereal cake because denatured protein was impossible to decompose by *Rhizopus* spp., but *Aspergillus oryzae* was impossible to assimilate non-heated starch in wheat flour [2,3]. In modern China, non-heated cereals such as barley or peas are used, whereby *Rhizopus spp.* or similar physiological features have *Mucor* spp., which can grow on non-cereals, predominant in it. It is difficult to decompose denatured cereal protein to en‐ hance their uptake for nutrition of micro-organisms, *Rhizopus* spp. has a weak protease or

It remains unclear whether Chinese type *KOJI* was used for *SAKE* production or not by an‐ cient Japanese. However, steamed rice is used for *SAKE* production where *Aspergillus* spp.

ing rice in the mouth. It was produced by that method until the eighth century [1].

among members as they cooperated in rice cultivation [1].

40 Food Industry

peptidase to grow on steamed cereals [4].

has been used since ancient times.

During the Heian period (8th century and thereafter), *SAKE* rose to importance for use in regional ceremonies or banquets. *SAKE* brewing by SAKE-NO-TSUKASA was both a *SAKE* brewery and a supervisory office of the imperial court. Some kinds of *SAKE* were brewed for emperor, imperial family, and the aristocracy for use at ceremonies or banquets. Brewing methods were described in *ENGISHIKI*, an ancient book of codes and procedures related to national rites and prayers. For example, *GOSYU* was specially brewed for the Emperor us‐ ing steamed rice, *KOJI*, and mother water. The mash was fermented using wild fermentative yeast for ten days; then the mash was filtrated. The resultant *SAKE* was used for subsequent brewing as mother water. The *SAKE* brewed steamed rice, *KOJI*, and strained *SAKE* brewing were repeated four times to produce *SAKE* with a very sweet taste [1]. The literature in this period described *SAKE* of more than two kinds. The minor aristocracy and many people were not able to drink *SAKE* because it was extremely expensive.

Between the later Heian Era and Muromachi Era via the Kamakura Era (12th–16th century) *SAKE* was produced and sold at Buddhist temples and private breweries. During that peri‐ od, it was a popular alcoholic beverage. In the 12th century, the feudal government issued alcohol prohibition laws many times to maintain security. Officers destroyed *SAKE* contain‐ ers throughout cities.

At the beginning of the Muromachi Era, according to the '*GOSHU-NO-NIKKI*', *SAKE* was brewed already using a modern process in which rice-*KOJI* and steamed rice and water were mashed successively step-by-step. Moreover, the techniques applied lactic-acid fer‐ mentation, which demonstrates protection of the mash from bacterial contamination and dominant growth of yeast during *SAKE* production [1, 5].

During the 16th century, the *TAMON-IN* Diary was written for 100 years. *TAMON-IN* were small temples belonging to the *KOFUKUJI* temple in Nara. The diary described heating methods used to kill contaminated germs already in this century. In Europe, Louis Pasteur announced low-temperature pasteurization of wine and milk in 1865. However, Japanese brewers had acquired experimentally pasteurized *SAKE* during the 16th century [5].

During the Edo Era, the brewing season extended from the autumnal equinox to the vernal. However, results show good tasting *SAKE* brewing conducted in midwinter using a method called '*KANZUKURI*'. The brewing techniques of those brewers in the Ikeda, Itami, and Na‐ da districts (Osaka City and Hyogo Prefecture) held the leadership in *SAKE* brewing at that time. During the Genroku period (end of the 17th Century), the total number of breweries was reported as greater than 27,000 [5].

After the Meiji Era, *SAKE* brewing methods changed drastically based on European science. Many improvements of *SAKE* brewing were accomplished by applying beer-brewing meth‐ ods directly. However, many special techniques are used in *SAKE* production. For example, mold culture is not required in beer brewing. Japanese brewers built the technology of *SAKE* production which mixed European beer brewing and old Japanese traditional techniques during the Meiji Era [5].

## **3.** *SAKE* **materials**

#### **3.1. Water**

Water is an important material used in *SAKE* brewing, accounting for about 80% (v/v) of *SAKE*. It is used not only as the material but also in many other procedures such as washing and steeping of rice, washing of bottles or *SAKE* tanks, and for boiling. Generally, approx. 20–30 kl of water is necessary to process one ton of rice for *SAKE* brewing [5, 6]. The water for *SAKE* brewing must be colorless, tasteless and odorless; it must also be neutral or weakly alkaline, containing only traces of iron, ammonia, nitrate, organic substances, and micro-or‐ ganisms. In particularly, iron ions are injurious to *SAKE*, giving it a color and engendering deterioration [5, 6]. Therefore, iron in brewing water is removed using appropriate treat‐ ments such as aeration, successive filtration, adsorption (with activated carbon or ion-ex‐ change resins) and flocculation (with a reagent of alum) [7, 8].

**Figure 1.** Rice grain size. Left side shows YAMADANISHI for variety of *SAKE* brewing rice. Right side shows HITOME‐

*SAKE* Alcoholic Beverage Production in Japanese Food Industry

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43

The scientific name of Japanese *KOJI* mold is *Aspergillus oryzae*. It grows on and within steamed rice grains. The mold accumulates various enzymes for *SAKE* production. Enzymes of about 50 kinds have been found in *KOJI*, the most important of which are amylases. α-Amylase (Endo- α- amylase, EC.3.2.1.1) and saccharifying amylase (Exo-α-glucosidase; E.C. 3.2.1.20) play important roles in amylolytic action [5, 11]. Furthermore, proteases of some kinds are also important enzymes: acid-proteases and alkaline-proteases are found in *KOJI*. In *SAKE* mash, the enzymes decompose protein to form amino acids and peptides (oligoamino acid) at low pH values such as pH 3–4 [5]. Furthermore, amino acids or peptide-sup‐ ported yeast grow with food or nutrition. The enzyme acts indirectly, decomposing rice

The taxonomy of mold was studied for *Aspergillus oryzae* by Ahlburg and Matsubara (1878) and Cohn (1883). A report by Wehmer (1895) was published, describing *KOJI* mold class *A. oryzae* in detailed mycological studies as an *A. flavus-oryzae* group. They are slight graded on variations in morphological and physiological properties [5]. Murakami et al. identified and reported that *KOJI* mold strains used for *SAKE* brewing belonged to *A. oryzae* and not *A. fla‐ vus*. Two species were distinguished based on mycological characteristics of each authentic type culture of the two species [13, 14]. It is noteworthy that no Japanese industrial strain of

In *SAKE* brewing, conidiospores produced over bran rice, so-called TANE-*KOJI*, are sprayed and inoculated on steamed rice. *KOJI* is prepared in an incubation room, a so-called *KOJI*-

BORE for variety of diverting rice.

**4. Microorganisms**

**4.1.** *KOJI* **mold (***Aspergillus oryzae***)**

protein while combining to an active site of the α-amylase [12].

*KOJI* mold is capable of aflatoxin production.

MURO.

#### **3.2. Rice**

The quality of rice, the principal raw material of *SAKE*, strongly affects the *SAKE* taste, but details of its effects are not clearly elucidated. Contrary to the other Asian alcohol produc‐ tion, Japonica short-grain varieties are used for *SAKE* production. In Korea and Taiwan, oth‐ er short-grain varieties might also be used for alcoholic beverages.

#### *3.2.1. Grain size*

Large grains are suitable for *SAKE* production. Figure 1 shows rice grain size. The grain size is generally reported as the weight of 1,000 kernels. A weight of more than 25.0 g has been quoted as a mean value of 101 selected varieties by scholars [5, 9]. The selected varieties have a white spot in the center known as *SHINPAKU*, which contains high levels of starch.

#### *3.2.2. Chemical constituents*

Rice contains 70–75% carbohydrates, 7–9% crude protein, 1.3–2.0% crude fat, and 1.0 ash, with 12–15% water. Other components such as proteins or lipids in rice, excepting starch, are unnecessary for *SAKE* production. In fact, *SAKE* produced with rice having excessive proteins or lipids does not have good flavor or taste. Their compounds exist on the endo‐ sperm surface, mainly around the aleurone layer. Therefore they are removed by rice polish‐ ing. Moreover, the following have close correlations among the weight of 1,000 kernels: crude protein contents, speed of adsorption of water during steeping, and formation of sug‐ ars by saccharification of rice with amylases [5, 10].

**Figure 1.** Rice grain size. Left side shows YAMADANISHI for variety of *SAKE* brewing rice. Right side shows HITOME‐ BORE for variety of diverting rice.

## **4. Microorganisms**

mold culture is not required in beer brewing. Japanese brewers built the technology of *SAKE* production which mixed European beer brewing and old Japanese traditional techniques

Water is an important material used in *SAKE* brewing, accounting for about 80% (v/v) of *SAKE*. It is used not only as the material but also in many other procedures such as washing and steeping of rice, washing of bottles or *SAKE* tanks, and for boiling. Generally, approx. 20–30 kl of water is necessary to process one ton of rice for *SAKE* brewing [5, 6]. The water for *SAKE* brewing must be colorless, tasteless and odorless; it must also be neutral or weakly alkaline, containing only traces of iron, ammonia, nitrate, organic substances, and micro-or‐ ganisms. In particularly, iron ions are injurious to *SAKE*, giving it a color and engendering deterioration [5, 6]. Therefore, iron in brewing water is removed using appropriate treat‐ ments such as aeration, successive filtration, adsorption (with activated carbon or ion-ex‐

The quality of rice, the principal raw material of *SAKE*, strongly affects the *SAKE* taste, but details of its effects are not clearly elucidated. Contrary to the other Asian alcohol produc‐ tion, Japonica short-grain varieties are used for *SAKE* production. In Korea and Taiwan, oth‐

Large grains are suitable for *SAKE* production. Figure 1 shows rice grain size. The grain size is generally reported as the weight of 1,000 kernels. A weight of more than 25.0 g has been quoted as a mean value of 101 selected varieties by scholars [5, 9]. The selected varieties have a white spot in the center known as *SHINPAKU*, which contains high levels of starch.

Rice contains 70–75% carbohydrates, 7–9% crude protein, 1.3–2.0% crude fat, and 1.0 ash, with 12–15% water. Other components such as proteins or lipids in rice, excepting starch, are unnecessary for *SAKE* production. In fact, *SAKE* produced with rice having excessive proteins or lipids does not have good flavor or taste. Their compounds exist on the endo‐ sperm surface, mainly around the aleurone layer. Therefore they are removed by rice polish‐ ing. Moreover, the following have close correlations among the weight of 1,000 kernels: crude protein contents, speed of adsorption of water during steeping, and formation of sug‐

change resins) and flocculation (with a reagent of alum) [7, 8].

er short-grain varieties might also be used for alcoholic beverages.

during the Meiji Era [5].

**3.** *SAKE* **materials**

**3.1. Water**

42 Food Industry

**3.2. Rice**

*3.2.1. Grain size*

*3.2.2. Chemical constituents*

ars by saccharification of rice with amylases [5, 10].

#### **4.1.** *KOJI* **mold (***Aspergillus oryzae***)**

The scientific name of Japanese *KOJI* mold is *Aspergillus oryzae*. It grows on and within steamed rice grains. The mold accumulates various enzymes for *SAKE* production. Enzymes of about 50 kinds have been found in *KOJI*, the most important of which are amylases. α-Amylase (Endo- α- amylase, EC.3.2.1.1) and saccharifying amylase (Exo-α-glucosidase; E.C. 3.2.1.20) play important roles in amylolytic action [5, 11]. Furthermore, proteases of some kinds are also important enzymes: acid-proteases and alkaline-proteases are found in *KOJI*. In *SAKE* mash, the enzymes decompose protein to form amino acids and peptides (oligoamino acid) at low pH values such as pH 3–4 [5]. Furthermore, amino acids or peptide-sup‐ ported yeast grow with food or nutrition. The enzyme acts indirectly, decomposing rice protein while combining to an active site of the α-amylase [12].

The taxonomy of mold was studied for *Aspergillus oryzae* by Ahlburg and Matsubara (1878) and Cohn (1883). A report by Wehmer (1895) was published, describing *KOJI* mold class *A. oryzae* in detailed mycological studies as an *A. flavus-oryzae* group. They are slight graded on variations in morphological and physiological properties [5]. Murakami et al. identified and reported that *KOJI* mold strains used for *SAKE* brewing belonged to *A. oryzae* and not *A. fla‐ vus*. Two species were distinguished based on mycological characteristics of each authentic type culture of the two species [13, 14]. It is noteworthy that no Japanese industrial strain of *KOJI* mold is capable of aflatoxin production.

In *SAKE* brewing, conidiospores produced over bran rice, so-called TANE-*KOJI*, are sprayed and inoculated on steamed rice. *KOJI* is prepared in an incubation room, a so-called *KOJI*-MURO.

#### **4.2. Yeast**

#### *4.2.1. Physiology of SAKE yeast*

Fermentative multi-budding yeast, *Saccharomyces cerevisiae*, which has been used not only in *SAKE* brewery, but also in beer brewry, winery and bakery, was discovered in ca. 1830 by J. Meyen; it was named by E.C. Hansen in 1882 [5]. *SAKE* yeast is classified taxonomically in the *Saccharomyces cerevisiae* group [15]. However, the yeast was distinguished from other strains of *S. cerevisiae* by additional properties such as vitamin requirements [16, 17], acid tolerance, sugar osmophilic character, and adaptability to anaerobic conditions. Additional‐ ly, *SAKE* yeast has advantageous features that enable its growth under high sugar contents and low pH conditions, to produce *SAKE* under open system fermentation.

fruit-like flavor is imparted to *SAKE* from yeast production because many Japanese consum‐ ers favor *SAKE* that has a fruit-like aroma. A yeast mutant producing fruity aromas was iso‐ lated for *SAKE* brewing. Their typical chemical components are ethyl caproate, which gives an apple-like aroma, and iso-amyl acetate or iso-amyl alcohol, which give a banana-like aro‐ ma. Before development of methods of breeding yeast to produce aromas, it was not easy for aromatic *SAKE* to be brewed and supplied stably for customers. Some competent *SAKE* brewers had controlled temperature severely to adjust enzymes that produced *KOJI* mold as amylase. Controlling the amounts of sugars as nutrient elements produced by amylase in mash adjusts the metabolisms of yeast growth and production of *SAKE* aromas as ethyl cap‐ roate and iso-amyl acetate. However, it is readily apparent that *SAKE* aroma synthesis by metabolic pathways or control mechanisms. The yeast producing fruity aroma was bred for

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45

use in commercial brewing [1, 5].

**Figure 3.** Flavor wheel of *SAKE.*

*SAKE* yeast formed a large amount of foam during main mash fermentation. Because onethird of the capacity of the fermentation vessel is occupied by foam during usual main fer‐ mentation, preventing foam formation would be greatly advantageous to breweries to save space occupied by the foam and scaling up the amount of *MOROMI* produced. Some largemolecular-weight compounds that arise from steamed rice grains are also regarded as tak‐ ing part in foam formation. Recently, foam formation has involved existing proteins, with foam formation on the yeast surface.

Ouchi and Akiyama obtained foam-less mutants that have the same characteristics as the parent yeast except for foam-formation [18, 19]. A foam-less mutant of *SAKE* yeast, a favor‐ ite strain of *Saccharomyces cerevisiae* (The Brewing Society of JAPAN is distributing it as *SAKE* yeast), has become available for *SAKE* brewing. Recently, foam protein in SAKE yeast, AWA 1, was cloned. TAKA-AWA foam has been obvious molecular biologically [20].

**Figure 2.** TAKA-AWA foam. (Photograph by Shiraki Tunesuke Co., Ltd.)

#### *4.2.2. Aroma production by SAKE yeast*

The *SAKE* aroma is produced by yeast mainly because rice, as a *SAKE* material, has weaker aroma than materials used for wine or beer. Furthermore, *SAKE* contains ethanol, higher concentrations of alcohol, and many aroma-producing compounds. Aromatic compounds are an important factor used to characterize *SAKE*. Recently, a flavor wheel for *SAKE* was produced similar to existing ones used for wine and beer [21,22]. According to this wheel, the aromas can be categorized as floral aroma, fruit-like nutty, caramel-like, and lipid-like. A

fruit-like flavor is imparted to *SAKE* from yeast production because many Japanese consum‐ ers favor *SAKE* that has a fruit-like aroma. A yeast mutant producing fruity aromas was iso‐ lated for *SAKE* brewing. Their typical chemical components are ethyl caproate, which gives an apple-like aroma, and iso-amyl acetate or iso-amyl alcohol, which give a banana-like aro‐ ma. Before development of methods of breeding yeast to produce aromas, it was not easy for aromatic *SAKE* to be brewed and supplied stably for customers. Some competent *SAKE* brewers had controlled temperature severely to adjust enzymes that produced *KOJI* mold as amylase. Controlling the amounts of sugars as nutrient elements produced by amylase in mash adjusts the metabolisms of yeast growth and production of *SAKE* aromas as ethyl cap‐ roate and iso-amyl acetate. However, it is readily apparent that *SAKE* aroma synthesis by metabolic pathways or control mechanisms. The yeast producing fruity aroma was bred for use in commercial brewing [1, 5].

**Figure 3.** Flavor wheel of *SAKE.*

**4.2. Yeast**

44 Food Industry

*4.2.1. Physiology of SAKE yeast*

foam formation on the yeast surface.

**Figure 2.** TAKA-AWA foam. (Photograph by Shiraki Tunesuke Co., Ltd.)

*4.2.2. Aroma production by SAKE yeast*

Fermentative multi-budding yeast, *Saccharomyces cerevisiae*, which has been used not only in *SAKE* brewery, but also in beer brewry, winery and bakery, was discovered in ca. 1830 by J. Meyen; it was named by E.C. Hansen in 1882 [5]. *SAKE* yeast is classified taxonomically in the *Saccharomyces cerevisiae* group [15]. However, the yeast was distinguished from other strains of *S. cerevisiae* by additional properties such as vitamin requirements [16, 17], acid tolerance, sugar osmophilic character, and adaptability to anaerobic conditions. Additional‐ ly, *SAKE* yeast has advantageous features that enable its growth under high sugar contents

*SAKE* yeast formed a large amount of foam during main mash fermentation. Because onethird of the capacity of the fermentation vessel is occupied by foam during usual main fer‐ mentation, preventing foam formation would be greatly advantageous to breweries to save space occupied by the foam and scaling up the amount of *MOROMI* produced. Some largemolecular-weight compounds that arise from steamed rice grains are also regarded as tak‐ ing part in foam formation. Recently, foam formation has involved existing proteins, with

Ouchi and Akiyama obtained foam-less mutants that have the same characteristics as the parent yeast except for foam-formation [18, 19]. A foam-less mutant of *SAKE* yeast, a favor‐ ite strain of *Saccharomyces cerevisiae* (The Brewing Society of JAPAN is distributing it as *SAKE* yeast), has become available for *SAKE* brewing. Recently, foam protein in SAKE yeast,

The *SAKE* aroma is produced by yeast mainly because rice, as a *SAKE* material, has weaker aroma than materials used for wine or beer. Furthermore, *SAKE* contains ethanol, higher concentrations of alcohol, and many aroma-producing compounds. Aromatic compounds are an important factor used to characterize *SAKE*. Recently, a flavor wheel for *SAKE* was produced similar to existing ones used for wine and beer [21,22]. According to this wheel, the aromas can be categorized as floral aroma, fruit-like nutty, caramel-like, and lipid-like. A

AWA 1, was cloned. TAKA-AWA foam has been obvious molecular biologically [20].

and low pH conditions, to produce *SAKE* under open system fermentation.

Typical yeast metabolic processes producing aromatic compounds are shown in Fig. 3.

**•** Higher alcohol metabolism pathway [1]

The higher alcohols as aromatic compounds are iso-amyl alcohol and iso-butyl alcohol. The alcohols are produced by two pathways by yeast as shown below.

Acetyl CoA + Alcohol →Acetyl ester + CoA-SH

zyme has a hydrophobic active site in it [26].

**3.** They are facultative anaerobic bacteria.

are defined as listed below.

**4.** They have no mobility.

**5.** They produce no spores.

both types of bacci lactic acid bacteria.

cose.

**4.3. Lactic acid bacteria** *(Lactobacillus sakei)* **[5, 27]**

**2.** Bacteria is Gram positive. Their shapes are cocci or bacci.

This enzyme, a microsomal enzyme, is an endogenous membrane protein dissolving by sur‐ factant. Furthermore, more than 70% of the activity exists in it. AATFase has two isozymes of molecular weight 56 k Da. Isozyme P1 is reacted mainly in the yeast cell. Its activity has ca. 70–80% overall activity. Its optimum temperature is 25°C (Isozyme P2 is 40°C), and the optimum pH is 8.0. The pH range of its reaction is pH 7.5–8.5 (Isozyme P2 is pH 7.0–8.5). Their enzyme inhibited phosphatidylserine and phosphatidylinositol, having interfacial ac‐ tivity, and oleic acid and linoleic acid. Accordingly, this phenomenon showed that this en‐

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Lactic acid bacteria are the most important bacteria in *SAKE* brewing. Lactic acid bacteria

**1.** Bacteria ferment glucose and producing more than 50% lactic acid per 1 molar of glu‐

Their fermentation types are two. One is homo type, 2 molar of lactic acid fermenting from 1 molar of glucose. The other is hetero type, 1 molar of lactic acid, 1 molar of ethanol and 1 molar carbon dioxide from 1 molar of glucose. Typical lactic acid bacteria for food process‐ ing are shown as the following: *Leuconostoc* spp. is a hetero-type cocci lactic acid bacteria, and *Pediococcus spp.* is a homo type cocci lactic acid bacteria. *Lactobacillus* spp. belongs to

In *SAKE* brewing, lactic acid bacteria are used in traditional seed mash, *KIMOTO* produc‐ tion for without sterilization safety open fermentation system without sterilization. In tradi‐ tional seed mash, *MOTO*, production, it is known that *Leuconostoc mesenteroides* as heterolactic acid fermentation grows the *MOTO* preparation earlier under extremely low

It is rarely that lactic acid bacteria spoil commercial *SAKE*. The bacteria are called *HIOCHI* bacteria, and have resistance to ethanol concentrations higher than 18% in *SAKE*. *SAKE*grown *HIOCHI* bacteria have turbidity and an uncomfortable cheese-like smell from diace‐ tyl [28]. Two types of lactic acid bacteria might be involved: one is *L. homohioch*i (homo lactic acid fermentation type); the other is *L. heterohiochi* (hetero-lactic acid fermentation type). Both bacteria have resistance to ethanol. The coefficient for growth of two bacteria in *SAKE* is mevalonic acid, which is produced by *KOJI* mold. Recently, mevalonic acid nonproduc‐

temperatures of less than 5°C. *L. sakei* as a hetero-lactic acid fermentation grows in it.

tive mutants have been bred for *SAKE*-*KOJI* production [1, 17, 29].

1. RCHNH2COOH⇔RCOCOOH→RCHO→RCH2OH

## 2. C6H12O6→RCOCOOH→RCHO→RCH2OH

In these two pathways, 2-oxo acid is produced as a precursor. In the 1 pathway, 2-oxo acid is produced by deamination reaction between Ehrlich pathways. In the 2 pathway presented above, 2-oxo acid is produced between production of amino acid pathway. Oxo acid is pro‐ duced by decarbonylation reaction and reduction reaction between both pathways, similarly as ethanol is produced from acetoaldehyde via pyruvic acid as oxo acid of one kind. For ex‐ ample, lacking amino acids as leucine and valine in *SAKE* mash, the yeast produces leucine and valine in *SAKE* mash. Furthermore, 2-oxo acid was transaminated from other amino acids. It is controlled by the amount of amino-acid-based amino bonds. Therefore, lacking extremely amino acid in mash, 2-oxo acid is converted to higher alcohol as iso-butyl alcohol and iso-amyl alcohol. Sufficing amino acid as leucine and valine in *SAKE* mash, the reaction of the 2 pathway inhibited by native feedback control and uptaken amino acid are converted by the 1 pathway.

**•** Fatty acid ethyl ester [1]

Ethyl caproate is a favorite flavor providing an apple-like aroma for Japanese consumers. This compound is produced by esterification from caproic acid as a precursor. Caproic acid is synthesized by fatty acid synthase between fatty acid synthesis pathway from acetyl-CoA and malonyl-CoA in *SAKE* yeast. Their synthase composes FAS 1 (Fas1p; β-subunit) and FAS2 (Fas2p:α-subunit), which are hexamer proteins (α6β6 subunit) [23]. Ichikawa reported a yeast breeding method that produces high levels of ethyl caproate that high levels of pre‐ cursor of ethyl caproate were producing in yeast cells [24]. Cerulenin, an antifungal antibiot‐ ic produced by *Cephalosporium caerulens*, inhibits beta-ketoacyl-ACP synthase as *fatty acid* synthetase. A mutant of cerulenin-resistant yeast strain decreases synthesis of long-chain fatty acids by mutating Gly1250 Ser in the gene. The strain can produce high levels of capro‐ ic acid [25].

**•** Ethyl acetate group

Higher alcohol and esterified fatty acid produce a fruity aroma in *SAKE*. Usually, *SAKE* has 0.1 ppm or higher concentrations of ester compounds. That slight amount of ester produces a fruity aroma and intensifies the *SAKE* flavor. Excessive esters destroy the balance of the *SAKE* flavor. Many ester compounds produced mainly by yeast are acetate ester groups that react and which are produced by an alcohol–acetyl transferase reaction that transfers an ace‐ tyl bond from acetyl CoA to alcohol. Alcohol acetyl transferase (AATFase; E.C. 23.1.84) cata‐ lyzes the following reaction.

## Acetyl CoA + Alcohol →Acetyl ester + CoA-SH

Typical yeast metabolic processes producing aromatic compounds are shown in Fig. 3.

alcohols are produced by two pathways by yeast as shown below.

1. RCHNH2COOH⇔RCOCOOH→RCHO→RCH2OH

The higher alcohols as aromatic compounds are iso-amyl alcohol and iso-butyl alcohol. The

In these two pathways, 2-oxo acid is produced as a precursor. In the 1 pathway, 2-oxo acid is produced by deamination reaction between Ehrlich pathways. In the 2 pathway presented above, 2-oxo acid is produced between production of amino acid pathway. Oxo acid is pro‐ duced by decarbonylation reaction and reduction reaction between both pathways, similarly as ethanol is produced from acetoaldehyde via pyruvic acid as oxo acid of one kind. For ex‐ ample, lacking amino acids as leucine and valine in *SAKE* mash, the yeast produces leucine and valine in *SAKE* mash. Furthermore, 2-oxo acid was transaminated from other amino acids. It is controlled by the amount of amino-acid-based amino bonds. Therefore, lacking extremely amino acid in mash, 2-oxo acid is converted to higher alcohol as iso-butyl alcohol and iso-amyl alcohol. Sufficing amino acid as leucine and valine in *SAKE* mash, the reaction of the 2 pathway inhibited by native feedback control and uptaken amino acid are converted

Ethyl caproate is a favorite flavor providing an apple-like aroma for Japanese consumers. This compound is produced by esterification from caproic acid as a precursor. Caproic acid is synthesized by fatty acid synthase between fatty acid synthesis pathway from acetyl-CoA and malonyl-CoA in *SAKE* yeast. Their synthase composes FAS 1 (Fas1p; β-subunit) and FAS2 (Fas2p:α-subunit), which are hexamer proteins (α6β6 subunit) [23]. Ichikawa reported a yeast breeding method that produces high levels of ethyl caproate that high levels of pre‐ cursor of ethyl caproate were producing in yeast cells [24]. Cerulenin, an antifungal antibiot‐ ic produced by *Cephalosporium caerulens*, inhibits beta-ketoacyl-ACP synthase as *fatty acid* synthetase. A mutant of cerulenin-resistant yeast strain decreases synthesis of long-chain fatty acids by mutating Gly1250 Ser in the gene. The strain can produce high levels of capro‐

Higher alcohol and esterified fatty acid produce a fruity aroma in *SAKE*. Usually, *SAKE* has 0.1 ppm or higher concentrations of ester compounds. That slight amount of ester produces a fruity aroma and intensifies the *SAKE* flavor. Excessive esters destroy the balance of the *SAKE* flavor. Many ester compounds produced mainly by yeast are acetate ester groups that react and which are produced by an alcohol–acetyl transferase reaction that transfers an ace‐ tyl bond from acetyl CoA to alcohol. Alcohol acetyl transferase (AATFase; E.C. 23.1.84) cata‐

**•** Higher alcohol metabolism pathway [1]

2. C6H12O6→RCOCOOH→RCHO→RCH2OH

by the 1 pathway.

46 Food Industry

ic acid [25].

**•** Ethyl acetate group

lyzes the following reaction.

**•** Fatty acid ethyl ester [1]

This enzyme, a microsomal enzyme, is an endogenous membrane protein dissolving by sur‐ factant. Furthermore, more than 70% of the activity exists in it. AATFase has two isozymes of molecular weight 56 k Da. Isozyme P1 is reacted mainly in the yeast cell. Its activity has ca. 70–80% overall activity. Its optimum temperature is 25°C (Isozyme P2 is 40°C), and the optimum pH is 8.0. The pH range of its reaction is pH 7.5–8.5 (Isozyme P2 is pH 7.0–8.5). Their enzyme inhibited phosphatidylserine and phosphatidylinositol, having interfacial ac‐ tivity, and oleic acid and linoleic acid. Accordingly, this phenomenon showed that this en‐ zyme has a hydrophobic active site in it [26].

### **4.3. Lactic acid bacteria** *(Lactobacillus sakei)* **[5, 27]**

Lactic acid bacteria are the most important bacteria in *SAKE* brewing. Lactic acid bacteria are defined as listed below.


Their fermentation types are two. One is homo type, 2 molar of lactic acid fermenting from 1 molar of glucose. The other is hetero type, 1 molar of lactic acid, 1 molar of ethanol and 1 molar carbon dioxide from 1 molar of glucose. Typical lactic acid bacteria for food process‐ ing are shown as the following: *Leuconostoc* spp. is a hetero-type cocci lactic acid bacteria, and *Pediococcus spp.* is a homo type cocci lactic acid bacteria. *Lactobacillus* spp. belongs to both types of bacci lactic acid bacteria.

In *SAKE* brewing, lactic acid bacteria are used in traditional seed mash, *KIMOTO* produc‐ tion for without sterilization safety open fermentation system without sterilization. In tradi‐ tional seed mash, *MOTO*, production, it is known that *Leuconostoc mesenteroides* as heterolactic acid fermentation grows the *MOTO* preparation earlier under extremely low temperatures of less than 5°C. *L. sakei* as a hetero-lactic acid fermentation grows in it.

It is rarely that lactic acid bacteria spoil commercial *SAKE*. The bacteria are called *HIOCHI* bacteria, and have resistance to ethanol concentrations higher than 18% in *SAKE*. *SAKE*grown *HIOCHI* bacteria have turbidity and an uncomfortable cheese-like smell from diace‐ tyl [28]. Two types of lactic acid bacteria might be involved: one is *L. homohioch*i (homo lactic acid fermentation type); the other is *L. heterohiochi* (hetero-lactic acid fermentation type). Both bacteria have resistance to ethanol. The coefficient for growth of two bacteria in *SAKE* is mevalonic acid, which is produced by *KOJI* mold. Recently, mevalonic acid nonproduc‐ tive mutants have been bred for *SAKE*-*KOJI* production [1, 17, 29].

**5.** *SAKE* **Production**

quired ratio [5].

**5.1. Rice treatment (polishing, washing, and steeping)**

does not change with increase of the polishing ratio [30].

**Table 1.** Changes in the contents of some rice grain components after polishing [5]

In contrast to the use of malt in brewing beer or producing spirits, in *SAKE* brewing, polish‐ ed rice is used. The main purpose of polishing is to remove unnecessary substances in rice aside from the starch, which are regarded as undesirable in *SAKE* brewing. Polishing re‐ moves surface layers of the rice grains, which contain proteins, lipids, and minerals. The ra‐ tio of percentages by weight of polished rice to the original brown rice is defined as the polishing ratio. Changes in the amounts of some constituents of the processed grain with various polishing ratios are presented in Table 1 (Research Institute of Brewing, Japan, 1964). Crude fat and ash contents decrease most rapidly, whereas the protein content de‐ creases gradually until the polishing ratio reaches 50%, after which it remains practically constant. In contrast to changes in the crude fat content, the lipid content (by hydrolysis)

Moisture 13.5 13.3 11.0 10.5 Crude protein 6.55 5.12 4.06 3.8 Crude fat 2.28 0.11 0.07 0.05 Ash 1.00 0.25 0.20 0.15 Starch 70.9 74.3 76.3 77.6

The lowest polishing ratio is strictly regulated under the Liquor Tax Law. In general, polish‐ ed rice of 75–70% ratio is used for reasonably priced *SAKE* brewing. In contrast, polished rice of a 60% ratio is used for special brewing brands such as GINJYO-SHU, and rice of less than 50% polishing ratio is used for Grand grade *SAKE*, DAIGINJYO-SHU. The latter is a prestige class of *SAKE*. Sometimes, the *SAKE* is brewed using rice of a 30% polishing ratio.

The rice polisher depicted in Fig. 6 is used for *SAKE* brewing. The roller made of carborun‐ dum and feldspar rotates around a vertical axis, and scrapes the surface of grains. Rice grains supplied from the hopper are polished and fall to the bottom of the basket conveyer. The grains go through the sieve to remove the rice bran. The rice is carried by the basket conveyer to the hopper. The operation continues until the grains are polished to the re‐

Generally, with a mill having a roller that is 40 cm in diameter, average times for polishing are 6–8 h for 89%, 7–10 h for 75%, 10–13 h for 70%, and 16–20 h for 60% polishing ratio [5].

