Rodrigo Patera Barcelos and Suzymeire Baroni

*Federal University of the Bahia Reconcavo / Center for Health Sciences*

The application of microorganisms, such as bacteria, yeasts and principally fungi, by the food industry has led to a highly diversified food industry with relevant economical assets. Fermentation, with special reference to the production of alcoholic beverages, ethyl alcohol, dairy products, organic acids and drugs which also comprise antibiotics are the most

The enzyme industry, as it is currently known, is the result of a rapid development of biotechnology, especially during the past four decades. Since ancient times, enzymes found in nature have been used in the production of food products such as cheese, beer, wine and

Enzymes which decompose complex molecules into smaller units, such as carbohydrates into sugars, are natural substances involved in all biochemical processes. Due to the

Although plants, fungi, bacteria and yeasts produce most enzymes, microbial sourcesproduced enzymes are more advantageous than their equivalents from animal or vegetable sources. The advantages assets comprise lower production costs, possibility of large-scale production in industrial fermentors, wide range of physical and chemical characteristics, possibility of genetic manipulation, absence of effects brought about by seasonality, rapid culture development and the use of non-burdensome methods. The above characteristics make microbial enzymes suitable biocatalysts for various industrial applications (Hasan et al., 2006). Therefore, the identification and the dissemination of new microbial sources, mainly those which are non-toxic to humans, are of high strategic interest. Besides guaranteeing enzyme supply to different industrial processes, the development of new enzymatic systems which cannot be obtained from plants or animals is made possible and important progress in the

Owing to progress in the knowledge of enzymes, fungi acquired great importance in several

In fact, the fungi kingdom has approximately 200 species of *Aspergillus* which produce enzymes. They are isolated from soil, decomposing plants and air. *Aspergillus* actually

enzymes' specificities, each substratum has a corresponding enzyme.

industries since they may improve various aspects of the final product.

**1. Introduction**

vinegar (Kirk et al., 2002).

food industry may be achieved.

**2. Fungus of industrial interest**

important examples of microbiological processes.

## **Microorganism-Produced Enzymes in the Food Industry**

Izabel Soares, Zacarias Távora, Rodrigo Patera Barcelos and Suzymeire Baroni *Federal University of the Bahia Reconcavo / Center for Health Sciences Brazil*

## **1. Introduction**

82 Scientific, Health and Social Aspects of the Food Industry

Malinowski, JM. & Gehret, MM. (2010). Phytosterols for Dyslipidemia. *American Journal of*

Niijar, PS., Burke, FM., Bloesch, A. & Rader, DJ. (2010). Role of Dietary Supplements in

Smith. (2000). Squalene: Potential Chemopreventive Agent. *Expert Opinion on Investigational*

Sugano, M., Koba, K. & Tsuji, E. (1999). Health Benefits of Rice Bran Oil. *Anticancer Research*,

Xiao-Wen, W. (2005). Leading Technology in the 21st Century "Supercritical Fluid

Yamauchi, Y. & Saito, M. (1990). Fractionation of Lemon-Peel Oil by Semi-Preparative

Zhao, HY. & Jiang, JG. (2010). Application of Chromatography Technology in the Separation

Supercritical Fluid Chromatography. *Journal of Chromatography*, Vol. 505 (No. 1):

of Active Components from Nature Derived Drugs. *Mini Reviews in Medicinal*

*System Pharmacists*, Vol. 67 (No. 14): 1165-1173.

Tsujiwaki, Y., Yamamoto, H. & Minami, K. (1995). Japan patent. 327687.

Extraction".*Shokuhin to Kaihatsu*, Vol. 40: 68-69.

*Chemistry*, Vol. 10 (No. 13): 1223-1234.

*Lipidology*, Vol. 4 (No. 4): 248-258.

*Drugs*, Vol. 9 (No. 8): 1841-1848.

Vol. 10 (No. 5A): 3651-3657.

237-246.

*Health-System Pharmacy : AJHP : Official Journal of the American Society of Health-*

Lowering Low-Density Lipoprotein Cholesterol: a review. *Journal of Clinical*

The application of microorganisms, such as bacteria, yeasts and principally fungi, by the food industry has led to a highly diversified food industry with relevant economical assets. Fermentation, with special reference to the production of alcoholic beverages, ethyl alcohol, dairy products, organic acids and drugs which also comprise antibiotics are the most important examples of microbiological processes.

The enzyme industry, as it is currently known, is the result of a rapid development of biotechnology, especially during the past four decades. Since ancient times, enzymes found in nature have been used in the production of food products such as cheese, beer, wine and vinegar (Kirk et al., 2002).

Enzymes which decompose complex molecules into smaller units, such as carbohydrates into sugars, are natural substances involved in all biochemical processes. Due to the enzymes' specificities, each substratum has a corresponding enzyme.

Although plants, fungi, bacteria and yeasts produce most enzymes, microbial sourcesproduced enzymes are more advantageous than their equivalents from animal or vegetable sources. The advantages assets comprise lower production costs, possibility of large-scale production in industrial fermentors, wide range of physical and chemical characteristics, possibility of genetic manipulation, absence of effects brought about by seasonality, rapid culture development and the use of non-burdensome methods. The above characteristics make microbial enzymes suitable biocatalysts for various industrial applications (Hasan et al., 2006).

Therefore, the identification and the dissemination of new microbial sources, mainly those which are non-toxic to humans, are of high strategic interest. Besides guaranteeing enzyme supply to different industrial processes, the development of new enzymatic systems which cannot be obtained from plants or animals is made possible and important progress in the food industry may be achieved.

## **2. Fungus of industrial interest**

Owing to progress in the knowledge of enzymes, fungi acquired great importance in several industries since they may improve various aspects of the final product.

In fact, the fungi kingdom has approximately 200 species of *Aspergillus* which produce enzymes. They are isolated from soil, decomposing plants and air. *Aspergillus* actually

Microorganism-Produced Enzymes in the Food Industry 85

and nuclear resonance have been used for a better understanding of regulatory secretion mechanisms of these enzymes and their catalytic activity. The biotechnological importance of microorganisms and their enzymes triggers a great interest toward the understanding of

Lipolytic enzymes such as lipases and esterases are an important group of enzymes associated with the metabolism of lipid degradation. Lipase-producing microorganisms such as *Penicillium restrictum* may be found in soil and various oil residues. The industries

Several microorganisms, such as *Candida rugosa, Candida antarctica, Pseudomonas alcaligenes, Pseudomonas mendocina* and *Burkholderia cepacia*, are lipase producers (Jaeger and Reetz, 1998). Other research works have also included *Geotrichum* sp. (Burkert et al., 2004), *Geotrichum candidum* DBM 4013 (Zarevúcka et al., 2005), *Pseudomonas cepacia, Bacillus stearothermophilus, Burkholderia cepacia* (Bradoo et al., 2002), *Candida lipolytica* (Tan et al., 2003) *Bacillus coagulans* (Alkan et al., 2007), *Bacillus coagulans* BTS-3 (Kumar et al., 2005), *Pseudomonas aeruginosa* PseA (Mahanta et al., 2008), *Clostridium thermocellum* 27405 (Chinn et al., 2008), *Yarrowia lipolytica* (Dominguez et al., 2003) and *Yarrowia lipolytica* CL180 (Kim et

The fungi of the genera *Rhizopus*, *Geotrichum*, *Rhizomucor*, *Aspergillus*, *Candida* and

The industrial demand for new lipase sources with different enzymatic characteristics and produced at low costs has motivated the isolation and selection of new lipolytic microorganisms. However, the production process may modify their gene expression and change their phenotypes, including growth, production of secondary metabolites and enzymes. Posterior to primary selection, the production of the enzyme should be evaluated during the growth of the promising strain in fermentation, in liquid medium and / or in the solid state (Colen et al., 2006). However, it is evident that each system will result in different proteins featuring specific characteristics with regard to reactions' catalysis and,

Popularly known as lactase, beta-galactosidases are enzymes classified as hydrolases. They catalyze the terminal residue of b-lactose galactopiranosil (Galb1 - 4Glc) and produce glucose and galactose (Carminatti, 2001). Lactase's production sources are peaches, almonds and certain species of wild roses; animal organisms, such as the intestine, the brain and skin tissues; yeasts, such as *Kluyveromyces lactis*, *K. fragilis* and *Candida pseudotropicalis*; bacteria, such as *Escherichia coli*, *Lactobacillus bulgaricus*, *Streptococcus lactis* and *Bacillus* sp; and fungi,

The b-galactosidase may be found in nature, or rather, in plants, particularly almonds, peaches, apricots, apples, animal organs such as the intestine, the brain, placenta and the

Lactase is produced by a widely diverse fungus population and by a large amount of microorganisms such as filamentous fungus, bacteria and yeast (Holsinger, 1997; Almeida

*Penicillium* have been reported to be producers of several commercially used lipases.

Novozymes, Amano and Gist Brocades already employ microbial lipases.

gene regulation and expression of extracellular enzymes.

consequently, to the products produced (Asther et al., 2002).

such as *Aspergillus foetidus*, *A. niger*, *A. oryzae* and *A. Phoenecia*.

**2.1.2 Lipases**

al., 2007).

**2.1.3 Lactase**

testis.

and Pastore, 2001).

produces a great number of extracellular enzymes, many of which are applied in biotechnology. *Aspergillus flavus*, *A. niger, A. oryzae, A. nidulans, A. fumigatus, A. clavatus, A. glaucus, A. ustus* and *A. versicolor* are the best known.

The remarkable interest in *Aspergillus niger*, a species of great commercial interest with a highly promising future and already widely applied in modern biotechnology, is due to its several and diverse reactions (Andersen et al, 2008).

Moreover, *A. niger* not only produces various enzymes but it is one of the few species of the fungus kingdom classified as GRAS (Generally Recognized as Safe) by the Food and Drug Administration (FDA). The species is used in the production of enzymes, its cell mass is used as a component in animal feed and its fermentation produces organic acids and other compounds of high economic value (Couto and Sanroman, 2006; Mulimania and Shankar, 2007).