**Polishing ratio (%)** 100 80 60 50

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**Figure 4.** Production of aroma compound mainly by yeast. Broken line shows inhibition of the reaction by amino acid

**Figure 5.** *SAKE* production.

## **5.** *SAKE* **Production**

CO2

Leu, thr

Leu


> Iso amylalcohol



Malonyl

CoA Asyl

**Figure 4.** Production of aroma compound mainly by yeast. Broken line shows inhibition of the reaction by amino acid

*KOJI*

Seed Mash (*MOTO*)

Water Yeast

Main Mash (*MOROMI*)

*SAKE* cake

Lees

Main Fermentation

Filtration

Fresh *SAKE*

Settling

Filtration

CoA


> Acetyl CoA


Isovaleric aldehyde

> Isoamyl acetate

Acetyl CoA

Storage/Aging

Blending

Adjusting Alcohol %

Pasteurization

*SAKE*

Bottling

Pasteurization

isobutyl alcohol

Isobutyl acetate

Pyruvic

Acetoald ehyde


Propion aldehyde

n-Propanol

48 Food Industry

acid Glucose dioxy isovaleric acid

Ile,Val,leu

Ethyl alcohol

> Fatty acid

Cooling

Steamed Rice

*KOJI*

Spore of

**Figure 5.** *SAKE* production.

*A.oryzae* Molding

Steaming

Washing

Polishing

Polishing Rice

Rice

Caproyl CoA

Caproic acid

HydroxyAceto lactic acid

 Dioxy methyl valeric

> -Aceto lactic acid

> > Ethyl Caproate

acid Methyl ethyl

Ile Val leu


acetoaldehyde

CO2

2-methyl-1 butanol

### **5.1. Rice treatment (polishing, washing, and steeping)**

In contrast to the use of malt in brewing beer or producing spirits, in *SAKE* brewing, polish‐ ed rice is used. The main purpose of polishing is to remove unnecessary substances in rice aside from the starch, which are regarded as undesirable in *SAKE* brewing. Polishing re‐ moves surface layers of the rice grains, which contain proteins, lipids, and minerals. The ra‐ tio of percentages by weight of polished rice to the original brown rice is defined as the polishing ratio. Changes in the amounts of some constituents of the processed grain with various polishing ratios are presented in Table 1 (Research Institute of Brewing, Japan, 1964). Crude fat and ash contents decrease most rapidly, whereas the protein content de‐ creases gradually until the polishing ratio reaches 50%, after which it remains practically constant. In contrast to changes in the crude fat content, the lipid content (by hydrolysis) does not change with increase of the polishing ratio [30].


**Table 1.** Changes in the contents of some rice grain components after polishing [5]

The lowest polishing ratio is strictly regulated under the Liquor Tax Law. In general, polish‐ ed rice of 75–70% ratio is used for reasonably priced *SAKE* brewing. In contrast, polished rice of a 60% ratio is used for special brewing brands such as GINJYO-SHU, and rice of less than 50% polishing ratio is used for Grand grade *SAKE*, DAIGINJYO-SHU. The latter is a prestige class of *SAKE*. Sometimes, the *SAKE* is brewed using rice of a 30% polishing ratio.

The rice polisher depicted in Fig. 6 is used for *SAKE* brewing. The roller made of carborun‐ dum and feldspar rotates around a vertical axis, and scrapes the surface of grains. Rice grains supplied from the hopper are polished and fall to the bottom of the basket conveyer. The grains go through the sieve to remove the rice bran. The rice is carried by the basket conveyer to the hopper. The operation continues until the grains are polished to the re‐ quired ratio [5].

Generally, with a mill having a roller that is 40 cm in diameter, average times for polishing are 6–8 h for 89%, 7–10 h for 75%, 10–13 h for 70%, and 16–20 h for 60% polishing ratio [5].

**Figure 6.** Diagram of a vertical type rice mill used in SAKE brewing [5]: A, the basket conveyor; B, rice hopper; C, the rice flow adjusting bulb; D, the polishing chamber; E, the roller; F, a resistance; G, exit; H, sieve and I, bran reservoir.

Fig.6

is absorbed to the extent of 7–12% of the weight of the starting rice grains, namely total wa‐ ter gain of about 35–40%. Historically, at many breweries, steaming processes usually gener‐ ated steam from water in a large pot. Today, boilers are often used in many breweries for steaming. A steamer is a shallow and wooden tub in which is bored a hole (1/20 diameter of bottom) at bottom. The steamer is put above the 1.5–2.0 kl caldron, and rice is permeated by large amounts of steam from the caldron. Recently, a modern apparatus for steaming rice as belt conveyor type apparatus is used in automated modern breweries. The steamed rice is cooled to nearly 40°C for *KOJI* production, and the rice used for preparing *MOTO* and *MO‐ ROMI*-mash is cooled to less than 10°C. Breweries usually use machines to cool the steamed rice with a draft of air as it moves on the screened belt. A pneumatic conveyer system is of‐

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A *KOJI* cultivates the *KOJI* mold, *Aspergillus oryzae* on and in steamed rice grains, and which accumulates various enzymes for *SAKE* production. For the preparation of *KOJI*, seed-molds are used at all breweries. The *Aspergillus oryzae* strains are cultivated in steamed bran rice dredging wood ash at 34–36°C for 5–6 days. This process results in abundant spore forma‐ tion. Cultivation conditions influence the enzyme production. In general, higher cultivation temperatures (approx. 42°C) develop the activities of amylases. Lower temperatures (ap‐

As cultivation times lengthen, more enzymic activities appear in the *KOJI* [32]. Nitrogenous substances and acids are accumulated more in *KOJI* that has been prepared from steamed rice of higher moisture contents [33]. They are regarded as related to the flavors and tastes of *SAKE*. After the steamed rice has been cooled to about 35°C by going through a cooling ap‐ paratus, it is transferred into the *KOJI*-*MURO*, a large incubating room, where temperature

After inoculating or spraying *TANE*-*KOJI* as seed mold in the proportion of 60–100 g/1,000 kg of rice, then the mixture is heaped in the center of a table for *KOJI* preparation. At this stage, the temperature of the material is 31–32°C. As the spores germinate and mycelia de‐ velop, the rice begins to smell moldy like sweet chestnut. After incubation for 10–12 h, the heap of rice grains is mixed to maintain uniformity of growth, temperature, and moisture contents. After another 10–12 h, with growth of the mold, mold mycelia can be observed dis‐ tinctly as small white spots on the grains. Furthermore, the material temperature has risen to 32–34°C. It is dispensed into wooden boxes, each with 15–45 kg of the grain. To control the rise in temperature and the moisture in the grain mass, the bottom of the box is made of wooden lattice or wire mesh. Temperature and moisture contents are also controlled by the thickness of the heaped grain layer in the box: 8 cm at the beginning, 6 cm at the first mix‐ ing, and 4 cm at the second mixing. Thereafter, at intervals of 6–8 h, the material is mixed and heaped again in the box. After incubation for about 40 h, the temperature of the materi‐ al rises to 40–42°C. The mycelium develops to cover and penetrate the grains which have sufficient enzymes, vitamins and various nutritive substances for mashing and growth of *SAKE* yeast. Then the *KOJI* is taken out of the room and spread on a clean cloth to be cooled

(26–28°C) and humidity are controlled at suitable levels to grow *KOJI* mold.

ten used to transfer steamed rice [1].

prox. 30°C) activate protease activities.

**5.4.** *KOJI* **preparation [5]**

#### **5.2. Washing and steeping**

Rice is washed and steeped in water before steaming. During washing, the grains are polish‐ ed further by collision of rice grains in water. During processing, the surface parts of the grains are removed, eliminating approx. 1–3% of the total grain weight [5]. Washed rice grains are passed into a vat and are steeped immediately in water. In washing and steeping procedures, the grains absorb water to about 25–30% of their original weight. The moisture promotes penetration of heat into the grain center during steaming and accelerates gelatini‐ zation of starch in the grains. Absorption of water is extremely important for preparing properly steamed rice, and controlling *KOJI* making and fermentation. The water absorption into grains differs according to the variety of rice and the polishing ratio [5, 10, 30]. General‐ ly, rice grains are steeped in water for 1–20 h, and soft rice absorbs water within 1–3 h. High‐ ly polished rice grains absorb water more rapidly. During washing and steeping, potassium ions and sugars are eluted from the grains [1, 31], whereas calcium and iron ions are absor‐ bed onto the grains [5]. After steeping, excess water is drained off from the grains for about 4–8 h before steaming.

#### **5.3. Steaming**

Starch is changed to the α-form, and protein is denatured by the steaming process. More‐ over, the grains are sterilized by steaming. The grains are usually steamed for 30–60 min, although previous reports show that steaming for as little as 15–20 min is sufficient to modi‐ fy the starch and protein of rice produced in Japan [1]. During steaming, the grain moisture is absorbed to the extent of 7–12% of the weight of the starting rice grains, namely total wa‐ ter gain of about 35–40%. Historically, at many breweries, steaming processes usually gener‐ ated steam from water in a large pot. Today, boilers are often used in many breweries for steaming. A steamer is a shallow and wooden tub in which is bored a hole (1/20 diameter of bottom) at bottom. The steamer is put above the 1.5–2.0 kl caldron, and rice is permeated by large amounts of steam from the caldron. Recently, a modern apparatus for steaming rice as belt conveyor type apparatus is used in automated modern breweries. The steamed rice is cooled to nearly 40°C for *KOJI* production, and the rice used for preparing *MOTO* and *MO‐ ROMI*-mash is cooled to less than 10°C. Breweries usually use machines to cool the steamed rice with a draft of air as it moves on the screened belt. A pneumatic conveyer system is of‐ ten used to transfer steamed rice [1].

### **5.4.** *KOJI* **preparation [5]**

A

Fig.6

B

<sup>C</sup> <sup>D</sup>

H

**Figure 6.** Diagram of a vertical type rice mill used in SAKE brewing [5]: A, the basket conveyor; B, rice hopper; C, the rice flow adjusting bulb; D, the polishing chamber; E, the roller; F, a resistance; G, exit; H, sieve and I, bran reservoir.

Rice is washed and steeped in water before steaming. During washing, the grains are polish‐ ed further by collision of rice grains in water. During processing, the surface parts of the grains are removed, eliminating approx. 1–3% of the total grain weight [5]. Washed rice grains are passed into a vat and are steeped immediately in water. In washing and steeping procedures, the grains absorb water to about 25–30% of their original weight. The moisture promotes penetration of heat into the grain center during steaming and accelerates gelatini‐ zation of starch in the grains. Absorption of water is extremely important for preparing properly steamed rice, and controlling *KOJI* making and fermentation. The water absorption into grains differs according to the variety of rice and the polishing ratio [5, 10, 30]. General‐ ly, rice grains are steeped in water for 1–20 h, and soft rice absorbs water within 1–3 h. High‐ ly polished rice grains absorb water more rapidly. During washing and steeping, potassium ions and sugars are eluted from the grains [1, 31], whereas calcium and iron ions are absor‐ bed onto the grains [5]. After steeping, excess water is drained off from the grains for about

Starch is changed to the α-form, and protein is denatured by the steaming process. More‐ over, the grains are sterilized by steaming. The grains are usually steamed for 30–60 min, although previous reports show that steaming for as little as 15–20 min is sufficient to modi‐ fy the starch and protein of rice produced in Japan [1]. During steaming, the grain moisture

I

<sup>E</sup> <sup>F</sup>

G

**5.2. Washing and steeping**

50 Food Industry

4–8 h before steaming.

**5.3. Steaming**

A *KOJI* cultivates the *KOJI* mold, *Aspergillus oryzae* on and in steamed rice grains, and which accumulates various enzymes for *SAKE* production. For the preparation of *KOJI*, seed-molds are used at all breweries. The *Aspergillus oryzae* strains are cultivated in steamed bran rice dredging wood ash at 34–36°C for 5–6 days. This process results in abundant spore forma‐ tion. Cultivation conditions influence the enzyme production. In general, higher cultivation temperatures (approx. 42°C) develop the activities of amylases. Lower temperatures (ap‐ prox. 30°C) activate protease activities.

As cultivation times lengthen, more enzymic activities appear in the *KOJI* [32]. Nitrogenous substances and acids are accumulated more in *KOJI* that has been prepared from steamed rice of higher moisture contents [33]. They are regarded as related to the flavors and tastes of *SAKE*. After the steamed rice has been cooled to about 35°C by going through a cooling ap‐ paratus, it is transferred into the *KOJI*-*MURO*, a large incubating room, where temperature (26–28°C) and humidity are controlled at suitable levels to grow *KOJI* mold.

After inoculating or spraying *TANE*-*KOJI* as seed mold in the proportion of 60–100 g/1,000 kg of rice, then the mixture is heaped in the center of a table for *KOJI* preparation. At this stage, the temperature of the material is 31–32°C. As the spores germinate and mycelia de‐ velop, the rice begins to smell moldy like sweet chestnut. After incubation for 10–12 h, the heap of rice grains is mixed to maintain uniformity of growth, temperature, and moisture contents. After another 10–12 h, with growth of the mold, mold mycelia can be observed dis‐ tinctly as small white spots on the grains. Furthermore, the material temperature has risen to 32–34°C. It is dispensed into wooden boxes, each with 15–45 kg of the grain. To control the rise in temperature and the moisture in the grain mass, the bottom of the box is made of wooden lattice or wire mesh. Temperature and moisture contents are also controlled by the thickness of the heaped grain layer in the box: 8 cm at the beginning, 6 cm at the first mix‐ ing, and 4 cm at the second mixing. Thereafter, at intervals of 6–8 h, the material is mixed and heaped again in the box. After incubation for about 40 h, the temperature of the materi‐ al rises to 40–42°C. The mycelium develops to cover and penetrate the grains which have sufficient enzymes, vitamins and various nutritive substances for mashing and growth of *SAKE* yeast. Then the *KOJI* is taken out of the room and spread on a clean cloth to be cooled until it is used for mashing. α-amylase and acid-protease activities increase during *KOJI* making. Carbohydrates are decomposed finally to water and carbon dioxide, which engen‐ ders the production of energy for growth of the mold.

*TO* mash. These bacteria multiply to reach a maximum count of about 107

and mash promotes the growth of lactic-acid bacteria in the early stages [36].

**•** Convenient *MOTO* preparation as *SOKUJYO-MOTO*

latter eventually predominate during the *MOTO* process [5].

Nitrite-reducing Bacteria

**Figure 7.** Changing numbers of micro-organisms in *KIMOTO* mash.

of lactic-acid bacteria [5].

1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08

reducing bacteria.

CFU (cells/ml)

other gram disappear before fermentation by *SAKE* yeast begins because of the accumula‐ tion of high concentrations of sugar and because of acidification resulting from the growth

The mixing process helps to dissolve the nutrients contained in the *KOJI* and steamed rice,

Recently, *SOKUJYO-MOTO* is popular for use in *SAKE* brewing. It was devised by Eda [37]. It is based on the principle that addition of pure lactic acid to *MOTO* can prevent contamina‐ tion by wild microorganisms. It takes a short time (7–15 days) to produce *MOTO* because of the time-saving lactic-acid formation by naturally occurring lactic acid bacteria, and saccha‐ rification of the mash proceeds quickly with the high initial mashing temperature (18–22°C). In this production, commercial lactic acid (75%, 650–700 m1/100 L of water) is added to the mash to adjust the pH value to 3.6–3.8. Although pure culture yeast is used as the inocu‐ lums, yeast grows more advantageously than do wild yeasts from *KOJI*. Furthermore, the

0 2 4 6 8 10 12 14 16 18 20

Lactic acid bacteria

(cocci) (bacci) *SAKE* yeast

Wild yeast Film yeast

Period (Days)

This predominance might be ascribed to the fact that the high mashing temperature and acidic conditions are close to the optimum for multiplication of both culture and wild yeasts. In addition, as opposed to the behavior in the classical process, no natural selection of wild yeasts by the toxic effect of nitrite occurs because the presence of lactic acid inhibits nitrate-

–108

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*SAKE* Alcoholic Beverage Production in Japanese Food Industry

/g. However,

53

### **5.5.** *SAKE* **mash fermentation**

### *5.5.1. 'MOTO' as yeast starter*

In *SAKE* brewing, *MOTO* is important as a yeast starter for the fermentation of *MOROMI*. *MOTO* is necessary to provide a pure and abundant yeast crop, and to supply sufficient lac‐ tic acid to prevent contamination of harmful wild yeast or bacteria during *MOTO* produc‐ tion or in the early stages of main fermentation.

In traditional *MOTO* preparation, lactic acid is produced by lactic-acid bacteria in the mash. In the modern method, pure lactic acid is added to the mash at the beginning of *MOTO* preparation. Lately, compression yeast cultivated using a method similar to that for baker's yeast used to ferment main mash safely with this yeast instead of *MOTO.* The amount of rice used for *MOTO* preparation is usually 7% of the total rice used for the entire *SAKE* mash.

**•** Traditional Seed Mash

*KIMOTO* is a traditional *MOTO.* Actually, *MOTO* has been handed down from early times, and the *MOTO* was modified to be simple and convenience by Kagi et al. [34]. The modified *MOTO* is called *KIMOTO.* The *YAMAHAIMOTO* is based on the same microbiological prin‐ ciple as that of *KIMOTO*, and has practically replaced *KIMOTO* because the related proce‐ dure is simpler [5].

Steamed rice (120 kg) is mixed with 60 kg of *KOJI* and 200 L of water in a vessel at an initial temperature of 13–14°C. It is then kept for 3–4 days with intermittent stirring and agitation. During this period, the rice grains are partially degraded and saccharified, and the tempera‐ ture falls gradually to 7–8°C. The mash is then warmed at a rate of 0.5–1.0°C/day by placing a wooden or metal cask filled with hot water in the mash after warming for an additional 10–15 days, after which the temperature reaches 14–15°C. In *KIMOTO* mash, some microorganisms grow successively to each other as Fig. 7, and mash brings acid condition to grow *SAKE* yeast easily without contamination [5].

In early stages, contaminating wild yeast or germs disappears within the first two weeks as a result of the toxic effect of nitrite, produced by nitrate-reducing bacteria from nitrate con‐ tained in or added to the water. Slight nitrate contained in the mother water is converted to nitrite, which has toxicity for micro-organisms by nitrate-reducing bacteria such as *Achromo‐ bacter* spp., *Flavobacterium* spp., *Pseudomonas* spp., and *Micrococcus* spp. (derived from *KOJI* and water). A toxic substance, nitrite, yeast of one kind from *KOJI* as *Pichia angusta* [35] was assimilated after oxidating nitrite during *MOTO* mash. Nitrite is toxic for lactic acid bacteria and fermentative yeast in traditional *MOTO.* Their utility micro-organisms are able to grow under the *MOTO* mash containing nitrite. After removing nitrite, lactic-acid bacteria includ‐ ing *Leuconostoc mesenteroides* and *Lactobacillus sakei* (derived from *KOJI*) can grow in the *MO‐* *TO* mash. These bacteria multiply to reach a maximum count of about 107 –108 /g. However, other gram disappear before fermentation by *SAKE* yeast begins because of the accumula‐ tion of high concentrations of sugar and because of acidification resulting from the growth of lactic-acid bacteria [5].

The mixing process helps to dissolve the nutrients contained in the *KOJI* and steamed rice, and mash promotes the growth of lactic-acid bacteria in the early stages [36].

**•** Convenient *MOTO* preparation as *SOKUJYO-MOTO*

until it is used for mashing. α-amylase and acid-protease activities increase during *KOJI* making. Carbohydrates are decomposed finally to water and carbon dioxide, which engen‐

In *SAKE* brewing, *MOTO* is important as a yeast starter for the fermentation of *MOROMI*. *MOTO* is necessary to provide a pure and abundant yeast crop, and to supply sufficient lac‐ tic acid to prevent contamination of harmful wild yeast or bacteria during *MOTO* produc‐

In traditional *MOTO* preparation, lactic acid is produced by lactic-acid bacteria in the mash. In the modern method, pure lactic acid is added to the mash at the beginning of *MOTO* preparation. Lately, compression yeast cultivated using a method similar to that for baker's yeast used to ferment main mash safely with this yeast instead of *MOTO.* The amount of rice used for *MOTO* preparation is usually 7% of the total rice used for the entire *SAKE*

*KIMOTO* is a traditional *MOTO.* Actually, *MOTO* has been handed down from early times, and the *MOTO* was modified to be simple and convenience by Kagi et al. [34]. The modified *MOTO* is called *KIMOTO.* The *YAMAHAIMOTO* is based on the same microbiological prin‐ ciple as that of *KIMOTO*, and has practically replaced *KIMOTO* because the related proce‐

Steamed rice (120 kg) is mixed with 60 kg of *KOJI* and 200 L of water in a vessel at an initial temperature of 13–14°C. It is then kept for 3–4 days with intermittent stirring and agitation. During this period, the rice grains are partially degraded and saccharified, and the tempera‐ ture falls gradually to 7–8°C. The mash is then warmed at a rate of 0.5–1.0°C/day by placing a wooden or metal cask filled with hot water in the mash after warming for an additional 10–15 days, after which the temperature reaches 14–15°C. In *KIMOTO* mash, some microorganisms grow successively to each other as Fig. 7, and mash brings acid condition to grow

In early stages, contaminating wild yeast or germs disappears within the first two weeks as a result of the toxic effect of nitrite, produced by nitrate-reducing bacteria from nitrate con‐ tained in or added to the water. Slight nitrate contained in the mother water is converted to nitrite, which has toxicity for micro-organisms by nitrate-reducing bacteria such as *Achromo‐ bacter* spp., *Flavobacterium* spp., *Pseudomonas* spp., and *Micrococcus* spp. (derived from *KOJI* and water). A toxic substance, nitrite, yeast of one kind from *KOJI* as *Pichia angusta* [35] was assimilated after oxidating nitrite during *MOTO* mash. Nitrite is toxic for lactic acid bacteria and fermentative yeast in traditional *MOTO.* Their utility micro-organisms are able to grow under the *MOTO* mash containing nitrite. After removing nitrite, lactic-acid bacteria includ‐ ing *Leuconostoc mesenteroides* and *Lactobacillus sakei* (derived from *KOJI*) can grow in the *MO‐*

ders the production of energy for growth of the mold.

tion or in the early stages of main fermentation.

*SAKE* yeast easily without contamination [5].

**5.5.** *SAKE* **mash fermentation**

*5.5.1. 'MOTO' as yeast starter*

**•** Traditional Seed Mash

dure is simpler [5].

mash.

52 Food Industry

Recently, *SOKUJYO-MOTO* is popular for use in *SAKE* brewing. It was devised by Eda [37]. It is based on the principle that addition of pure lactic acid to *MOTO* can prevent contamina‐ tion by wild microorganisms. It takes a short time (7–15 days) to produce *MOTO* because of the time-saving lactic-acid formation by naturally occurring lactic acid bacteria, and saccha‐ rification of the mash proceeds quickly with the high initial mashing temperature (18–22°C). In this production, commercial lactic acid (75%, 650–700 m1/100 L of water) is added to the mash to adjust the pH value to 3.6–3.8. Although pure culture yeast is used as the inocu‐ lums, yeast grows more advantageously than do wild yeasts from *KOJI*. Furthermore, the latter eventually predominate during the *MOTO* process [5].

**Figure 7.** Changing numbers of micro-organisms in *KIMOTO* mash.

This predominance might be ascribed to the fact that the high mashing temperature and acidic conditions are close to the optimum for multiplication of both culture and wild yeasts. In addition, as opposed to the behavior in the classical process, no natural selection of wild yeasts by the toxic effect of nitrite occurs because the presence of lactic acid inhibits nitratereducing bacteria.

An example of the preparation of *SOKUJYO-MOTO* is the following: *KOJI* (60 kg) is added with 200 L of water and 140 ml of lactic acid (75%). A pure culture of *SAKE* yeast is inoculated to the mash (105 – 106 /g). Its temperature is about 12°C. Steamed rice (140 kg) is added to the mixture, cooling it sufficiently to give a temperature of about 18–20°C. After keeping the mash for 1–2 days with intermittent stirring and agitation, it is warmed gradually in the same way as *YAMAHAI-MOTO* by increasing the temperature at a rate of approx. 1.0–1.5°C/day. As the temperature rises to about 15°C, *SAKE* yeast reaches its peak and fermentation begins.

tion. In *SAKE* brewing, temperature control is also extremely important to balance saccharification and fermentation, both of which occur simultaneously in *MOROMI*. There‐ fore, we call it 'Parallel Fermentation'. Small quantities of sugars released from steamed rice and *KOJI* are fermented gradually by *SAKE* yeast until the alcohol content reaches nearly 20% (v/v). Accumulated alcohol of 20% v/v in the mash from 40% (w/v) of sugars. If such a high concentration of sugars is supplied at once, then *SAKE* yeast would not ferment alcohol in the mash. Instead, the mash fermentation at a low temperature (below 10–18°C) is also a characteristic of *SAKE* brewing which gives the mash a balanced flavor and taste as well as a high alcohol concentration. After the third addition of materials, the mash is agitated, usual‐

A foam resembles soap suds. Furthermore, it spreads gradually over the surface, and subse‐ quently increases to form a thick layer. A fresh fruit-like aroma at this stage indicates healthy fermentation. The fermentation gradually becomes more vigorous with a rise in mash temperature, and a rather viscous foam rises to form *TAKA-AWA* (a deep layer of foam, shown in Fig. 2), which reaches to the brim of the vessel. In some breweries, it is bro‐ ken down with a small electric agitator. At this stage, the yeast cell count reaches a maxi‐

becomes less dense, and is easily dispersed. The fermentation finishes usually during 20–25 days. In some breweries, pure alcohol (30–40%) is added to the mash to adjust the final con‐

Quite often, to sweeten the mash, 7–10% of the total amount of steamed rice is added during the final stage of the *MOROMI* process to produce glucose from starch by the saccharifying

After alcohol fermentation, the mash is divided into *SAKE* and solids by filtration. The mash is poured into bags of about 5 L capacity made of synthetic fiber, which are laid in a rectan‐ gular box. *SAKE* is squeezed out under hydraulic pressure. After complete filtration, the sol‐ ids pressed in a sheet are stripped out of the bags. Recently several automatic filter presses for filtering *MOROMI* mash have been used. The *SAKE* lees or *SAKE* cake, residue squeez‐ ing *SAKE* as cake was called '*SAKE*-KASU', contains starch, protein, yeast cells and various enzymes, *SAKE* lees is used traditionally for making foodstuffs such as pickles and soup. In general, regarding 3 kl of *SAKE* containing 20% ethanol and 200–250 kg of KASU are ob‐ tained from one ton of polished rice. The slightly turbid *SAKE* is clarified to separate lees by

After settling the clarified *SAKE* for a further 30–40 days, The *SAKE* is pasteurized, killing yeasts, harmful lactic acid bacteria, and enzymes. The *SAKE* is heated to 60–65°C, passing it through a helical tube type heat exchanger for a short time. Recently plate-type heat ex‐

/g [38]. Because the alcohol concentrations increase, the foam

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55

ly twice a day. The mash density then reaches maximum levels 3–4 days later.

mum of about 2.5 × 108

*5.5.3. Filtration [1,5]*

centration to about 20–22% (v/v).

action of *KOJI* that accumulates in the mash.

standing in a vessel for 5–10 days at a low temperature.

changers with high efficiency of heat transfer have become available.

*5.5.4. Storage (aging) and bottling [5]*

The cultivation period can be shortened further by starting the mashing at 25°C and by keeping the temperature of *MOTO* over 18°C. Moreover, the variety of *SOKUJYO-MOTO* as *KOONTOKA-MOTO* (hot-mashed *MOTO*) is used by Japanese brewers. This mashing meth‐ od is conducted at 56–60°C during several hours with subsequent inoculation of pure cul‐ tured *SAKE* yeast. To prevent excessive accumulation of sugars and the development of a high viscosity, the ratio of water to rice used is raised to 150–160 L/100 kg [5].

### *5.5.2. Main fermentation [5]*

*MOROMI*, as main mash, is fermented in a large open vessel with a capacity ranging from 6–20 kl without special sterilization, in an open fermentation system. The weight of polished rice (1.5 t) was used for mashing one lot as standard. However, recently, larger vessels as 3– 7 tons or sometimes over 10 tons have been used for mashing one lot. The *MOROMI* mash is brewed steamed rice, *KOJI* and water. Table 2 shows proportions of various raw materials used for a typical *MOROMI* mash. The preparation of stepwise mashing as three steps is one characteristic of *MOROMI* mash production. First, steamed rice, *KOJI* and water are added to the *MOTO.* Consequently, the total acid and yeast population in *MOTO* are diluted to about one-half. The temperature of the first mash is about 12°C, and the yeast propagates gradually. After two days, the yeast grows until 108 /g, which reaches the same order as that in *MOTO.* As a second addition, the materials are added in an amount that is nearly twice as much as the first addition. The yeast population and total acids are diluted by about half too. The temperature of the second addition is lowered to 9–10°C. In a third addition, mate‐ rials are added in a larger amount.


**Table 2.** Proportions of raw materials used in a typical *SAKEMOROMI* [1]

The amount of *MOROMI* bring 14 folds as same as *MOTO* mash. Whereby yeast cells are diluted. This stepwise addition of material plays an important role in suppressing the inva‐ sion of wild micro-organisms together with lowering the mashing temperature in each addi‐ tion. In *SAKE* brewing, temperature control is also extremely important to balance saccharification and fermentation, both of which occur simultaneously in *MOROMI*. There‐ fore, we call it 'Parallel Fermentation'. Small quantities of sugars released from steamed rice and *KOJI* are fermented gradually by *SAKE* yeast until the alcohol content reaches nearly 20% (v/v). Accumulated alcohol of 20% v/v in the mash from 40% (w/v) of sugars. If such a high concentration of sugars is supplied at once, then *SAKE* yeast would not ferment alcohol in the mash. Instead, the mash fermentation at a low temperature (below 10–18°C) is also a characteristic of *SAKE* brewing which gives the mash a balanced flavor and taste as well as a high alcohol concentration. After the third addition of materials, the mash is agitated, usual‐ ly twice a day. The mash density then reaches maximum levels 3–4 days later.

A foam resembles soap suds. Furthermore, it spreads gradually over the surface, and subse‐ quently increases to form a thick layer. A fresh fruit-like aroma at this stage indicates healthy fermentation. The fermentation gradually becomes more vigorous with a rise in mash temperature, and a rather viscous foam rises to form *TAKA-AWA* (a deep layer of foam, shown in Fig. 2), which reaches to the brim of the vessel. In some breweries, it is bro‐ ken down with a small electric agitator. At this stage, the yeast cell count reaches a maxi‐ mum of about 2.5 × 108 /g [38]. Because the alcohol concentrations increase, the foam becomes less dense, and is easily dispersed. The fermentation finishes usually during 20–25 days. In some breweries, pure alcohol (30–40%) is added to the mash to adjust the final con‐ centration to about 20–22% (v/v).

Quite often, to sweeten the mash, 7–10% of the total amount of steamed rice is added during the final stage of the *MOROMI* process to produce glucose from starch by the saccharifying action of *KOJI* that accumulates in the mash.

#### *5.5.3. Filtration [1,5]*

An example of the preparation of *SOKUJYO-MOTO* is the following: *KOJI* (60 kg) is added with 200 L of water and 140 ml of lactic acid (75%). A pure culture of *SAKE* yeast is inoculated

mixture, cooling it sufficiently to give a temperature of about 18–20°C. After keeping the mash for 1–2 days with intermittent stirring and agitation, it is warmed gradually in the same way as *YAMAHAI-MOTO* by increasing the temperature at a rate of approx. 1.0–1.5°C/day. As the

The cultivation period can be shortened further by starting the mashing at 25°C and by keeping the temperature of *MOTO* over 18°C. Moreover, the variety of *SOKUJYO-MOTO* as *KOONTOKA-MOTO* (hot-mashed *MOTO*) is used by Japanese brewers. This mashing meth‐ od is conducted at 56–60°C during several hours with subsequent inoculation of pure cul‐ tured *SAKE* yeast. To prevent excessive accumulation of sugars and the development of a

*MOROMI*, as main mash, is fermented in a large open vessel with a capacity ranging from 6–20 kl without special sterilization, in an open fermentation system. The weight of polished rice (1.5 t) was used for mashing one lot as standard. However, recently, larger vessels as 3– 7 tons or sometimes over 10 tons have been used for mashing one lot. The *MOROMI* mash is brewed steamed rice, *KOJI* and water. Table 2 shows proportions of various raw materials used for a typical *MOROMI* mash. The preparation of stepwise mashing as three steps is one characteristic of *MOROMI* mash production. First, steamed rice, *KOJI* and water are added to the *MOTO.* Consequently, the total acid and yeast population in *MOTO* are diluted to about one-half. The temperature of the first mash is about 12°C, and the yeast propagates

in *MOTO.* As a second addition, the materials are added in an amount that is nearly twice as much as the first addition. The yeast population and total acids are diluted by about half too. The temperature of the second addition is lowered to 9–10°C. In a third addition, mate‐

Total rice (kg) 140 280 890 160 2000 Steamed rice (kg) 95 200 720 160 1580 *KOJI* rice (kg) 45 80 170 420 Water (liter) 155 250 1260 160 2460

The amount of *MOROMI* bring 14 folds as same as *MOTO* mash. Whereby yeast cells are diluted. This stepwise addition of material plays an important role in suppressing the inva‐ sion of wild micro-organisms together with lowering the mashing temperature in each addi‐

**1st addition 2nd addition 3rd addition 4th addition Total**

temperature rises to about 15°C, *SAKE* yeast reaches its peak and fermentation begins.

high viscosity, the ratio of water to rice used is raised to 150–160 L/100 kg [5].

/g). Its temperature is about 12°C. Steamed rice (140 kg) is added to the

/g, which reaches the same order as that

to the mash (105

54 Food Industry

*5.5.2. Main fermentation [5]*

gradually. After two days, the yeast grows until 108

**Table 2.** Proportions of raw materials used in a typical *SAKEMOROMI* [1]

rials are added in a larger amount.

– 106

After alcohol fermentation, the mash is divided into *SAKE* and solids by filtration. The mash is poured into bags of about 5 L capacity made of synthetic fiber, which are laid in a rectan‐ gular box. *SAKE* is squeezed out under hydraulic pressure. After complete filtration, the sol‐ ids pressed in a sheet are stripped out of the bags. Recently several automatic filter presses for filtering *MOROMI* mash have been used. The *SAKE* lees or *SAKE* cake, residue squeez‐ ing *SAKE* as cake was called '*SAKE*-KASU', contains starch, protein, yeast cells and various enzymes, *SAKE* lees is used traditionally for making foodstuffs such as pickles and soup. In general, regarding 3 kl of *SAKE* containing 20% ethanol and 200–250 kg of KASU are ob‐ tained from one ton of polished rice. The slightly turbid *SAKE* is clarified to separate lees by standing in a vessel for 5–10 days at a low temperature.

#### *5.5.4. Storage (aging) and bottling [5]*

After settling the clarified *SAKE* for a further 30–40 days, The *SAKE* is pasteurized, killing yeasts, harmful lactic acid bacteria, and enzymes. The *SAKE* is heated to 60–65°C, passing it through a helical tube type heat exchanger for a short time. Recently plate-type heat ex‐ changers with high efficiency of heat transfer have become available.

produced in *SAKE* during aging. Furthermore, the *SAKE* taste is smooth and less stimulated by ethanol because of molecules of ethanol and water flocculate in the *SAKE* during aging.

Recently, aging of *SAKE* to add value has been attempted by some breweries with so-called *KOSYU* as old vintage *SAKE*. *KOSYU*-*SAKE* has rich and complex flavors and tastes like those of cherry wine and a brown color by amino-carbonyl reaction as shown in Fig. 9. Aged

> 6 Months

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57

Fig.9

3 Years

However, research of *SAKE* aging has been conducted by many researchers [1].

5 Years

In Japan, *SAKE* production and labeling are regulated strictly by the Liquor Tax Law. Ac‐ cording to this law, *SAKE* is made from defined raw materials and methods of production as follows: 1, *SAKE* is an alcoholic beverage produced by fermenting materials such as rice, rice-*KOJI*, and water, with subsequent filtering of the material mixture. 2, *SAKE* is an alco‐ holic beverage fermenting a material such as rice, water, *SAKE* lees, rice-*KOJI*, and other ma‐ terial as authorized by government ordinance and filtering the material mixture. 3, *SAKE* is an alcoholic beverage filtrate of a mixture of *SAKE* and *SAKE* lees. Moreover, *SAKE* has been categorized as grand, first and second class, by alcohol concentration, and sensory

10 Years

**Figure 9.** Changes of color of sake during aging.

**6. Varieties of** *SAKE* **[39]**

*SAKE* can even have a chocolate color.

**Figure 8.** SAKE squeezer (Photograph by Hamada Co. Ltd)

As described in this chapter, the history of *SAKE* pasteurization began in the 16th century, before Pasteur's discoveries. After pasteurization, *SAKE* is transferred to sealed vessels for storage with or without addition of activated carbon. Pasteurization and the high content of alcohol in *SAKE* (usually 20%) prevent microbial infection. The blended *SAKE* is diluted with water to the appropriate alcohol content, usually 15.0–16.5% (v/v), and is filtered through activated carbon to improve the flavor and taste and to adjust the color and clarity. In modern procedures, filtration through activated carbon is followed by filtration through membranes or sheets having numerous pores of micrometer size, thereby removing minute particles including micro-organisms if any are present. This procedure enables the *SAKE* producer to omit pasteurization in the bottling procedure and therefore to prevent deterio‐ ration of quality caused by heating *SAKE*. The spoilage of *SAKE* is sometimes encountered, off-flavors and tastes are attributed mainly to the formation of diacetyl and acetic acid by *HIOCHI* bacteria.

*SAKE* is usually sold in a pale blue bottle of 1.8 l capacity, which is pervious to short and medium wavelengths in sunlight, as are beer, wine, and other alcoholic beverages. Coloring is spoilage of *SAKE* by sunlight, deferriferrichrysin precipitates, and tyrosine or tryptophan, kynurenic acid or flavin precipitates as precursors of colorants. Usually *SAKE* is aged and stored for a short time. It does not age for a long time of several years or longer. Vintage wine is aged much longer than *SAKE*. During storage, *SAKE* matures gradually. The matu‐ ration process is probably the result of oxidation reactions and physicochemical changes. *SAKE* changes and adopts a smoother taste. The storage temperature should be maintained carefully at 13–18°C, with consideration being devoted to the rate of maturation and the time of bottling.

*SAKE* is browned not only by amino-carbonyl reactions but also by still unknown reactions during aging. Long-aged *SAKE* has a sherry wine-like aroma that is attributable to furfurals

Fig.9

produced in *SAKE* during aging. Furthermore, the *SAKE* taste is smooth and less stimulated by ethanol because of molecules of ethanol and water flocculate in the *SAKE* during aging. However, research of *SAKE* aging has been conducted by many researchers [1].

Recently, aging of *SAKE* to add value has been attempted by some breweries with so-called *KOSYU* as old vintage *SAKE*. *KOSYU*-*SAKE* has rich and complex flavors and tastes like those of cherry wine and a brown color by amino-carbonyl reaction as shown in Fig. 9. Aged *SAKE* can even have a chocolate color.

**Figure 9.** Changes of color of sake during aging.

## **6. Varieties of** *SAKE* **[39]**

**Figure 8.** SAKE squeezer (Photograph by Hamada Co. Ltd)

*HIOCHI* bacteria.

56 Food Industry

time of bottling.

As described in this chapter, the history of *SAKE* pasteurization began in the 16th century, before Pasteur's discoveries. After pasteurization, *SAKE* is transferred to sealed vessels for storage with or without addition of activated carbon. Pasteurization and the high content of alcohol in *SAKE* (usually 20%) prevent microbial infection. The blended *SAKE* is diluted with water to the appropriate alcohol content, usually 15.0–16.5% (v/v), and is filtered through activated carbon to improve the flavor and taste and to adjust the color and clarity. In modern procedures, filtration through activated carbon is followed by filtration through membranes or sheets having numerous pores of micrometer size, thereby removing minute particles including micro-organisms if any are present. This procedure enables the *SAKE* producer to omit pasteurization in the bottling procedure and therefore to prevent deterio‐ ration of quality caused by heating *SAKE*. The spoilage of *SAKE* is sometimes encountered, off-flavors and tastes are attributed mainly to the formation of diacetyl and acetic acid by

*SAKE* is usually sold in a pale blue bottle of 1.8 l capacity, which is pervious to short and medium wavelengths in sunlight, as are beer, wine, and other alcoholic beverages. Coloring is spoilage of *SAKE* by sunlight, deferriferrichrysin precipitates, and tyrosine or tryptophan, kynurenic acid or flavin precipitates as precursors of colorants. Usually *SAKE* is aged and stored for a short time. It does not age for a long time of several years or longer. Vintage wine is aged much longer than *SAKE*. During storage, *SAKE* matures gradually. The matu‐ ration process is probably the result of oxidation reactions and physicochemical changes. *SAKE* changes and adopts a smoother taste. The storage temperature should be maintained carefully at 13–18°C, with consideration being devoted to the rate of maturation and the

*SAKE* is browned not only by amino-carbonyl reactions but also by still unknown reactions during aging. Long-aged *SAKE* has a sherry wine-like aroma that is attributable to furfurals In Japan, *SAKE* production and labeling are regulated strictly by the Liquor Tax Law. Ac‐ cording to this law, *SAKE* is made from defined raw materials and methods of production as follows: 1, *SAKE* is an alcoholic beverage produced by fermenting materials such as rice, rice-*KOJI*, and water, with subsequent filtering of the material mixture. 2, *SAKE* is an alco‐ holic beverage fermenting a material such as rice, water, *SAKE* lees, rice-*KOJI*, and other ma‐ terial as authorized by government ordinance and filtering the material mixture. 3, *SAKE* is an alcoholic beverage filtrate of a mixture of *SAKE* and *SAKE* lees. Moreover, *SAKE* has been categorized as grand, first and second class, by alcohol concentration, and sensory evaluation by officers until 1992. However, labels and names of *SAKE* have not been regulat‐ ed by law. For various reasons, many commercial products, *SAKE* which labels producing method or excessive name, was sold in the market and low-quality *SAKE* also was sold. Fur‐ thermore, many consumers were confused and purchased it mistakenly. Whereby, they were regulated by law in 1992.

**7.** *SAKE* **tastes**

Appearance 1.000

Intensity of aroma

Appeal of aroma

Intensity of sourness

Intensity of bitterness

Intensity of sweetness

Preference of consumer

Appeal (Balance) of tastes

As explained in this chapter, *SAKE* is a favorite food and beverages and individual favored *SAKE* and components of *SAKE* are important factors for purchasing *SAKE*. Over 500 chemi‐

*SAKE* consumption has decreased since the 1970s, it is 1.7 million kL. Recently, in 2009, *SAKE* consumption is about one-third that of the 1970s. According to a survey of household spending conducted by the Public Management Ministry in Japan [40], consumers in their 20s spend 1100 yen per month for *SAKE*, those in their 30s spend 2500 yen per month, and those in their 60s spend 3800 yen per month. Elder consumers spend three times as much as young consumers. To examine favorite tastes of young consumers (20s–30s) play a role to

> **Intensity of sourness**

**Intensity of bitterness**

**Intensity of sweetness**

*SAKE* Alcoholic Beverage Production in Japanese Food Industry

http://dx.doi.org/10.5772/53153

59

**Appeal (Balance) of tastes**

**Preference of consumer**

cal compounds exist in *SAKE*, producing a complex flavor and taste in *SAKE*.

development of new *SAKE* for them and to increase *SAKE* consumption in Japan.

**Appeal of aroma**

**Appearance Intensity**

0.450 1.000

(Correlations are significant; \*\*, *P* < 0.01, \*, *P* < 0.05)



**Table 4.** Correlations for Evaluating *SAKE* using Sensory Evaluation Methods


0.030 -0.380 \*\* 0.210 0.030 -0.570 1.000


Suzuki and co-authors [41] investigated the opinions and preferences of panelists (22 persons, 20s–30s) to conduct a *SAKE* sensory evaluation for research into favorite tastes and consumer preferences. The correlation of sensory evaluations of *SAKE* are presented in Table 4. Correla‐


**of aroma**


**Table 3.** Classification of *SAKE* types by law [39]

Instead of *SAKE* grades such as grand grade, fiesta grade and second grade that had been used until 1992, *SAKE* is categorized as *DAIGINJYO*-*SHU*, *GINJYO*-*SHU*, *JYUNMAI*-*SHU*, *JYUNMAI*-*DAIGINJYOU*-*SHU*, *JYUNMAI*-*GINJYOU*-*SYU*, *TOKUBETSU*-*JYUNMAI*-*SYU*, *HONJYOZO*-*SYU*, and *TOKUBETSU*-*HONJYOZO*-*SHU*, and the labeling *SAKE* is regulated by the law as shown in Table 3.

The polishing rice ratio and using *KOJI* ratio regulated by the law to sell their categorized *SAKE*. Then they must be shown on the label. JYUNMAI means that *SAKE* is brewed using only rice and rice-*KOJI* and mother water, and *GINJYO* means special brewing. *DAI*-*GINJYO* means special brewing and prestige class in the *SAKE* brewery. Consequently, *DAI*-*GINJYO* tends to be expensive, but the price of *SAKE* is decided by the policy of the brewery. Addi‐ tionally, *KOSYU* as aged vintage *SAKE* or *NAMAZAKE* as non-pasteurized *SAKE* is dis‐ played on the *SAKE* label. It is necessary that some method or public organization manage other *SAKE* label items.

## **7.** *SAKE* **tastes**

evaluation by officers until 1992. However, labels and names of *SAKE* have not been regulat‐ ed by law. For various reasons, many commercial products, *SAKE* which labels producing method or excessive name, was sold in the market and low-quality *SAKE* also was sold. Fur‐ thermore, many consumers were confused and purchased it mistakenly. Whereby, they

**Polishing ratio**

Rice, rice *KOJI* - Good flavor; good and clear appearance

Rice, rice *KOJI* Less than 60% Especially good flavor; good and clear

Instead of *SAKE* grades such as grand grade, fiesta grade and second grade that had been used until 1992, *SAKE* is categorized as *DAIGINJYO*-*SHU*, *GINJYO*-*SHU*, *JYUNMAI*-*SHU*, *JYUNMAI*-*DAIGINJYOU*-*SHU*, *JYUNMAI*-*GINJYOU*-*SYU*, *TOKUBETSU*-*JYUNMAI*-*SYU*, *HONJYOZO*-*SYU*, and *TOKUBETSU*-*HONJYOZO*-*SHU*, and the labeling *SAKE* is regulated

The polishing rice ratio and using *KOJI* ratio regulated by the law to sell their categorized *SAKE*. Then they must be shown on the label. JYUNMAI means that *SAKE* is brewed using only rice and rice-*KOJI* and mother water, and *GINJYO* means special brewing. *DAI*-*GINJYO* means special brewing and prestige class in the *SAKE* brewery. Consequently, *DAI*-*GINJYO* tends to be expensive, but the price of *SAKE* is decided by the policy of the brewery. Addi‐ tionally, *KOSYU* as aged vintage *SAKE* or *NAMAZAKE* as non-pasteurized *SAKE* is dis‐ played on the *SAKE* label. It is necessary that some method or public organization manage

Rice, rice *KOJI* Less than 60% Fermentation at low temperature; fruity- flavor;

Rice, rice *KOJI* Less than 50% Fermentation at low temperature; fruity- flavor;

appearance

appearance

**Requirement**

Less than 60% Fermentation at low temperature; fruity- flavor; good and clear appearance

Less than 50% Fermentation at low temperature; fruity- flavor; good and clear appearance

good and clear appearance

good and clear appearance

Less than 70% Good flavor; good and clear appearance

Less than 60% Especially good flavor; good and clear

were regulated by law in 1992.

GINJYO -SYU

58 Food Industry

DAI-GINJYO-SYU

JYUNMAI-SYU

JYUNMAI-GINJYO-SYU

JYUNMAI-DAI-GINJYO-SYU

TOKUBETSU-JYUNMAI

HONJYOZO-SYU

TOKUBETSU-JYUNMAI

*SAKE* **type Used material Used rice**

Rice, rice *KOJI* and pure distilled alcohol

Rice, rice *KOJI* and pure distilled alcohol

Rice, rice *KOJI* and pure distilled alcohol

Rice, rice *KOJI* and pure distilled alcohol

**Table 3.** Classification of *SAKE* types by law [39]

by the law as shown in Table 3.

other *SAKE* label items.

As explained in this chapter, *SAKE* is a favorite food and beverages and individual favored *SAKE* and components of *SAKE* are important factors for purchasing *SAKE*. Over 500 chemi‐ cal compounds exist in *SAKE*, producing a complex flavor and taste in *SAKE*.

*SAKE* consumption has decreased since the 1970s, it is 1.7 million kL. Recently, in 2009, *SAKE* consumption is about one-third that of the 1970s. According to a survey of household spending conducted by the Public Management Ministry in Japan [40], consumers in their 20s spend 1100 yen per month for *SAKE*, those in their 30s spend 2500 yen per month, and those in their 60s spend 3800 yen per month. Elder consumers spend three times as much as young consumers. To examine favorite tastes of young consumers (20s–30s) play a role to development of new *SAKE* for them and to increase *SAKE* consumption in Japan.


**Table 4.** Correlations for Evaluating *SAKE* using Sensory Evaluation Methods

Suzuki and co-authors [41] investigated the opinions and preferences of panelists (22 persons, 20s–30s) to conduct a *SAKE* sensory evaluation for research into favorite tastes and consumer preferences. The correlation of sensory evaluations of *SAKE* are presented in Table 4. Correla‐ tion was found between 'Intensity of aroma' and 'Balance of taste', for which the correlation factor is 0.55, and the relation between 'Appetite of consumers', with a correlation factor of 0.65. However, it showed a negative relation with 'bitterness', and the correlation factor was -0.430. Young consumers hope to buy and drink *SAKE* having a favorite flavor. Furthermore, correlation was found between 'Preferences of consumers', and 'Balance of taste', with a corre‐ lation factor is 0.88. 'Preferences of consumers', showed a relation with 'Intensity of bitter‐ ness', with close correlation factor of -0.80. Furthermore, a negative and close statistical correlation was found between 'Balance of *SAKE* tastes' and 'Intensity of bitterness', for which the correlation factor was -0.86. These data show that consumers hope to buy or drink *SAKE* with no bitter taste. 'Bitter' is a decreased balance of *SAKE* test and consumer appetite. Al‐ though bitter taste has played an important role in giving richness-taste to *SAKE* for a long time, young consumers are sensitive to bitter tastes in *SAKE*. It is therefore considered that the control of bitter taste must be undertaken in brewing processes.

**Author details**

Makoto Kanauchi

**References**

Miyagi University, Japan

kyo, Japan; 1995.

Soc. Jpn. (1998). , 93, 910-915.

Soc. Brew. Jpn. 973;68: 767-771.

Japanese) J. Brew. Soc. Jpn. (1982). , 77(11), 831-835.

721-729.

York; 1977.

[1] Yoshizawa K. SAKE NO KAGAKU (Science of Alcoholic Beverages). Asakura, To‐

*SAKE* Alcoholic Beverage Production in Japanese Food Industry

http://dx.doi.org/10.5772/53153

61

[2] Kanauchi, M., Shindo, H., Suzuki, M., Kakuta, T., Yoshizawa, K., & Koizumi, T. Characteristics of traditional wheat-qu (koji) described in the classic literature, "Chi min yao shu", of the ancient Chinese. (in Japanese) J. Brew. Soc. Jpn. (1998). , 93,

[3] Kanauchi, M., Shindo, H., Suzuki, M., Kakuta, T., Yoshizawa, K., & Koizumi, T. Role of extract from cockleburr [*Xanthium strumarium*] leaves used for wheat-qu (koji) making described in Chinese old literature Chi min yao shu. (in Japanese) J. Brew.

[4] Tanaka, T., Okazaki, N., & Kitani, M. Comparison of Growth and Enzyme Produc‐ tion between *A. oryzae* and *Rhizopus* spp. Growth of Mold on Uncooked Grain (II), (in

[5] Rose AH. Economic Microbiology. Vol. 1, Alcoholic Beverages. Academic Press, New

[6] Nojiro K, Kamata K, Tadenuka M, Yoshizawa K, Mizunuma T. JOZO NO JITEN (En‐ cyclopedia of Brewage, Fermentation and Enology), Asakura, Tokyo, Japan; 1988.

[7] Nanba Y, Momose H, Ooba T. SEISYU SEIZOU GIJYUTU (Techniques of SAKE

[8] Totuka A, Namba Y, Kobuyama Y. Removal of Metal ion from water by Poly Alumi‐

[9] Sato J, Yamada M. Research of Brewer's Rice using Physical and Chemical Methods, (in Japanese) Reports of the Research Institute of Brewing, Japan 1925;93: 506-642.

[10] Yoshizawa K, Ishikawa T, Hamada Y. Sutadied on brewer's Rice (III), (in Japanese) J.

[11] Shimada S, Sugita O. in Mizumoto K. Studies of Aspergillus oryzae Strains for Sake-Brewing (IV) : On he Amylase Actions of "Koji" for Sake-Brewing. (in Japanese) Jour‐

Brewing), (in Japanese) Society of Brewing, Japan Tokyo; 1979.

num Chloride. (in Japanese) J. Brew. Soc. Jpn. 1971;67: 162-166.

nal of Fermentation Technology. Osaka 1953;31: 498-501.

## **8. Conclusions**

*SAKE* brewing necessitates the use of high-quality techniques that have been developed ex‐ perimentally without acquaintance with scientific method. Furthermore, unique techniques have been researched, as fermenting under low temperature, more than 18% of high alcohol concentration without distillation, open fermentation system without sterilization, and hav‐ ing a fruity aroma in *SAKE*. *SAKE* brewing using only rice as a material can yet produce fruity aromas such as those of apple, melon, or banana. Specially brewed *SAKE* for Japanese *SAKE* contests includes 6–7 ppm of ethyl caproate [42], which is a very high amount for al‐ coholic beverages, which is one reason that producing ethyl caproate yeast has been devel‐ oped and fostered at public institutes in many Japanese prefectures. However, strong doubts persist that their *SAKE* has been adequately adapted to favor consumers. In ques‐ tionnaire investigation, young consumers (20–30s) bring up the image that *SAKE* is a bever‐ age for elderly people [41]. This is one reason for their image that *SAKE* is a cheap alcoholic beverage also. It is expected that *SAKE* consumption will decrease because the Japanese population is decreasing as result of the nation's low birthrate and high longevity.

All brewers and researchers of the *SAKE* field must make efforts to brew high-quality *SAKE* and suitable *SAKE* for consumers or for *SAKE* not only in Japan but also in foreign countries exporting it. Furthermore, *SAKE* can be highly appreciated by connoisseurs, just as 'Cha‐ teaux' wines, Grand cru, are in European countries.

## **Acknowledgments**

I thank Hamada Co., Ltd. and Shiraki Tunesuke Co., Ltd. for supplying part of the photo‐ graphs.

## **Author details**

tion was found between 'Intensity of aroma' and 'Balance of taste', for which the correlation factor is 0.55, and the relation between 'Appetite of consumers', with a correlation factor of 0.65. However, it showed a negative relation with 'bitterness', and the correlation factor was -0.430. Young consumers hope to buy and drink *SAKE* having a favorite flavor. Furthermore, correlation was found between 'Preferences of consumers', and 'Balance of taste', with a corre‐ lation factor is 0.88. 'Preferences of consumers', showed a relation with 'Intensity of bitter‐ ness', with close correlation factor of -0.80. Furthermore, a negative and close statistical correlation was found between 'Balance of *SAKE* tastes' and 'Intensity of bitterness', for which the correlation factor was -0.86. These data show that consumers hope to buy or drink *SAKE* with no bitter taste. 'Bitter' is a decreased balance of *SAKE* test and consumer appetite. Al‐ though bitter taste has played an important role in giving richness-taste to *SAKE* for a long time, young consumers are sensitive to bitter tastes in *SAKE*. It is therefore considered that the

*SAKE* brewing necessitates the use of high-quality techniques that have been developed ex‐ perimentally without acquaintance with scientific method. Furthermore, unique techniques have been researched, as fermenting under low temperature, more than 18% of high alcohol concentration without distillation, open fermentation system without sterilization, and hav‐ ing a fruity aroma in *SAKE*. *SAKE* brewing using only rice as a material can yet produce fruity aromas such as those of apple, melon, or banana. Specially brewed *SAKE* for Japanese *SAKE* contests includes 6–7 ppm of ethyl caproate [42], which is a very high amount for al‐ coholic beverages, which is one reason that producing ethyl caproate yeast has been devel‐ oped and fostered at public institutes in many Japanese prefectures. However, strong doubts persist that their *SAKE* has been adequately adapted to favor consumers. In ques‐ tionnaire investigation, young consumers (20–30s) bring up the image that *SAKE* is a bever‐ age for elderly people [41]. This is one reason for their image that *SAKE* is a cheap alcoholic beverage also. It is expected that *SAKE* consumption will decrease because the Japanese

population is decreasing as result of the nation's low birthrate and high longevity.

teaux' wines, Grand cru, are in European countries.

**Acknowledgments**

graphs.

All brewers and researchers of the *SAKE* field must make efforts to brew high-quality *SAKE* and suitable *SAKE* for consumers or for *SAKE* not only in Japan but also in foreign countries exporting it. Furthermore, *SAKE* can be highly appreciated by connoisseurs, just as 'Cha‐

I thank Hamada Co., Ltd. and Shiraki Tunesuke Co., Ltd. for supplying part of the photo‐

control of bitter taste must be undertaken in brewing processes.

**8. Conclusions**

60 Food Industry

Makoto Kanauchi

Miyagi University, Japan

## **References**


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[28] Tomiyasu S. The flavor of spoilage HIOCHI-SAKE. (in Japanese) Journal of Fermen‐

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[29] Sugama S, Iguchi T. A study of the prevention of sake spoilage – Development of S.I. medium and its applications to prediction of hiochi phenomena. (in Japanese) J.

[30] Yoshizawa K, Ishikawa T, Noshiro K. Studies of brewer's Rice (I). (in Japanese) J.

[31] Yoshizawa K, Ishikawa T, Unemoto F, Noshiro K. Studies of Brewer's Rice (II), (in

[32] Suzuki M, Nunokawa Y, Imajuku I, Teruuchi Y, Uruma M. Studies of brewage KOJI – Comparison of temperature, period and each enzyme activity Preparation of KOJI

[33] Nunokawa Y. Studies of protease in Koji (IIII): the specificity of substrate of acid pro‐ tease and lkaline protease. (in Japanese) J. Agri. Chem. Soc. Jpn. 1962;36: 884-890.

[34] Kagi K, Otake I, Moriyama Y, Ando F, Eda K, Yamamoto T. Research of Brewing YA‐ MAHAIMOTO, (in Japanese) Reports of the Research Institute of Brewing. Japan

[35] Shimaoka Y, Kanauchi M, Kasahara S, Yoshizawa K. The Elimination of Nitrite by Pi‐ chia angusta Y-11393 Isolated from Sake Koji (in Japanese) J. Brew. Soc. Jpn.

[36] Ashizawa C. Studies on Micro-flour in YAMAHAI MOTO (10) – Cocci and Bacci Lac‐

[38] Nojiro K. Sprinkle of SAKE yeast in SAKE mash and growth of the yeast in it. (in Jap‐

[39] National Tax Agency; [http://www.nta.go.jp/shiraberu/senmonjoho/sake/hyoji/

[40] Public Management Ministry :(http://www.e-stat.go.jp/SG1/estat/List.do?

[41] Suzuki Y, Kanauchi-Kamiya H, Kanauchi M, Ishido T, Morita A, Tsubota Y. The fac‐ tors of taste determining consumer preference for Sake by consumers in their 20s or

tic acid Bacteria –. (in Japanese) J. Brew. Soc. Jpn. 1965;60(10): 900-903.

[37] Eda K. SOKUJYOMOTO. J. Brew. Soc. Jpn. 1909;4: 5-12.

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[15] Kodama K. In 'The Yeasts', (A. H. Rose and J. S. Harrison, eds.), volume 3, Academic

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[17] Nojiro K, Kosaki M, Yoshii H. JOZOGAKU (Brewing, Fermentation and Oenology),

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[19] Nunokawa Y, Ouchi K. Sake brewing using foamless mutantsof sake yeast Kyokai

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2153-2154.


**Chapter 4**

**Structuring Fat Foods**

Rene Maria Ignácio

**1. Introduction**

**1.1. Fat roles**

http://dx.doi.org/10.5772/53369

Suzana Caetano da Silva Lannes and

Additional information is available at the end of the chapter

pression of genes, and regulation of cellular signaling [1].

essing, such as cooking, grinding, and pressing processes [2].

mals, e.g. lard, tallow and fish oil [3].

Food fat provides taste, consistency, and helps us feel full. Fat is a major source of energy for the body, and aids in the absorption of lipid soluble substances including vitamins A, D, E, and K. Dietary fat is essential for normal growth, development, and maintenance, and serves a number of important functions. Increasing evidence indicates that fatty acids and their derived substances may mediate critical cellular events, including activation and ex‐

When and how humans learned to use fats and oils is unknown, but it is known that primi‐ tive people in all climates used them for food, medicine, cosmetics, lighting, preservatives, lubricants, and other purposes. The use of fats as food was probably instinctive, whereas the other applications most likely resulted from observations of their properties and behavior under various environmental conditions. More than likely, the first fats used by humans were of animal origin and were separated from the tissue by heating or boiling. Recovery of oil from small seeds or nuts required the development of more advanced methods of proc‐

The total global oil and fat market is a huge economic factor. The rise of affluence in devel‐ oping countries, this market is increasing and can be expected to increase further. The main fats commonly consumed are vegetable oils and fats, dairy fat and fats derived from ani‐

Refining edible oils such as neutralization, bleaching, and deodorization, has been practiced for just over a century, but it has had a great impact on eating habits. Whereas the refining processes have increased the availability of sufficiently palatable oils, the oil modification

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

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

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

**Chapter 4**

## **Structuring Fat Foods**

Suzana Caetano da Silva Lannes and Rene Maria Ignácio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53369

## **1. Introduction**

#### **1.1. Fat roles**

Food fat provides taste, consistency, and helps us feel full. Fat is a major source of energy for the body, and aids in the absorption of lipid soluble substances including vitamins A, D, E, and K. Dietary fat is essential for normal growth, development, and maintenance, and serves a number of important functions. Increasing evidence indicates that fatty acids and their derived substances may mediate critical cellular events, including activation and ex‐ pression of genes, and regulation of cellular signaling [1].

When and how humans learned to use fats and oils is unknown, but it is known that primi‐ tive people in all climates used them for food, medicine, cosmetics, lighting, preservatives, lubricants, and other purposes. The use of fats as food was probably instinctive, whereas the other applications most likely resulted from observations of their properties and behavior under various environmental conditions. More than likely, the first fats used by humans were of animal origin and were separated from the tissue by heating or boiling. Recovery of oil from small seeds or nuts required the development of more advanced methods of proc‐ essing, such as cooking, grinding, and pressing processes [2].

The total global oil and fat market is a huge economic factor. The rise of affluence in devel‐ oping countries, this market is increasing and can be expected to increase further. The main fats commonly consumed are vegetable oils and fats, dairy fat and fats derived from ani‐ mals, e.g. lard, tallow and fish oil [3].

Refining edible oils such as neutralization, bleaching, and deodorization, has been practiced for just over a century, but it has had a great impact on eating habits. Whereas the refining processes have increased the availability of sufficiently palatable oils, the oil modification

processes (hydrogenation, interesterification, and fractionation) have increased the useful‐ ness of edible oils by increasing their interchangeability [4].

lengths having a lower melting point than the preceding even chain acid [4]. Most of the satu‐ rated fatty acids occurring in nature have unbranched structures with an even number of car‐ bon atoms. These acids range from short-chain-length volatile liquids to waxy solids having chain lengths of ten or more carbon atoms. Fatty acids from 2 to 30 carbons (or longer) do occur, but the most common and important acids contain between 12 and 22 carbons and are found in many different plant and animal fats.Saturated fatty acids are also functionally divided into short- and long-chain acids and are most widely known by their trivial names. The short-chain saturated acids (4:0–10:0) are known to occur in milk fats and in a few seed fats [1]. Medium chain fatty acids (8:0, 10:0, 12:0, and 14:0) occur together in coconut and palm kernel oils, both tropical commodity oils. In both of these oils, lauric acid (12:0) predominates (45 to 55%). Pal‐ mitic acid (16:0) is the most abundant and widespread natural saturated acid, present in plants, animals, and microorganisms. Palm oil is a rich commodity oil source and contains over 40% of palmitic acid. Stearic acid (18:0) is also ubiquitous, usually at low levels, but is abundant in co‐ coa butter (~34%) and some animal fats, e.g., lard (5 to 24%) and beef tallow (6 to 40%). A few tropical plant species contain around 50 to 60% of 18:0 [4]. The long-chain saturated acids (19:0

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Unsaturated fatty acids contain one or more carbon-to-carbon double bonds and are liquid at room temperature with substantially lower melting points than their saturated fatty acid counterparts. Monounsaturated fatty acids have only one double bond in the carbon chain and polyunsaturated fatty acids have two or more double bonds in the carbon chain [2]. Pol‐ yunsaturated fatty acids, sometimes referred to as PUFAs or polyalkenoic acids, can be div‐ ided into conjugated (double-bonded carbon atoms alternate with single bonds) and unconjugated (double bonds are separated by one or more carbon atoms with only single

The most common monounsaturated is oleic acid (18:1 9c). Oleic acid is found in most plant and animal lipids and is the major fatty acid in olive oil (70 to 75%) and several nut oils, e.g., macadamia, pistachio, pecan, almond, and hazelnut (filbert) contain 50 to over 70%. High oleic varieties of sunflower and safflower contain 75 to 80% oleic acid. *Cis*-monounsaturated with 18 or less carbons are liquids at room temperature or low-melting solids; higher homo‐ logues are low-melting solids. *Trans*-monounsaturated are higher melting, closer to the cor‐ responding saturated acids. Double bond position also influences the melting point; both *cis*- and *trans*-C18 monounsaturated are higher melting when the double bond is at even po‐ sitions than at odd positions [4]. Saturated fatty acids are very stable, but unsaturated acids are susceptible to oxidation; the more double bonds the greater the susceptibility. Unsaturat‐ ed fatty acids, therefore, have to be handled under an atmosphere of inert gas (e.g. nitrogen)

Monosaturated and methylene-interrupted polyunsaturated fatty acids are predominantly *cis*. *Trans* isomers, mainly monosaturated, are produced during catalytic partial hydrogenation,

and kept away from oxidants or substances giving rise to free radicals [5].

and greater) are major components in only a few uncommon seed oils.

*2.1.3. Unsaturated fatty acids*

bonds) [1].