#### **2.1 Microbial enzymes for industries**

#### **2.1.1 Pectinase enzyme**

Plants, filamentous fungi, bacteria and yeasts produce the pectinase enzymes group with wide use in the food and beverages industries. The enzyme is employed in the food industries for fruit ripening, viscosity clarification and reduction of fruit juices, preliminary treatment of grape juice for wine industries, extraction of tomato pulp (Adams et al., 2005), tea and chocolate fermentation (Almeida et al. 2005; da Silva et al., 2005), vegetal wastes treatment, fiber degumming in the textile and paper industries (Sorensen, et al. 2004; Kaur, et al. 2004, Taragano, et al., 1999, Lima, et al., 2000), animal nutrition, protein enrichment of baby food and oil extraction (Da Silva et al., 2005, Lima, et al., 2000).

The main application of the above mentioned enzyme group lies within the juice processing industry during the extraction, clarification and concentration stages (Martin, 2007). The enzymes are also used to reduce excessive bitterness in citrus peel, restore flavor lost during drying and improve the stableness of processed peaches and pickles. Pectinase and βglucosidase infusion enhances the scent and volatile substances of fruits and vegetables, increases the amount of antioxidants in extra virgin olive oil and reduces rancidity.

The advantages of pectinase in juices include, for example, the clarification of juices, concentrated products, pulps and purees; a decrease in total time in their extraction; improvement in the production of juices and stable concentrated products and reduction in waste pulp; decrease of production costs; and the possibility of processing different types of fruit (Uenojo and Pastore 2007). For instance, in the production of passion fruit juice, the enzymes are added prior to filtration when the plant structure's enzymatic hydrolysis occurs. This results in the degradation of suspended solids and in viscosity decrease, speeding up the entire process (Paula, et al., 2004).

Several species of microorganisms such as *Bacillus, Erwinia, Kluyveromyces, Aspergillus, Rhizopus, Trichoderma, Pseudomonas, Penicillium* and *Fusarium* are good producers of pectinases (De Gregorio, et al., 2002). Among the microorganisms which synthesize pectinolytic enzymes, fungi, especially filamentous fungi, such as *Aspergillus niger* and *Aspergillus carbonarius* and *Lentinus edodes*, are preferred in industries since approximately 90% of produced enzymes may be secreted into the culture medium (Blandino et al., 2001).

In fact, several studies have been undertaken to isolate, select, produce and characterize these specific enzymes so that pectinolytic enzymes could be employed not only in food processing but also in industrial ones. High resolution techniques such as crystallography and nuclear resonance have been used for a better understanding of regulatory secretion mechanisms of these enzymes and their catalytic activity. The biotechnological importance of microorganisms and their enzymes triggers a great interest toward the understanding of gene regulation and expression of extracellular enzymes.

### **2.1.2 Lipases**

84 Scientific, Health and Social Aspects of the Food Industry

produces a great number of extracellular enzymes, many of which are applied in biotechnology. *Aspergillus flavus*, *A. niger, A. oryzae, A. nidulans, A. fumigatus, A. clavatus, A.*

The remarkable interest in *Aspergillus niger*, a species of great commercial interest with a highly promising future and already widely applied in modern biotechnology, is due to its

Moreover, *A. niger* not only produces various enzymes but it is one of the few species of the fungus kingdom classified as GRAS (Generally Recognized as Safe) by the Food and Drug Administration (FDA). The species is used in the production of enzymes, its cell mass is used as a component in animal feed and its fermentation produces organic acids and other compounds of high economic value (Couto and Sanroman, 2006; Mulimania and Shankar,

Plants, filamentous fungi, bacteria and yeasts produce the pectinase enzymes group with wide use in the food and beverages industries. The enzyme is employed in the food industries for fruit ripening, viscosity clarification and reduction of fruit juices, preliminary treatment of grape juice for wine industries, extraction of tomato pulp (Adams et al., 2005), tea and chocolate fermentation (Almeida et al. 2005; da Silva et al., 2005), vegetal wastes treatment, fiber degumming in the textile and paper industries (Sorensen, et al. 2004; Kaur, et al. 2004, Taragano, et al., 1999, Lima, et al., 2000), animal nutrition, protein enrichment of

The main application of the above mentioned enzyme group lies within the juice processing industry during the extraction, clarification and concentration stages (Martin, 2007). The enzymes are also used to reduce excessive bitterness in citrus peel, restore flavor lost during drying and improve the stableness of processed peaches and pickles. Pectinase and βglucosidase infusion enhances the scent and volatile substances of fruits and vegetables,

The advantages of pectinase in juices include, for example, the clarification of juices, concentrated products, pulps and purees; a decrease in total time in their extraction; improvement in the production of juices and stable concentrated products and reduction in waste pulp; decrease of production costs; and the possibility of processing different types of fruit (Uenojo and Pastore 2007). For instance, in the production of passion fruit juice, the enzymes are added prior to filtration when the plant structure's enzymatic hydrolysis occurs. This results in the degradation of suspended solids and in viscosity decrease,

Several species of microorganisms such as *Bacillus, Erwinia, Kluyveromyces, Aspergillus, Rhizopus, Trichoderma, Pseudomonas, Penicillium* and *Fusarium* are good producers of pectinases (De Gregorio, et al., 2002). Among the microorganisms which synthesize pectinolytic enzymes, fungi, especially filamentous fungi, such as *Aspergillus niger* and *Aspergillus carbonarius* and *Lentinus edodes*, are preferred in industries since approximately 90% of produced enzymes may be secreted into the culture medium (Blandino et al., 2001). In fact, several studies have been undertaken to isolate, select, produce and characterize these specific enzymes so that pectinolytic enzymes could be employed not only in food processing but also in industrial ones. High resolution techniques such as crystallography

increases the amount of antioxidants in extra virgin olive oil and reduces rancidity.

baby food and oil extraction (Da Silva et al., 2005, Lima, et al., 2000).

speeding up the entire process (Paula, et al., 2004).

*glaucus, A. ustus* and *A. versicolor* are the best known.

several and diverse reactions (Andersen et al, 2008).

**2.1 Microbial enzymes for industries**

**2.1.1 Pectinase enzyme**

2007).

Lipolytic enzymes such as lipases and esterases are an important group of enzymes associated with the metabolism of lipid degradation. Lipase-producing microorganisms such as *Penicillium restrictum* may be found in soil and various oil residues. The industries Novozymes, Amano and Gist Brocades already employ microbial lipases.

Several microorganisms, such as *Candida rugosa, Candida antarctica, Pseudomonas alcaligenes, Pseudomonas mendocina* and *Burkholderia cepacia*, are lipase producers (Jaeger and Reetz, 1998).

The fungi of the genera *Rhizopus*, *Geotrichum*, *Rhizomucor*, *Aspergillus*, *Candida* and *Penicillium* have been reported to be producers of several commercially used lipases.

The industrial demand for new lipase sources with different enzymatic characteristics and produced at low costs has motivated the isolation and selection of new lipolytic microorganisms. However, the production process may modify their gene expression and change their phenotypes, including growth, production of secondary metabolites and enzymes. Posterior to primary selection, the production of the enzyme should be evaluated during the growth of the promising strain in fermentation, in liquid medium and / or in the solid state (Colen et al., 2006). However, it is evident that each system will result in different proteins featuring specific characteristics with regard to reactions' catalysis and, consequently, to the products produced (Asther et al., 2002).

## **2.1.3 Lactase**

Popularly known as lactase, beta-galactosidases are enzymes classified as hydrolases. They catalyze the terminal residue of b-lactose galactopiranosil (Galb1 - 4Glc) and produce glucose and galactose (Carminatti, 2001). Lactase's production sources are peaches, almonds and certain species of wild roses; animal organisms, such as the intestine, the brain and skin tissues; yeasts, such as *Kluyveromyces lactis*, *K. fragilis* and *Candida pseudotropicalis*; bacteria, such as *Escherichia coli*, *Lactobacillus bulgaricus*, *Streptococcus lactis* and *Bacillus* sp; and fungi, such as *Aspergillus foetidus*, *A. niger*, *A. oryzae* and *A. Phoenecia*.

The b-galactosidase may be found in nature, or rather, in plants, particularly almonds, peaches, apricots, apples, animal organs such as the intestine, the brain, placenta and the testis.

Lactase is produced by a widely diverse fungus population and by a large amount of microorganisms such as filamentous fungus, bacteria and yeast (Holsinger, 1997; Almeida and Pastore, 2001).

Microorganism-Produced Enzymes in the Food Industry 87

amyloglucosidase, the species *Aspergillus niger*, *A. oryzae, A. awamori, Fusarum oxysporum*, *Humicola insolens*, *Mucor pusillus*, *Trichoderma viride* . Species Are producing α-amylase. *Aspergillus niger*, *A. fumigatus*, *A. saitri, A. terreus*, *A. foetidus foetidus*, *Rhizopus, R. delemar* (Pandey et al. 2005), with special emphasis on the species of the genera *Aspergillus* sp.,

In fact, filamentous fungi and the enzymes produced thereby have been used in food and in the food-processing industries for decades. In fact, their GRAS (Generally Recognized as Safe) status is acknowledged by the U.S. Food and Drug Administration in the case of some

The food industry use amylases for the conversion of starch into dextrins. The latter are employed in clinical formulas as stabilizers and thickeners; in the conversion of starch into maltose, in confectioneries and in the manufacture of soft drinks, beer, jellies and ice cream; in the conversion of starch into glucose with applications in the soft-drinks industry, bakery, brewery and as a subsidy for ethanol production and other bioproducts; in the conversion of glucose into fructose, used in soft drinks, jams and yoghurts (Aquino et al., 2003, Nguyen *et*

Amylases provide better bread color, volume and texture in the baking industry. The use of these enzymes in bread production retards its aging process and maintains fresh bread for a longer period. Whereas fungal α-amylase provides greater fermentation potential, amyloglucosidase improves flavor and taste and a better bread crust color (Novozymes, 2005). Amylases are the most used enzymes in bread baking (Giménez et al. 2007; Haros;

Amylases have an important role in carbon cycling contained in starch by hydrolyzing the starch molecule in several products such as dextrins and glucose. Dextrins are mainly applied in clinical formulas and in material for enzymatic saccharification. Whereas maltose is used in confectioneries and in soft drinks, beer, jam and ice cream industries, glucose is employed as a sweetener in fermentations for the production of ethanol and other

The above amylases break the glycosidic bonds in the amylose and amylopectin chains. Thus, amylases have an important role in commercial enzymes. They are mainly applied in food, drugs, textiles and paper industries and in detergent formulas (Peixoto et al. 2003;

Results from strains tested for the potential production of amylases, kept at 4°C during 10 days, indicated that the wild and mutant strains still removed the nutrients required from the medium by using the available substrate. This fact showed that cooling maintained intact the amylase's activities or that a stressful condition for the fungus caused its

The best enzyme activity of microbial enzymes occurs in the same conditions that produce the microorganisms' maximum growth. Most studies on the production of amylases were undertaken from mesophilic fungi between 25 and 37°C. Best yields for α-amylase were achieved between 30 and 37°C for *Aspergillus* sp.; 30°C for *A. niger* in the production of amyloglucosidase 30°C in the production of α-amylase by *A. oryzae* (Tunga, R.; Tunga B.S, 2003), 55°C by thermophile fungus *Thermomonospora*, and 50°C by *T. lanuginosus* in the production of α-amylase (Gupta et al., 2003). However, no reports exist whether increase in enzyme activity after growth of fungus in ideal conditions and kept refrigerated at 4°C for

degradation and thus consumed more compounds than normal (Smith, et al., 2010).