*2.1.4. Trans fatty acids*

## **2. Fats and oils**

Fats and oils are water insoluble substances that are a combination of glycerin and fatty acids called triacylglycerols. Fats appear solid at ambient temperatures and oils appear liquid. Seeds, fruits, animal, and marine sources provide oils and/or fats; however, only a few of these sour‐ ces are of economic importance. Fats and oils are the most concentrated source of energy of the three basic foods (carbohydrates, proteins, and fats), and many contain fatty acids essential for health that are not manufactured by the human body. Fats and oils are commonly referred to as triacylglycerols because the glycerin molecule has three hydroxyl groups where a fatty acid can be attached. The triacylglycerol structure is affected by the present and the position of at‐ tachment (alpha, sn-1; middle, sn-2; outer, sn-3) of each fatty acid to the glycerin. The chemical and physical properties of fats and oils are largely determined by the fatty acids that they con‐ tain and their position within the triacylglycerol molecule [2].

### **2.1. Fatty acids**

Fatty acids consist of elements, such as carbon, hydrogen, and oxygen, which are arranged as a linear carbon chain skeleton of variable length with a carboxyl group at one end. Fatty acids can be saturated (no double bond), monounsaturated (one double bond), or polyunsa‐ turated (two or more double bonds), and are essential for energetic, metabolic, and structur‐ al activities. An unsaturated fatty acid with a double bond can have two possible configurations, either *cis* or *trans*, depending on the relative positions of the alkyl groups.

#### *2.1.1. Fatty acids occurrence*

The fatty-acid carbon-chain lengths vary between 4 and 24 carbon atoms with up to three dou‐ ble bonds, with C18 the most common. Over 1000 fatty acids are known with different chain lengths, positions, configurations and types of unsaturation, and a range of additional sub‐ stituents along the aliphatic chain. However, only around 20 fatty acids occur widely in nature; of these, palmitic, oleic, and linoleic acids make up ~80% of commodity oils and fats[4].

The most prevalent saturated fatty acids are lauric (C-12:0), myristic (C-14:0), palmitic (C-16:0), stearic (C-18:0), arachidic (C-20:0), behenic (C-22:0), and lignoceric (C-24:0). The most important monounsaturated fatty acids are oleic (C-18:1) and erucic (C-22:1). The es‐ sential polyunsaturated fatty acids are linoleic (C-18:2) and linolenic (C-18:3) [2].

#### *2.1.2. Saturated fatty acids*

Saturated fatty acids contain only single carbon-to-carbon bonds and are the least reactive chemically [2]. Saturated acids with 10 or more carbons are solids, and melting points increase with chain length. Melting points alternate between odd and even chain length, with odd chain lengths having a lower melting point than the preceding even chain acid [4]. Most of the satu‐ rated fatty acids occurring in nature have unbranched structures with an even number of car‐ bon atoms. These acids range from short-chain-length volatile liquids to waxy solids having chain lengths of ten or more carbon atoms. Fatty acids from 2 to 30 carbons (or longer) do occur, but the most common and important acids contain between 12 and 22 carbons and are found in many different plant and animal fats.Saturated fatty acids are also functionally divided into short- and long-chain acids and are most widely known by their trivial names. The short-chain saturated acids (4:0–10:0) are known to occur in milk fats and in a few seed fats [1]. Medium chain fatty acids (8:0, 10:0, 12:0, and 14:0) occur together in coconut and palm kernel oils, both tropical commodity oils. In both of these oils, lauric acid (12:0) predominates (45 to 55%). Pal‐ mitic acid (16:0) is the most abundant and widespread natural saturated acid, present in plants, animals, and microorganisms. Palm oil is a rich commodity oil source and contains over 40% of palmitic acid. Stearic acid (18:0) is also ubiquitous, usually at low levels, but is abundant in co‐ coa butter (~34%) and some animal fats, e.g., lard (5 to 24%) and beef tallow (6 to 40%). A few tropical plant species contain around 50 to 60% of 18:0 [4]. The long-chain saturated acids (19:0 and greater) are major components in only a few uncommon seed oils.

#### *2.1.3. Unsaturated fatty acids*

processes (hydrogenation, interesterification, and fractionation) have increased the useful‐

Fats and oils are water insoluble substances that are a combination of glycerin and fatty acids called triacylglycerols. Fats appear solid at ambient temperatures and oils appear liquid. Seeds, fruits, animal, and marine sources provide oils and/or fats; however, only a few of these sour‐ ces are of economic importance. Fats and oils are the most concentrated source of energy of the three basic foods (carbohydrates, proteins, and fats), and many contain fatty acids essential for health that are not manufactured by the human body. Fats and oils are commonly referred to as triacylglycerols because the glycerin molecule has three hydroxyl groups where a fatty acid can be attached. The triacylglycerol structure is affected by the present and the position of at‐ tachment (alpha, sn-1; middle, sn-2; outer, sn-3) of each fatty acid to the glycerin. The chemical and physical properties of fats and oils are largely determined by the fatty acids that they con‐

Fatty acids consist of elements, such as carbon, hydrogen, and oxygen, which are arranged as a linear carbon chain skeleton of variable length with a carboxyl group at one end. Fatty acids can be saturated (no double bond), monounsaturated (one double bond), or polyunsa‐ turated (two or more double bonds), and are essential for energetic, metabolic, and structur‐ al activities. An unsaturated fatty acid with a double bond can have two possible configurations, either *cis* or *trans*, depending on the relative positions of the alkyl groups.

The fatty-acid carbon-chain lengths vary between 4 and 24 carbon atoms with up to three dou‐ ble bonds, with C18 the most common. Over 1000 fatty acids are known with different chain lengths, positions, configurations and types of unsaturation, and a range of additional sub‐ stituents along the aliphatic chain. However, only around 20 fatty acids occur widely in nature;

The most prevalent saturated fatty acids are lauric (C-12:0), myristic (C-14:0), palmitic (C-16:0), stearic (C-18:0), arachidic (C-20:0), behenic (C-22:0), and lignoceric (C-24:0). The most important monounsaturated fatty acids are oleic (C-18:1) and erucic (C-22:1). The es‐

Saturated fatty acids contain only single carbon-to-carbon bonds and are the least reactive chemically [2]. Saturated acids with 10 or more carbons are solids, and melting points increase with chain length. Melting points alternate between odd and even chain length, with odd chain

of these, palmitic, oleic, and linoleic acids make up ~80% of commodity oils and fats[4].

sential polyunsaturated fatty acids are linoleic (C-18:2) and linolenic (C-18:3) [2].

ness of edible oils by increasing their interchangeability [4].

tain and their position within the triacylglycerol molecule [2].

**2. Fats and oils**

66 Food Industry

**2.1. Fatty acids**

*2.1.1. Fatty acids occurrence*

*2.1.2. Saturated fatty acids*

Unsaturated fatty acids contain one or more carbon-to-carbon double bonds and are liquid at room temperature with substantially lower melting points than their saturated fatty acid counterparts. Monounsaturated fatty acids have only one double bond in the carbon chain and polyunsaturated fatty acids have two or more double bonds in the carbon chain [2]. Pol‐ yunsaturated fatty acids, sometimes referred to as PUFAs or polyalkenoic acids, can be div‐ ided into conjugated (double-bonded carbon atoms alternate with single bonds) and unconjugated (double bonds are separated by one or more carbon atoms with only single bonds) [1].

The most common monounsaturated is oleic acid (18:1 9c). Oleic acid is found in most plant and animal lipids and is the major fatty acid in olive oil (70 to 75%) and several nut oils, e.g., macadamia, pistachio, pecan, almond, and hazelnut (filbert) contain 50 to over 70%. High oleic varieties of sunflower and safflower contain 75 to 80% oleic acid. *Cis*-monounsaturated with 18 or less carbons are liquids at room temperature or low-melting solids; higher homo‐ logues are low-melting solids. *Trans*-monounsaturated are higher melting, closer to the cor‐ responding saturated acids. Double bond position also influences the melting point; both *cis*- and *trans*-C18 monounsaturated are higher melting when the double bond is at even po‐ sitions than at odd positions [4]. Saturated fatty acids are very stable, but unsaturated acids are susceptible to oxidation; the more double bonds the greater the susceptibility. Unsaturat‐ ed fatty acids, therefore, have to be handled under an atmosphere of inert gas (e.g. nitrogen) and kept away from oxidants or substances giving rise to free radicals [5].

#### *2.1.4. Trans fatty acids*

Monosaturated and methylene-interrupted polyunsaturated fatty acids are predominantly *cis*. *Trans* isomers, mainly monosaturated, are produced during catalytic partial hydrogenation, and can be present in substantial amounts in hardened fats, generally as a mixture of positional isomers. Heat treatment during deodorization of commodity oils may result in low levels of *trans* isomers, particularly of polyunsaturated. The undesirable nutritional properties of *trans* fatty acids have led to alternative ways of producing hardened fats, such as interesterification or blending with fully saturated fats, and to the use of milder deodorization procedures [4]. It is important to note that *trans* double bonds do occur in natural fats, as well as in industrially processed fats, but generally much less abundantly than *cis* bonds. Thus some seed oils have a significant content of fatty acids with *trans* unsaturation [5]. Saturated and *trans* fatty acids have a higher melting point than unsaturated and *cis* fatty acids [1].

*2.1.6. Low-trans*

*trans*-free fats and oils in their products [1].

**2.2. Structural characteristics**

*2.2.1. Crystals*

pearance, and eating properties [4, 12, 13].

The new rules about *trans* fatty acids promise to strongly affect what is acceptable to con‐ sumers and food manufacturers. It will be difficult to meet all the demands for low-*trans* fats and other traits that are important to consumers with the current technology, especially for frying fats and oils. Seed suppliers are busy trying to furnish seeds with compositions that will meet these needs and find farmers to grow these crops. Contracts with oilseed process‐ ors have been made to process the harvest. Some food companies have pledged to use only

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*Trans*-free fat blends can be constructed by blending oils with fully hardened oils, or indeed where the entire blend has been randomized through interesterification. Blending vegetable oil types from different sources is an efficient alternative to hydrogenated vegetable oils, and still provides the appropriate physicochemical properties and nutritional requirements demand‐ ed. Such fat blends can also be rich in polyunsaturated fatty acids as well as being *trans*- free. *Trans*-free options are commercially available in the form of a blend of tailored emulsifiers and oil blends where they meet demands for shelf-life, processing and distribution requirements. These *trans*-free options are available for a wide range of products covering, snacks, cakes, breads, tortillas, nutrition bars, cookies and breakfast cereals. *Trans*-free oil blends are also rou‐ tinely designed for margarines, where they impart structure and texture, and shortenings where they provide firmness and contribute to crumb structure [10]. The combination of *trans*free modification techniques (full hydrogenation, interesterification and fractionation) and the availability of a variety of different feedstocks can be used to produce virtually *trans*-free hard‐ stocks with a range of physical properties such as solid phase lines determining melting per‐ formances. Liquid seed oils, low in solids, are first fully hydrogenated to generate solids combined with a very low *trans* level (< 1.25%). These fully hydrogenated oils may subsequent‐ ly be interesterified with non-hydrogenated liquid oil to reduce the solid fat content at high

temperature (> 40°C). This solid fat content can be further reduced by fractionation [11].

Fats are the main structural components in many food products such as chocolate, confec‐ tionery coatings, dairy products, butter, cream shortenings, margarine, and spreads. The sensory characteristics of fat-structured materials such as spreadability, hardness, and mouth feel are highly dependent on the structure of the underlying fat crystal network. This fat crystal network is built by the interaction of polycrystalline fat particles. The amount, ge‐ ometry, and spatial distribution of solid fat crystals as well as their interactions at different levels within the network all affect the rheological properties of fats and fat-structured food products. Fat crystallization largely determines consistency, physical stability, visual ap‐

A crystal consists of a material in a solid state in which the building entities—molecules, atoms or ions—are closely packed so that the free energy of the material is at minimum. As

#### *2.1.5. Health problems*

Concerns about possible toxic effects of fatty acids with *trans*-unsaturation began with the publication of results of experiments with pigs given diets containing hydrogenated vegeta‐ ble fat for 8 months. They had more extensive arterial disease than those given otherwise equivalent diets devoid of *trans*-unsaturated fatty acids. Subsequently, numerous animal feeding trials, epidemiological studies of human populations and controlled dietary experi‐ ments with human subjects have been reported [5]. In January 2003 the US Food and Drug Administration (FDA) instituted a requirement to list *trans* fat content as a separate item on the Nutrition Facts label on packaged foods from 2006. This change in labeling requirements has served as a catalyst to accelerate food product reformulation. On a voluntary basis, many food manufacturers and restaurants have reformulated their products and modified their operations to reduce *trans* fats in their offerings [6, 7].

Consumption of *trans* fatty acids raise the level of low density lipoprotein (LDL) cholester‐ ol and decrease the level of high density lipoprotein (HDL) cholesterol. Based on results of epidemiological and intervention studies it is clear that these changes in blood profiles in‐ crease the risk of coronary heart diseases. The main food sources for *trans* fatty acids are cookies and confectionary, snacks, and frying fats [8]. Consumption of significant amounts of *trans* fatty acids has been a major health concern for consumers and regulatory agen‐ cies over the past decade. The major dietary sources of *trans* fatty acids are products for‐ mulated with partially hydrogenated fats. Examples include margarines, shortenings, bakery products, and fast foods. The regulatory mandate from FDA and consumer con‐ cerns have led to the development of alternative processes to produce foods with zero or reduced *trans* fatty acids contents [7, 9].

Fats and oils can be formulated as *trans*-acid-free products, but saturates are required for the solids contents that provide the functionality for plastic and liquid products. Reduced satu‐ rates may be an option in some cases, but a saturate-free product is probably impossible if functionality is to be maintained [2]. Obviously, if the fat is completely hydrogenated there will be no double bonds and hence no problem; however, partially hydrogenated fats have *trans* double bonds. *Trans* double bonds are rare in naturally occurring fats, the major natu‐ ral source is milk fat because they are formed by bacterial action in the rumen. So, most nat‐ urally occurring oils and fats have *cis* double bonds; however, some *trans* double bonds are found in milk fat and some marine oils.

### *2.1.6. Low-trans*

and can be present in substantial amounts in hardened fats, generally as a mixture of positional isomers. Heat treatment during deodorization of commodity oils may result in low levels of *trans* isomers, particularly of polyunsaturated. The undesirable nutritional properties of *trans* fatty acids have led to alternative ways of producing hardened fats, such as interesterification or blending with fully saturated fats, and to the use of milder deodorization procedures [4]. It is important to note that *trans* double bonds do occur in natural fats, as well as in industrially processed fats, but generally much less abundantly than *cis* bonds. Thus some seed oils have a significant content of fatty acids with *trans* unsaturation [5]. Saturated and *trans* fatty acids

Concerns about possible toxic effects of fatty acids with *trans*-unsaturation began with the publication of results of experiments with pigs given diets containing hydrogenated vegeta‐ ble fat for 8 months. They had more extensive arterial disease than those given otherwise equivalent diets devoid of *trans*-unsaturated fatty acids. Subsequently, numerous animal feeding trials, epidemiological studies of human populations and controlled dietary experi‐ ments with human subjects have been reported [5]. In January 2003 the US Food and Drug Administration (FDA) instituted a requirement to list *trans* fat content as a separate item on the Nutrition Facts label on packaged foods from 2006. This change in labeling requirements has served as a catalyst to accelerate food product reformulation. On a voluntary basis, many food manufacturers and restaurants have reformulated their products and modified

Consumption of *trans* fatty acids raise the level of low density lipoprotein (LDL) cholester‐ ol and decrease the level of high density lipoprotein (HDL) cholesterol. Based on results of epidemiological and intervention studies it is clear that these changes in blood profiles in‐ crease the risk of coronary heart diseases. The main food sources for *trans* fatty acids are cookies and confectionary, snacks, and frying fats [8]. Consumption of significant amounts of *trans* fatty acids has been a major health concern for consumers and regulatory agen‐ cies over the past decade. The major dietary sources of *trans* fatty acids are products for‐ mulated with partially hydrogenated fats. Examples include margarines, shortenings, bakery products, and fast foods. The regulatory mandate from FDA and consumer con‐ cerns have led to the development of alternative processes to produce foods with zero or

Fats and oils can be formulated as *trans*-acid-free products, but saturates are required for the solids contents that provide the functionality for plastic and liquid products. Reduced satu‐ rates may be an option in some cases, but a saturate-free product is probably impossible if functionality is to be maintained [2]. Obviously, if the fat is completely hydrogenated there will be no double bonds and hence no problem; however, partially hydrogenated fats have *trans* double bonds. *Trans* double bonds are rare in naturally occurring fats, the major natu‐ ral source is milk fat because they are formed by bacterial action in the rumen. So, most nat‐ urally occurring oils and fats have *cis* double bonds; however, some *trans* double bonds are

have a higher melting point than unsaturated and *cis* fatty acids [1].

their operations to reduce *trans* fats in their offerings [6, 7].

reduced *trans* fatty acids contents [7, 9].

found in milk fat and some marine oils.

*2.1.5. Health problems*

68 Food Industry

The new rules about *trans* fatty acids promise to strongly affect what is acceptable to con‐ sumers and food manufacturers. It will be difficult to meet all the demands for low-*trans* fats and other traits that are important to consumers with the current technology, especially for frying fats and oils. Seed suppliers are busy trying to furnish seeds with compositions that will meet these needs and find farmers to grow these crops. Contracts with oilseed process‐ ors have been made to process the harvest. Some food companies have pledged to use only *trans*-free fats and oils in their products [1].

*Trans*-free fat blends can be constructed by blending oils with fully hardened oils, or indeed where the entire blend has been randomized through interesterification. Blending vegetable oil types from different sources is an efficient alternative to hydrogenated vegetable oils, and still provides the appropriate physicochemical properties and nutritional requirements demand‐ ed. Such fat blends can also be rich in polyunsaturated fatty acids as well as being *trans*- free. *Trans*-free options are commercially available in the form of a blend of tailored emulsifiers and oil blends where they meet demands for shelf-life, processing and distribution requirements. These *trans*-free options are available for a wide range of products covering, snacks, cakes, breads, tortillas, nutrition bars, cookies and breakfast cereals. *Trans*-free oil blends are also rou‐ tinely designed for margarines, where they impart structure and texture, and shortenings where they provide firmness and contribute to crumb structure [10]. The combination of *trans*free modification techniques (full hydrogenation, interesterification and fractionation) and the availability of a variety of different feedstocks can be used to produce virtually *trans*-free hard‐ stocks with a range of physical properties such as solid phase lines determining melting per‐ formances. Liquid seed oils, low in solids, are first fully hydrogenated to generate solids combined with a very low *trans* level (< 1.25%). These fully hydrogenated oils may subsequent‐ ly be interesterified with non-hydrogenated liquid oil to reduce the solid fat content at high temperature (> 40°C). This solid fat content can be further reduced by fractionation [11].

#### **2.2. Structural characteristics**

Fats are the main structural components in many food products such as chocolate, confec‐ tionery coatings, dairy products, butter, cream shortenings, margarine, and spreads. The sensory characteristics of fat-structured materials such as spreadability, hardness, and mouth feel are highly dependent on the structure of the underlying fat crystal network. This fat crystal network is built by the interaction of polycrystalline fat particles. The amount, ge‐ ometry, and spatial distribution of solid fat crystals as well as their interactions at different levels within the network all affect the rheological properties of fats and fat-structured food products. Fat crystallization largely determines consistency, physical stability, visual ap‐ pearance, and eating properties [4, 12, 13].

#### *2.2.1. Crystals*

A crystal consists of a material in a solid state in which the building entities—molecules, atoms or ions—are closely packed so that the free energy of the material is at minimum. As a result the entities are arranged in a regularly repeating pattern or lattice and are affected by the following points [14]: the molecules, or atoms, or ions are subject to heat motion; only the average positions will be fixed; diffusion can occur in a crystalline material, but the time scales involved are centuries rather than seconds; incorporation of a foreign molecule lead‐ ing to a dislocation in the crystal lattice (Figure 1b); some solid materials are ''polycrystal‐ line'', i.e., they are composites of many small crystalline domains of various orientations (Figure 1c).

Crystals show enormous variation in external shape or habit caused by variation in the growth rate of the various faces of a crystal, which rates often depend on the composition of the solution. Corners and edges are rounded; curved faces can appear in large crystals; some needlelike crystals have a slight twist; some faces can grow faster than others (Figure 4). A noncrystalline solid is often referred to as an amorphous solid. Whether a material is crystal‐ line or not can be established by x-ray diffraction. X-rays have a very small wavelength, of the order of 0.1 nm, which implies that individual atoms may cause scattering. If the atoms (or small molecules) occur at regular distances, sharp diffraction maxima occur, and the

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Many of the sensory attributes such as spreadability, mouthfeel, snap of chocolate, texture, etc., are dependent on the mechanical strength of the underlying fat crystal network. In ad‐ dition to this obvious industrial importance, fat crystal networks form a particular class of soft materials, which demonstrate a yield stress and viscoelastic properties, rendering these plastic materials. The levels of structure in a typical fat network are defined as the fat crys‐ tallizes from the melt. The growth of a fat crystal network can be visualized thus: the triacyl‐ glycerols present in the sample crystallize from the melt into particular polymorphic/ polytypic states. These crystals grow into larger microstructural elements (≈ 6 mm) which then aggregate via a mass- and heat-transfer limited process into larger microstructures (≈ 100 mm). The aggregation process continues until a continuous three-dimensional network is formed by the collection of microstructures. Trapped within this solid network structure

The crystallization process consists of two steps: nucleation and crystal growth. Nucleation can be described as a process in which molecules come into contact, orient and interact to form highly ordered structures, called nuclei. Crystal growth is the enlargement of these nu‐ clei. Nucleation and crystal growth are not mutually exclusive: nucleation may take place

Nucleation can only be achieved via supersaturation or supercooling. A solution is supersa‐ turated if it contains more of a component than can be theoretically dissolved within it at a particular temperature. Supercooling refers to the degree to which the solution is cooled with respect to the melting temperature of the crystallized solution. It is very difficult to de‐ termine the parameters of supersaturation and supercooling for a crystallizing system, and therefore, as a good approximation, in practice only supercooling is usually considered for

When the temperature of a fat melt is decreased below its maximum melting temperature, it becomes supersaturated in the higher-melting triacylglycerol species present in the mixture. This so-called undercooling or supercooling represents the thermodynamic driving force for the change in state from liquid to solid. Fats usually have to be undercooled by at least 5-10ºC before they begin to crystallize. For a few degrees below the melting point, the melt exists in a so-called metastable region. In this region, molecules begin to aggregate into tiny clusters called embryos. At these low degrees of undercooling, embryos continuously form

crystal structure can be derived from the diffraction pattern [14].

*2.2.2. Crystallization*

is the liquid phase of the fat [12].

while crystals grow on existing nuclei [16].

crystallization of triacylglycerol molecules from the melt [17].

**Figure 1.** Two-dimensional illustration of crystalline order: (a) crystal lattice with perfect order, (b) a defect in the crys‐ tal leading to a dislocation in the lattice, (c) a polycrystalline material [14].

Different lattice arrangements and unit cells (Figure 2) can be constructed in terms of the lat‐ tice parameters, also known as Bravais lattices: three spatial dimensions - a, b, and c; and three angles - α, β, and γ. For example, cubic systems all must have equal lengths (a=b=c) and angles equal to 90° [15].

**Figure 2.** Variation in crystal morphology for identical unit cells: (a) rhombohedral, (b) cubic, and (c) monoclinic [15].

Crystals show enormous variation in external shape or habit caused by variation in the growth rate of the various faces of a crystal, which rates often depend on the composition of the solution. Corners and edges are rounded; curved faces can appear in large crystals; some needlelike crystals have a slight twist; some faces can grow faster than others (Figure 4). A noncrystalline solid is often referred to as an amorphous solid. Whether a material is crystal‐ line or not can be established by x-ray diffraction. X-rays have a very small wavelength, of the order of 0.1 nm, which implies that individual atoms may cause scattering. If the atoms (or small molecules) occur at regular distances, sharp diffraction maxima occur, and the crystal structure can be derived from the diffraction pattern [14].

### *2.2.2. Crystallization*

a result the entities are arranged in a regularly repeating pattern or lattice and are affected by the following points [14]: the molecules, or atoms, or ions are subject to heat motion; only the average positions will be fixed; diffusion can occur in a crystalline material, but the time scales involved are centuries rather than seconds; incorporation of a foreign molecule lead‐ ing to a dislocation in the crystal lattice (Figure 1b); some solid materials are ''polycrystal‐ line'', i.e., they are composites of many small crystalline domains of various orientations

**Figure 1.** Two-dimensional illustration of crystalline order: (a) crystal lattice with perfect order, (b) a defect in the crys‐

Different lattice arrangements and unit cells (Figure 2) can be constructed in terms of the lat‐ tice parameters, also known as Bravais lattices: three spatial dimensions - a, b, and c; and three angles - α, β, and γ. For example, cubic systems all must have equal lengths (a=b=c)

**Figure 2.** Variation in crystal morphology for identical unit cells: (a) rhombohedral, (b) cubic, and (c) monoclinic [15].

tal leading to a dislocation in the lattice, (c) a polycrystalline material [14].

and angles equal to 90° [15].

(Figure 1c).

70 Food Industry

Many of the sensory attributes such as spreadability, mouthfeel, snap of chocolate, texture, etc., are dependent on the mechanical strength of the underlying fat crystal network. In ad‐ dition to this obvious industrial importance, fat crystal networks form a particular class of soft materials, which demonstrate a yield stress and viscoelastic properties, rendering these plastic materials. The levels of structure in a typical fat network are defined as the fat crys‐ tallizes from the melt. The growth of a fat crystal network can be visualized thus: the triacyl‐ glycerols present in the sample crystallize from the melt into particular polymorphic/ polytypic states. These crystals grow into larger microstructural elements (≈ 6 mm) which then aggregate via a mass- and heat-transfer limited process into larger microstructures (≈ 100 mm). The aggregation process continues until a continuous three-dimensional network is formed by the collection of microstructures. Trapped within this solid network structure is the liquid phase of the fat [12].

The crystallization process consists of two steps: nucleation and crystal growth. Nucleation can be described as a process in which molecules come into contact, orient and interact to form highly ordered structures, called nuclei. Crystal growth is the enlargement of these nu‐ clei. Nucleation and crystal growth are not mutually exclusive: nucleation may take place while crystals grow on existing nuclei [16].

Nucleation can only be achieved via supersaturation or supercooling. A solution is supersa‐ turated if it contains more of a component than can be theoretically dissolved within it at a particular temperature. Supercooling refers to the degree to which the solution is cooled with respect to the melting temperature of the crystallized solution. It is very difficult to de‐ termine the parameters of supersaturation and supercooling for a crystallizing system, and therefore, as a good approximation, in practice only supercooling is usually considered for crystallization of triacylglycerol molecules from the melt [17].

When the temperature of a fat melt is decreased below its maximum melting temperature, it becomes supersaturated in the higher-melting triacylglycerol species present in the mixture. This so-called undercooling or supercooling represents the thermodynamic driving force for the change in state from liquid to solid. Fats usually have to be undercooled by at least 5-10ºC before they begin to crystallize. For a few degrees below the melting point, the melt exists in a so-called metastable region. In this region, molecules begin to aggregate into tiny clusters called embryos. At these low degrees of undercooling, embryos continuously form and breakdown, but do not persist to form stable nuclei. The energy of interaction between triacylglycerol molecules has to be greater than the kinetic energy of the molecules in the melt so as to overcome Brownian effects. For these flexible molecules, it is not sufficient to simply interact; molecules have to adopt a specific conformation in order to form a stable nucleus. The adoption of this more stable conformation is relatively slow, thus explaining the existence of a metastable region. As the undercooling is increased (i.e., at lower tempera‐ tures) stable nuclei of a specific critical size are formed [18].

is not the measuring temperature. This step is referred to as a tempering step. For confec‐ tionery fats, a tempering step of 40 hours at 26°C is mentioned in the standard methods to ensure that cocoa butter and similar fats like cocoa butter equivalents (CBEs) are converted

Structuring Fat Foods

73

http://dx.doi.org/10.5772/53369

Tempering is a technique of controlled pre-crystallization employed to induce the most sta‐ ble solid form of cocoa butter, a polymorphic fat in finished chocolates. The process consists of shearing chocolate mass at controlled temperatures to promote crystallization of triacyl‐ glycerols in cocoa butter to effect good setting characteristics, foam stability, demoulding properties, product snap, contraction, gloss and shelf-life characteristics. Time–temperature protocols and shearing are employed to induce nucleation of stable polymorphs with the formation of three-dimensional crystal network structure influencing the microstructure, mechanical properties and appearance of products. The crystal network organization and the polymorphic state of the triacylglycerols crystals as affected by the crystallization condi‐ tions are major factors determining rheological and textural properties of crystallized tria‐

The solid fat content (SFC) is a measure of the percentage of solid, crystalline fat in a sample at a selected temperature. Often, the SFC is measured at selected points within a temperature range. A measure of the SFC can be determined by a variety of methods: dilatometry, pulsed nuclear magnetic resonance (p-NMR), or differential scanning calorimetry (DSC). The method

Each application area requires its proper fat. The specifications of the fat depend on: recipe, equipment, procedure, temperature of fat and other ingredients, ambient temperature, stor‐ age and distribution temperature of the final product. Some conditions to attend a satisfato‐ ry fat design must be the compatibility among the components of the mixture: equivalent thermal properties (solid fat content, melting point and range); similar molecular size, shape and packing (to allow isomorphous replacement or formation of a single lattice unit in mix‐ tures); similar polymorphism (transformation from stable to unstable forms should occur as

Edible fats and oils have been separated from animal tissues, oilseeds, and oil-bearing fruits for thousands of years. The combined largest source of vegetable oils is the seeds of annual plants grown in relatively temperate climates. The oilseeds are processed by expeller or screw press extraction, by prepress solvent extraction, or bay expander–solvent extraction. A second source of vegetable oil is the oil-bearing tree fruits and kernels. Oil-bearing fruits are pressed to obtain oil, sometimes after drying or sterilizing, or are cold pressed to preserve flavor and odor. Animal tissues may be wet- or dry-rendered (cooking processes) to sepa‐

used and differences in the way it is executed can seriously affect the final result [4].

readily for binary mixtures as with individual components) (Figure 3).

to their β-polymorph before the SFC is measured [4].

cylglycerols systems [19].

*2.2.5. Solid fat content*

**2.3. Fat design**

*2.3.1. Processing*

### *2.2.3. Polymorphism*

An important way to characterize fats and oils is through the predominant crystalline phase, or polymorph, that tend to form upon crystallization. When the same ensemble of molecules can pack in different arrangements on crystallization, depending on the processing condi‐ tions, the substance is said to demonstrate polymorphism. The different polymorphic states of a particular substance often demonstrate quite different physical properties (such as melt‐ ing behavior and hardness), but on melting yield identical liquids [17].

Polymorphism is the ability of long-chain compounds such as fatty acids to exist in more than one crystal form, and this results from different patterns of molecular packing in the crystals. Triacylglycerols may occur in three main forms, namely, α, β', and β in order of in‐ creasing stability and melting point. When fats are cooled, crystals of a lower melting form may be produced. These may change slowly or rapidly into a more stable form. The change is monotropic, that is, it always proceeds from lower to higher stability. Polymorphism re‐ sults in the phenomenon of multiple melting points. When a fat is crystallized in an unstable form and heated to a temperature slightly above its melting point, it may resolidify into a more stable form [1]. The polymorphs differ in stability, melting point, melting enthalpy, and density. The α-polymorph is the least stable and has the lowest melting point, melting enthalpy, and density. The β-polymorph is the most stable and has the highest melting point, melting enthalpy, and density. The β′-polymorph has intermediate properties [4].

Under rapid cooling conditions, triacylglycerol molecules usually crystallize in metastable polymorphic forms, which subsequently transform into polymorphs of higher stability. On the other hand, at slow cooling rates, triacylglycerol molecules of similar chain lengths have time to associate with each other in more stable geometrical arrangements, resulting in the formation of a more stable polymorphic form. Due to the dependence of fat crystallization on the degree of undercooling and the cooling rate used, different results will be observed when using different cooling rates [18].

#### *2.2.4. Tempering*

Before its solid fat content can be determined, the fat must be exposed to a prescribed tem‐ perature profile: first it has to be melted completely to destroy all traces of crystals, and then cooled to achieve virtually complete crystallization, and finally it has to be held at the meas‐ uring temperature to come to equilibrium at that temperature. Sometimes, depending on the fat used, an extra step is introduced where the fat is held at a particular temperature, which is not the measuring temperature. This step is referred to as a tempering step. For confec‐ tionery fats, a tempering step of 40 hours at 26°C is mentioned in the standard methods to ensure that cocoa butter and similar fats like cocoa butter equivalents (CBEs) are converted to their β-polymorph before the SFC is measured [4].

Tempering is a technique of controlled pre-crystallization employed to induce the most sta‐ ble solid form of cocoa butter, a polymorphic fat in finished chocolates. The process consists of shearing chocolate mass at controlled temperatures to promote crystallization of triacyl‐ glycerols in cocoa butter to effect good setting characteristics, foam stability, demoulding properties, product snap, contraction, gloss and shelf-life characteristics. Time–temperature protocols and shearing are employed to induce nucleation of stable polymorphs with the formation of three-dimensional crystal network structure influencing the microstructure, mechanical properties and appearance of products. The crystal network organization and the polymorphic state of the triacylglycerols crystals as affected by the crystallization condi‐ tions are major factors determining rheological and textural properties of crystallized tria‐ cylglycerols systems [19].