Najafpour, Gupta et al., 2002; Asghar et al. 2006; Mitidieri et al., 2006).

*Rhizopus* sp. and *Endomyces* sp (Soccol et al. 2003).

species such as *Aspergillus niger* and *Aspergillus oryzae*.

*al.,* 2002).

Rosell, Leon; Durán).

10 days has ever been tested.

bioproducts.

Beta-galactosidase is highly important in the dairy industry, in the hydrolysis of lactose into glucose and galactose with an improvement in the solubility and digestibility of milk and dairy products. Food with low lactose contents, ideal for lactose-intolerant consumers, is thus obtained (Mahoney, 1997; Kardel et al. 1995; Pivarnik et al., 1995). It also favors consumers who are less tolerant to dairy products' crystallization, such as milk candy, condensed milk, frozen concentrated milk, yoghurt and ice cream mixtures, (Mahoney, 1998; Kardel et al., 1995). It also produces oligosaccharides (Almeida and Pastore, 2001), the best biodegradability of whey second to lactose hydrolysis (Mlichová; Rosenberg, 2006).

## **2.1.4 Cellulases**

Cellulases are enzymes that break the glucosidic bonds of cellulose microfibrils, releasing oligosaccharides, cellobiose and glucose (Dillon, 2004). These hydrolytic enzymes are not only used in food, drug, cosmetics, detergent and textile industries, but also in wood pulp and paper industry, in waste management and in the medical-pharmaceutical industry (Bhat and Bhat, 1997).

In the food industry, cellulases are employed in the extraction of components from green tea, soy protein, essential oils, aromatic products and sweet potato starch. Coupled to hemicellulases and pectinases they are used in the extraction and clarification of fruit juices. After fruit crushing, the enzymes are used to increase liquefaction through the degradation of the solid phase.

The above enzymes are also employed in the production process of orange vinegar and agar and in the extraction and clarification of citrus fruit juices (Orberg 1981). Cellulases supplement pectinases in juice and wine industries as extraction, clarification and filtration aids, with an increase in yield, flavor and the durability of filters and finishers (Pretel, 1997).

Cellulase is produced by a vast and diverse fungus population, such as the genera *Trichoderma*, *Chaetomium*, *Penicillium*, *Aspergillus*, *Fusarium* and *Phoma*; aerobic bacteria, such as *Acidothermus*, *Bacillus*, *Celvibrio*, *pseudonoma*, *Staphylococcus*, *Streptomyces* and *Xanthomonas*; and anaerobic bacteria, such as *Acetovibrio*, *Bacteroides*, *Butyrivibrio*, *Caldocellum*, *Clostridium*, *Erwinia*, *Eubacterium*, *Pseudonocardia*, *Ruminococcus* and *Thermoanaerobacter* (Moreira & Siqueira, 2006; Zhang et al., 2006). *Aspergillus* filamentous fungi stand out as major producers of cellulolytic enzymes. It is worth underscoring the filamentous fungus *Aspergillus niger*, a fermenting microorganism, which has been to produce of cellulolytic enzymes, organic acids and other products with high added value by solid-state fermentation processes. (Castro, 2006, Chandra et. al., 2007, Castro & Pereira Jr. 2010)

## **2.1.5 Amylases**

Amylases started to be produced during the last century due to their great industrial importance. In fact, they are the most important industrial enzymes with high biotechnological relevance. Their use ranges from textiles, beer, liquor, bakery, infant feeding cereals, starch liquefaction-saccharification and animal feed industries to the chemical and pharmaceutical ones.

Currently, large quantities of microbial amylases are commercially available and are almost entirely applied in starch hydrolysis in the starch-processing industries.

The species *Aspergillus* and *Rhizopus* are highly important among the filamentous fungus for the production of amylases (Pandey et al., 1999, 2005). In the production of

Beta-galactosidase is highly important in the dairy industry, in the hydrolysis of lactose into glucose and galactose with an improvement in the solubility and digestibility of milk and dairy products. Food with low lactose contents, ideal for lactose-intolerant consumers, is thus obtained (Mahoney, 1997; Kardel et al. 1995; Pivarnik et al., 1995). It also favors consumers who are less tolerant to dairy products' crystallization, such as milk candy, condensed milk, frozen concentrated milk, yoghurt and ice cream mixtures, (Mahoney, 1998; Kardel et al., 1995). It also produces oligosaccharides (Almeida and Pastore, 2001), the best biodegradability of whey second to lactose hydrolysis (Mlichová; Rosenberg, 2006).

Cellulases are enzymes that break the glucosidic bonds of cellulose microfibrils, releasing oligosaccharides, cellobiose and glucose (Dillon, 2004). These hydrolytic enzymes are not only used in food, drug, cosmetics, detergent and textile industries, but also in wood pulp and paper industry, in waste management and in the medical-pharmaceutical industry

In the food industry, cellulases are employed in the extraction of components from green tea, soy protein, essential oils, aromatic products and sweet potato starch. Coupled to hemicellulases and pectinases they are used in the extraction and clarification of fruit juices. After fruit crushing, the enzymes are used to increase liquefaction through the degradation

The above enzymes are also employed in the production process of orange vinegar and agar and in the extraction and clarification of citrus fruit juices (Orberg 1981). Cellulases supplement pectinases in juice and wine industries as extraction, clarification and filtration aids, with an increase in yield, flavor and the durability of filters and finishers (Pretel, 1997). Cellulase is produced by a vast and diverse fungus population, such as the genera *Trichoderma*, *Chaetomium*, *Penicillium*, *Aspergillus*, *Fusarium* and *Phoma*; aerobic bacteria, such as *Acidothermus*, *Bacillus*, *Celvibrio*, *pseudonoma*, *Staphylococcus*, *Streptomyces* and *Xanthomonas*; and anaerobic bacteria, such as *Acetovibrio*, *Bacteroides*, *Butyrivibrio*, *Caldocellum*, *Clostridium*, *Erwinia*, *Eubacterium*, *Pseudonocardia*, *Ruminococcus* and *Thermoanaerobacter* (Moreira & Siqueira, 2006; Zhang et al., 2006). *Aspergillus* filamentous fungi stand out as major producers of cellulolytic enzymes. It is worth underscoring the filamentous fungus *Aspergillus niger*, a fermenting microorganism, which has been to produce of cellulolytic enzymes, organic acids and other products with high added value by solid-state

fermentation processes. (Castro, 2006, Chandra et. al., 2007, Castro & Pereira Jr. 2010)

Amylases started to be produced during the last century due to their great industrial importance. In fact, they are the most important industrial enzymes with high biotechnological relevance. Their use ranges from textiles, beer, liquor, bakery, infant feeding cereals, starch liquefaction-saccharification and animal feed industries to the

Currently, large quantities of microbial amylases are commercially available and are almost

The species *Aspergillus* and *Rhizopus* are highly important among the filamentous fungus for the production of amylases (Pandey et al., 1999, 2005). In the production of

entirely applied in starch hydrolysis in the starch-processing industries.

**2.1.4 Cellulases**

(Bhat and Bhat, 1997).

of the solid phase.

**2.1.5 Amylases**

chemical and pharmaceutical ones.

amyloglucosidase, the species *Aspergillus niger*, *A. oryzae, A. awamori, Fusarum oxysporum*, *Humicola insolens*, *Mucor pusillus*, *Trichoderma viride* . Species Are producing α-amylase. *Aspergillus niger*, *A. fumigatus*, *A. saitri, A. terreus*, *A. foetidus foetidus*, *Rhizopus, R. delemar* (Pandey et al. 2005), with special emphasis on the species of the genera *Aspergillus* sp., *Rhizopus* sp. and *Endomyces* sp (Soccol et al. 2003).

In fact, filamentous fungi and the enzymes produced thereby have been used in food and in the food-processing industries for decades. In fact, their GRAS (Generally Recognized as Safe) status is acknowledged by the U.S. Food and Drug Administration in the case of some species such as *Aspergillus niger* and *Aspergillus oryzae*.

The food industry use amylases for the conversion of starch into dextrins. The latter are employed in clinical formulas as stabilizers and thickeners; in the conversion of starch into maltose, in confectioneries and in the manufacture of soft drinks, beer, jellies and ice cream; in the conversion of starch into glucose with applications in the soft-drinks industry, bakery, brewery and as a subsidy for ethanol production and other bioproducts; in the conversion of glucose into fructose, used in soft drinks, jams and yoghurts (Aquino et al., 2003, Nguyen *et al.,* 2002).

Amylases provide better bread color, volume and texture in the baking industry. The use of these enzymes in bread production retards its aging process and maintains fresh bread for a longer period. Whereas fungal α-amylase provides greater fermentation potential, amyloglucosidase improves flavor and taste and a better bread crust color (Novozymes, 2005). Amylases are the most used enzymes in bread baking (Giménez et al. 2007; Haros; Rosell, Leon; Durán).

Amylases have an important role in carbon cycling contained in starch by hydrolyzing the starch molecule in several products such as dextrins and glucose. Dextrins are mainly applied in clinical formulas and in material for enzymatic saccharification. Whereas maltose is used in confectioneries and in soft drinks, beer, jam and ice cream industries, glucose is employed as a sweetener in fermentations for the production of ethanol and other bioproducts.