#### *2.2.5. Solid fat content*

and breakdown, but do not persist to form stable nuclei. The energy of interaction between triacylglycerol molecules has to be greater than the kinetic energy of the molecules in the melt so as to overcome Brownian effects. For these flexible molecules, it is not sufficient to simply interact; molecules have to adopt a specific conformation in order to form a stable nucleus. The adoption of this more stable conformation is relatively slow, thus explaining the existence of a metastable region. As the undercooling is increased (i.e., at lower tempera‐

An important way to characterize fats and oils is through the predominant crystalline phase, or polymorph, that tend to form upon crystallization. When the same ensemble of molecules can pack in different arrangements on crystallization, depending on the processing condi‐ tions, the substance is said to demonstrate polymorphism. The different polymorphic states of a particular substance often demonstrate quite different physical properties (such as melt‐

Polymorphism is the ability of long-chain compounds such as fatty acids to exist in more than one crystal form, and this results from different patterns of molecular packing in the crystals. Triacylglycerols may occur in three main forms, namely, α, β', and β in order of in‐ creasing stability and melting point. When fats are cooled, crystals of a lower melting form may be produced. These may change slowly or rapidly into a more stable form. The change is monotropic, that is, it always proceeds from lower to higher stability. Polymorphism re‐ sults in the phenomenon of multiple melting points. When a fat is crystallized in an unstable form and heated to a temperature slightly above its melting point, it may resolidify into a more stable form [1]. The polymorphs differ in stability, melting point, melting enthalpy, and density. The α-polymorph is the least stable and has the lowest melting point, melting enthalpy, and density. The β-polymorph is the most stable and has the highest melting point, melting enthalpy, and density. The β′-polymorph has intermediate properties [4].

Under rapid cooling conditions, triacylglycerol molecules usually crystallize in metastable polymorphic forms, which subsequently transform into polymorphs of higher stability. On the other hand, at slow cooling rates, triacylglycerol molecules of similar chain lengths have time to associate with each other in more stable geometrical arrangements, resulting in the formation of a more stable polymorphic form. Due to the dependence of fat crystallization on the degree of undercooling and the cooling rate used, different results will be observed

Before its solid fat content can be determined, the fat must be exposed to a prescribed tem‐ perature profile: first it has to be melted completely to destroy all traces of crystals, and then cooled to achieve virtually complete crystallization, and finally it has to be held at the meas‐ uring temperature to come to equilibrium at that temperature. Sometimes, depending on the fat used, an extra step is introduced where the fat is held at a particular temperature, which

tures) stable nuclei of a specific critical size are formed [18].

ing behavior and hardness), but on melting yield identical liquids [17].

*2.2.3. Polymorphism*

72 Food Industry

when using different cooling rates [18].

*2.2.4. Tempering*

The solid fat content (SFC) is a measure of the percentage of solid, crystalline fat in a sample at a selected temperature. Often, the SFC is measured at selected points within a temperature range. A measure of the SFC can be determined by a variety of methods: dilatometry, pulsed nuclear magnetic resonance (p-NMR), or differential scanning calorimetry (DSC). The method used and differences in the way it is executed can seriously affect the final result [4].

#### **2.3. Fat design**

Each application area requires its proper fat. The specifications of the fat depend on: recipe, equipment, procedure, temperature of fat and other ingredients, ambient temperature, stor‐ age and distribution temperature of the final product. Some conditions to attend a satisfato‐ ry fat design must be the compatibility among the components of the mixture: equivalent thermal properties (solid fat content, melting point and range); similar molecular size, shape and packing (to allow isomorphous replacement or formation of a single lattice unit in mix‐ tures); similar polymorphism (transformation from stable to unstable forms should occur as readily for binary mixtures as with individual components) (Figure 3).

#### *2.3.1. Processing*

Edible fats and oils have been separated from animal tissues, oilseeds, and oil-bearing fruits for thousands of years. The combined largest source of vegetable oils is the seeds of annual plants grown in relatively temperate climates. The oilseeds are processed by expeller or screw press extraction, by prepress solvent extraction, or bay expander–solvent extraction. A second source of vegetable oil is the oil-bearing tree fruits and kernels. Oil-bearing fruits are pressed to obtain oil, sometimes after drying or sterilizing, or are cold pressed to preserve flavor and odor. Animal tissues may be wet- or dry-rendered (cooking processes) to sepa‐ rate the fats. Edible meat fats are supplied by lard from pigs, tallow from cattle and sheep, and milk fat or butter from cows. After recovering, fats and oils can be physically and/or chemically refined. Chemical refining removes most impurities with an alkaline solution, whereas physical refining removes them by distillation [2].

time was nickel, and it has practically remained the same in the current hydrogenation pro‐ cedures. Complete reduction of all double bonds in the oil would yield 100% saturated fatty acids, whereas reduction of only a fraction of the double bonds results in partially hydro‐ genated fats. During the process of hydrogenation the *cis* double bond can open up and re‐ form into a *trans* double bond, as well as shift positions along the fatty acid carbon chain. Structurally, *cis* double bonds in unsaturated fatty acids produce a bend in the chain that prevents unsaturated fatty acids from packing as tightly as saturated fatty acids. As a conse‐ quence, a *cis* unsaturated fatty acid has a lower melting point than a saturated fatty acids with the same molecular weight. Conversely, the *trans* double bonds do not create a bend on the fatty acid chain. Therefore, *trans* unsaturated fatty acid chains are virtually straight, re‐ sembling saturated fatty acids, and display higher melting points than the corresponding *cis*

Structuring Fat Foods

75

http://dx.doi.org/10.5772/53369

The aim of the hydrogenation process is the total or partial saturation of the double bonds of unsaturated fats to obtain hard or plastic fats or to improve the stability to oxidation of an oil. The obtained product depends on the nature of the starting oil, the type and concentra‐ tion of the catalyst used, the concentration of hydrogen, and the experimental conditions un‐ der which the reaction takes place. Nickel catalyst was reported to catalyze undesirable side reactions such as *cis*, *trans* isomerization and positional isomerization of double bonds. The position of the double bonds affects the melting point of the fatty acid to a limited extent. The presence of different geometric isomers of fatty acids influences the physical character‐

Interesterification has been developed as an alternative to hydrogenation, with the specific aim of eliminating the formation of *trans* fatty acids. The process rearranges the distribution of the fatty acids either chemically or enzymatically, within and between the triacylglycer‐ ols, thus the fatty acid distribution is altered, but the fatty acid composition remains un‐ changed – this rearrangement can be done either in a random or controlled manner. The technique is effective and can be used to produce fat products for spreads that are soft and spreadable and also *trans*-free. Interesterification is nothing new, having been around for

Fractionation is a fully reversible modification process; it is basically a thermo-mechanical separation process in which a multi-component mixture is physically separated into two or more fractions with distinct physical and chemical properties. The separation can be based on differences in solidification, solubility, or volatility of the different compounds: fractional crystallization, fractional distillation, short-path distillation, supercritical extraction, liquidliquid extraction, adsorption, complexation, membrane separation, etc. are the main techni‐ ques practiced. Fractional crystallization refers to a separation process in which the fatty material is crystallized, after which the liquid phase is separated from the solid. It is based on differences in solubility of the solid triacylglycerol in the liquid phase, depending on

some time, and the basic principles were first documented in 1969 [10].

isomers [21].

istics of the fat to a greater extent [22].

*2.3.1.3. Interesterification*

*2.3.1.4. Fractionation*

**Figure 3.** Physical and chemical functions of fats.

#### *2.3.1.1. Industrialization*

Searching for fat substitutes started in France during the Industrial Revolution. Large popu‐ lation shifts from farms to factories and, in France, a depression and an imminent war with Prussia, created a demand for butter that the milk supply could not meet, escalating butter prices. The first acceptable butter substitute, named "margarine", was produced by the French chemist Mege Mouries in 1869, on commission from Emperor Napolean. Soon after the introduction of the first butter substitute on the market, several inventors patented vari‐ ous modifications of Mouries' process [2, 4, 17]. Before 1900, animal fats were used as sour‐ ces of fat with a high content of solids in margarine production. This led to a shortage of animal fats since they were also the main feedstock for soap making [3]. The best known modification processes applied today in the edible oil industry are hydrogenation, interes‐ terification (chemical or enzymatic) and fractionation. The main purpose of these processes is to change the physicochemical properties of the oil or fat, by reducing the degree of unsa‐ turation of the acyl groups (hydrogenation), by redistributing the fatty acids chains (interes‐ terification) or by a physical separation of the triacylglycerols through selective crystallization and filtration (fractionation) [20].

#### *2.3.1.2. Hydrogenation*

Based on work done by the French chemist Paul Sabatier on the metal-catalyzed hydrogena‐ tion of unsaturated organic compounds, German chemist Wilhelm Normann developed the method for hydrogenation of edible oils in 1903. Chemically, the hydrogenation of oils is the reduction of the double bonds in unsaturated fatty acids to single saturated bonds, by the reaction of hydrogen gas in the presence of a metal catalyst. The metal catalyst used at the time was nickel, and it has practically remained the same in the current hydrogenation pro‐ cedures. Complete reduction of all double bonds in the oil would yield 100% saturated fatty acids, whereas reduction of only a fraction of the double bonds results in partially hydro‐ genated fats. During the process of hydrogenation the *cis* double bond can open up and re‐ form into a *trans* double bond, as well as shift positions along the fatty acid carbon chain. Structurally, *cis* double bonds in unsaturated fatty acids produce a bend in the chain that prevents unsaturated fatty acids from packing as tightly as saturated fatty acids. As a conse‐ quence, a *cis* unsaturated fatty acid has a lower melting point than a saturated fatty acids with the same molecular weight. Conversely, the *trans* double bonds do not create a bend on the fatty acid chain. Therefore, *trans* unsaturated fatty acid chains are virtually straight, re‐ sembling saturated fatty acids, and display higher melting points than the corresponding *cis* isomers [21].

The aim of the hydrogenation process is the total or partial saturation of the double bonds of unsaturated fats to obtain hard or plastic fats or to improve the stability to oxidation of an oil. The obtained product depends on the nature of the starting oil, the type and concentra‐ tion of the catalyst used, the concentration of hydrogen, and the experimental conditions un‐ der which the reaction takes place. Nickel catalyst was reported to catalyze undesirable side reactions such as *cis*, *trans* isomerization and positional isomerization of double bonds. The position of the double bonds affects the melting point of the fatty acid to a limited extent. The presence of different geometric isomers of fatty acids influences the physical character‐ istics of the fat to a greater extent [22].

#### *2.3.1.3. Interesterification*

rate the fats. Edible meat fats are supplied by lard from pigs, tallow from cattle and sheep, and milk fat or butter from cows. After recovering, fats and oils can be physically and/or chemically refined. Chemical refining removes most impurities with an alkaline solution,

Searching for fat substitutes started in France during the Industrial Revolution. Large popu‐ lation shifts from farms to factories and, in France, a depression and an imminent war with Prussia, created a demand for butter that the milk supply could not meet, escalating butter prices. The first acceptable butter substitute, named "margarine", was produced by the French chemist Mege Mouries in 1869, on commission from Emperor Napolean. Soon after the introduction of the first butter substitute on the market, several inventors patented vari‐ ous modifications of Mouries' process [2, 4, 17]. Before 1900, animal fats were used as sour‐ ces of fat with a high content of solids in margarine production. This led to a shortage of animal fats since they were also the main feedstock for soap making [3]. The best known modification processes applied today in the edible oil industry are hydrogenation, interes‐ terification (chemical or enzymatic) and fractionation. The main purpose of these processes is to change the physicochemical properties of the oil or fat, by reducing the degree of unsa‐ turation of the acyl groups (hydrogenation), by redistributing the fatty acids chains (interes‐ terification) or by a physical separation of the triacylglycerols through selective

Based on work done by the French chemist Paul Sabatier on the metal-catalyzed hydrogena‐ tion of unsaturated organic compounds, German chemist Wilhelm Normann developed the method for hydrogenation of edible oils in 1903. Chemically, the hydrogenation of oils is the reduction of the double bonds in unsaturated fatty acids to single saturated bonds, by the reaction of hydrogen gas in the presence of a metal catalyst. The metal catalyst used at the

whereas physical refining removes them by distillation [2].

**Figure 3.** Physical and chemical functions of fats.

crystallization and filtration (fractionation) [20].

*2.3.1.1. Industrialization*

74 Food Industry

*2.3.1.2. Hydrogenation*

Interesterification has been developed as an alternative to hydrogenation, with the specific aim of eliminating the formation of *trans* fatty acids. The process rearranges the distribution of the fatty acids either chemically or enzymatically, within and between the triacylglycer‐ ols, thus the fatty acid distribution is altered, but the fatty acid composition remains un‐ changed – this rearrangement can be done either in a random or controlled manner. The technique is effective and can be used to produce fat products for spreads that are soft and spreadable and also *trans*-free. Interesterification is nothing new, having been around for some time, and the basic principles were first documented in 1969 [10].

#### *2.3.1.4. Fractionation*

Fractionation is a fully reversible modification process; it is basically a thermo-mechanical separation process in which a multi-component mixture is physically separated into two or more fractions with distinct physical and chemical properties. The separation can be based on differences in solidification, solubility, or volatility of the different compounds: fractional crystallization, fractional distillation, short-path distillation, supercritical extraction, liquidliquid extraction, adsorption, complexation, membrane separation, etc. are the main techni‐ ques practiced. Fractional crystallization refers to a separation process in which the fatty material is crystallized, after which the liquid phase is separated from the solid. It is based on differences in solubility of the solid triacylglycerol in the liquid phase, depending on their molecular weight and degree of unsaturation; this is a consequence of the ability of fats to produce crystals. On an industrial scale, crystals can be obtained according to three main technologies: detergent fractionation, solvent fractionation and dry fractionation [20].

Plastic shortening describes fats that are readily spread, mixed or worked. The property of plasticity is highly important in fats used as shortening agents in baked products. Com‐ mercially, these are prepared by hydrogenation of oils, during which, some of the double bonds are isomerised into *trans* fatty acids from their *cis* configuration. *Trans* fatty acids have higher melting points and greater stability against oxidative rancidity than their *cis*isomers and are important contributors to the functional properties of hydrogenated prod‐ ucts. To meet the requirements of health-conscious consumers fats having a wide melting range which crystallize in the b' polymorphic form without the formation of *trans* fatty

Structuring Fat Foods

77

http://dx.doi.org/10.5772/53369

Palm oil, because of its naturally β' tending nature, is favoured for shortening applications, such that it can impart stability to the emulsion, smooth consistency and provide good aera‐

When triacylglycerols are cooled from the melt to a temperature below their melting point, i.e., when they are supercooled, they undergo a liquid–solid transformation to form primary crystals with characteristic polymorphism. These primary crystals aggregate, or grow into each other, to form clusters, which further interact, resulting in the formation of a continu‐ ous three-dimensional network. The mechanical properties of a fat, can be influenced by all these levels of structure; however, most directly by the level of structure closest to the mac‐

It is during crystallization that the template for the final physical properties of the result‐ ing fat crystal network is created. Hence, the mechanical properties of a fat crystal net‐ work are determined by the different levels of structure, such as chemical composition, solid fat content (SFC), and crystal habit (polymorphism and microstructure). To study the mechanical properties of fat crystal networks, rheologic tests are used, which meas‐ ure how the crystallized material responds to applied forces (stress) and deformations

Foods are edible structures created as a result of the responses of proteins, polysaccharides, and lipids in aqueous media to different processing methods, such as thermal processing, homogenization, and other physical treatments. The processing operations to which foods are subjected affect their structure and microstructure. Most, if not all, of the responses are physical in nature. By definition, rheology is the study of deformation and flow of materials. In foods, measured rheological responses are those at the macroscopic level. However, they are directly affected by the changes and properties at the microscopic level. Fractal dimen‐ sion has been used to characterize food particles in addition to microscopic and size distri‐ bution data. The fractal dimension can be estimated by several techniques such as

acids are needed [23].

tion properties [10].

(strain) [18].

viscoelastic behavior [25].

**3. Mechanical properties**

roscopic world, namely the microstructure [24].

### *2.3.2. Fat replacers*

Fat replacers are called by many synonyms with various nuances in their usage: fat *replacers* can provide some or all of the functions of fat; fat *substitutes* resemble conventional fats and oils and provide all food functions of fat; fat *analogs* provide food with many of the charac‐ teristics of fat; fat *extenders* optimize the functionality of fat; fat *mimetics* mimic one or more of the sensory and physical functions of fat in the food.

Fat replacers are most frequently used to replace fat in products with a high fat content and are used in a variety of food products, including frozen desserts, processed meats, cheese, sour cream, salad dressings, snack chips and baked goods. At the height of the interest in low-fat foods, more than 1000 fat-modified foods were introduced, with fat modified snacks being the fastest growing category of products in supermarkets at the time [11]. Normal fat contains nine calories per gram compared with five calories per gram for the sugar and pro‐ tein components. If the proportion of fat is reduced the calorific value will fall. Corn starch, maltodextrin, pectin, gelatin, xanthan gum, guar gum, carrageenan, and soy protein were all commonly used ingredients in reduced fat products launched in the period 2008–10. Low in saturated fatty acids, sunflower oil was commonly used in new reduced fat foods. Fat re‐ placers of the future will need to meet some important criteria, including reducing or replac‐ ing the target fat effectively, being available at a cost appropriate to the benefits provided, and being safe and legal with no appreciable side effects.

#### *2.3.3. Shortening*

Shortening was the term used to describe the function performed by naturally occurring sol‐ id fats such as lard and butter in baked products. These fats contributed a "short" (or ten‐ derizing) quality to baked products by preventing the cohesion of the flour gluten during mixing and baking. Shortening later became the term used by all-vegetable oil processors when they abandoned the lard-substitute concept. Shortening has become virtually synony‐ mous with fat and includes many other types of edible fats designed for purposes other than baking. Currently, a description for shortening would be processed fats and oils products that affect the stability, flavor, storage quality, eating characteristics, and eye appeal of pre‐ pared foods by providing emulsification, lubricity, structure, aeration, a moisture barrier, a flavor medium, or heat transfer [2].

Fats and oils added to breads, cakes and similar baked goods are often referred to as shortenings that contribute to tenderness, improve volume gain of bread dough, enhance texture, crumb structure and shelf-life of the products. In order to produce a satisfactory shortening, one has to pay specific attention to the crystal structure, and similarly the con‐ sistency of the shortening will depend on the ratio of solid to liquid fat present at differ‐ ent temperatures [10].

Plastic shortening describes fats that are readily spread, mixed or worked. The property of plasticity is highly important in fats used as shortening agents in baked products. Com‐ mercially, these are prepared by hydrogenation of oils, during which, some of the double bonds are isomerised into *trans* fatty acids from their *cis* configuration. *Trans* fatty acids have higher melting points and greater stability against oxidative rancidity than their *cis*isomers and are important contributors to the functional properties of hydrogenated prod‐ ucts. To meet the requirements of health-conscious consumers fats having a wide melting range which crystallize in the b' polymorphic form without the formation of *trans* fatty acids are needed [23].

Palm oil, because of its naturally β' tending nature, is favoured for shortening applications, such that it can impart stability to the emulsion, smooth consistency and provide good aera‐ tion properties [10].

## **3. Mechanical properties**

their molecular weight and degree of unsaturation; this is a consequence of the ability of fats to produce crystals. On an industrial scale, crystals can be obtained according to three main

Fat replacers are called by many synonyms with various nuances in their usage: fat *replacers* can provide some or all of the functions of fat; fat *substitutes* resemble conventional fats and oils and provide all food functions of fat; fat *analogs* provide food with many of the charac‐ teristics of fat; fat *extenders* optimize the functionality of fat; fat *mimetics* mimic one or more

Fat replacers are most frequently used to replace fat in products with a high fat content and are used in a variety of food products, including frozen desserts, processed meats, cheese, sour cream, salad dressings, snack chips and baked goods. At the height of the interest in low-fat foods, more than 1000 fat-modified foods were introduced, with fat modified snacks being the fastest growing category of products in supermarkets at the time [11]. Normal fat contains nine calories per gram compared with five calories per gram for the sugar and pro‐ tein components. If the proportion of fat is reduced the calorific value will fall. Corn starch, maltodextrin, pectin, gelatin, xanthan gum, guar gum, carrageenan, and soy protein were all commonly used ingredients in reduced fat products launched in the period 2008–10. Low in saturated fatty acids, sunflower oil was commonly used in new reduced fat foods. Fat re‐ placers of the future will need to meet some important criteria, including reducing or replac‐ ing the target fat effectively, being available at a cost appropriate to the benefits provided,

Shortening was the term used to describe the function performed by naturally occurring sol‐ id fats such as lard and butter in baked products. These fats contributed a "short" (or ten‐ derizing) quality to baked products by preventing the cohesion of the flour gluten during mixing and baking. Shortening later became the term used by all-vegetable oil processors when they abandoned the lard-substitute concept. Shortening has become virtually synony‐ mous with fat and includes many other types of edible fats designed for purposes other than baking. Currently, a description for shortening would be processed fats and oils products that affect the stability, flavor, storage quality, eating characteristics, and eye appeal of pre‐ pared foods by providing emulsification, lubricity, structure, aeration, a moisture barrier, a

Fats and oils added to breads, cakes and similar baked goods are often referred to as shortenings that contribute to tenderness, improve volume gain of bread dough, enhance texture, crumb structure and shelf-life of the products. In order to produce a satisfactory shortening, one has to pay specific attention to the crystal structure, and similarly the con‐ sistency of the shortening will depend on the ratio of solid to liquid fat present at differ‐

technologies: detergent fractionation, solvent fractionation and dry fractionation [20].

of the sensory and physical functions of fat in the food.

and being safe and legal with no appreciable side effects.

*2.3.2. Fat replacers*

76 Food Industry

*2.3.3. Shortening*

flavor medium, or heat transfer [2].

ent temperatures [10].

When triacylglycerols are cooled from the melt to a temperature below their melting point, i.e., when they are supercooled, they undergo a liquid–solid transformation to form primary crystals with characteristic polymorphism. These primary crystals aggregate, or grow into each other, to form clusters, which further interact, resulting in the formation of a continu‐ ous three-dimensional network. The mechanical properties of a fat, can be influenced by all these levels of structure; however, most directly by the level of structure closest to the mac‐ roscopic world, namely the microstructure [24].

It is during crystallization that the template for the final physical properties of the result‐ ing fat crystal network is created. Hence, the mechanical properties of a fat crystal net‐ work are determined by the different levels of structure, such as chemical composition, solid fat content (SFC), and crystal habit (polymorphism and microstructure). To study the mechanical properties of fat crystal networks, rheologic tests are used, which meas‐ ure how the crystallized material responds to applied forces (stress) and deformations (strain) [18].

Foods are edible structures created as a result of the responses of proteins, polysaccharides, and lipids in aqueous media to different processing methods, such as thermal processing, homogenization, and other physical treatments. The processing operations to which foods are subjected affect their structure and microstructure. Most, if not all, of the responses are physical in nature. By definition, rheology is the study of deformation and flow of materials. In foods, measured rheological responses are those at the macroscopic level. However, they are directly affected by the changes and properties at the microscopic level. Fractal dimen‐ sion has been used to characterize food particles in addition to microscopic and size distri‐ bution data. The fractal dimension can be estimated by several techniques such as viscoelastic behavior [25].

#### **3.1. Rheology and texture**

Rheology has been defined as the study of the flow and deformation of materials, with spe‐ cial emphasis being usually placed on the former. In flow, elements of the liquid are deform‐ ing resisted by viscosity. Solids when stressed creep, i.e. continue to deform very slowly over a very long time scale. In structured liquids there is a natural rest condition of the mi‐ crostructure that represents a minimum-energy state. When these liquids are deformed, thermodynamic forces immediately begin to operate to restore this rest state. This kind of energy is the origin of elasticity in structured liquids. Alongside these elastic forces are the ever-present viscous forces that produce viscoelastic effects [26].

Rheological methods can be divided into small and large deformation rheology. *Small defor‐ mation rheology* does not cause structural damage to the sample. They are performed in the linear viscoelastic region (LVR), in which the stress is directly proportional to the strain. *Large deformation rheology* is based on the deformation of a sample at a constant rate to the point where the force exceeds the structural capacity of the sample, causing it to permanent‐ ly deform and break [18]. Sometimes, oscillatory testing is referred to as small amplitude os‐ cillatory testing because small deformations must be employed to maintain linear viscoelastic behavior. Many processes, such as mastication and swallowing, are only accom‐ plished with very large deformations. Collecting viscoelastic data relevant to this type of problem involves testing in the non-linear range behavior [27].

**Figure 4.** The various regions of an oscillatory test of structured liquids [26].

The penetrometry method and the two-plate compression method are large-deformation tests and are widely used to determine the yield stress or the firmness of a plastic fat. The large-deformation method has been widely used to study the physical properties of fat products, such as the spreadability of shortenings and the hardness of chocolate and milk

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Texture has been defined as the way in which various constituents and structural ele‐ ments are arranged and combined into a micro- and macrostructure and this structure is externally manifested in terms of flow and deformation [29]. The structural elements of fats consist of solid fat crystals. They are suspended in liquid oil and when present in suf‐ ficient quantity form a three-dimensional network that imparts plastic properties to the fat. The external manifestations of this network structure include a number of physical and mechanical properties such as hardness, softness, spreadability, brittleness, shortening power, and aeration properties. The texture of fats is influenced by a number of factors, including the solids content, the fatty acid and triacylglycerol composition of the solids, the polymorphic behavior of the fat crystals, the size and shape of the crystals, the nature of the crystal network, mechanical treatment, and temperature history. Many of these fac‐ tors are interrelated, making it difficult to establish the effect of each independently [30]. Crystallization usually results in harder materials with higher solid fat contents. In addi‐ tion, microstructural differences must be taken into account when evaluating the function‐ al properties of lipids. The possibility of different polymorphic forms must not be neglected either because it can influence the texture and sensory profile. The texture of plastic fats can be determined by three main methods such as: cone penetrometry, pene‐ tration by a probe, compression between parallel plates. The analyses and the evaluation of food texture are very important in food processing. Some of the attributes such as

fat, and the results have been found to correlate well with sensory tests [28].

hardness and adhesiveness can be evaluated by texture analysis.

A frequently used method of measuring linear viscoelastic response is oscillatory testing, i.e. applying an oscillating stress or strain as an input to the liquid and monitoring the resulting oscillatory strain or stress output. Oscillatory tests are performed over a range of frequency. Short times correspond to high frequencies, and long times relate to low frequencies. In a sine-wave-shaped input of either stress or strain the resulting sinusoidal strain or stress out‐ put is separated into solid-like response, which is in phase with the input, and a correspond‐ ing liquid-like response which is π/2 (i.e. 90°) out of phase with the input. The solid-like component at any particular frequency is characterized by the storage modulus, G', and the liquid-like response is described by the complementary loss modulus, G''. The behavior nor‐ mally seen for typical viscoelastic liquids is an initial elastic response, thereafter, a delayed elastic response where the deformation rate becomes slower and slower, ending up as a very slow but steady-state deformation at the longest times, i.e. the material is in steady flow. The overall G', G'' response of structured liquids is shown in Figure 4 [26].

G' expresses the magnitude of the energy that is stored in the material or recoverable per cycle of deformation, while G'' is a measure of the energy which is lost as viscous dissipa‐ tion per cycle of deformation. For a viscoelastic material the resultant stress is also sinusoi‐ dal but shows a phase lag of δ radians when compared with the strain. The phase angle covers the range of 0 to π/2 as the viscous component increases. If G' is much greater than G'', the material will behave more like a solid, i.e., the deformations will be essentially elas‐ tic or recoverable. The loss tangent, tan δ, is the ratio of the energy dissipated to that stored per cycle of deformation. When G'' is much greater than G', the energy used to deform the material is dissipated viscously and the materials behavior is liquid like [25].

**Figure 4.** The various regions of an oscillatory test of structured liquids [26].

**3.1. Rheology and texture**

78 Food Industry

Rheology has been defined as the study of the flow and deformation of materials, with spe‐ cial emphasis being usually placed on the former. In flow, elements of the liquid are deform‐ ing resisted by viscosity. Solids when stressed creep, i.e. continue to deform very slowly over a very long time scale. In structured liquids there is a natural rest condition of the mi‐ crostructure that represents a minimum-energy state. When these liquids are deformed, thermodynamic forces immediately begin to operate to restore this rest state. This kind of energy is the origin of elasticity in structured liquids. Alongside these elastic forces are the

Rheological methods can be divided into small and large deformation rheology. *Small defor‐ mation rheology* does not cause structural damage to the sample. They are performed in the linear viscoelastic region (LVR), in which the stress is directly proportional to the strain. *Large deformation rheology* is based on the deformation of a sample at a constant rate to the point where the force exceeds the structural capacity of the sample, causing it to permanent‐ ly deform and break [18]. Sometimes, oscillatory testing is referred to as small amplitude os‐ cillatory testing because small deformations must be employed to maintain linear viscoelastic behavior. Many processes, such as mastication and swallowing, are only accom‐ plished with very large deformations. Collecting viscoelastic data relevant to this type of

A frequently used method of measuring linear viscoelastic response is oscillatory testing, i.e. applying an oscillating stress or strain as an input to the liquid and monitoring the resulting oscillatory strain or stress output. Oscillatory tests are performed over a range of frequency. Short times correspond to high frequencies, and long times relate to low frequencies. In a sine-wave-shaped input of either stress or strain the resulting sinusoidal strain or stress out‐ put is separated into solid-like response, which is in phase with the input, and a correspond‐ ing liquid-like response which is π/2 (i.e. 90°) out of phase with the input. The solid-like component at any particular frequency is characterized by the storage modulus, G', and the liquid-like response is described by the complementary loss modulus, G''. The behavior nor‐ mally seen for typical viscoelastic liquids is an initial elastic response, thereafter, a delayed elastic response where the deformation rate becomes slower and slower, ending up as a very slow but steady-state deformation at the longest times, i.e. the material is in steady flow. The

G' expresses the magnitude of the energy that is stored in the material or recoverable per cycle of deformation, while G'' is a measure of the energy which is lost as viscous dissipa‐ tion per cycle of deformation. For a viscoelastic material the resultant stress is also sinusoi‐ dal but shows a phase lag of δ radians when compared with the strain. The phase angle covers the range of 0 to π/2 as the viscous component increases. If G' is much greater than G'', the material will behave more like a solid, i.e., the deformations will be essentially elas‐ tic or recoverable. The loss tangent, tan δ, is the ratio of the energy dissipated to that stored per cycle of deformation. When G'' is much greater than G', the energy used to deform the

ever-present viscous forces that produce viscoelastic effects [26].

problem involves testing in the non-linear range behavior [27].

overall G', G'' response of structured liquids is shown in Figure 4 [26].

material is dissipated viscously and the materials behavior is liquid like [25].

The penetrometry method and the two-plate compression method are large-deformation tests and are widely used to determine the yield stress or the firmness of a plastic fat. The large-deformation method has been widely used to study the physical properties of fat products, such as the spreadability of shortenings and the hardness of chocolate and milk fat, and the results have been found to correlate well with sensory tests [28].

Texture has been defined as the way in which various constituents and structural ele‐ ments are arranged and combined into a micro- and macrostructure and this structure is externally manifested in terms of flow and deformation [29]. The structural elements of fats consist of solid fat crystals. They are suspended in liquid oil and when present in suf‐ ficient quantity form a three-dimensional network that imparts plastic properties to the fat. The external manifestations of this network structure include a number of physical and mechanical properties such as hardness, softness, spreadability, brittleness, shortening power, and aeration properties. The texture of fats is influenced by a number of factors, including the solids content, the fatty acid and triacylglycerol composition of the solids, the polymorphic behavior of the fat crystals, the size and shape of the crystals, the nature of the crystal network, mechanical treatment, and temperature history. Many of these fac‐ tors are interrelated, making it difficult to establish the effect of each independently [30]. Crystallization usually results in harder materials with higher solid fat contents. In addi‐ tion, microstructural differences must be taken into account when evaluating the function‐ al properties of lipids. The possibility of different polymorphic forms must not be neglected either because it can influence the texture and sensory profile. The texture of plastic fats can be determined by three main methods such as: cone penetrometry, pene‐ tration by a probe, compression between parallel plates. The analyses and the evaluation of food texture are very important in food processing. Some of the attributes such as hardness and adhesiveness can be evaluated by texture analysis.

### **3.2. Fractal**

Once the attraction forces have become larger than the repulsion, and also larger than Brow‐ nian motion, particles can remain together when they collide. The resulting aggregates or flocs have a very complex structure and most of the flocs do not have homogeneous internal structures. The center is usually denser than the outer regions; hence the mass does not change with the third power of the radius as in normal objects with constant density [31].

link to build up a 3-D fat crystal network. The shape of the fat crystal clusters can be spherulitic, feather-like, blade or needle-shaped. The size of fat crystal clusters can vary from several micrometers to more than 200 µm. Processing conditions can affect the size of

Structuring Fat Foods

81

http://dx.doi.org/10.5772/53369

Fats are the main structural constituents of many food products including margarine, chocolate, butter and spreads. The sensory textural characteristics (i.e., spreadability, hard‐ ness, snap) of fat structured foods are dependent on their macroscopic rheological proper‐ ties, which are a consequence of the structure of their underlying fat crystal network. This network arises from the interactions between polycrystalline fat particles, and provides the elastic component, or the solid-like behavior, of a plastic fat. The sensory properties of the fat-structured foods are dependent not only on the amount of solid fat crystals present and their polymorphism, but also their geometry and the spatial distribution of crystalline

The microstructure of fat crystal networks can be quantified by fractal dimensions, which can describes the combined effects of morphology and spatial distribution patterns of the crystal clusters in fat crystal networks. The usefulness in the quantification of the micro‐ structure of fats using the concept of fractal dimension arises from the possibility of relating structure to physical properties [28]. The fractal model of fat crystal networks explains the scaling behavior of rheological properties of semi-solid fat products to their solid fat content by their microstructure, which can be quantified using fractal dimensions. In general, differ‐ ent microscopy fractal dimensions reflect different aspects of the micro- structure and thus have different physical meanings. An unambiguous agreement between physical fractal di‐

Fat crystal networks are statically self-similar, which means that the microstructure in a fat crystal network looks similar at different magnifications. Fractal structures are created by agglomeration, or clustering, of small particles to form a larger object in a random, iterative fashion under some constraint. In a similar fashion, fat crystal networks are built from clus‐ ters of polycrystalline particles (crystallites) that aggregate in a diffusionally limited, fractal fashion. Fractal mathematics have been used to relate the elastic properties of fat crystal net‐ works to the spatial distribution of the network mass and to link crystallization kinetics and phase behavior to microstructure. The fractal dimension defines the cluster size and has been evaluated by rheology techniques. Rheology is the most common technique for the quantification of microstructure in fat crystal networks and utilizes the relationship of the shear storage modulus (G′) to the volume fraction of network solid mass via the mass fractal

The shape, size, and the strength of the fat crystal flocs making up the fat crystal network are always different. The weakest floc will become a flaw and acts as a stress concentrator. The elastic properties of the network depend on the number of connections between neigh‐ boring structural clusters, rather than on the amount of apparent solids. This implies that the connectivity of the networks increases with an increasing volume fraction of solids. An idealized view of the structure of a fat crystal network showing the one dimensional defor‐ mation of the links between crystal clusters [35]. In this same work, by using a modified

mensions and microscopy fractal dimensions is required [25].

the fat crystal clusters [28].

dimension of the network [18].

material [34].

Many patterns in nature such as the geometry of coastlines, mountains, trees, and vegeta‐ bles, for instance, cannot always be defined adequately by using the familiar straight lines, circles, conic sections, polygons, spheres, quadratic surfaces, etc. Fractal geometry was born out of this lack of geometrical tools. A geometric shape belongs to standard ge‐ ometry when smaller and smaller portions of it become increasingly smooth. For example, a generic curve becomes a straight line, and a generic surface becomes a plane. Fractals are shapes whose roughness and fragmentation neither tend to vanish, nor fluctuate up and down, but remain essentially unchanged as one zooms in continually and examina‐ tion is refined. Hence, the structure of every piece holds the key to the whole structure. Fractals are characterized by two types of symmetries: self-similar and self-affine. In selfsimilar each part is a linear geometric reduction of the whole, with the same reduction ra‐ tios in all directions. In self-affine, the reductions are still linear but the reduction ratios in different directions are different [32].

A fractal dimension is a powerful means of quantifying the structure of non-Euclidean ob‐ jects by capturing the complexity of a structure's geometry in a single number. The chal‐ lenge, however, is to give physical meaning to the number obtained [24].

The macroscopic rheological properties of the network are influenced by all levels of struc‐ ture defined during the formation of the network, i.e. the structure of the individual triacyl‐ glycerols, the structure of the individual crystalline units formed, or the polymorphic nature of the network, and the microstructural level of structure. The microstructural aggregate or microstructural network present in fat crystal networks scale in a fractal manner in the range between the size of the individual particles composing the sample (microstructural el‐ ements) and the size of the microstructures. For colloidal aggregates and other fractal sys‐ tems (such as fat crystal networks), fractal concept quantifies the way in which the mass of the sample/system increases with its size, according to the fractal dimension [33].

#### **3.3. Fat crystal network**

Early nucleation and crystal growth events lead to the formation of submicron primary crys‐ tallites from the melt. These crystallites associate into micron-range particles, which further aggregate into clusters, until a continuous three-dimensional network with voids filled with liquid fat is formed [18].

A structural hierarchy exists within fat crystal networks. Polymorphism has to do with dif‐ ferent molecular packing arrangements of tryacylglicerol molecules, at the nanostructural range, within the primary fat crystals. Once the primary fat crystals are formed, they aggre‐ gate, or grow into each other, to form fat crystal clusters (or aggregate), which in turn crosslink to build up a 3-D fat crystal network. The shape of the fat crystal clusters can be spherulitic, feather-like, blade or needle-shaped. The size of fat crystal clusters can vary from several micrometers to more than 200 µm. Processing conditions can affect the size of the fat crystal clusters [28].

**3.2. Fractal**

80 Food Industry

different directions are different [32].

**3.3. Fat crystal network**

liquid fat is formed [18].

Once the attraction forces have become larger than the repulsion, and also larger than Brow‐ nian motion, particles can remain together when they collide. The resulting aggregates or flocs have a very complex structure and most of the flocs do not have homogeneous internal structures. The center is usually denser than the outer regions; hence the mass does not change with the third power of the radius as in normal objects with constant density [31]. Many patterns in nature such as the geometry of coastlines, mountains, trees, and vegeta‐ bles, for instance, cannot always be defined adequately by using the familiar straight lines, circles, conic sections, polygons, spheres, quadratic surfaces, etc. Fractal geometry was born out of this lack of geometrical tools. A geometric shape belongs to standard ge‐ ometry when smaller and smaller portions of it become increasingly smooth. For example, a generic curve becomes a straight line, and a generic surface becomes a plane. Fractals are shapes whose roughness and fragmentation neither tend to vanish, nor fluctuate up and down, but remain essentially unchanged as one zooms in continually and examina‐ tion is refined. Hence, the structure of every piece holds the key to the whole structure. Fractals are characterized by two types of symmetries: self-similar and self-affine. In selfsimilar each part is a linear geometric reduction of the whole, with the same reduction ra‐ tios in all directions. In self-affine, the reductions are still linear but the reduction ratios in

A fractal dimension is a powerful means of quantifying the structure of non-Euclidean ob‐ jects by capturing the complexity of a structure's geometry in a single number. The chal‐

The macroscopic rheological properties of the network are influenced by all levels of struc‐ ture defined during the formation of the network, i.e. the structure of the individual triacyl‐ glycerols, the structure of the individual crystalline units formed, or the polymorphic nature of the network, and the microstructural level of structure. The microstructural aggregate or microstructural network present in fat crystal networks scale in a fractal manner in the range between the size of the individual particles composing the sample (microstructural el‐ ements) and the size of the microstructures. For colloidal aggregates and other fractal sys‐ tems (such as fat crystal networks), fractal concept quantifies the way in which the mass of

Early nucleation and crystal growth events lead to the formation of submicron primary crys‐ tallites from the melt. These crystallites associate into micron-range particles, which further aggregate into clusters, until a continuous three-dimensional network with voids filled with

A structural hierarchy exists within fat crystal networks. Polymorphism has to do with dif‐ ferent molecular packing arrangements of tryacylglicerol molecules, at the nanostructural range, within the primary fat crystals. Once the primary fat crystals are formed, they aggre‐ gate, or grow into each other, to form fat crystal clusters (or aggregate), which in turn cross-

lenge, however, is to give physical meaning to the number obtained [24].

the sample/system increases with its size, according to the fractal dimension [33].

Fats are the main structural constituents of many food products including margarine, chocolate, butter and spreads. The sensory textural characteristics (i.e., spreadability, hard‐ ness, snap) of fat structured foods are dependent on their macroscopic rheological proper‐ ties, which are a consequence of the structure of their underlying fat crystal network. This network arises from the interactions between polycrystalline fat particles, and provides the elastic component, or the solid-like behavior, of a plastic fat. The sensory properties of the fat-structured foods are dependent not only on the amount of solid fat crystals present and their polymorphism, but also their geometry and the spatial distribution of crystalline material [34].

The microstructure of fat crystal networks can be quantified by fractal dimensions, which can describes the combined effects of morphology and spatial distribution patterns of the crystal clusters in fat crystal networks. The usefulness in the quantification of the micro‐ structure of fats using the concept of fractal dimension arises from the possibility of relating structure to physical properties [28]. The fractal model of fat crystal networks explains the scaling behavior of rheological properties of semi-solid fat products to their solid fat content by their microstructure, which can be quantified using fractal dimensions. In general, differ‐ ent microscopy fractal dimensions reflect different aspects of the micro- structure and thus have different physical meanings. An unambiguous agreement between physical fractal di‐ mensions and microscopy fractal dimensions is required [25].

Fat crystal networks are statically self-similar, which means that the microstructure in a fat crystal network looks similar at different magnifications. Fractal structures are created by agglomeration, or clustering, of small particles to form a larger object in a random, iterative fashion under some constraint. In a similar fashion, fat crystal networks are built from clus‐ ters of polycrystalline particles (crystallites) that aggregate in a diffusionally limited, fractal fashion. Fractal mathematics have been used to relate the elastic properties of fat crystal net‐ works to the spatial distribution of the network mass and to link crystallization kinetics and phase behavior to microstructure. The fractal dimension defines the cluster size and has been evaluated by rheology techniques. Rheology is the most common technique for the quantification of microstructure in fat crystal networks and utilizes the relationship of the shear storage modulus (G′) to the volume fraction of network solid mass via the mass fractal dimension of the network [18].

The shape, size, and the strength of the fat crystal flocs making up the fat crystal network are always different. The weakest floc will become a flaw and acts as a stress concentrator. The elastic properties of the network depend on the number of connections between neigh‐ boring structural clusters, rather than on the amount of apparent solids. This implies that the connectivity of the networks increases with an increasing volume fraction of solids. An idealized view of the structure of a fat crystal network showing the one dimensional defor‐ mation of the links between crystal clusters [35]. In this same work, by using a modified fractal model, which describes the increase of G′ with SFC well, show the idea that the stress-carrying mechanism in fat crystal networks is heterogeneous, i.e. since real networks are not fully connected and that connectivity of networks increases with the volume fraction of solids, the load-bearing volume fraction of solids in real networks increases in an expo‐ nential fashion with the apparent volume fraction of solids.

the only continuous phase in chocolate, thus responsible for melting behavior and the dis‐ persion of all other constituents. A careful tempering of the chocolate is necessary in or‐ der to obtain the fine crystals in the correct form (β-modification). Cupuassu fat, a similar cocoa butter fat, shows polymorphic behavior like cocoa butter (β form) and needs to be tempered like cocoa butter; at 24-25ºC an α (alpha) form is present. The melting profiles of cocoa butter and cupuassu fat are similar as shown. At all temperatures, cocoa butter has a higher solid fat content than cupuassu fat. This suggests that cupuassu fat would be useful in filled chocolate manufacture as a softer filling fat compatible with cocoa butter. The fatty acid and triacylglycerol compositions of cupuassu fat in comparison with cocoa butter show that palmitic acid in cupuassu fat is present in much smaller amount (7.8%) than in cocoa butter (26.1%); stearic acid is about the same; oleic acid is higher in cupuas‐ su. Particularly notable is the high amount of arachidic acid (20:0) in cupuassu fat. The tri‐ acylglycerol compositions reflect the fatty acid compositions, but give more useful information. Although cupuassu has a higher SOS content than cocoa butter, its contents of POP and POS are much lower reflecting its low level of palmitic acid. Total SOS-type triacylglycerols, i.e. POP+POS+SOS+SOA, is 57% in cupuassu and 83% in cocoa butter. Fractionation, as applied to fats such as shea and sal, would be needed to bring the total SOS-type content to the same as in cocoa butter. Fractionation could be used to modify cupuassu fat to make it more similar to cocoa butter for use as a CBE (cocoa butter equiv‐

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Modified lipids are used in the majority of chocolate and confectionery applications, such as chocolate compounds, filling fats in pralines, aerated products and cold products such as ice cream toppings. Production economics is often related to price, speed of production and equipment requirements, which in turn are related to the raw materials and their ability to

The quality is related to the capacity of the fat to remain stable in terms of appearance, tex‐ ture and taste; and the sensory properties can briefly be described as appearance, smell, taste and the role that fat plays in mouth feel with regard to texture and melt off properties. In chocolate industry fat bloom is still a problem. It modifies (shortens) the shelf life of the end products and makes life difficult for product development. Fat migration is one of the

In chocolate industry, for processing and texture reasons, however, it is not possible to re‐ duce the level too much below 25%. This is insufficient to make a low calorie claim on the product, so two manufacturers have produced fats that melt like cocoa butter but have a lower calorific value. Like lauric fats they are incompatible with cocoa butter and so the products have to be made with cocoa powder [36]. Some fats go into confectionery as a com‐ ponent of other ingredients. The common example is nuts, which contain fats, often of types such as lauric or unsaturated fats. These fats are sometimes the origin of spoilage problems. Studies correlating chocolate composition and textural or rheological properties are com‐ monly found due to the source of new fat or cocoa butter replacers which strongly affect rheological parameters on chocolate manufacture and final product texture. According to that, adaptations on manufacturing scale have to be done in order to keep the desirable sen‐

alent), with 65% minimum of total SOS-type triacylglycerols [38].

causes of bloom, but it will also soften the products during storage [39].

crystallize rapidly.

## **4. Fat foods**

Fats and oils are the raw materials for liquid oils, shortenings, margarines, and other special‐ ty or tailored products that are functional ingredients in food products prepared by food processors and restaurants and in the home. Humans have used fats and oils for food and a variety of other applications since prehistoric times, as they were easily isolated from their source. Fats and oils found utility because of their unique properties. These ingredients were found to add flavor, lubricity, texture, and satiety to foods. They have also been found to have a major role in human nutrition. Fats and oils are the highest energy source of the three basic foods (carbohydrates, proteins, and fats), and many contain fatty acids essential for health that are not manufactured by the human body [2].

While vegetable fats were used originally as a cheaper substitute for milk fat the ability to specify the properties of vegetable fat has considerable advantages. This ability arises be‐ cause of the science and technology available to the fat processing industry. Some vegetable fats used in foods are not tailor-made but are simply a vegetable fat of known origin and treatment. The commonest example is palm kernel oil (HPKO), which is often used in foods.

#### **4.1. Chocolate products**

Chocolate can be described as a suspension consisting of nonfat particles (sugar and cocoa solids and, eventually, milk powder particles) dispersed in cocoa butter as a continuous phase. Molten chocolates represent a dense blend of phospholipid-coated sucrose and co‐ coa particles in liquid fat. Milk chocolate usually contains about 12 g of cocoa mass, 19 g whole milk powder, 48.5 g sugar and, additionally, 20 g added cocoa butter per 100 g chocolate [36].

The characteristic flavor of chocolate has to be developed in several processing steps. Dur‐ ing processing, the components are mixed, refined, and conched to attain desired rheologi‐ cal properties for a final defined product texture and melting characteristics. A conche is a scraped-surface mixer that optimizes flavor development and turns chocolate mass into a flowable liquid. Through shear and longitudinal mixing, acidic flavors and moisture in the cocoa mass are reduced. Upon entering the conche, not all sugar and cocoa particles will be coated with cocoa butter. Fat in the chocolate will be released from the agglomerated choco‐ late mass and spread to cover these particles so that they can flow easily. The final chocolate mass viscosity should be deemed optimal for the ensuing tempering [37].

Cocoa butter, which amounts to 25-36% in finished chocolate, is responsible for the smooth texture, contractability, flavor release, and gloss of the product. The fat phase is the only continuous phase in chocolate, thus responsible for melting behavior and the dis‐ persion of all other constituents. A careful tempering of the chocolate is necessary in or‐ der to obtain the fine crystals in the correct form (β-modification). Cupuassu fat, a similar cocoa butter fat, shows polymorphic behavior like cocoa butter (β form) and needs to be tempered like cocoa butter; at 24-25ºC an α (alpha) form is present. The melting profiles of cocoa butter and cupuassu fat are similar as shown. At all temperatures, cocoa butter has a higher solid fat content than cupuassu fat. This suggests that cupuassu fat would be useful in filled chocolate manufacture as a softer filling fat compatible with cocoa butter. The fatty acid and triacylglycerol compositions of cupuassu fat in comparison with cocoa butter show that palmitic acid in cupuassu fat is present in much smaller amount (7.8%) than in cocoa butter (26.1%); stearic acid is about the same; oleic acid is higher in cupuas‐ su. Particularly notable is the high amount of arachidic acid (20:0) in cupuassu fat. The tri‐ acylglycerol compositions reflect the fatty acid compositions, but give more useful information. Although cupuassu has a higher SOS content than cocoa butter, its contents of POP and POS are much lower reflecting its low level of palmitic acid. Total SOS-type triacylglycerols, i.e. POP+POS+SOS+SOA, is 57% in cupuassu and 83% in cocoa butter. Fractionation, as applied to fats such as shea and sal, would be needed to bring the total SOS-type content to the same as in cocoa butter. Fractionation could be used to modify cupuassu fat to make it more similar to cocoa butter for use as a CBE (cocoa butter equiv‐ alent), with 65% minimum of total SOS-type triacylglycerols [38].

fractal model, which describes the increase of G′ with SFC well, show the idea that the stress-carrying mechanism in fat crystal networks is heterogeneous, i.e. since real networks are not fully connected and that connectivity of networks increases with the volume fraction of solids, the load-bearing volume fraction of solids in real networks increases in an expo‐

Fats and oils are the raw materials for liquid oils, shortenings, margarines, and other special‐ ty or tailored products that are functional ingredients in food products prepared by food processors and restaurants and in the home. Humans have used fats and oils for food and a variety of other applications since prehistoric times, as they were easily isolated from their source. Fats and oils found utility because of their unique properties. These ingredients were found to add flavor, lubricity, texture, and satiety to foods. They have also been found to have a major role in human nutrition. Fats and oils are the highest energy source of the three basic foods (carbohydrates, proteins, and fats), and many contain fatty acids essential for

While vegetable fats were used originally as a cheaper substitute for milk fat the ability to specify the properties of vegetable fat has considerable advantages. This ability arises be‐ cause of the science and technology available to the fat processing industry. Some vegetable fats used in foods are not tailor-made but are simply a vegetable fat of known origin and treatment. The commonest example is palm kernel oil (HPKO), which is often used in foods.

Chocolate can be described as a suspension consisting of nonfat particles (sugar and cocoa solids and, eventually, milk powder particles) dispersed in cocoa butter as a continuous phase. Molten chocolates represent a dense blend of phospholipid-coated sucrose and co‐ coa particles in liquid fat. Milk chocolate usually contains about 12 g of cocoa mass, 19 g whole milk powder, 48.5 g sugar and, additionally, 20 g added cocoa butter per 100 g

The characteristic flavor of chocolate has to be developed in several processing steps. Dur‐ ing processing, the components are mixed, refined, and conched to attain desired rheologi‐ cal properties for a final defined product texture and melting characteristics. A conche is a scraped-surface mixer that optimizes flavor development and turns chocolate mass into a flowable liquid. Through shear and longitudinal mixing, acidic flavors and moisture in the cocoa mass are reduced. Upon entering the conche, not all sugar and cocoa particles will be coated with cocoa butter. Fat in the chocolate will be released from the agglomerated choco‐ late mass and spread to cover these particles so that they can flow easily. The final chocolate

Cocoa butter, which amounts to 25-36% in finished chocolate, is responsible for the smooth texture, contractability, flavor release, and gloss of the product. The fat phase is

mass viscosity should be deemed optimal for the ensuing tempering [37].

nential fashion with the apparent volume fraction of solids.

health that are not manufactured by the human body [2].

**4. Fat foods**

82 Food Industry

**4.1. Chocolate products**

chocolate [36].

Modified lipids are used in the majority of chocolate and confectionery applications, such as chocolate compounds, filling fats in pralines, aerated products and cold products such as ice cream toppings. Production economics is often related to price, speed of production and equipment requirements, which in turn are related to the raw materials and their ability to crystallize rapidly.

The quality is related to the capacity of the fat to remain stable in terms of appearance, tex‐ ture and taste; and the sensory properties can briefly be described as appearance, smell, taste and the role that fat plays in mouth feel with regard to texture and melt off properties. In chocolate industry fat bloom is still a problem. It modifies (shortens) the shelf life of the end products and makes life difficult for product development. Fat migration is one of the causes of bloom, but it will also soften the products during storage [39].

In chocolate industry, for processing and texture reasons, however, it is not possible to re‐ duce the level too much below 25%. This is insufficient to make a low calorie claim on the product, so two manufacturers have produced fats that melt like cocoa butter but have a lower calorific value. Like lauric fats they are incompatible with cocoa butter and so the products have to be made with cocoa powder [36]. Some fats go into confectionery as a com‐ ponent of other ingredients. The common example is nuts, which contain fats, often of types such as lauric or unsaturated fats. These fats are sometimes the origin of spoilage problems.

Studies correlating chocolate composition and textural or rheological properties are com‐ monly found due to the source of new fat or cocoa butter replacers which strongly affect rheological parameters on chocolate manufacture and final product texture. According to that, adaptations on manufacturing scale have to be done in order to keep the desirable sen‐ sory characteristics in the final product. Rheology is a useful feature on setting those issues. Several works have been conducted to study and understand rheological properties of choc‐ olates. The various fats used in chocolate can contain different levels of trisaturated triacyl‐ glycerols. Since these can crystallize out early in the tempering process, they can, in some instances, have an effect on the rheology of the chocolate. Six basic source oils are permitted as non-cocoa vegetable fats (CBE - Cocoa Butter Equivalent) in chocolate throughout Euro‐ pean Union - palm oil, shea oil, illipe butter, sal oil, kokum gurgi, and mango kernel oil. Among these six oils, four (palm, shea, sal, and mango kernel) usually have to undergo some form of fractionation process to concentrate the SOS type of triacylglycerol necessary for equivalence to cocoa butter. Palm oil is even more complicated since it contains a signifi‐ cant quantity of trisaturated triacylglycerols which also have to be removed [40].

tween phases, the rheology, and structure of individual component phase. In order to im‐ prove the quality of this very appreciated foodstuff, ingredients research and their impact on formulations are very desirable. In [43] was investigated the potential of a chemically modified polysaccharide (N-succinil chitosan hydrogel) when applied as structuring agent in colloidal systems. It was found that the mixes resulting by combination among chitosan and palm fat presented good characteristics; the enormous structuring power presented by this biomolecule can be very useful to elaborate low-fat formulations with good textural properties. Moreover, taking in account the physiological activity, it can be employed in or‐ der to promote best nutritional quality in foods; this biopolymer and their derivatives, can

Structuring Fat Foods

85

http://dx.doi.org/10.5772/53369

In [41] was found in study that the replacement of hydrogenated vegetable fat by palm fat caused changes in melting ranges of formulations. Higher melting rate was observed by combination between palm fat and fructose syrup. In addition to effects expected on melting behavior and solids content, sugar blends employed in this study affected the air incorpora‐ tion. There is consense that greater air content increase the melting resistance. However, de‐ spite to lower overrun, ice creams made with fructose syrup melted more slowly. Thus, the levels of air added into the products not allow safe conclusions about the influence of this parameter on physical behavior of assessed ice creams in this study. In [44] was evaluated also the influence of the substitution of hydrogenated fat in the manufacture of ice cream formulation with palm fat through rheology analysis, and compare the results obtained with the melting test. The rheological and the melting tests showed a better response from the ageing process, and a better formed structure with the formulation produced with hydro‐ genated fat. It was suggested that formulations produced with palm fat suffers a poorer par‐ tial coalescence by its crystallization profile and less membrane destabilization by the

In another study was evaluated the influence of the substitution of hydrogenated fat in the manufacture of ice cream formulation with cupuassu fat through rheology analysis, and compare the results obtained with the melting test. The rheological tests showed similar re‐ sponse from the ageing process to both formulations, and the melting tests showed a slower meltdown of the structure with the ice cream produced with cupuassu fat. The results ob‐ tained demonstrated that cupuassu fat is a good substitute for hydrogenated vegetable fat

The functionality of fats in bakery products can be explained as: development of the struc‐ ture; lubrication; aeration; heat transfer; moisture retention; improved shelf-life, volume, texture and flavor. In some cases the function of a fat can be either partially or completely

Fats shorten the texture of baked products by preventing cohesion of gluten strands during mixing, hence the term shortening. All-purpose shortenings are used primarily for cookies but are also common ingredients in cakes, breads, and icings and are also used for frying applications. The quality of cakes and icings is highly dependent upon aeration; therefore, a

be extensively explored, since appear do not has limitations in its potentialities.

emulsifiers.

**4.3. Bakery**

for using in ice cream formulations [45].

replaced by some other ingredient, typically an emulsifier.

#### **4.2. Ice cream**

Ice cream has been identified as three component foam made up of a network of fat globules and ice crystals dispersed in a high viscosity aqueous phase. The composition of ice cream varies depending on the market requirements and processing conditions. Although the quality of the final product depends largely on the processing and freezing parameters, the ingredients also play an important role. The physical structure of ice cream affects its melt‐ ing rate and hardness, although the specific relationships have not all been worked out. Structure development in ice cream often is attributed to the macromolecules present in the ice cream mix – milk fat, protein, and complex carbohydrates. Milk fat interacts with other ingredients to develop the texture, mouthfeel, creaminess, and overall sensations of lubrici‐ ty. Typically, ice cream contains 10 to 16% fat and its type and amount influence the charac‐ teristics of the resultant products by affecting their rheological properties. The fat content can influence the size of the ice crystals. Fat globules could mechanically impede the ice crystal growth. Since each type of fat exhibits a specific polymorphism function of its triacyl‐ glycerol composition, the thermal behavior of fats during ice cream processing should influ‐ ence the physicochemical properties of the intermediate and final products [41].

A typical ice cream formulation has fat (7-15%), lactose (5-7%), other sugars (12-16%), stabil‐ izers, emulsifiers and flavours (0.5%), total solids (28-40%), water (60-72%), milk protein (4-5%). Fat performs several functions in ice cream: it helps to stabilize the foam, it is largely responsible for the creamy texture, it slows down the rate at which ice cream melts and it is necessary to deliver flavour molecules that are soluble in fat but not water. The major sour‐ ces of fat used in industrial ice cream production are butterfat, cream and vegetable fat [42].

Ice creams are metastable systems created from an emulsion o/w employing several unit op‐ erations: mixing, heating, cooling, freezing, aerating and packaging. While the ingredients combination is responsible by chemical characteristics, a sophisticated microstructural ar‐ rangement constituted by fat globules, ice crystals and air bubbles supported in a highly vis‐ cous matrix dictates mechanical, thermal and sensorial properties.

There are many factors within the microstructure of products, which determine the rheolog‐ ical properties, such as colloidal interactions between disperses components, the junctions between structural elements, the properties of this elements, the interfacial behavior be‐ tween phases, the rheology, and structure of individual component phase. In order to im‐ prove the quality of this very appreciated foodstuff, ingredients research and their impact on formulations are very desirable. In [43] was investigated the potential of a chemically modified polysaccharide (N-succinil chitosan hydrogel) when applied as structuring agent in colloidal systems. It was found that the mixes resulting by combination among chitosan and palm fat presented good characteristics; the enormous structuring power presented by this biomolecule can be very useful to elaborate low-fat formulations with good textural properties. Moreover, taking in account the physiological activity, it can be employed in or‐ der to promote best nutritional quality in foods; this biopolymer and their derivatives, can be extensively explored, since appear do not has limitations in its potentialities.

In [41] was found in study that the replacement of hydrogenated vegetable fat by palm fat caused changes in melting ranges of formulations. Higher melting rate was observed by combination between palm fat and fructose syrup. In addition to effects expected on melting behavior and solids content, sugar blends employed in this study affected the air incorpora‐ tion. There is consense that greater air content increase the melting resistance. However, de‐ spite to lower overrun, ice creams made with fructose syrup melted more slowly. Thus, the levels of air added into the products not allow safe conclusions about the influence of this parameter on physical behavior of assessed ice creams in this study. In [44] was evaluated also the influence of the substitution of hydrogenated fat in the manufacture of ice cream formulation with palm fat through rheology analysis, and compare the results obtained with the melting test. The rheological and the melting tests showed a better response from the ageing process, and a better formed structure with the formulation produced with hydro‐ genated fat. It was suggested that formulations produced with palm fat suffers a poorer par‐ tial coalescence by its crystallization profile and less membrane destabilization by the emulsifiers.

In another study was evaluated the influence of the substitution of hydrogenated fat in the manufacture of ice cream formulation with cupuassu fat through rheology analysis, and compare the results obtained with the melting test. The rheological tests showed similar re‐ sponse from the ageing process to both formulations, and the melting tests showed a slower meltdown of the structure with the ice cream produced with cupuassu fat. The results ob‐ tained demonstrated that cupuassu fat is a good substitute for hydrogenated vegetable fat for using in ice cream formulations [45].

#### **4.3. Bakery**

sory characteristics in the final product. Rheology is a useful feature on setting those issues. Several works have been conducted to study and understand rheological properties of choc‐ olates. The various fats used in chocolate can contain different levels of trisaturated triacyl‐ glycerols. Since these can crystallize out early in the tempering process, they can, in some instances, have an effect on the rheology of the chocolate. Six basic source oils are permitted as non-cocoa vegetable fats (CBE - Cocoa Butter Equivalent) in chocolate throughout Euro‐ pean Union - palm oil, shea oil, illipe butter, sal oil, kokum gurgi, and mango kernel oil. Among these six oils, four (palm, shea, sal, and mango kernel) usually have to undergo some form of fractionation process to concentrate the SOS type of triacylglycerol necessary for equivalence to cocoa butter. Palm oil is even more complicated since it contains a signifi‐

cant quantity of trisaturated triacylglycerols which also have to be removed [40].

ence the physicochemical properties of the intermediate and final products [41].

cous matrix dictates mechanical, thermal and sensorial properties.

A typical ice cream formulation has fat (7-15%), lactose (5-7%), other sugars (12-16%), stabil‐ izers, emulsifiers and flavours (0.5%), total solids (28-40%), water (60-72%), milk protein (4-5%). Fat performs several functions in ice cream: it helps to stabilize the foam, it is largely responsible for the creamy texture, it slows down the rate at which ice cream melts and it is necessary to deliver flavour molecules that are soluble in fat but not water. The major sour‐ ces of fat used in industrial ice cream production are butterfat, cream and vegetable fat [42]. Ice creams are metastable systems created from an emulsion o/w employing several unit op‐ erations: mixing, heating, cooling, freezing, aerating and packaging. While the ingredients combination is responsible by chemical characteristics, a sophisticated microstructural ar‐ rangement constituted by fat globules, ice crystals and air bubbles supported in a highly vis‐

There are many factors within the microstructure of products, which determine the rheolog‐ ical properties, such as colloidal interactions between disperses components, the junctions between structural elements, the properties of this elements, the interfacial behavior be‐

Ice cream has been identified as three component foam made up of a network of fat globules and ice crystals dispersed in a high viscosity aqueous phase. The composition of ice cream varies depending on the market requirements and processing conditions. Although the quality of the final product depends largely on the processing and freezing parameters, the ingredients also play an important role. The physical structure of ice cream affects its melt‐ ing rate and hardness, although the specific relationships have not all been worked out. Structure development in ice cream often is attributed to the macromolecules present in the ice cream mix – milk fat, protein, and complex carbohydrates. Milk fat interacts with other ingredients to develop the texture, mouthfeel, creaminess, and overall sensations of lubrici‐ ty. Typically, ice cream contains 10 to 16% fat and its type and amount influence the charac‐ teristics of the resultant products by affecting their rheological properties. The fat content can influence the size of the ice crystals. Fat globules could mechanically impede the ice crystal growth. Since each type of fat exhibits a specific polymorphism function of its triacyl‐ glycerol composition, the thermal behavior of fats during ice cream processing should influ‐

**4.2. Ice cream**

84 Food Industry

The functionality of fats in bakery products can be explained as: development of the struc‐ ture; lubrication; aeration; heat transfer; moisture retention; improved shelf-life, volume, texture and flavor. In some cases the function of a fat can be either partially or completely replaced by some other ingredient, typically an emulsifier.

Fats shorten the texture of baked products by preventing cohesion of gluten strands during mixing, hence the term shortening. All-purpose shortenings are used primarily for cookies but are also common ingredients in cakes, breads, and icings and are also used for frying applications. The quality of cakes and icings is highly dependent upon aeration; therefore, a variety of very specialized shortenings has been developed over the years to satisfy that de‐ mand. High ratio shortenings (containing mono and diglycerides), designed primarily for cakes, began to appear in the '30s. Fluid cake shortenings were commercialized in the '60s and offer many advantages including pumpability, ease of handling and the option of bulk delivery and storage [46].

Emulsion is a thermodynamically unstable system due to flocculation, creaming, coales‐ cence, phase inversion and Ostwald ripping. Emulsifier is a surfactant which can stabilize the emulsion by absorption at the interface, thereby lowering the interfacial tension. It is usually used to improve the emulsion stability. Proteins and polysaccharides are often ap‐ plied in emulsion as emulsifier. Proteins are usually used for their surfactant and gelling properties to improve the textural characteristics and stability of emulsion, while polysac‐ charides are usually added to increase the viscosity or to obtain a gel-like product. It was studied the impact of the use of a biomaterial (N-succinil chitosan hydrogel) in elaboration and structuration of food emulsion, and in substitution of a part of the oil phase. Chitosan showed to be a versatile ingredient since that was capable to modify the rheological proper‐ ties, acting as emulsifying agent, besides its already known antimicrobial and nutritional

Structuring Fat Foods

87

http://dx.doi.org/10.5772/53369

Margarine and spreads are prepared by blending fats and/or oils with other ingredients such as water and/or milk products, suitable edible proteins, salt, flavoring and coloring ma‐ terials and Vitamins A and D. Margarine must contain at least 80% fat by federal regulation, however, "diet" margarines and spreads may contain 0-80% fat. These products may be for‐ mulated from vegetable oils and/or animal fats, however, the vast preponderance are all vegetable. Non hydrogenated oils typically represent the majority of the fat phase. Lesser amounts of partially hydrogenated fats, that are naturally semisolid at room temperature, and/or hard fractions of certain fats are added to the blend as required to deliver the desired structure and melting properties [46]. At the moment, interesterification technics have been employed to produce tailor fats. Margarine originated as a substitute for butter. The big ad‐ vantage of margarine is that as a manufactured product the properties can be tailored to suit

An acceptable margarine must be a soft plastic at room temperature; the ratio of solid or crystalline fat to liquid oil in the mixture must be such that when the fat crystals are of the proper size and well dispersed, the mass will offer some resistance to deformation and sepa‐ ration of solid and liquid fats will be negligible; all the fat crystals must melt completely at body temperature and leave a pasty sensation in the mouth; the fat crystals must not melt

The major brands are today sold all over the world. The economic impact of any bad image on these super brands has led the major companies to focus on brand image. The increase in obesity has focused on health aspects of foods in general but also on chocolate and confec‐ tionery products. Fat replacers of the future will need to meet some important criteria, in‐ cluding reducing or replacing the target fat effectively, being available at a cost appropriate to the benefits provided, and being safe and legal with no appreciable side effects [39, 52].

qualities [50].