The above amylases break the glycosidic bonds in the amylose and amylopectin chains. Thus, amylases have an important role in commercial enzymes. They are mainly applied in food, drugs, textiles and paper industries and in detergent formulas (Peixoto et al. 2003; Najafpour, Gupta et al., 2002; Asghar et al. 2006; Mitidieri et al., 2006).

Results from strains tested for the potential production of amylases, kept at 4°C during 10 days, indicated that the wild and mutant strains still removed the nutrients required from the medium by using the available substrate. This fact showed that cooling maintained intact the amylase's activities or that a stressful condition for the fungus caused its degradation and thus consumed more compounds than normal (Smith, et al., 2010).

The best enzyme activity of microbial enzymes occurs in the same conditions that produce the microorganisms' maximum growth. Most studies on the production of amylases were undertaken from mesophilic fungi between 25 and 37°C. Best yields for α-amylase were achieved between 30 and 37°C for *Aspergillus* sp.; 30°C for *A. niger* in the production of amyloglucosidase 30°C in the production of α-amylase by *A. oryzae* (Tunga, R.; Tunga B.S, 2003), 55°C by thermophile fungus *Thermomonospora*, and 50°C by *T. lanuginosus* in the production of α-amylase (Gupta et al., 2003). However, no reports exist whether increase in enzyme activity after growth of fungus in ideal conditions and kept refrigerated at 4°C for 10 days has ever been tested.

Microorganism-Produced Enzymes in the Food Industry 89

The enzyme is used in the food industry for the removing of harmful oxygen. Packaging materials and storage conditions are vital for the quality of products containing probiotic microorganisms since the microbial group's metabolism is essentially anaerobic or microaerophilic (MattilaSandholm et al., 2002). Oxygen level during storage should be consequently minimal to avoid toxicity, the organism's death and the consequent loss of the

Glucose oxidase may be a biotechnological asset to increase stability of probiotic bacteria in

Glucose isomerase (GI) (D-xylose ketol isomerase; EC <IP_ADDRESS>) catalyzes the reversible isomerase from D-glucose and D-xylose into D-fructose and D-xylulose, respectively. The enzyme is highly important in the food industry due to its application in the production of

Interconversion of xylose into xylulose by GI is a nutritional requirement of saprophytic bacteria and has a potential application in the bioconversion of hemicellulose into ethanol. The enzyme is widely distributed among prokaryotes and several studies have been

The isolation of GI in *Arthrobacter* strains was performed by Smith et al. (1991), whereas Walfridsson et al. (1996) cloned gene xylA of *Thermus themophilus* and introduced it into *Saccharomyces cerevisiae* to be expressed under the control of the yeast PGK1 promoter. The search for GI thermostable enzymes has been the target of protein engineering (Hartley et

In fact, biotechnology has an important role in obtaining mutants with promising prospects

The development of microbial strains which use xylan with prime matters for the growth or selection of GI-constituted mutants should lead towards the discontinuation of the use of

Invertase is an S-bD-fructofuranosidase obtained from *Saccharomyces cerevisiae* and other microorganisms. The enzyme catalyzes the hydrolysis from sucrose to fructose and glucose. The manufacture of inverted sugar is one of invertase's several applications. Owing to its sweetening effects which are higher than sucrose's, it has high industrial importance and

Invertase is more active at temperatures and pH ranging respectively between 40o and 60o C and between 3.0 and 5.0. When invertase-S is applied at 0.6% rate in a solution of sucrose

When Cardoso et al. (1998) added invertases to banana juice to assess its sweetness potential, they reported an increase in juice viscosity besides an increase in sweetness. *Alternaria* sp isolates from soybean seed were inoculated in a semi-solid culture and the microorganism accumulated large amounts of extracellular invertase, which was produced

Microorganisms, such as filamentous fungi, are good producers of invertase with potential

Gould et al. (2003) cultivated the filamentous fungus *Rhizopus* sp in wheat bran medium, and obtained invertase identified as polyacrylamide gel. Another potentially producing

yoghurt without chemical additives. It may thus be a biotechnology alternative.

undertaken to enhance its industrial application (Bhosale et al, 1996).

for the commercialization of glucose isomerase enzyme.

there are good prospects for its use in biotechnology.

constitutively without the need for an inductor.

application in various industrial sectors.

40% w / w at 40C, it inverts 80% of sucrose after 4h. 20min.

xylose as an enzyme production inducer.

product's functionality.

**2.1.8 Glucose isomerase**

fructose-rich corn syrup.

al., 2000).

**2.1.9 Invertase**

## **2.1.6 Proteases**

Proteases are enzymes produced by several microorganisms, namely, *Aspergillus niger, A. oryzae, Bacillus amyloliquefaciens*, *B. stearotermophilus*, *Mucor miehei, M. pusillus*. Proteases have important roles in baking, brewing and in the production of various oriental foods such as soy sauce, miso, meat tenderization and cheese manufacture.

Man's first contact with proteases activities occurred when he started producing milk curd. Desert nomads from the East used to carry milk in bags made of the goat's stomach. After long journeys, they realized that the milk became denser and sour, without understanding the process's cause. Curds became thus a food source and a delicacy. Renin, an animalproduced enzyme, is the protease which caused the hydrolysis of milk protein.

Proteases, enzymes that catalyze the cleavage of peptide bonds in proteins, are Class 3 enzymes, hydrolases, and sub-class 3.4, peptide-hydrolases. Proteases may be classified as exopeptidases and endopeptidases, according to the peptide bond to be chain-cleaved.

Recently proteases represent 60% of industrial enzymes on the market, whereas microbial proteases, particularly fungal infections, are advantageous because they are easy to obtain and to recover (Smith et al, 2009).

An enzyme extract (Neves-Souza, 2005), which coagulates milk and which is derived from the fungus *Aspergillus niger* var. *awamori*, is already produced industrially.

Although the bovine-derived protease called renin has been widely used in the manufacture of different types of cheese, the microbial-originated proteases are better for coagulant (CA) and proteolytic (PA) activities (PA). The relationship AC / AP has been a parameter to select potentially renin-producing microbial samples. The higher the ratio AC / AP, the most promising is the strain. It features high coagulation activity, with fewer risks in providing undesirable characteristics from enhanced proteolysis (Melo et al, 2002).

The microbial proteases have also been important in brewery. Beer contains poorly soluble protein complexes at lower temperature, causing turbidity when cold. The use of proteolytic enzymes to hydrolyze proteins involved in turbidity is an alternative for solving this problem.

Most commercial serine proteinases (Rawling et al, 1994), mainly neutral and alkaline, are produced by organisms belonging to the genus *Bacillus*. Whereas subtilisin enzymes are representatives of this group, similar enzymes are also produced by other bacteria such as *Thermus caldophilus*, *Desulfurococcus mucosus* and *Streptomyces* and by the genera *Aeromonas* and *Escherichia coli*.

In their studies and observations on the activities of proteases from *Bacillus*, Singh and Patel (2005), Silva, and Martin Delaney (2007); Sheri and Al-Mostafa (2004) and others evaluated their properties for a better performance in pH and temperature ranges.

## **2.1.7 Glucose oxidase**

Glucose oxidase [E.C. <IP_ADDRESS>] (GOx) is an enzyme that catalyzes the oxidation of beta-Dglucose with the formation of D-gluconolactone. The enzyme contains the prosthetic group flavin adenine dinucleotide (FAD) which enables the protein to catalyze oxidation-reduction reactions.

Guimarães et al. (2006) performed a screening of filamentous fungi which could potentially produce glucose-oxidase. Their results showed high levels of GOx in *Aspergillus versicolor* and *Rhizopus stolonifer*. The literature already suggests that the genus *Aspergillus* is a major GOx producer.

The enzyme is used in the food industry for the removing of harmful oxygen. Packaging materials and storage conditions are vital for the quality of products containing probiotic microorganisms since the microbial group's metabolism is essentially anaerobic or microaerophilic (MattilaSandholm et al., 2002). Oxygen level during storage should be consequently minimal to avoid toxicity, the organism's death and the consequent loss of the product's functionality.

Glucose oxidase may be a biotechnological asset to increase stability of probiotic bacteria in yoghurt without chemical additives. It may thus be a biotechnology alternative.

## **2.1.8 Glucose isomerase**

88 Scientific, Health and Social Aspects of the Food Industry

Proteases are enzymes produced by several microorganisms, namely, *Aspergillus niger, A. oryzae, Bacillus amyloliquefaciens*, *B. stearotermophilus*, *Mucor miehei, M. pusillus*. Proteases have important roles in baking, brewing and in the production of various oriental foods such as

Man's first contact with proteases activities occurred when he started producing milk curd. Desert nomads from the East used to carry milk in bags made of the goat's stomach. After long journeys, they realized that the milk became denser and sour, without understanding the process's cause. Curds became thus a food source and a delicacy. Renin, an animal-

Proteases, enzymes that catalyze the cleavage of peptide bonds in proteins, are Class 3 enzymes, hydrolases, and sub-class 3.4, peptide-hydrolases. Proteases may be classified as exopeptidases and endopeptidases, according to the peptide bond to be chain-cleaved. Recently proteases represent 60% of industrial enzymes on the market, whereas microbial proteases, particularly fungal infections, are advantageous because they are easy to obtain

An enzyme extract (Neves-Souza, 2005), which coagulates milk and which is derived from

Although the bovine-derived protease called renin has been widely used in the manufacture of different types of cheese, the microbial-originated proteases are better for coagulant (CA) and proteolytic (PA) activities (PA). The relationship AC / AP has been a parameter to select potentially renin-producing microbial samples. The higher the ratio AC / AP, the most promising is the strain. It features high coagulation activity, with fewer risks in

The microbial proteases have also been important in brewery. Beer contains poorly soluble protein complexes at lower temperature, causing turbidity when cold. The use of proteolytic enzymes to hydrolyze proteins involved in turbidity is an alternative for solving this

Most commercial serine proteinases (Rawling et al, 1994), mainly neutral and alkaline, are produced by organisms belonging to the genus *Bacillus*. Whereas subtilisin enzymes are representatives of this group, similar enzymes are also produced by other bacteria such as *Thermus caldophilus*, *Desulfurococcus mucosus* and *Streptomyces* and by the genera *Aeromonas*

In their studies and observations on the activities of proteases from *Bacillus*, Singh and Patel (2005), Silva, and Martin Delaney (2007); Sheri and Al-Mostafa (2004) and others evaluated

Glucose oxidase [E.C. <IP_ADDRESS>] (GOx) is an enzyme that catalyzes the oxidation of beta-Dglucose with the formation of D-gluconolactone. The enzyme contains the prosthetic group flavin adenine dinucleotide (FAD) which enables the protein to catalyze oxidation-reduction

Guimarães et al. (2006) performed a screening of filamentous fungi which could potentially produce glucose-oxidase. Their results showed high levels of GOx in *Aspergillus versicolor* and *Rhizopus stolonifer*. The literature already suggests that the genus *Aspergillus* is a major

their properties for a better performance in pH and temperature ranges.

produced enzyme, is the protease which caused the hydrolysis of milk protein.

the fungus *Aspergillus niger* var. *awamori*, is already produced industrially.

providing undesirable characteristics from enhanced proteolysis (Melo et al, 2002).

soy sauce, miso, meat tenderization and cheese manufacture.