*4.4.2. Margarine*

a particular use.

abruptely [51].

**5. Prospective**

Cake is a baked batter made from wheat flour, sugar, eggs, shortening, leavening agents, salt, nonfat dry milk, flavors, and water. Cake batters are essentially a 'foam', that is a sys‐ tem in which air bubbles are trapped and held in an aqueous phase. The main function of fat in cake making is to assist with the incorporation of air into the batter during mixing, and the air bubble size and stability. High-ratio cakes, rich in sugar and fat, are extensively used in the baking industry [47].

Margarine has always had the advantage over butter in that the properties of the product can be tailored to give the best performance in a particular system. For puff pastry, i.e., spe‐ cialized margarines are easier to work with than butter. Various bakery margarines are manufactured to meet the technical requirements of particular uses.

The effect of different fats and margarines on the physical properties of cakes was investi‐ gated. The low *trans* fat suggested: greater volume and firmness; resilience comparable to hydrogenated fat; elasticity and chewiness were comparable to other formulations, as well the color parameters of the crumbs [48].

Textural properties are important quality parameters for this type of product. Physical and structure changes during aerated batters processing may alter their performance during baking or the quality of the final product. It is possible to test materials with particles and fiber in suspensions, since flushing effects may reduce sedimentation problems. In [49] was examined the influence of different types of fats (hydrogenated fat, margarine and vegetable oil) in formulation of cake batter, evaluating textural properties by Herschel-Bulkley equa‐ tion using back extrusion analysis; and it were observed values close in all parameters to samples prepared with margarine and hydrogenated fat. It can be mentioned the break point values could be consider by the industry as an important parameter, pointing the need of less energy in their processes of pumping, i.e.

#### **4.4. Food emulsions**

#### *4.4.1. Mayonnaise and salad dressing*

Mayonnaise and salad dressing are emulsified, semi-solid fatty foods that by federal regu‐ lation must contain not less than 65% and 30% vegetable oil, respectively, and dried whole eggs or egg yolks. Salt, sugar, spices, seasoning, vinegar, lemon juice, and other in‐ gredients complete these products. Pourable and spoonable dressings may be two phase (e.g., vinegar and oil) or the emulsified viscous type (e.g., French). There is a great variety of products available of varying compositions with a wide range in their oil content. Sal‐ ad oils exclusively are used for dressing products; typical choices include soybean, canola and olive oils [46].

Emulsion is a thermodynamically unstable system due to flocculation, creaming, coales‐ cence, phase inversion and Ostwald ripping. Emulsifier is a surfactant which can stabilize the emulsion by absorption at the interface, thereby lowering the interfacial tension. It is usually used to improve the emulsion stability. Proteins and polysaccharides are often ap‐ plied in emulsion as emulsifier. Proteins are usually used for their surfactant and gelling properties to improve the textural characteristics and stability of emulsion, while polysac‐ charides are usually added to increase the viscosity or to obtain a gel-like product. It was studied the impact of the use of a biomaterial (N-succinil chitosan hydrogel) in elaboration and structuration of food emulsion, and in substitution of a part of the oil phase. Chitosan showed to be a versatile ingredient since that was capable to modify the rheological proper‐ ties, acting as emulsifying agent, besides its already known antimicrobial and nutritional qualities [50].

### *4.4.2. Margarine*

variety of very specialized shortenings has been developed over the years to satisfy that de‐ mand. High ratio shortenings (containing mono and diglycerides), designed primarily for cakes, began to appear in the '30s. Fluid cake shortenings were commercialized in the '60s and offer many advantages including pumpability, ease of handling and the option of bulk

Cake is a baked batter made from wheat flour, sugar, eggs, shortening, leavening agents, salt, nonfat dry milk, flavors, and water. Cake batters are essentially a 'foam', that is a sys‐ tem in which air bubbles are trapped and held in an aqueous phase. The main function of fat in cake making is to assist with the incorporation of air into the batter during mixing, and the air bubble size and stability. High-ratio cakes, rich in sugar and fat, are extensively used

Margarine has always had the advantage over butter in that the properties of the product can be tailored to give the best performance in a particular system. For puff pastry, i.e., spe‐ cialized margarines are easier to work with than butter. Various bakery margarines are

The effect of different fats and margarines on the physical properties of cakes was investi‐ gated. The low *trans* fat suggested: greater volume and firmness; resilience comparable to hydrogenated fat; elasticity and chewiness were comparable to other formulations, as well

Textural properties are important quality parameters for this type of product. Physical and structure changes during aerated batters processing may alter their performance during baking or the quality of the final product. It is possible to test materials with particles and fiber in suspensions, since flushing effects may reduce sedimentation problems. In [49] was examined the influence of different types of fats (hydrogenated fat, margarine and vegetable oil) in formulation of cake batter, evaluating textural properties by Herschel-Bulkley equa‐ tion using back extrusion analysis; and it were observed values close in all parameters to samples prepared with margarine and hydrogenated fat. It can be mentioned the break point values could be consider by the industry as an important parameter, pointing the need

Mayonnaise and salad dressing are emulsified, semi-solid fatty foods that by federal regu‐ lation must contain not less than 65% and 30% vegetable oil, respectively, and dried whole eggs or egg yolks. Salt, sugar, spices, seasoning, vinegar, lemon juice, and other in‐ gredients complete these products. Pourable and spoonable dressings may be two phase (e.g., vinegar and oil) or the emulsified viscous type (e.g., French). There is a great variety of products available of varying compositions with a wide range in their oil content. Sal‐ ad oils exclusively are used for dressing products; typical choices include soybean, canola

manufactured to meet the technical requirements of particular uses.

delivery and storage [46].

86 Food Industry

in the baking industry [47].

the color parameters of the crumbs [48].

of less energy in their processes of pumping, i.e.

**4.4. Food emulsions**

and olive oils [46].

*4.4.1. Mayonnaise and salad dressing*

Margarine and spreads are prepared by blending fats and/or oils with other ingredients such as water and/or milk products, suitable edible proteins, salt, flavoring and coloring ma‐ terials and Vitamins A and D. Margarine must contain at least 80% fat by federal regulation, however, "diet" margarines and spreads may contain 0-80% fat. These products may be for‐ mulated from vegetable oils and/or animal fats, however, the vast preponderance are all vegetable. Non hydrogenated oils typically represent the majority of the fat phase. Lesser amounts of partially hydrogenated fats, that are naturally semisolid at room temperature, and/or hard fractions of certain fats are added to the blend as required to deliver the desired structure and melting properties [46]. At the moment, interesterification technics have been employed to produce tailor fats. Margarine originated as a substitute for butter. The big ad‐ vantage of margarine is that as a manufactured product the properties can be tailored to suit a particular use.

An acceptable margarine must be a soft plastic at room temperature; the ratio of solid or crystalline fat to liquid oil in the mixture must be such that when the fat crystals are of the proper size and well dispersed, the mass will offer some resistance to deformation and sepa‐ ration of solid and liquid fats will be negligible; all the fat crystals must melt completely at body temperature and leave a pasty sensation in the mouth; the fat crystals must not melt abruptely [51].

## **5. Prospective**

The major brands are today sold all over the world. The economic impact of any bad image on these super brands has led the major companies to focus on brand image. The increase in obesity has focused on health aspects of foods in general but also on chocolate and confec‐ tionery products. Fat replacers of the future will need to meet some important criteria, in‐ cluding reducing or replacing the target fat effectively, being available at a cost appropriate to the benefits provided, and being safe and legal with no appreciable side effects [39, 52].

## **Author details**

Suzana Caetano da Silva Lannes and Rene Maria Ignácio

\*Address all correspondence to: scslan@usp.br

Biochemical-Pharmaceutical Technology Department, Pharmaceutical Sciences Faculty, Sao Paulo University, São Paulo, Brazil

[12] Narine S.S., Marangoni A.G. Relating structure of fat crystal networks to mechanical

Structuring Fat Foods

89

http://dx.doi.org/10.5772/53369

[13] Awad T.S., Rogers M.A., Marangoni A.G. Scaling behavior of the elastic modulus in colloidal networks of fat crystals. Journal of Physical Chemistry B 2004; 108: 171-179.

[15] Myerson A.S. Handbook of Industrial Crystallization. Elsevier Science & Technology

[16] Foubert I., Vanrolleghem P. A., Vanhoutte B.; Dewettinck K.. Dynamic mathematical model of the crystallization kinetics of fats. Food Research International 2002; 35:

[17] Ghotra B.S., Dyal S.D., Narine S.S. Lipid shortenings: a review. Food Research Inter‐

[19] Afoakwa E.O., Paterson A., Fowler M., Vieira J. Effects of tempering and fat crystalli‐ sation behaviour on microstructure, mechanical properties and appearance in dark

[20] Kellens M. , Gibon V., Hendrix M., De Greyt W. Palm oil fractionation. European

[21] Tarrago-Trani M.T., Phillips K. M., Lemar L. E. , Holden J. M. New and existing oils and fats used in products with reduced *trans*-fatty acid content. Journal of the *A*meri‐

[22] Herrera M.L , Falabella C., Melgarejo M., Añón M.C. Isothermal crystallization of hy‐ drogenated sunflower oil: I — Nucleation. Journal of the American Oil Chemistys'

[23] Jeyarani T., Khan M.I., Khatoon S. *Trans*-free plastic shortenings from coconut stearin

[24] Marangoni A.G. The nature of fractality in fat crystal networks. Trends in Food Sci‐

[25] Mcclements D.J. Understanding and controlling the microstructure of complex foods.

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**Author details**

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Biochemical-Pharmaceutical Technology Department, Pharmaceutical Sciences Faculty,

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Zurich, Swiss. Lappersdorf: Kerschensteiner Verlag, 2012.

foodfats.htm (Accessed August 2012)

do Conjunto das Químicas, 2010; 46: 82-82.

tute of Food Science and Technology, 2012.

meeting, 2009, Anaheim, CA. Chicago: IFT, 2009.

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[29] Deman J. M. Principles of food chemistry. 3rd ed. Aspen Publishing:Gaithersburg;

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[39] Norberg S. Chocolate and confectionery fats. In: Gunstone F.D. ( ed.) Modifying lip‐

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[41] Silva Junior E. , Lannes S. C. S. Effect of different sweetener blends and fat types on ice cream properties. Ciência e Tecnologia de Alimentos 2011; 31: 217-220.

[43] Silva Junior E., Mello K. G. P. C., Polakiewicz B., Lannes S. C. S. Ice cream mixes for‐ mulated with n-succinil chitosan hydrogel characterized by rheo-optic techniques. In: 14th World Congress of Food Science & Technology-IUFoST: proceedings of the 14th World Congress of Food Science & Technology - IUFoST, 2008, Beijing, China.

[44] Su F., Lannes S. C. S.. Structural evaluation of ice cream produced with palm fat through rheology. In: International Symposium on Food Rheology and Structure:

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**Chapter 5**

**Mineral Composition of Blood Sausages – A Two-Case**

It is well known that a balanced diet is essential in maintaining a good health; hence, the nutritional value of foods is an important aspect of food quality [1]. In this context, more and more people are becoming very concerned about the chemistry of what they eat. Conse‐ quently, food industry is interested in maintaining a high standard of quality of their manu‐ factured products which could meet the demands of an increasingly sophisticated consumer. Therefore, an important issue of food industry is the determination of food com‐

Food scientists and food industry have long since been paying great attention to minerals in food, which has been mainly devoted to its essential role in human nutrition, i.e., physiolog‐ ical functions, humans' nutritional requirements, and mineral implication on safeness is‐ sues, i.e., mineral toxicity. There are more than 60 minerals in the human body, but only a few are considered to be essential, namely, iron, calcium, zinc, magnesium, phosphorus, so‐ dium, potassium, manganese, selenium, copper. These minerals are absolutely essential to a host of vital processes, from bone and tooth formation, to the functioning of neurological, circulatory, renal and digestive systems, and some of them are necessary for regulation of

Minerals deficiencies in human are common world-wide and there are evidences which sug‐ gest that deficiencies may play a main negative role in children's development, pregnancy

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

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

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

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

Daphne D. Ramos, Luz H. Villalobos-Delgado,

Additional information is available at the end of the chapter

**1.1. Relevance of the assessment of mineral content in food**

position and the establishment of analytical controls [2].

Enrique A. Cabeza, Irma Caro,

http://dx.doi.org/10.5772/53591

**1. Introduction**

enzyme systems [2,3].

Ana Fernández-Diez and Javier Mateo

**Study**

## **Mineral Composition of Blood Sausages – A Two-Case Study**

Daphne D. Ramos, Luz H. Villalobos-Delgado, Enrique A. Cabeza, Irma Caro, Ana Fernández-Diez and Javier Mateo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53591

## **1. Introduction**

### **1.1. Relevance of the assessment of mineral content in food**

It is well known that a balanced diet is essential in maintaining a good health; hence, the nutritional value of foods is an important aspect of food quality [1]. In this context, more and more people are becoming very concerned about the chemistry of what they eat. Conse‐ quently, food industry is interested in maintaining a high standard of quality of their manu‐ factured products which could meet the demands of an increasingly sophisticated consumer. Therefore, an important issue of food industry is the determination of food com‐ position and the establishment of analytical controls [2].

Food scientists and food industry have long since been paying great attention to minerals in food, which has been mainly devoted to its essential role in human nutrition, i.e., physiolog‐ ical functions, humans' nutritional requirements, and mineral implication on safeness is‐ sues, i.e., mineral toxicity. There are more than 60 minerals in the human body, but only a few are considered to be essential, namely, iron, calcium, zinc, magnesium, phosphorus, so‐ dium, potassium, manganese, selenium, copper. These minerals are absolutely essential to a host of vital processes, from bone and tooth formation, to the functioning of neurological, circulatory, renal and digestive systems, and some of them are necessary for regulation of enzyme systems [2,3].

Minerals deficiencies in human are common world-wide and there are evidences which sug‐ gest that deficiencies may play a main negative role in children's development, pregnancy

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

and elderly health [3]. In this context, Ca, K, Mg and Fe are the most commonly under-con‐ sumed minerals in humans' diet [4]. Fe deficiency is the most common and widespread nu‐ tritional disorder in the world affecting both developing and industrialized nations [5]. Insufficient intakes of Fe cause anemia, fatigue, poor growth, rickets and impaired cognitive performance in humans [3]. On the other hand, the concentration of non-desired minerals in food can be increased by the persistent release of hazardous pollutants to the environment mainly derived from human industrial activity. This contamination of food supply can re‐ sult in an increase of exposure of consumers to toxic metals such as lead, cadmium, arsenic and mercury, to levels higher than the tolerable daily intake [6].

number of countries, where meat by-products are usually linked to traditional or ethnic foods. Meat by-products are traditionally sold to the lower income market however, by dif‐ ferent reasons – one of them could be the increase in tourism – their consumption seems to be increasing and some of the by-products are becoming delicacies in niche markets. Advan‐ tageously, meat by-products consumption contributes to increase the edible portion of slaughter animals, Furthermore, meat edible by-products constitute an excellent source of nutrients like essentials amino acids, minerals and vitamins [15,16]. Due to the great variety and specificity of edible meat by-products and their peculiar consumption patterns and their relative low economic value, there is relatively scarce information on their making process

Mineral Composition of Blood Sausages – A Two-Case Study

http://dx.doi.org/10.5772/53591

95

In some areas of the world, and to different degrees, blood is utilized as an edible meat byproduct. For example, for several ethnic groups of Africa and India, blood is the primary source of animal protein, where it holds ritualistic importance. However, in some cultures (Islamic and Jews), blood consumption is seen as a taboo [17,18]. In Europe and Asia, animal blood has been traditionally used in making a variety of foods such as blood sausages, blood

From the nutritional point of view, blood is a good source of dietary protein, lysine and iron [19,21]. The high iron content of blood (approximately between 400-500 mg of iron per liter), coupled with the high absorption of heme iron compared to non-heme iron, is particularly useful for food based strategies designed to combat iron deficiency anemia. Furthermore, the environmental concern associated with blood disposal at slaughterhouses, together with blood nutritive value, has fostered research and industrial efforts to recover blood or blood components, to be used into a wide range of food products or as dietary supplements [22]. For example, blood or blood proteins (plasma or cellular fractions) are being used in meat products, primarily to increase protein levels and enhance water binding and emulsifying

Blood sausages are very popular traditional meat products in many parts of the world such as Europe, Latin America or Asia [23-26]. In Europe, blood sausages are normally called morcilla and morcella in Spain and Portugal, black pudding in Great Britain, blutwurst and Thuringer blood sausage in Germany, *blodpϕlse* in Denmark, *boudin noir* in France, *bloed worst* in Belgium, blood-tongue sausage and black pudding in Austria, *caltabosi cu singe* in Hungary, *vaerevorst i*n Estonia, *kaszanka* in Poland, *biroldo* in Italy. In Latin American coun‐ tries, blood sausages are also produced and are named as *relleno, prieta, moronga, mocillón* in Mexico, Colombia, Peru or Argentina, and Morcela in Brazil; these sausages from Latin America show characteristics similar to those from Europe, especially to those of Iberian Peninsula [25]. In this sense, blood sausages from Latin America can be included into the group of creole meat products, which means that they were originated from the adaptation of former Iberian meat products (brought to America by immigrants) to local condition and

Nowadays, blood sausages are currently receiving worldwide increasing attention because they have become gourmet products in several countries, thus leading to an increase in their production and potential markets [27]. Furthermore, increasing consumer demand for eth‐

pudding, biscuits and bread, as well as blood soups and crackers [19,20].

circumstances, thus, involving an innovation process at that time.

and chemical composition.

capacity.

The assessment of the mineral content in food is not only interesting from the nutritional and toxicological points of view. Since a few decades ago, instrumental analytical techni‐ ques based on atomic absorption or emission spectrometry applied to the determination of the mineral content coupled to multivariate statistical analysis have been proved to produce suitable methods to characterise food products, discriminate between food quality catego‐ ries and control food authenticity, i.e., determination of the geographical origin of food, dis‐ crimination between cultivation methods (e.g. organic vs convenience crops), varieties of fruits and vegetables, or food processing practices [7-10].

The analysis of minerals in foods is challenging due to the wide range of concentrations present, which may vary from ppb to percent levels. The situation is further complicated by naturally occurring seasonal and varietal differences in concentrations within the same food [11]. Official methods by de AOAC offers many single element methods based on colorimet‐ ric techniques: UV/Visible spectrophotometry, and flame and graphite furnace atomic ab‐ sorption spectrophotometry. However, although no AOAC food methods currently employ Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), it is a well-estab‐ lished multi-element technique that no requires the use of dangerous solvents from the envi‐ ronmental point of view [11]. Its high specificity, multi-element detection capability and good detection limits result in the use of this technique in a large variety of applications. De‐ tection limits typically range from parts per million (ppm) to parts per billion (ppb), al‐ though depending on the element and instrument, it can sometimes achieve even less than ppb detection [12]. ICP-AES provides higher reproducibility and quantitative linear range compared to conventional AES, and reduces molecular interferences due to a higher temper‐ ature (7000-8000 K) in the excitation source (plasma). On the other hand, ICP-AES is more expensive than conventional AES, and in complex samples, emission patters can be of diffi‐ cult interpretation [13].

#### **1.2. Blood sausages, making process and chemical composition**

Meat products are generally made from various raw materials (from different origins and suppliers), which are combined at the formulation stage in obedience to criteria of composi‐ tion, technological factors, sensory characteristics, legal regulations and also economic effi‐ ciency and profit [14].

Among meat and meat products, muscle foods are the most commonly consumed. However several edible meat by-products and their derivatives are also importantly consumed in a number of countries, where meat by-products are usually linked to traditional or ethnic foods. Meat by-products are traditionally sold to the lower income market however, by dif‐ ferent reasons – one of them could be the increase in tourism – their consumption seems to be increasing and some of the by-products are becoming delicacies in niche markets. Advan‐ tageously, meat by-products consumption contributes to increase the edible portion of slaughter animals, Furthermore, meat edible by-products constitute an excellent source of nutrients like essentials amino acids, minerals and vitamins [15,16]. Due to the great variety and specificity of edible meat by-products and their peculiar consumption patterns and their relative low economic value, there is relatively scarce information on their making process and chemical composition.

and elderly health [3]. In this context, Ca, K, Mg and Fe are the most commonly under-con‐ sumed minerals in humans' diet [4]. Fe deficiency is the most common and widespread nu‐ tritional disorder in the world affecting both developing and industrialized nations [5]. Insufficient intakes of Fe cause anemia, fatigue, poor growth, rickets and impaired cognitive performance in humans [3]. On the other hand, the concentration of non-desired minerals in food can be increased by the persistent release of hazardous pollutants to the environment mainly derived from human industrial activity. This contamination of food supply can re‐ sult in an increase of exposure of consumers to toxic metals such as lead, cadmium, arsenic

The assessment of the mineral content in food is not only interesting from the nutritional and toxicological points of view. Since a few decades ago, instrumental analytical techni‐ ques based on atomic absorption or emission spectrometry applied to the determination of the mineral content coupled to multivariate statistical analysis have been proved to produce suitable methods to characterise food products, discriminate between food quality catego‐ ries and control food authenticity, i.e., determination of the geographical origin of food, dis‐ crimination between cultivation methods (e.g. organic vs convenience crops), varieties of

The analysis of minerals in foods is challenging due to the wide range of concentrations present, which may vary from ppb to percent levels. The situation is further complicated by naturally occurring seasonal and varietal differences in concentrations within the same food [11]. Official methods by de AOAC offers many single element methods based on colorimet‐ ric techniques: UV/Visible spectrophotometry, and flame and graphite furnace atomic ab‐ sorption spectrophotometry. However, although no AOAC food methods currently employ Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), it is a well-estab‐ lished multi-element technique that no requires the use of dangerous solvents from the envi‐ ronmental point of view [11]. Its high specificity, multi-element detection capability and good detection limits result in the use of this technique in a large variety of applications. De‐ tection limits typically range from parts per million (ppm) to parts per billion (ppb), al‐ though depending on the element and instrument, it can sometimes achieve even less than ppb detection [12]. ICP-AES provides higher reproducibility and quantitative linear range compared to conventional AES, and reduces molecular interferences due to a higher temper‐ ature (7000-8000 K) in the excitation source (plasma). On the other hand, ICP-AES is more expensive than conventional AES, and in complex samples, emission patters can be of diffi‐

Meat products are generally made from various raw materials (from different origins and suppliers), which are combined at the formulation stage in obedience to criteria of composi‐ tion, technological factors, sensory characteristics, legal regulations and also economic effi‐

Among meat and meat products, muscle foods are the most commonly consumed. However several edible meat by-products and their derivatives are also importantly consumed in a

and mercury, to levels higher than the tolerable daily intake [6].

fruits and vegetables, or food processing practices [7-10].

**1.2. Blood sausages, making process and chemical composition**

cult interpretation [13].

94 Food Industry

ciency and profit [14].

In some areas of the world, and to different degrees, blood is utilized as an edible meat byproduct. For example, for several ethnic groups of Africa and India, blood is the primary source of animal protein, where it holds ritualistic importance. However, in some cultures (Islamic and Jews), blood consumption is seen as a taboo [17,18]. In Europe and Asia, animal blood has been traditionally used in making a variety of foods such as blood sausages, blood pudding, biscuits and bread, as well as blood soups and crackers [19,20].

From the nutritional point of view, blood is a good source of dietary protein, lysine and iron [19,21]. The high iron content of blood (approximately between 400-500 mg of iron per liter), coupled with the high absorption of heme iron compared to non-heme iron, is particularly useful for food based strategies designed to combat iron deficiency anemia. Furthermore, the environmental concern associated with blood disposal at slaughterhouses, together with blood nutritive value, has fostered research and industrial efforts to recover blood or blood components, to be used into a wide range of food products or as dietary supplements [22]. For example, blood or blood proteins (plasma or cellular fractions) are being used in meat products, primarily to increase protein levels and enhance water binding and emulsifying capacity.

Blood sausages are very popular traditional meat products in many parts of the world such as Europe, Latin America or Asia [23-26]. In Europe, blood sausages are normally called morcilla and morcella in Spain and Portugal, black pudding in Great Britain, blutwurst and Thuringer blood sausage in Germany, *blodpϕlse* in Denmark, *boudin noir* in France, *bloed worst* in Belgium, blood-tongue sausage and black pudding in Austria, *caltabosi cu singe* in Hungary, *vaerevorst i*n Estonia, *kaszanka* in Poland, *biroldo* in Italy. In Latin American coun‐ tries, blood sausages are also produced and are named as *relleno, prieta, moronga, mocillón* in Mexico, Colombia, Peru or Argentina, and Morcela in Brazil; these sausages from Latin America show characteristics similar to those from Europe, especially to those of Iberian Peninsula [25]. In this sense, blood sausages from Latin America can be included into the group of creole meat products, which means that they were originated from the adaptation of former Iberian meat products (brought to America by immigrants) to local condition and circumstances, thus, involving an innovation process at that time.

Nowadays, blood sausages are currently receiving worldwide increasing attention because they have become gourmet products in several countries, thus leading to an increase in their production and potential markets [27]. Furthermore, increasing consumer demand for eth‐ nic specialties has renewed interest in such products, leading to a consequent need to assure safety and longer shelf-lives in an expanding market. Moreover, the Governmental Institu‐ tions, e.g., European Union, are getting more involved in the protection of high-quality tra‐ ditional foods from specific regions or areas, which reflects a policy of supporting the inhabitants of rural areas and promoting regional products [23,27].

In general, meat and meat products are generally recognized as good sources of high biolog‐ ical-value proteins, group B vitamins, minerals as well as some other bioactive compounds [15]. The composition of meat products depends on their formulation. Thus, the chemical composition of blood sausages is diverse and would depend on the ingredients and manu‐ facturing process used. As a matter of reference, Table 2 shows the proximate composition of several blood sausages from Europe and Latin America. Moisture is expressed as percent‐ age of fresh weight, and values of protein, fat, available carbohydrate, fibre and ash are ex‐ pressed as percentage of dry matter. The literature sources for the data are the following

[36], b

De Burgos, Spaina 62.2 13.1 28.7 51.1 1.7 4.3 Asturiana, Spainb 38.5 7.0 69.1 8.0 - 2.9 With onion, Spainb 46.0 20.9 59.4 23.2 0.0 - Blutwurst, Germanyc 55.9 27.4 65.8 0.0 - - Thueringer, Germanyc 66.2 58.9 32.3 0.0 - - Verivanukas, Finlandd 61.1 19.3 22.6 43.9 9.8 - Verimakkara, Finlandd 54.7 28.7 42.0 21.2 6.2 - Blodpølse, Denmarke 43.7 19.0 36.9 32.0 8.9 3.2 With rice, Portugalf 62.0 28.9 38.9 24.6 - - Boudin noir, Franceg 62.0 26.8 58.1 10.8 - -

Blood Sausage, USAh 47.3 27.7 65.5 2.5 0.0 4.4 Traditional, Chilei 77.8 47.3 38.3 0.0 5.9 8.6 Traditional, Boliviaj 44.5 31.7 57.3 10.8 - 1.8 With tongue, Boliviaj 48.8 41.2 55.5 0.0 - 3.3 Stege, Boliviaj 41.2 31.2 56.8 6.5 - 5.4

Moisture content of blood sausages would depend inversely on the fat content and directly on the amount of moisture evaporated during an eventual drying/smoking stage. As can be seen in Table 2, the ranges of fat, available carbohydrate and protein in dry matter vary from 22.6 to 69.1, 0 to 51.1 and 7 to 58.9, respectively. There are great variations in dry matter composition between sausage types, which can be attributable to differences in the quanti‐

[40], c

[38], d[41], e

**Available carbohydrate**

[42], f

Mineral Composition of Blood Sausages – A Two-Case Study

http://dx.doi.org/10.5772/53591

97

[35], g[37], h[16],

**Fibre Ash**

(see Table for superscripts correspondence): a

**name Moisture Protein Fat**

**Table 2.** Proximate composition of several blood sausages from Europe and America

i [43], j

**Europe**

**America**

[44].

**Location and blood sausage**

Blood sausages are basically made with blood, fat and a variety of vegetable origin food; Moreover the use of meat, pork skin or offal (e.g., liver, intestine) is common, mainly in Ger‐ man blood sausages [28-31]. The vegetable-origin food used is enormously diverse so that, apart from spices and condiments, blood sausages can contain as main ingredients onion, leeks, cereals (rice, oat, flour, bread, etc.), sugar, fruits (apple, plum, etc.), nuts, etc. [32-34]. Other ingredients such as eggs, cream, milk are used in some types of blood sausages in France [32]. Moreover, as any meat product, blood sausages are added with common salt. The NaCl used in blood sausages from Mediterranean Europe tend to be between 1.2 and 1.5 % [35-37], and in blood sausages from Germany [38] and USA [16] tend to be higher, close to 2%. NaCl has a direct effect on the flavour and also increases the shelf-life, decreas‐ ing the water activity. Finally, several additives such as curing agents (nitrate and nitrite salts), pH modifiers (such as lactate or acetate salts) or emulsifiers can be also used [30,39].

The making process of blood sausages differs as a result of type, region and manufacturer. However, a common flow chart of the process of most of the blood sausages is depicted in Table 1 [21]. The initial mixture of blood sausages is complex by the number of ingredients used and pre-treatments to which those ingredients have been undergone. For example, meat can be cured previously to the mixture preparation, or pork rind can be cooked and emulsified. Similarly, several ingredients, such as fat, onion or rice, can be cooked before the mixture is prepared. Once prepared, the initial mixture is normally stuffed into natural or artificial casings and the sausage is cooked in hot water until a temperature of 65-75 ºC is reached in the inner part of it [31], and then the sausage is chilled before refrigeration stor‐ age. Some varieties of blood sausages are dried and/or smoked after cooking. Once cooked and chilled, most of blood sausages present a dark-red to black colour and a rather firm and sliceable texture [30] due to the formation of a gel structure from the interaction of collagen, starch, blood proteins, etc.; nonetheless, some blood sausages are soft and spreadable.


**Table 1.** General flow chart of blood sausage making process

In general, meat and meat products are generally recognized as good sources of high biolog‐ ical-value proteins, group B vitamins, minerals as well as some other bioactive compounds [15]. The composition of meat products depends on their formulation. Thus, the chemical composition of blood sausages is diverse and would depend on the ingredients and manu‐ facturing process used. As a matter of reference, Table 2 shows the proximate composition of several blood sausages from Europe and Latin America. Moisture is expressed as percent‐ age of fresh weight, and values of protein, fat, available carbohydrate, fibre and ash are ex‐ pressed as percentage of dry matter. The literature sources for the data are the following (see Table for superscripts correspondence): a [36], b [40], c [38], d[41], e [42], f [35], g[37], h[16], i [43], j [44].

nic specialties has renewed interest in such products, leading to a consequent need to assure safety and longer shelf-lives in an expanding market. Moreover, the Governmental Institu‐ tions, e.g., European Union, are getting more involved in the protection of high-quality tra‐ ditional foods from specific regions or areas, which reflects a policy of supporting the

Blood sausages are basically made with blood, fat and a variety of vegetable origin food; Moreover the use of meat, pork skin or offal (e.g., liver, intestine) is common, mainly in Ger‐ man blood sausages [28-31]. The vegetable-origin food used is enormously diverse so that, apart from spices and condiments, blood sausages can contain as main ingredients onion, leeks, cereals (rice, oat, flour, bread, etc.), sugar, fruits (apple, plum, etc.), nuts, etc. [32-34]. Other ingredients such as eggs, cream, milk are used in some types of blood sausages in France [32]. Moreover, as any meat product, blood sausages are added with common salt. The NaCl used in blood sausages from Mediterranean Europe tend to be between 1.2 and 1.5 % [35-37], and in blood sausages from Germany [38] and USA [16] tend to be higher, close to 2%. NaCl has a direct effect on the flavour and also increases the shelf-life, decreas‐ ing the water activity. Finally, several additives such as curing agents (nitrate and nitrite salts), pH modifiers (such as lactate or acetate salts) or emulsifiers can be also used [30,39].

The making process of blood sausages differs as a result of type, region and manufacturer. However, a common flow chart of the process of most of the blood sausages is depicted in Table 1 [21]. The initial mixture of blood sausages is complex by the number of ingredients used and pre-treatments to which those ingredients have been undergone. For example, meat can be cured previously to the mixture preparation, or pork rind can be cooked and emulsified. Similarly, several ingredients, such as fat, onion or rice, can be cooked before the mixture is prepared. Once prepared, the initial mixture is normally stuffed into natural or artificial casings and the sausage is cooked in hot water until a temperature of 65-75 ºC is reached in the inner part of it [31], and then the sausage is chilled before refrigeration stor‐ age. Some varieties of blood sausages are dried and/or smoked after cooking. Once cooked and chilled, most of blood sausages present a dark-red to black colour and a rather firm and sliceable texture [30] due to the formation of a gel structure from the interaction of collagen,

starch, blood proteins, etc.; nonetheless, some blood sausages are soft and spreadable.