**2.1.6 Proteases**

and to recover (Smith et al, 2009).

problem.

reactions.

GOx producer.

and *Escherichia coli*.

**2.1.7 Glucose oxidase**

Glucose isomerase (GI) (D-xylose ketol isomerase; EC <IP_ADDRESS>) catalyzes the reversible isomerase from D-glucose and D-xylose into D-fructose and D-xylulose, respectively. The enzyme is highly important in the food industry due to its application in the production of fructose-rich corn syrup.

Interconversion of xylose into xylulose by GI is a nutritional requirement of saprophytic bacteria and has a potential application in the bioconversion of hemicellulose into ethanol. The enzyme is widely distributed among prokaryotes and several studies have been undertaken to enhance its industrial application (Bhosale et al, 1996).

The isolation of GI in *Arthrobacter* strains was performed by Smith et al. (1991), whereas Walfridsson et al. (1996) cloned gene xylA of *Thermus themophilus* and introduced it into *Saccharomyces cerevisiae* to be expressed under the control of the yeast PGK1 promoter. The search for GI thermostable enzymes has been the target of protein engineering (Hartley et al., 2000).

In fact, biotechnology has an important role in obtaining mutants with promising prospects for the commercialization of glucose isomerase enzyme.

The development of microbial strains which use xylan with prime matters for the growth or selection of GI-constituted mutants should lead towards the discontinuation of the use of xylose as an enzyme production inducer.

#### **2.1.9 Invertase**

Invertase is an S-bD-fructofuranosidase obtained from *Saccharomyces cerevisiae* and other microorganisms. The enzyme catalyzes the hydrolysis from sucrose to fructose and glucose. The manufacture of inverted sugar is one of invertase's several applications. Owing to its sweetening effects which are higher than sucrose's, it has high industrial importance and there are good prospects for its use in biotechnology.

Invertase is more active at temperatures and pH ranging respectively between 40o and 60o C and between 3.0 and 5.0. When invertase-S is applied at 0.6% rate in a solution of sucrose 40% w / w at 40C, it inverts 80% of sucrose after 4h. 20min.

When Cardoso et al. (1998) added invertases to banana juice to assess its sweetness potential, they reported an increase in juice viscosity besides an increase in sweetness.

*Alternaria* sp isolates from soybean seed were inoculated in a semi-solid culture and the microorganism accumulated large amounts of extracellular invertase, which was produced constitutively without the need for an inductor.

Microorganisms, such as filamentous fungi, are good producers of invertase with potential application in various industrial sectors.

Gould et al. (2003) cultivated the filamentous fungus *Rhizopus* sp in wheat bran medium, and obtained invertase identified as polyacrylamide gel. Another potentially producing

Microorganism-Produced Enzymes in the Food Industry 91

Alkan, H., Baisal, Z., Uyar, F., Dogru, M. (2007). Production of Lipase by a Newly Isolated

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fungus invertase is *Aspergillus casiellus*. It was inoculated in soybean meal medium and after 72 hours its crude extract was isolated (Novak et al., 2010). Since most invertases used in industry are produced by yeasts, underscoring the search for fungi that produce it in great amounts is a must.

## **3. Final considerations**

Perspectives for biotechnological production of enzymes by microorganisms.

Biotechnology is an important tool for a more refined search for microorganisms with commercial assets. Microorganisms have existed on the planet Earth during millions of years and are a source of biotechnological possibilities due to their genetic plasticity and adaptation.

The isolation of new species from several and different habitats, such as saltwater and freshwater, soils, hot springs, contaminated soils, caves and hostile environments is required. Microorganisms adapted to these conditions may have great biotechnological potential.

Methods such as the selection of mutants are simple ways to obtain strains or strains with enzymatic possibilities and these methods are widely used by researchers in academic pure science laboratories.

Geneticists also employ the recombination and selection of mutants, which feature promising characteristics, in new strains. This method consists of transferring genetic material among contrasting genotypes, obtain recombinants and use the selection for the desired need.

The recombinant DNA technology (TDR) is a very useful method under three aspects: it increases the production of a microbial enzyme during the fermentation process; it provides enzymes with new properties suitable for industrial processes, such as thermostability and ability to function outside the normal pH range; it produces enzymes from animal- and vegetable-derived microorganisms.

Extremophile microorganisms are potentially producing enzymes with useful characteristics for high temperature industrial processes.

Microorganisms that grow at low temperatures have important biotechnological assets since their enzymes are more effective at low temperatures and enables contamination risks in continuous fermentation processes. This will shorten fermentation time and enhance energy saving.

The DNA sequencing technologies have advanced greatly in recent years and important progress on genes that synthesize proteins and thus determine their function in organisms has been achieved.

Genomes of several microorganisms have been sequenced, including those which are important for the food industry, such as *Saccharomyces cerevisiae*, *Bacillus subtilis*, *Lactococcus Latis*, *Lactobacillus acidophilus*, *Lactobacillus* sp, and *Streptococcus thermophilus*. These genomes have revealed several new genes, most of which codify enzymes.

Microorganisms are potential producers of enzymes useful for the food industry. Biotechnological tools are available for the selection and obtaining of strains and for strains which increase enzymes' production on a large scale.

Progress and achievements in this area will bring improvements in the food industry and, consequently, a better health quality for mankind.

#### **4. References**

90 Scientific, Health and Social Aspects of the Food Industry

fungus invertase is *Aspergillus casiellus*. It was inoculated in soybean meal medium and after 72 hours its crude extract was isolated (Novak et al., 2010). Since most invertases used in industry are produced by yeasts, underscoring the search for fungi that produce it in great

Biotechnology is an important tool for a more refined search for microorganisms with commercial assets. Microorganisms have existed on the planet Earth during millions of years and are a source of biotechnological possibilities due to their genetic plasticity and

The isolation of new species from several and different habitats, such as saltwater and freshwater, soils, hot springs, contaminated soils, caves and hostile environments is required. Microorganisms adapted to these conditions may have great biotechnological

Methods such as the selection of mutants are simple ways to obtain strains or strains with enzymatic possibilities and these methods are widely used by researchers in academic pure

Geneticists also employ the recombination and selection of mutants, which feature promising characteristics, in new strains. This method consists of transferring genetic material among contrasting genotypes, obtain recombinants and use the selection for the

The recombinant DNA technology (TDR) is a very useful method under three aspects: it increases the production of a microbial enzyme during the fermentation process; it provides enzymes with new properties suitable for industrial processes, such as thermostability and ability to function outside the normal pH range; it produces enzymes from animal- and

Extremophile microorganisms are potentially producing enzymes with useful characteristics

Microorganisms that grow at low temperatures have important biotechnological assets since their enzymes are more effective at low temperatures and enables contamination risks in continuous fermentation processes. This will shorten fermentation time and enhance energy

The DNA sequencing technologies have advanced greatly in recent years and important progress on genes that synthesize proteins and thus determine their function in organisms

Genomes of several microorganisms have been sequenced, including those which are important for the food industry, such as *Saccharomyces cerevisiae*, *Bacillus subtilis*, *Lactococcus Latis*, *Lactobacillus acidophilus*, *Lactobacillus* sp, and *Streptococcus thermophilus*. These genomes

Microorganisms are potential producers of enzymes useful for the food industry. Biotechnological tools are available for the selection and obtaining of strains and for strains

Progress and achievements in this area will bring improvements in the food industry and,

have revealed several new genes, most of which codify enzymes.

which increase enzymes' production on a large scale.

consequently, a better health quality for mankind.

Perspectives for biotechnological production of enzymes by microorganisms.

amounts is a must.

adaptation.

potential.

science laboratories.

vegetable-derived microorganisms.

for high temperature industrial processes.

desired need.

saving.

has been achieved.

**3. Final considerations**

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Kashyap, D. R.; Chandra, S.; Kaul, A.; Tewari, R. (2000). *World J. Microbiol. Biotechnol. 16*, 277. Kardel, G.; Furtado, M.M.; Neto, J.P.M.L. (1995). Lactase na Indústria de Laticínios (Parte 1).

*Revista do Instituto de Laticínios "Cândido Tostes".* Juiz de Fora, Vol.50, No.294, pp.15-

*Enzyme and Microbial technology*, Vol.39, No.2, pp. 235-251.

foods containing lactose. *Food Technology*, Vol.45, No.1, pp. 94-95.

SCP and crude pectinase production by slurry-state fermentation of lemon pulps.

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(2005). Wheat flour constituents: how they impact bread quality, and how to impact their functionality. *Trends in Food* Science *& Technology*, Cambridge, Vol.16,

Production of amylases by *Aspergillus fumigatus*. *Revista de Microbiologia*, v.29, p.99-

bread texture and bread staling. *Eur. Food Res. Technol*., Berlin,Vol.215, No. 5, pp.

insights into protein engineering for increased thermostability. *Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology*, Vol. 1543, Issue

*Res. 5*, 859.

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2, p.294-335.

17.

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Barking, Vol.18, No.2, pp. 196-204.

*Biochemistry*, Vol.38, No.11, p. 1-18.

*biotecnológicos*. Ribeirão Preto: *Legis Summa*, p. 243-270.

Kaur, G.; Kumar, S.; Satyanarayama, T. (2004). *Bioresour. Technol. 94*, 239.

*Valladolid España*

**Nanotechnology and Food Industry**

Francisco Javier Gutiérrez, Mª Luisa Mussons,

*Centro Tecnológico CARTIF. Parque Tecnológico de Boecillo,*

Human population will reach 9,100 million by 2,050, which supposes an increase of 34% respect present situation. This growth will occur in emerging countries mainly. As a consequence of that, there will be an increase in global demand for foods, feed and energy. Initial estimation on the increment of this world demands are in the order of 70%. Accordingly, the pressure on resources (in special water and crops) will be higher. World surface devoted to crop production will be increased, in order to meet demands on food energy and other industrial uses and so, the environmental impact is some areas could be

In order to obtain commodities and other feedstock in a sustainable way it is necessary to improve the current working methods and control the environmental impact, acting on:

In a broad context, some factors affecting living standards are the water availability and food. In fact, life expectancy and health are determined by both. Considering the expected demand on food (and water) as well as the global situation, several technological challenges should be overcome to make a rational use of resources possible. In this sense,

Where is the interest of nanotechnology? Which are the possibilities of nanotechnology in relation to foods and food production? Is it possible to improve crops using nanotechnology? Which are the advantages concerning water and use management? How is

A general view on the nanotechnology concerning foods and water is presented in this

According to Chaudhry et al., 2011, in food nanotechnology different categories can be

nanotechnology could suppose a great tool in solving that situation.

it envisaged to achieve those potential benefits at a global scale?

chapter and some answers to the former questions are proposed.

materials intelligent packaging and nanosensors.

**1. Introduction**

specified:

high.

Paloma Gatón and Ruth Rojo

## **Nanotechnology and Food Industry**

Francisco Javier Gutiérrez, Mª Luisa Mussons, Paloma Gatón and Ruth Rojo *Centro Tecnológico CARTIF. Parque Tecnológico de Boecillo, Valladolid España*

## **1. Introduction**

94 Scientific, Health and Social Aspects of the Food Industry

Peixoto, S.C.; jorge, J.A.; Terenzi, H.F.; Polizeli, M.L.T.M. (2003). Rhizopus microsporus var.

Pretel, M.T.(1997). Pectic enzymes in fresh fruit processing: optimization of enzymic peeling

Pivarnik, L. F.; Senegal, A.G.; Rand, A.G. (1995). Hydrolytic and transgalactosil activities of

Rawling, N.D., Barret, A. (1994). Families of serine peptidases. *Meth. Enzymol*., Vol.244,

Shankar, S.K.; Mulimania, V.H. (2007). β-Galactosidase production by *Aspergillus* oryzae

Silva, C.R.; Delatorre, A. B.; Martins, M. L. L. (2007). Effect of the culture conditions on the

Silva, G.A.B; Almeida, W.E.S; Cortes, M.S; Martins, E.S. (2009). Produção e caracterização de

Smith, C. A.; Rangarajan, M.; Hartley, B. S. (1991). D-Xylose (D-glucose) isomerase from

Soccol, C.R. & Vandenberghe, L.P.S., (2003). Overview of applied solidstate fermentation in

Sorensen, J. F.; Krag, K. M.; Sibbesen, O.; Delcur, J.; Goesaert, H.; Svensson, B.; Tahir, T. A.;

Tan, T., Zhang, M., Wang, B., Ying, C., Deng, Li. (2003). Screening of high lipase producing

Tunga, R.; Tunga, B. S. (2003).Extra-cellular Amylase Production by *Aspergillus oryzae* Under

Uenojo, M., Pastore, G. M. (2007). Pectinases: Aplicações Industriais e Perspectivas. *Química*

Walfridsson, M.; Bao, X.; Anderlund, M.; Lilius, G. Bulow, L.; Hahn-Hagerdal, B. (1996).

Zarevúcka, M., Kejík, Z., Saman, D., Wimmer, Z., Demnerová, K. (2005). Enantioselective

isomerase. *Appl. Environ. Microbiol.,* Vol.62, No.12, pp. 4648-4651.

Giovane, A.; Bonnin, E.; Juge, N. (2004). *Biochim. Biophys. Acta*, *1696*, 275.

Brazil. *Biochemical Engineering Journal*, Vol. 13, pp. 205-218.

Taragano, V. M.; Pilosof, A. M. R. (1999). *Enzyme Microb. Technol. 25*, 411.

thermostable amylases. *Int. Microbiol*., Vol.6, pp.269-273.

of oranges. *Process Biochemistry*. Vol.32, No.1, pp. 43-49.

*Nutrition Research*, New York, Vol.38, p. 33.

Vol.03, No.01, pp. 28-41. ISSN: 1981-3686.

*Journal,* Vol.1; No.277, pp. 255–261.

Systems Biology, Vol.4, No.178, 1-13.

pp.459-465.

University, 12 p.

Vol.37, pp.481-486.

*Nova*, Vol.30, No. 2, pp. 388-394.

*Bioresource Technology*, Vol.98, No.4, pp. 958-961.

pp.18-61.

258.

rhizopodiformis: a thermotolerant fungus with potential for production of

commercial -galactosidase (lactase) in food processing. *Advances in Food and*

production of an extracellular protease by thermophilic *Bacillus* sp. and some properties of the enzymatic activity. *Brazilian Journal of Microbiology*, Vol.38, pp.253-

protease obtida por *gliocladium verticilloides* através da fermentação em estado sólido de subprodutos agroindustriais. *Revista Brasileira de Tecnologia Agroindustrial,*

Arthrobacter strain N.R.R.L. B3728. Purification and properties. *Biochemestry*

Brufau, J.; Perez-Vendrell, A. M.; Bellincamp, D.; D'Ovidio, R.; Camardella, L.;

*Candida* sp. And production of lipase by fermentation. *Process Biochemistry*, Vol.39,

Solid State Fermentation. Japan: *International Center for Biotechnology*, Osaka

Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the *Thermus thermophilus* xylA gene, which expresses an active xylose (glucose)

properties of induced lipases from *Geotrichum*. *Enzyme and Microbial Technology*,

Human population will reach 9,100 million by 2,050, which supposes an increase of 34% respect present situation. This growth will occur in emerging countries mainly. As a consequence of that, there will be an increase in global demand for foods, feed and energy. Initial estimation on the increment of this world demands are in the order of 70%. Accordingly, the pressure on resources (in special water and crops) will be higher. World surface devoted to crop production will be increased, in order to meet demands on food energy and other industrial uses and so, the environmental impact is some areas could be high.

In order to obtain commodities and other feedstock in a sustainable way it is necessary to improve the current working methods and control the environmental impact, acting on:

In a broad context, some factors affecting living standards are the water availability and food. In fact, life expectancy and health are determined by both. Considering the expected demand on food (and water) as well as the global situation, several technological challenges should be overcome to make a rational use of resources possible. In this sense, nanotechnology could suppose a great tool in solving that situation.

Where is the interest of nanotechnology? Which are the possibilities of nanotechnology in relation to foods and food production? Is it possible to improve crops using nanotechnology? Which are the advantages concerning water and use management? How is it envisaged to achieve those potential benefits at a global scale?

A general view on the nanotechnology concerning foods and water is presented in this chapter and some answers to the former questions are proposed.

According to Chaudhry et al., 2011, in food nanotechnology different categories can be specified:

Nanotechnology and Food Industry 97

hundred of nanometres showing activity can be found, it seem reasonable to extent the

Despite that the potential of nanotechnology has been already recognised and applied in other industrial sectors (electronic, medical, pharmaceutical energetic sector and material sciences), the application on the agro-food sector has been limited up to now. The most promising applications of nanotechnology in foods includes: Enhancement of activity and bioavailability of nutrients and activity principles of foods, improvement of organoleptic features (colour, flavour), better consistence of food matrix, new packaging development,

In the case of the water use, the high nanoparticle activity allows the use of new purification techniques and removal of unwanted substances. In agriculture, the increase of bioavailability and the particles behaviour could boost the reduction of the side-effects in

The pressure on crops for agricultural production will increase in the future (both the area available for cultivation and the consumption of water required for this purpose). This is related to the fact that soil is used not only for food production but also for other products of industrial interest such as biofuels. To give some figures, the annual cereal production will have to increase from 2.1 billion to 3 billion while the annual meat production will have to

The scientific community trust in nanotechnology as a tool which could help to solve the challenge faced by the farmer: get highly productive crops while minimizing the use of synthetic chemicals. Despite the promising use of this technology in agriculture, most applications are still under research: nanotechnology and the genetic improvement of crops and production of more selective, effective and easier to dose plant protection products (FAO / WHO, 2009; Nair et al., 2010). Nanotechnology will likely have something to say about the search of alternative energy sources and cleaning and decontamination of water or soil resources. Researches also work in the application of nanotechnology in surveillance and control systems to determine when is the best moment to harvest or to monitor crop

But the crops will not only be benefited from the potential advantages of this technology,

This technology, combined with others such as biotechnology, can make genetic manipulation of plants easier. It allows that nanoparticles, nanofibers or nanocapsules are used as vectors of new genetic material instead of conventional viral vectors. These new vehicles could carry a larger number of genes as well as substances able to trigger gene expression (Miller & Senjen, 2008; Nair et al., 2010) or to control the release of genetic

Chitosan is one of the most studied non-viral gene vectors since its positive charge density allows the condensation of DNA by electrostatic interaction, protecting the entrapped genes

**2. Potential applications of nanotechnology in the agro-food industries**

food traceability, safety and food monitoring during transport and storage.

**2.1 Can nanotechnology enhance the access to the crops?**

but also could be used as organic producers of nanoparticles.

**2.1.1 Nanotechnology and crops genetic improvement**

definition up to 1,000 nm.

environment.

double.

safety (Joseph & Morrison, 2006).

material throughout time.

The state of the nanotechnology in relation with food production and water use is presented in this chapter through some examples. Why the nanotechnology shows a great application potential will be explained presenting some of the most recent findings. Therefore, a general view on how nanotechnology could be a solution for the improvement of methods used in the food production and water uses is provided.

## **1.1 Basic notions on nanotechnology**

Nanotechnology can be briefly defined as 'the engineering of very small systems and structures'. Actually, nanotechnology consists in a set of technologies than can be developed and used in several activities and agro-food sector it is not an exception.