**Table 1.** General flow chart of blood sausage making process

1. Raw matter selection 2. Preliminary preparation of raw materials (weighting, size reduction, premixing, precooking, curing, etc.) 3. Mixing 4. Stuffing 5. Cooking 6. Chilling

inhabitants of rural areas and promoting regional products [23,27].

96 Food Industry


**Table 2.** Proximate composition of several blood sausages from Europe and America

Moisture content of blood sausages would depend inversely on the fat content and directly on the amount of moisture evaporated during an eventual drying/smoking stage. As can be seen in Table 2, the ranges of fat, available carbohydrate and protein in dry matter vary from 22.6 to 69.1, 0 to 51.1 and 7 to 58.9, respectively. There are great variations in dry matter composition between sausage types, which can be attributable to differences in the quanti‐ ties of the main ingredients used, i.e., pork fat, cereals, vegetables, meat or blood. Thus, the presence and levels of fibre are the result of the use of vegetables, namely onion, leek, fruits, etc. Finally, ash content is related to the amount of common salt used in the making process. Regarding to the mineral content of blood sausages, the Fe content is the most reported in literature. Fe content of blood sausages is high due to the use of blood, and amounts report‐ ed vary from 6 to 16 mg per 100 g [16,36,42,45].

small local producers and retail stores in Tumbes City (north-western Peru) and small vil‐ lages around the city. For each sausage sampled, a 300 g sample was packaged individually in a bag and transported in refrigerated containers to the laboratory in Tumbes. Subsequent‐ ly, samples were frozen at -40° C and were transported to the laboratory at University of Leon where upon arrival at laboratory the samples were kept frozen at -40 °C until the anal‐

Mineral Composition of Blood Sausages – A Two-Case Study

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99

Determinations of moisture, fat, protein and ash contents in the sausage samples were per‐ formed in duplicate according to methods recommended by the AOAC International [46] – Official Methods nos. 950.46, 991.36, 981.10 and 920.153, respectively. Total dietary fibre was analysed following the AOAC 991.43 standard method [46], using the K-ACHDF 11/06 en‐ zymatic kit (Megazyme, Wicklow, Ireland). Finally, the percentage of available carbohy‐

The analysis of mineral composition of sausages was performed by ICP-AES on wet digest‐ ed samples. Duplicate aliquots of approximately 1 g (±0.01) of the previously homogenised samples were digested with 10 ml of concentrated HNO3 in tightly closed screw cap glass tubes for 18 h at room temperature, and then for a further 4 h at 90 °C. For the analysis of sodium, potassium, sulphur and phosphorus, 1 ml of the mineralized solution was added with 8 ml of deionized water and 1 ml of scandium solution as internal standard. In order to determine the levels of calcium, copper, iron, magnesium, manganese and zinc, 3 ml of the

The instrumental analysis was performed with an Optima 2000 DV ICP optical emission spectrometer (PerkinElmer, Waltham, MA, USA). Instrument operating conditions were: ra‐ diofrequency power, 1400 W; plasma gas flow, 15.0 l/min; auxiliary gas flow, 0.2 l/min; neb‐ ulizer gas flow 0.75 l/min, crossed flow; standard axial torch with 2.0 mm i.d. injector of silica; peristaltic pump flow, 1 ml/min; no. of replicates, 2. The spectrometer was calibrated for Cu, Mn, Zn, Fe, Ca and Mg determinations (at 224.7, 257.61, 213.9, 238.2, 393.4 and 279.6 nm, respectively) with nitric acid/water (1:1, v/v) standard solutions of 2, 5 and 10 ppm of each element, and for Na, P, S and K (at 589.6, 213.6, 182.0 and 766.5, respectively) with ni‐

The software STATISTICA for Windows [47] was used for the statistical treatment of data. Furthermore, a principal component (PC) analysis, unrotated method, using the mineral

The Morcilla de Leon (Figure 1), typically produced in the region of Leon (north-western Spain), is made from a mixture of chopped onion (used at amounts between 65 and 75 % of

drates was calculated by difference (100 – the percentage of the rest of components).

digested solution was added with 6 ml of deionized water and 1 ml of Sc solution.

tric acid/water (1:9, v/v) standard solutions of 30, 50 and 100 ppm, respectively.

composition as expressed as non-fat dry matter, was also performed.

ysis was performed.

**2.3. Statistical analysis**

**3.1. Making process**

**3. Results and discussion**

#### **1.3. Aim of the study**

In spite of their popularity and increasing interest, literature on the composition and quality of blood sausages is to our knowledge scarce. The knowledge of the chemical composition of blood sausages presents potential usefulness regarding nutritional, product characteriza‐ tion and quality control aspects. Among the chemical composition, the mineral content of blood sausages seems to be a key point in those aspects.Therefore, the main aim of the present study is to describe and determine, as case studies, the manufacturing process and the chemical composition with particular interest on the mineral content, of two typical blood sausages produced in two different parts of the world: a typical blood sausage with white onion (*Allium cepa*), from the region of Leon (north-western Spain), known as Morcilla de Leon; and typical blood sausage with white cabbage (*Brassica Oleracea* var. *capitata*), from the region of Tumbes (north-western Peru), known as Relleno de Tumbes.

## **2. Material and methods**

#### **2.1. Making process of the blood sausages**

In order to collect information about the making process of the blood sausage Morcilla de Leon, four interviews were conducted with the correspondent production managers at the four main local companies producing this sausage in Leon city. The two-member interview panel asked a set of questions regarding general company characteristics, raw materials used, making process and storage conditions. Moreover, collecting data on the making proc‐ ess of Relleno de Tumbes was carried out by standardized open-ended interviews conduct‐ ed with 15 homemade manufacturers at the region of Tumbes (Tumbes city and small villages at Zarumilla province). The questions asked were to know information on the raw materials used and the making process followed. In both cases, the interviews were fol‐ lowed by the observation of the sausage making process.

#### **2.2. Chemical analysis**

A total of 8 samples of Morcilla de Leon were manufactured by local producers (city of Leon, north-western Spain) and were purchased from local markets. The sample weights were approximately 250 g. Once taken, the sausages were transported under refrigeration (<4 °C) to the laboratory of Department of Food Hygiene and Technology (University of Leon). On the other hand, a total of 12 samples of Relleno de Tumbes were obtained from small local producers and retail stores in Tumbes City (north-western Peru) and small vil‐ lages around the city. For each sausage sampled, a 300 g sample was packaged individually in a bag and transported in refrigerated containers to the laboratory in Tumbes. Subsequent‐ ly, samples were frozen at -40° C and were transported to the laboratory at University of Leon where upon arrival at laboratory the samples were kept frozen at -40 °C until the anal‐ ysis was performed.

Determinations of moisture, fat, protein and ash contents in the sausage samples were per‐ formed in duplicate according to methods recommended by the AOAC International [46] – Official Methods nos. 950.46, 991.36, 981.10 and 920.153, respectively. Total dietary fibre was analysed following the AOAC 991.43 standard method [46], using the K-ACHDF 11/06 en‐ zymatic kit (Megazyme, Wicklow, Ireland). Finally, the percentage of available carbohy‐ drates was calculated by difference (100 – the percentage of the rest of components).

The analysis of mineral composition of sausages was performed by ICP-AES on wet digest‐ ed samples. Duplicate aliquots of approximately 1 g (±0.01) of the previously homogenised samples were digested with 10 ml of concentrated HNO3 in tightly closed screw cap glass tubes for 18 h at room temperature, and then for a further 4 h at 90 °C. For the analysis of sodium, potassium, sulphur and phosphorus, 1 ml of the mineralized solution was added with 8 ml of deionized water and 1 ml of scandium solution as internal standard. In order to determine the levels of calcium, copper, iron, magnesium, manganese and zinc, 3 ml of the digested solution was added with 6 ml of deionized water and 1 ml of Sc solution.

The instrumental analysis was performed with an Optima 2000 DV ICP optical emission spectrometer (PerkinElmer, Waltham, MA, USA). Instrument operating conditions were: ra‐ diofrequency power, 1400 W; plasma gas flow, 15.0 l/min; auxiliary gas flow, 0.2 l/min; neb‐ ulizer gas flow 0.75 l/min, crossed flow; standard axial torch with 2.0 mm i.d. injector of silica; peristaltic pump flow, 1 ml/min; no. of replicates, 2. The spectrometer was calibrated for Cu, Mn, Zn, Fe, Ca and Mg determinations (at 224.7, 257.61, 213.9, 238.2, 393.4 and 279.6 nm, respectively) with nitric acid/water (1:1, v/v) standard solutions of 2, 5 and 10 ppm of each element, and for Na, P, S and K (at 589.6, 213.6, 182.0 and 766.5, respectively) with ni‐ tric acid/water (1:9, v/v) standard solutions of 30, 50 and 100 ppm, respectively.

### **2.3. Statistical analysis**

ties of the main ingredients used, i.e., pork fat, cereals, vegetables, meat or blood. Thus, the presence and levels of fibre are the result of the use of vegetables, namely onion, leek, fruits, etc. Finally, ash content is related to the amount of common salt used in the making process. Regarding to the mineral content of blood sausages, the Fe content is the most reported in literature. Fe content of blood sausages is high due to the use of blood, and amounts report‐

In spite of their popularity and increasing interest, literature on the composition and quality of blood sausages is to our knowledge scarce. The knowledge of the chemical composition of blood sausages presents potential usefulness regarding nutritional, product characteriza‐ tion and quality control aspects. Among the chemical composition, the mineral content of blood sausages seems to be a key point in those aspects.Therefore, the main aim of the present study is to describe and determine, as case studies, the manufacturing process and the chemical composition with particular interest on the mineral content, of two typical blood sausages produced in two different parts of the world: a typical blood sausage with white onion (*Allium cepa*), from the region of Leon (north-western Spain), known as Morcilla de Leon; and typical blood sausage with white cabbage (*Brassica Oleracea* var. *capitata*), from

In order to collect information about the making process of the blood sausage Morcilla de Leon, four interviews were conducted with the correspondent production managers at the four main local companies producing this sausage in Leon city. The two-member interview panel asked a set of questions regarding general company characteristics, raw materials used, making process and storage conditions. Moreover, collecting data on the making proc‐ ess of Relleno de Tumbes was carried out by standardized open-ended interviews conduct‐ ed with 15 homemade manufacturers at the region of Tumbes (Tumbes city and small villages at Zarumilla province). The questions asked were to know information on the raw materials used and the making process followed. In both cases, the interviews were fol‐

A total of 8 samples of Morcilla de Leon were manufactured by local producers (city of Leon, north-western Spain) and were purchased from local markets. The sample weights were approximately 250 g. Once taken, the sausages were transported under refrigeration (<4 °C) to the laboratory of Department of Food Hygiene and Technology (University of Leon). On the other hand, a total of 12 samples of Relleno de Tumbes were obtained from

the region of Tumbes (north-western Peru), known as Relleno de Tumbes.

ed vary from 6 to 16 mg per 100 g [16,36,42,45].

**1.3. Aim of the study**

98 Food Industry

**2. Material and methods**

**2.2. Chemical analysis**

**2.1. Making process of the blood sausages**

lowed by the observation of the sausage making process.

The software STATISTICA for Windows [47] was used for the statistical treatment of data. Furthermore, a principal component (PC) analysis, unrotated method, using the mineral composition as expressed as non-fat dry matter, was also performed.

## **3. Results and discussion**

#### **3.1. Making process**

The Morcilla de Leon (Figure 1), typically produced in the region of Leon (north-western Spain), is made from a mixture of chopped onion (used at amounts between 65 and 75 % of total weight), animal fat (lard and/or tallow; 10-20 %), blood (normally from pigs, 10-20 %), rice or breadcrumbs (2-10 %), salt (1-1.5 %), dry powdered paprika (1-2 %; including hot and sweet paprika), garlic and a mixture of spices (usually up to 1 g/kg) composed of several of the following: oregano, cumin, anis, cinnamon or pepper. Normally, onion and rice are pre‐ cooked with the lard or tallow for 1-2 hours (until the onion becomes soft and tender). At the end of cooking, the condiments, spices and blood (liquid) are added and the mix is stir‐ red from some minutes. Nevertheless, one manufacturer did not precook the onion and fat, and thus all ingredients (raw) were cold-mixed. The mixture, (hot if it was precooked or cold if not precooked) is stuffed in natural pork or beef casings of around 45 mm of diame‐ ter, tied or clipped forming 20-cm pieces. After the stuffing of the mix, the sausages are cooked in hot water at 80-90 °C for 20-45 min. After this step, sausages are drained hung at room temperature for a few hours and then chill-stored. This product is usually stored with‐ out packaging, and the shelf–life is around 12 days at refrigeration temperatures.

coagulated, sometimes precooked and shredded) is manually mixed with the lard, chopped vegetables and salt. Then, the mix is manually stuffed into natural pork casings (large intes‐ tine). The blood sausages are cooked in boiling water for approximately half an hour. After

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101

The proximate composition of Morcilla de Leon and Relleno de Tumbes are shown in Tables 3 and 4, respectively. Moisture is expressed as percentage of fresh weight, and values of pro‐ tein, fat, available carbohydrate, fibre and ash as percentage of fresh and dry matter weights. Moisture content variability would mainly depend on fat content and the degree of drying loss during cooling and storage. Furthermore, the presence and variability of protein, fat, available carbohydrates and total dietary fibre would be respectively explained mainly by the amounts of blood, lard or tallow, rice or breadcrumbs, onion or cabbage (plus other vegetal condiments and species) used in the formulation. In fresh weight basis, both types of blood sausages have a similar percentage of moisture. However, Morcilla de Leon shows lower amount of protein and higher of fat, fibre and available carbohydrates, than Relleno de Tumbes, both in fresh and dry weight basis. This is explained by a higher amount of

cooking, the blood sausages are cooled and then drained hung.

**Figure 2.** Peruvian blood sausage Relleno de Tumbes.

**3.2. Chemical composition**

*3.2.1. Proximate composition*

**Figure 1.** Spanish blood sausage Morcilla de León.

Relleno de Tumbes (Figure 2) is a typical blood sausage from Northern Peru, which consists of a mixture of blood (approximately 30%), pork lard fat (10%), chopped cabbage (40%), chopped red and Chinese onion (5%), chopped fresh paprika (2%; including sweet and hot paprika local varieties), common salt (1.5%) and a number of herbs and spices at low quanti‐ ties (spearmint, coriander, garlic, cumin, pepper) and a in-situ-prepared annatto oil extract; furthermore, the addition of glutamate is common. The amounts indicated above are rough‐ ly estimated because the manufacturers did not use scales and the interviewers did not carry a scale in order to weight the ingredients used in the making process. The blood (liquid or coagulated, sometimes precooked and shredded) is manually mixed with the lard, chopped vegetables and salt. Then, the mix is manually stuffed into natural pork casings (large intes‐ tine). The blood sausages are cooked in boiling water for approximately half an hour. After cooking, the blood sausages are cooled and then drained hung.

**Figure 2.** Peruvian blood sausage Relleno de Tumbes.

#### **3.2. Chemical composition**

total weight), animal fat (lard and/or tallow; 10-20 %), blood (normally from pigs, 10-20 %), rice or breadcrumbs (2-10 %), salt (1-1.5 %), dry powdered paprika (1-2 %; including hot and sweet paprika), garlic and a mixture of spices (usually up to 1 g/kg) composed of several of the following: oregano, cumin, anis, cinnamon or pepper. Normally, onion and rice are pre‐ cooked with the lard or tallow for 1-2 hours (until the onion becomes soft and tender). At the end of cooking, the condiments, spices and blood (liquid) are added and the mix is stir‐ red from some minutes. Nevertheless, one manufacturer did not precook the onion and fat, and thus all ingredients (raw) were cold-mixed. The mixture, (hot if it was precooked or cold if not precooked) is stuffed in natural pork or beef casings of around 45 mm of diame‐ ter, tied or clipped forming 20-cm pieces. After the stuffing of the mix, the sausages are cooked in hot water at 80-90 °C for 20-45 min. After this step, sausages are drained hung at room temperature for a few hours and then chill-stored. This product is usually stored with‐

out packaging, and the shelf–life is around 12 days at refrigeration temperatures.

Relleno de Tumbes (Figure 2) is a typical blood sausage from Northern Peru, which consists of a mixture of blood (approximately 30%), pork lard fat (10%), chopped cabbage (40%), chopped red and Chinese onion (5%), chopped fresh paprika (2%; including sweet and hot paprika local varieties), common salt (1.5%) and a number of herbs and spices at low quanti‐ ties (spearmint, coriander, garlic, cumin, pepper) and a in-situ-prepared annatto oil extract; furthermore, the addition of glutamate is common. The amounts indicated above are rough‐ ly estimated because the manufacturers did not use scales and the interviewers did not carry a scale in order to weight the ingredients used in the making process. The blood (liquid or

**Figure 1.** Spanish blood sausage Morcilla de León.

100 Food Industry

#### *3.2.1. Proximate composition*

The proximate composition of Morcilla de Leon and Relleno de Tumbes are shown in Tables 3 and 4, respectively. Moisture is expressed as percentage of fresh weight, and values of pro‐ tein, fat, available carbohydrate, fibre and ash as percentage of fresh and dry matter weights. Moisture content variability would mainly depend on fat content and the degree of drying loss during cooling and storage. Furthermore, the presence and variability of protein, fat, available carbohydrates and total dietary fibre would be respectively explained mainly by the amounts of blood, lard or tallow, rice or breadcrumbs, onion or cabbage (plus other vegetal condiments and species) used in the formulation. In fresh weight basis, both types of blood sausages have a similar percentage of moisture. However, Morcilla de Leon shows lower amount of protein and higher of fat, fibre and available carbohydrates, than Relleno de Tumbes, both in fresh and dry weight basis. This is explained by a higher amount of blood and lower of vegetables and fat, being used in the Relleno de Tumbes making process, with respect to those being used for Morcilla de Leon.

blood sausages, it can be notice that blood appears to be the main source of Fe and Cu to blood sausages. On the other hand, onion and specially cabbage would be the main sources of K, Ca and Mn. Furthermore, S, P, Mg and Zn are importantly provided by both blood and vegetables, with the the high S content of cabbage being remarkable. Finally, lard seems not to be a good source of minerals and common salt, added at amounts of 1-2% to the sausage mixture, is the major source of Na in sausages (not shown in tables). In this context, the higher content of Fe and Cu in Relleno de Tumbes can be associated to the higher quantity of blood used. Similarly, the high content of Ca and S in cabbage together with the high quantity used in Relleno de Tumbes would account for the higher levels of those minerals

Na 623 ± 131 1900 ± 615 3315 ± 1038 K 149 ± 27 452 ± 83 795 ± 121 S 76 ± 9 240 ± 61 402 ± 101 P 45 ± 13 136 ± 21 229 ± 35 Ca 29 ± 8 86 ± 21 146 ± 36 Mg 15 ± 2 48 ± 9 78 ± 16

Fe 10.96 ± 3.30 33.24 ± 10.96 58.71 ± 23.33 Zn 0.37 ± 0.12 1.14 ± 0.35 1.88 ± 0.41 Mn 0.20 ± 0.05 0.59 ± 0.10 1.00 ± 0.18 Cu 0.08 ± 0.02 0.26 ± 0.05 0.42 ± 0.12

From the nutritional point of view, comparing the mineral content of blood sausages (fresh weight basis) with that of pork meat or muscle meat products, such as frankfurters or chori‐ zos [16-51], the blood sausages had considerably higher levels of Ca (more than three times), and Mn, Fe and Cu (more than ten times). On the contrary, amounts of K, P, S and Zn are slightly lower in blood sausages (up to 60% lower than those in meat). The levels of Mg were roughly comparable in meat and blood sausages, and those of Na depends on the quantities of common salt added. Having into account the Reference Labelling Values (RLVs) reported by the Scientific Committee of Food from the European Union [52], which are the following: K (2000 mg), Ca (1000 mg), P (700 mg), Na (600 mg), Mg (375 mg), Fe (14 mg), Zn (10 mg), Mn (2 mg) and Cu (1 mg); interestingly, a portion of 100 g of blood sausage equals or exceeds the recommended daily intake of Fe and contributes with 10-15% the rec‐ ommended daily intake of Mn and Cu. Thus, the high iron content of blood, coupled with

**Table 5.** Essential mineral content (mg/100 g) of the Spanish blood sausage Morcilla de Leon (n = 8).

**Fresh weight Dry matter Non-fat dry matter**

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with respect to Morcilla de Leon.

**Macroelements**

**Microelements**


**Table 3.** Proximate composition of the Spanish blood sausage Morcilla de Leon (n = 8).


**Table 4.** Proximate composition of the Peruvian blood sausage Relleno de Tumbes (n = 12).

#### *3.2.2. Mineral composition*

The mineral contents of Morcilla de Leon and Relleno de Tumbes are shown in Tables 5 and 6, respectively. Values (expressed as mg/100 g) are given in all fresh, dry and nonfat dry weight basis. Na is the mineral with the highest concentration, and the mean value seems slightly lower in Morcilla de León than in Relleno de Tumbes, where Na concentration shows a great variability between samples (high standard deviation). In average, Relleno de Tumbes contained higher amounts (approximately twice as much) of Ca and S macroele‐ ments and of Fe, Zn and Cu microelements.

The mineral content of blood sausages is the result of the sum of the contributions from all the ingredients used. In order to better ascertain the eventual contribution of ingredients to the mineral content of blood sausages, the mineral composition of the main ingredients used in Morcilla de Leon and/or Relleno de Tumbes is shown Table 7 [16,41,48-50]. From Table 7 and taking into account the quantities of the ingredients used in the making processes of the blood sausages, it can be notice that blood appears to be the main source of Fe and Cu to blood sausages. On the other hand, onion and specially cabbage would be the main sources of K, Ca and Mn. Furthermore, S, P, Mg and Zn are importantly provided by both blood and vegetables, with the the high S content of cabbage being remarkable. Finally, lard seems not to be a good source of minerals and common salt, added at amounts of 1-2% to the sausage mixture, is the major source of Na in sausages (not shown in tables). In this context, the higher content of Fe and Cu in Relleno de Tumbes can be associated to the higher quantity of blood used. Similarly, the high content of Ca and S in cabbage together with the high quantity used in Relleno de Tumbes would account for the higher levels of those minerals with respect to Morcilla de Leon.

blood and lower of vegetables and fat, being used in the Relleno de Tumbes making process,

Moisture 67.1 ± 5.8 - Protein 5.2 ± 0.9 16.3 ± 3.6 Fat 14.2 ± 3.9 42.9 ± 7.6 Ash 1.9 ± 0.1 5.9 ± 1.2 Total dietary fibre 3.4 ± 1.5 10.1 ± 3.1 Available carbohydrate 8.2 ± 3.4 25.0 ± 6.3

Moisture 71.8 ± 6.9 - Protein 11.9 ± 2.8 42.4 ± 10.2 Fat 9.4 ± 4.0 33.3 ± 13.9 Ash 2.1 ± 0.9 7.6 ± 3.2 Total dietary fibre 1.1 ± 0.4 3.9 ± 1.3 Available carbohydrate 3.6 ± 1.6 13.8 ± 6.3

**Table 4.** Proximate composition of the Peruvian blood sausage Relleno de Tumbes (n = 12).

The mineral contents of Morcilla de Leon and Relleno de Tumbes are shown in Tables 5 and 6, respectively. Values (expressed as mg/100 g) are given in all fresh, dry and nonfat dry weight basis. Na is the mineral with the highest concentration, and the mean value seems slightly lower in Morcilla de León than in Relleno de Tumbes, where Na concentration shows a great variability between samples (high standard deviation). In average, Relleno de Tumbes contained higher amounts (approximately twice as much) of Ca and S macroele‐

The mineral content of blood sausages is the result of the sum of the contributions from all the ingredients used. In order to better ascertain the eventual contribution of ingredients to the mineral content of blood sausages, the mineral composition of the main ingredients used in Morcilla de Leon and/or Relleno de Tumbes is shown Table 7 [16,41,48-50]. From Table 7 and taking into account the quantities of the ingredients used in the making processes of the

*3.2.2. Mineral composition*

ments and of Fe, Zn and Cu microelements.

**Table 3.** Proximate composition of the Spanish blood sausage Morcilla de Leon (n = 8).

Mean ± SD (% of fresh weight)

Mean ± SD (% of fresh weight)

Mean ± SD (% of dry weight)

Mean ± SD (% of dry weight)

with respect to those being used for Morcilla de Leon.

102 Food Industry


**Table 5.** Essential mineral content (mg/100 g) of the Spanish blood sausage Morcilla de Leon (n = 8).

From the nutritional point of view, comparing the mineral content of blood sausages (fresh weight basis) with that of pork meat or muscle meat products, such as frankfurters or chori‐ zos [16-51], the blood sausages had considerably higher levels of Ca (more than three times), and Mn, Fe and Cu (more than ten times). On the contrary, amounts of K, P, S and Zn are slightly lower in blood sausages (up to 60% lower than those in meat). The levels of Mg were roughly comparable in meat and blood sausages, and those of Na depends on the quantities of common salt added. Having into account the Reference Labelling Values (RLVs) reported by the Scientific Committee of Food from the European Union [52], which are the following: K (2000 mg), Ca (1000 mg), P (700 mg), Na (600 mg), Mg (375 mg), Fe (14 mg), Zn (10 mg), Mn (2 mg) and Cu (1 mg); interestingly, a portion of 100 g of blood sausage equals or exceeds the recommended daily intake of Fe and contributes with 10-15% the rec‐ ommended daily intake of Mn and Cu. Thus, the high iron content of blood, coupled with the high absorption of heme iron compared to non-heme iron, is particularly useful for food based strategies designed to combat iron deficiency anemia a major global malnutrition problem.

small homemade sausage producing facilities in small villages. This reason could be respon‐

Na 300 11 3 41 K 50 65 166 161 S 140 - 51 300 P 100 38 35 32 Ca 7 2 22 53 Mg 6 2 11 15

Fe 50 0.2 0.2 0.6 Zn 0.5 0.4 0.2 0.2 Mn 0.0 0.0 0.2 0.2 Cu 0.7 0.0 0.1 0.0

**Table 7.** Essential mineral content (mg/100 g) of the main ingredients used in Morcilla de Leon and/or Relleno de

**Figure 3.** Principal component score plot (two first principal components or factors), considering mineral composition on non-fat dry matter basis, and showing samples according to sausage type: L, Morcilla de Leon; T, Relleno de

**Blood, pork Pork fat Onion Cabbage**

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105

sible for part of the distance between the Fe and Cu points.

**Macroelements**

**Microelements**

Tumbes

Tumbes


**Table 6.** Essential mineral content (mg/100 g) of the Peruvian blood sausage Relleno de Tumbes (n = 12)

Results of principal component analysis are shown in Figures 3 and 4. Principal component analysis was carried out with the mineral content expressed as mg per 100 g of nonfat dry matter for all the blood sausages analysed in this study. The first principal component (fac‐ tor 1) accounted for a variance of 42.77% and the second of 19.89%. Figure 3 shows that sam‐ ples from each type of blood sausage are located in two defined sets of results, which corroborate the differences in mineral contents found between both blood sausages. Figure 4 shows the projection of the variables (mineral contents) on the plane formed by the two principal components. The minerals with higher influence (factor loadings higher than 0.8) on factor 1 are Mn, Zn, and Ca. The mineral with higher influence on factor 2 was K (factor loading > 0.8).

Moreover, in Figure 4 it can be seen that the most correlated mineral contents, as indicated by the highest proximity of points in the plain, were S with Ca and Mn with Zn. The first relation could be explained by the significant contribution of cabbage and onion to the S and Ca content in the sausage mixture. However, the second relation is difficult to explain from the contribution of ingredients. Other remarkable correlation is that of Fe with Cu, with blood being the main source of both of them. This correlation could be not as strong as ex‐ pected due to the feasible migration of Fe ions to ingredients and sausage mixture from the surfaces of cast iron equipment, i.e., pans, knives, etc. [53], which are frequently present at


small homemade sausage producing facilities in small villages. This reason could be respon‐ sible for part of the distance between the Fe and Cu points.

the high absorption of heme iron compared to non-heme iron, is particularly useful for food based strategies designed to combat iron deficiency anemia a major global malnutrition

Na 706 ± 335 2821 ± 1343 3951 ± 1640 K 142 ± 56 565 ± 223 798 ± 254 S 116 ± 22 411 ± 102 590 ± 108 P 48 ± 19 190 ± 67 271 ± 83 Ca 50 ± 18 180 ± 65 257 ± 83 Mg 14 ± 6 50 ± 21 71 ± 28

Fe 29.01 ± 8.55 101.03 ± 25.39 146.50 ± 36.81 Zn 0.70 ± 0.10 2.44 ± 0.51 3.51 ± 0.52 Mn 0.14 ± 0.07 0.52 ± 0.25 0.74 ± 0.36 Cu 0.13 ± 0.06 0.49 ± 0.25 0.69 ± 0.32

**Table 6.** Essential mineral content (mg/100 g) of the Peruvian blood sausage Relleno de Tumbes (n = 12)

Results of principal component analysis are shown in Figures 3 and 4. Principal component analysis was carried out with the mineral content expressed as mg per 100 g of nonfat dry matter for all the blood sausages analysed in this study. The first principal component (fac‐ tor 1) accounted for a variance of 42.77% and the second of 19.89%. Figure 3 shows that sam‐ ples from each type of blood sausage are located in two defined sets of results, which corroborate the differences in mineral contents found between both blood sausages. Figure 4 shows the projection of the variables (mineral contents) on the plane formed by the two principal components. The minerals with higher influence (factor loadings higher than 0.8) on factor 1 are Mn, Zn, and Ca. The mineral with higher influence on factor 2 was K (factor

Moreover, in Figure 4 it can be seen that the most correlated mineral contents, as indicated by the highest proximity of points in the plain, were S with Ca and Mn with Zn. The first relation could be explained by the significant contribution of cabbage and onion to the S and Ca content in the sausage mixture. However, the second relation is difficult to explain from the contribution of ingredients. Other remarkable correlation is that of Fe with Cu, with blood being the main source of both of them. This correlation could be not as strong as ex‐ pected due to the feasible migration of Fe ions to ingredients and sausage mixture from the surfaces of cast iron equipment, i.e., pans, knives, etc. [53], which are frequently present at

**Fresh weight Dry matter Non-fat dry matter**

problem.

104 Food Industry

**Macroelements**

**Microelements**

loading > 0.8).

**Table 7.** Essential mineral content (mg/100 g) of the main ingredients used in Morcilla de Leon and/or Relleno de Tumbes

**Figure 3.** Principal component score plot (two first principal components or factors), considering mineral composition on non-fat dry matter basis, and showing samples according to sausage type: L, Morcilla de Leon; T, Relleno de Tumbes

**Author details**

Daphne D. Ramos1

**References**

86: 49-55.

166-173.

383, 59-69.

Ana Fernández-Diez4

, Luz H. Villalobos-Delgado3

1 Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru

2 Department of Microbiology, University of Pamplona, Pamplona, Colombia

3 Institute of Agroindustry, Technological University of the Mixteca, Oaxaca, Mexico

4 Department of Food Hygiene and Technology, University of León, Campus León, Spain

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[2] Castro FMM, Morgano MA, Nascimiento de Queiroz SC, Mantovani MDB. Relation‐ ships of the Minerals and Fatty Acid Contents in Processed Turkey Meat Products.

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and Javier Mateo4

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, Enrique A. Cabeza2

, Irma Caro4

Mineral Composition of Blood Sausages – A Two-Case Study

,

http://dx.doi.org/10.5772/53591

107

**Figure 4.** Projection of the normalised factor coordinates of variables (mineral contents) in the 1 x 2 factor plane ob‐ tained by the principal component analysis

### **4. Conclusion**

The mineral content of two traditional blood sausages from different parts of the world: Morcilla de Leon and Relleno de Tumbes, as well as the proximate composition and gen‐ eral guidelines of the making process have been described in this study, which thus con‐ tribute to the chemical characterisation, diffusion and protection of these two traditional meat products.

The variety and quantities of ingredients used for blood sausage production have a signifi‐ cant relevance on their mineral content. Blood provides important quantities of Fe, Cu and Mn to the blood sausages from the nutritional point of view. The content of Fe of 100 g of Morcilla de Leon practically equals the daily requirements for adults and that of Relleno de Tumbes exceeds those requirements.

## **Author details**

Daphne D. Ramos1 , Luz H. Villalobos-Delgado3 , Enrique A. Cabeza2 , Irma Caro4 , Ana Fernández-Diez4 and Javier Mateo4

\*Address all correspondence to: jmato@unileon.es

1 Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru

2 Department of Microbiology, University of Pamplona, Pamplona, Colombia

3 Institute of Agroindustry, Technological University of the Mixteca, Oaxaca, Mexico

4 Department of Food Hygiene and Technology, University of León, Campus León, Spain

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**Figure 4.** Projection of the normalised factor coordinates of variables (mineral contents) in the 1 x 2 factor plane ob‐

The mineral content of two traditional blood sausages from different parts of the world: Morcilla de Leon and Relleno de Tumbes, as well as the proximate composition and gen‐ eral guidelines of the making process have been described in this study, which thus con‐ tribute to the chemical characterisation, diffusion and protection of these two traditional

The variety and quantities of ingredients used for blood sausage production have a signifi‐ cant relevance on their mineral content. Blood provides important quantities of Fe, Cu and Mn to the blood sausages from the nutritional point of view. The content of Fe of 100 g of Morcilla de Leon practically equals the daily requirements for adults and that of Relleno de

tained by the principal component analysis

Tumbes exceeds those requirements.

**4. Conclusion**

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**Section 2**

**Food Quality**

**Section 2**