The main feature of nanotechnology is defined by the size of the systems of work: the 'nanoscale'. At this scale the matter presents properties which are different (and new) than the observed at macroscopic level. These properties emerge as a consequence of the size of the structures that produces the material and their interactions. Somehow, in nanotechnology, atomic and molecular forces turn on determinant factors over other effects of relevance at higher scales. Other properties and possibilities of nanotechnology, which have great interest in food technology, are high reactivity, enhanced bioavailability and bioactivity, adherence effects and surface effects of nanoparticles.

According to the former, which is the magnitude order defining nanoscale?. Although there is a consensus about considering this as a range between 10 and 100 nm, there is growing evidence on particles and systems of several hundred of nanometres with activity that can be related with a typical behaviour of nanomaterial (Ashwood et al., 2007).

The definition 'working under 100 nm' could be considered a common approach when defining nanotechnology, but this approach could be too restrictive in the agro-food sector, as well as when the effects on health and environment are considered.

At present several definitions about the term 'nano' are presented by organisations at international level. For instance, Commonwealth Scientific and Industrial Research Organisation (CSIRO) or the Food and Drug Administration (FDA) define nanomaterial as that material under 1,000 nm.

According to the ISO/TS 8004-1:2010 norm, nanotechnology is: 'The application of scientific knowledge to control and utilize matter in the nanoscale, where properties and phenomena related to size or structure can emerge'. And 'nanoscale' is defined as: 'Size range from approximately 10 nm to 100 nm'.

Regardless of the former, in this chapter nanoscale will be considered when the working range is less than 1,000 nm. This decision comes from the fact that a clear distinction can be made when considering the production of foods and water use:

So, since the final aim is the search for a kind of functionality (mainly physiological type) through the control of the size of the matter and, considering the fact that particles of several

The state of the nanotechnology in relation with food production and water use is presented in this chapter through some examples. Why the nanotechnology shows a great application potential will be explained presenting some of the most recent findings. Therefore, a general view on how nanotechnology could be a solution for the improvement of methods used in

Nanotechnology can be briefly defined as 'the engineering of very small systems and structures'. Actually, nanotechnology consists in a set of technologies than can be developed

The main feature of nanotechnology is defined by the size of the systems of work: the 'nanoscale'. At this scale the matter presents properties which are different (and new) than the observed at macroscopic level. These properties emerge as a consequence of the size of the structures that produces the material and their interactions. Somehow, in nanotechnology, atomic and molecular forces turn on determinant factors over other effects of relevance at higher scales. Other properties and possibilities of nanotechnology, which have great interest in food technology, are high reactivity, enhanced bioavailability and

According to the former, which is the magnitude order defining nanoscale?. Although there is a consensus about considering this as a range between 10 and 100 nm, there is growing evidence on particles and systems of several hundred of nanometres with activity that can

The definition 'working under 100 nm' could be considered a common approach when defining nanotechnology, but this approach could be too restrictive in the agro-food sector,

At present several definitions about the term 'nano' are presented by organisations at international level. For instance, Commonwealth Scientific and Industrial Research Organisation (CSIRO) or the Food and Drug Administration (FDA) define nanomaterial as

According to the ISO/TS 8004-1:2010 norm, nanotechnology is: 'The application of scientific knowledge to control and utilize matter in the nanoscale, where properties and phenomena related to size or structure can emerge'. And 'nanoscale' is defined as: 'Size range from

Regardless of the former, in this chapter nanoscale will be considered when the working range is less than 1,000 nm. This decision comes from the fact that a clear distinction can be

So, since the final aim is the search for a kind of functionality (mainly physiological type) through the control of the size of the matter and, considering the fact that particles of several

or other agrochemical aimed at production improvement.

and used in several activities and agro-food sector it is not an exception.

bioactivity, adherence effects and surface effects of nanoparticles.

be related with a typical behaviour of nanomaterial (Ashwood et al., 2007).

as well as when the effects on health and environment are considered.

made when considering the production of foods and water use:

the food production and water uses is provided.

**1.1 Basic notions on nanotechnology**

that material under 1,000 nm.

approximately 10 nm to 100 nm'.

packaging)

water.

hundred of nanometres showing activity can be found, it seem reasonable to extent the definition up to 1,000 nm.

Despite that the potential of nanotechnology has been already recognised and applied in other industrial sectors (electronic, medical, pharmaceutical energetic sector and material sciences), the application on the agro-food sector has been limited up to now. The most promising applications of nanotechnology in foods includes: Enhancement of activity and bioavailability of nutrients and activity principles of foods, improvement of organoleptic features (colour, flavour), better consistence of food matrix, new packaging development, food traceability, safety and food monitoring during transport and storage.

In the case of the water use, the high nanoparticle activity allows the use of new purification techniques and removal of unwanted substances. In agriculture, the increase of bioavailability and the particles behaviour could boost the reduction of the side-effects in environment.

## **2. Potential applications of nanotechnology in the agro-food industries**

## **2.1 Can nanotechnology enhance the access to the crops?**

The pressure on crops for agricultural production will increase in the future (both the area available for cultivation and the consumption of water required for this purpose). This is related to the fact that soil is used not only for food production but also for other products of industrial interest such as biofuels. To give some figures, the annual cereal production will have to increase from 2.1 billion to 3 billion while the annual meat production will have to double.

The scientific community trust in nanotechnology as a tool which could help to solve the challenge faced by the farmer: get highly productive crops while minimizing the use of synthetic chemicals. Despite the promising use of this technology in agriculture, most applications are still under research: nanotechnology and the genetic improvement of crops and production of more selective, effective and easier to dose plant protection products (FAO / WHO, 2009; Nair et al., 2010). Nanotechnology will likely have something to say about the search of alternative energy sources and cleaning and decontamination of water or soil resources. Researches also work in the application of nanotechnology in surveillance and control systems to determine when is the best moment to harvest or to monitor crop safety (Joseph & Morrison, 2006).

But the crops will not only be benefited from the potential advantages of this technology, but also could be used as organic producers of nanoparticles.

#### **2.1.1 Nanotechnology and crops genetic improvement**

This technology, combined with others such as biotechnology, can make genetic manipulation of plants easier. It allows that nanoparticles, nanofibers or nanocapsules are used as vectors of new genetic material instead of conventional viral vectors. These new vehicles could carry a larger number of genes as well as substances able to trigger gene expression (Miller & Senjen, 2008; Nair et al., 2010) or to control the release of genetic material throughout time.

Chitosan is one of the most studied non-viral gene vectors since its positive charge density allows the condensation of DNA by electrostatic interaction, protecting the entrapped genes

Nanotechnology and Food Industry 99

chemicals are the nanoparticles functionalized with the aim of being absorbed through insects' cuticle. The protective wax layer of insects is damaged and leads them to death by desiccation. This approach is safe for plants and entails less environmental damage. Nair et al., 2010 reported that certain nanoparticles are able to reach sap, and Corredor et al., 2009 found that they can move towards several sites in pumpkins. In other words, nanoparticles

Substances in their nano form can affect cellular metabolism in a specific manner; for instance Ursache-Oprisan et al., 2011 reported an inhibition in chlorophyll biosynthesis caused by magnetic nanoparticles. But nanomaterials not only influence plants, but also animals like *Eisenia fetida* earthworms which avoid silver nanoparticles enriched soils as was

One of the biggest challenges in this century is the search for new and feasible energy sources different from fuel, nuclear or hydroelectric power. Alternative energy should maintain socio-economic development without jeopardizing the environment. According to U.S. Energy Information Administration, more than 90% of the total energy produced during the first eight months of 2010 in the USA was obtained from coal, gas, nuclear, oil and wood. This energetic mix finds explanation in the fact that renewable energies (solar,

Although, it is not the aim of this chapter to talk about the connection between energy and agriculture, it is worth to mention that agricultural sector requires important energy inputs (direct but also indirect). We think any step forward in the production; storage or use of new energy sources could be easier and faster with the help of nanotechnology. Then society will be moving towards a more sustainable, affordable and less dependent

Furthermore being vehicle for active substances (pesticides, plant growth regulators or fertilizers), nanoparticles can also be synthesized with a catalytic oxidation-reduction objective. The latter application would reduce the amount of these active substances present in the environment and also the time during which it is exposed to their action (Knauer & Bucheli, 2009). This section focuses on the application of reduction-oxidation catalytic nanoparticles as soil decontaminants, while the role of nanotechnology in water remediation

The research focus is twofold in this issue: first, try to accelerate the degradation of residual pesticides in the soil, and secondly, improving these pollutants' detection and quantification

Between those who try to accelerate the decomposition of these contaminants in soil, are Shen et al., 2007 who synthesized magnetic Fe3O4-C18 composite nanoparticles (5-10 nm) more effective than conventional C18 materials as cleaner substances of organophosphorous pesticides. Zeng et al., 2010 proved that TiO2 nanoparticles can enhance

Currently, several authors are working in pesticide analysis methods' optimization. Magnetic composite nanoparticle-modified screen printed carbon electrodes (Gan et al.,

organophosphorous and carbamates' degradation rate (30% faster) in crop fields.

have the ability to act in sites different from their application point.

**2.1.3 Alternative energy sources and nanotechnology**

wind, geothermal and tidal) are still non cost-effective.

**2.1.4 Water and soil resources remediation**

is developed in section 2.3. Nanotechnology and water supplies.

evidenced by Shoults-Wilson et al., 2011.

on fuel agriculture.

methods.

from nuclease action. However, chitosan nanocarriers have still not replaced conventional vectors. These vehicles are often associated with low transfection efficiencies. Authors like Zhao et al., 2011 are recently working on the enhancement of this property through modification of chitosan nanocomplexes with an octapeptide. They have condensed DNA into spherical nanoparticles of around 100-200 nm size with higher transfection efficiencies and lower cytotoxicity than those of DNA complexed with unmodified chitosan. Their transfection capacity varies depending on cell type. Authors like Mao et al., 2001 when they used as a genetic vehicle chitosan nanoparticles, found higher levels of gene expression in human kidney cells and bronchial cells than in cancerous cells. Wang et al., 2011 worked with *Jatropha curcas* callus cells and demonstrated the integration of DNA carried by this type of nanoparticles in their genome.

Chitosan nanoparticles are quite versatile, as well as their transfection efficiency can be modified, they can be PEGylated in order to control the release of genetic material as time goes by (Mao et al., 2001). This effect of time controlled genetic material release can be achieved by encapsulating pDNA into poly (DL-lactide-co-glycolide) particles. Specifically speaking, Cohen et al., 2000 achieved sustained release of pDNA over a month. Nonetheless the major advantage of nanobiotechnology is the simultaneous delivery of both DNA and effector molecules to specific sites. This effect has been achieved in intact tobacco and maize tissues when using gold-capped mesoporous silica nanoparticles (MSNs) as plasmid DNA transfers (Nair et al., 2010).

#### **2.1.2 Production of nano-agrochemicals**

Nanotechnology can help significantly to improve crop management techniques and it seems to be particularly useful in the use and handling of agrochemicals. Usually, only a very small amount of a given active compound is needed for treatment. Nowadays larger amount of chemicals, which are applied several times, are used in the field and nanotechnology could help to increase efficacy and to prevent losses. These losses can be explained for several reasons: UV degradation, hydrolysis, microbiota interaction or leaching. This way of handling chemicals brings negative effects to the environment (soil degradation, water pollution and side effects in other species). It is postulated that active substances in their nano form will allow to formulate more effective products, with time controlled action, active only under certain environmental conditions and against specific organisms, or able to reach and act on specific sites inducing changes in plant metabolism.

Research in this field is carried out by groups such as Wang et al., 2007 who formulated beta-cypermethrin nanoemulsions. They were more effective than commercial microemulsions of the same compound. The surface-functionalized silica nanoparticle (SNP) developed by Debnath et al., 2011 (about 15-30 nm) caused 90% mortality in *Sitophilus oryzae* when comparing its effectiveness with conventional silica (> 1 µm). Qian et al., 2011 achieved 2 weeks of validamycin sustained release when nano-sized calcium carbonate (nano-CC) was used. Guan et al., 2010 encapsulated imidacloprid with a coating of chitosan and sodium alginate through layer-by-layer self-assembly, increasing its speed rate in soil applications.

In the synthesis of products active only under specific environmental conditions work among others, Song et al., 2009 group, who showed that triazophos can be effectively protected from hydrolysis in acidic and neutral media by including it in a nano-emulsion, while its release was very easily achieved in alkaline media. Other examples of selective

from nuclease action. However, chitosan nanocarriers have still not replaced conventional vectors. These vehicles are often associated with low transfection efficiencies. Authors like Zhao et al., 2011 are recently working on the enhancement of this property through modification of chitosan nanocomplexes with an octapeptide. They have condensed DNA into spherical nanoparticles of around 100-200 nm size with higher transfection efficiencies and lower cytotoxicity than those of DNA complexed with unmodified chitosan. Their transfection capacity varies depending on cell type. Authors like Mao et al., 2001 when they used as a genetic vehicle chitosan nanoparticles, found higher levels of gene expression in human kidney cells and bronchial cells than in cancerous cells. Wang et al., 2011 worked with *Jatropha curcas* callus cells and demonstrated the integration of DNA carried by this

Chitosan nanoparticles are quite versatile, as well as their transfection efficiency can be modified, they can be PEGylated in order to control the release of genetic material as time goes by (Mao et al., 2001). This effect of time controlled genetic material release can be achieved by encapsulating pDNA into poly (DL-lactide-co-glycolide) particles. Specifically speaking, Cohen et al., 2000 achieved sustained release of pDNA over a month. Nonetheless the major advantage of nanobiotechnology is the simultaneous delivery of both DNA and effector molecules to specific sites. This effect has been achieved in intact tobacco and maize tissues when using gold-capped mesoporous silica nanoparticles (MSNs) as

Nanotechnology can help significantly to improve crop management techniques and it seems to be particularly useful in the use and handling of agrochemicals. Usually, only a very small amount of a given active compound is needed for treatment. Nowadays larger amount of chemicals, which are applied several times, are used in the field and nanotechnology could help to increase efficacy and to prevent losses. These losses can be explained for several reasons: UV degradation, hydrolysis, microbiota interaction or leaching. This way of handling chemicals brings negative effects to the environment (soil degradation, water pollution and side effects in other species). It is postulated that active substances in their nano form will allow to formulate more effective products, with time controlled action, active only under certain environmental conditions and against specific organisms, or able to reach and act on specific sites inducing changes in plant metabolism. Research in this field is carried out by groups such as Wang et al., 2007 who formulated beta-cypermethrin nanoemulsions. They were more effective than commercial microemulsions of the same compound. The surface-functionalized silica nanoparticle (SNP) developed by Debnath et al., 2011 (about 15-30 nm) caused 90% mortality in *Sitophilus oryzae* when comparing its effectiveness with conventional silica (> 1 µm). Qian et al., 2011 achieved 2 weeks of validamycin sustained release when nano-sized calcium carbonate (nano-CC) was used. Guan et al., 2010 encapsulated imidacloprid with a coating of chitosan and sodium alginate through layer-by-layer self-assembly, increasing its speed rate in soil

In the synthesis of products active only under specific environmental conditions work among others, Song et al., 2009 group, who showed that triazophos can be effectively protected from hydrolysis in acidic and neutral media by including it in a nano-emulsion, while its release was very easily achieved in alkaline media. Other examples of selective

type of nanoparticles in their genome.

plasmid DNA transfers (Nair et al., 2010).

**2.1.2 Production of nano-agrochemicals**

applications.

chemicals are the nanoparticles functionalized with the aim of being absorbed through insects' cuticle. The protective wax layer of insects is damaged and leads them to death by desiccation. This approach is safe for plants and entails less environmental damage. Nair et al., 2010 reported that certain nanoparticles are able to reach sap, and Corredor et al., 2009 found that they can move towards several sites in pumpkins. In other words, nanoparticles have the ability to act in sites different from their application point.

Substances in their nano form can affect cellular metabolism in a specific manner; for instance Ursache-Oprisan et al., 2011 reported an inhibition in chlorophyll biosynthesis caused by magnetic nanoparticles. But nanomaterials not only influence plants, but also animals like *Eisenia fetida* earthworms which avoid silver nanoparticles enriched soils as was evidenced by Shoults-Wilson et al., 2011.

#### **2.1.3 Alternative energy sources and nanotechnology**

One of the biggest challenges in this century is the search for new and feasible energy sources different from fuel, nuclear or hydroelectric power. Alternative energy should maintain socio-economic development without jeopardizing the environment. According to U.S. Energy Information Administration, more than 90% of the total energy produced during the first eight months of 2010 in the USA was obtained from coal, gas, nuclear, oil and wood. This energetic mix finds explanation in the fact that renewable energies (solar, wind, geothermal and tidal) are still non cost-effective.

Although, it is not the aim of this chapter to talk about the connection between energy and agriculture, it is worth to mention that agricultural sector requires important energy inputs (direct but also indirect). We think any step forward in the production; storage or use of new energy sources could be easier and faster with the help of nanotechnology. Then society will be moving towards a more sustainable, affordable and less dependent on fuel agriculture.

#### **2.1.4 Water and soil resources remediation**

Furthermore being vehicle for active substances (pesticides, plant growth regulators or fertilizers), nanoparticles can also be synthesized with a catalytic oxidation-reduction objective. The latter application would reduce the amount of these active substances present in the environment and also the time during which it is exposed to their action (Knauer & Bucheli, 2009). This section focuses on the application of reduction-oxidation catalytic nanoparticles as soil decontaminants, while the role of nanotechnology in water remediation is developed in section 2.3. Nanotechnology and water supplies.

The research focus is twofold in this issue: first, try to accelerate the degradation of residual pesticides in the soil, and secondly, improving these pollutants' detection and quantification methods.

Between those who try to accelerate the decomposition of these contaminants in soil, are Shen et al., 2007 who synthesized magnetic Fe3O4-C18 composite nanoparticles (5-10 nm) more effective than conventional C18 materials as cleaner substances of organophosphorous pesticides. Zeng et al., 2010 proved that TiO2 nanoparticles can enhance organophosphorous and carbamates' degradation rate (30% faster) in crop fields.

Currently, several authors are working in pesticide analysis methods' optimization. Magnetic composite nanoparticle-modified screen printed carbon electrodes (Gan et al.,

Nanotechnology and Food Industry 101

growing surpassing 100 million tonnes of produced meat in Asia in 2007 for instance (FAO,

In this way, nanotechnology will be able to solve these problems in the animal production sector. In order to explain this issue, we will speak of the different areas within the animal production where nanotechnology can give support and provide some important solutions. These fields can be classified within 5 categories (Kuzma, 2010) being currently all of them

This first category includes the use of nanodetectors not only to detect pathogens but to bind and remove them. An example will be the case of *S. typhi* detection in chickens skins, which uses magnetic particles functionalised with antibodies that after binding the pathogen, removes them by means of the introduction of magnetic forces. Other case is the study against foot-and mouth disease (FMD) virus in chickens, with the development of surfaces based on nanostructured gold films with topography matched to that of the size of FMDV. These surfaces are functionalized with a single chain antibody specific for FMDV in such a way that liquid crystals will uniformly anchor on these surfaces. FMDV will bind to these surfaces in such a way that it will give rise to easily visualized changes in the appearance of liquid crystals anchored on these surfaces and so virus presence is detected (Platypus

Therefore, some of the challenges the animal production sector will have to deal with are: - Look for an environmentally friendly sustainable production system that contributes to maintain environment preventing further degradation (livestock is responsible for 18 percent of global greenhouse gas emissions, (FAO, 2009) and livestock grazing occupies 26 percent of the earth's ice-free land surface), this joint to the increasing supply of meat products to a growing population (food demand is foreseen to be twice by 2050 due to the socio-economic and population growths, FAO, 2009). However, this growing population has not always higher economic resources (FAO estimates that between 2003–05, 75 million more people were added to the total number of undernourished and high food prices share part of the blame and this number increased in 2007 up to

2009).

923 million (FAO,2008).

fraud in this sector.

under research and development. - Pathogen detection and removal

**2.2.1 Pathogen detection and removal**

Technologies LLC, 2002).

exacerbate poverty being essential its minimisation.

2010), electrodes coated with multiwalled carbon nanotubes (Sundari & Manisankar, 2011) or nano TiO2 (Kumaravel & Chandrasekaran, 2011) are some examples of nanomaterials which have being successfully used. They all act as sensors of pesticides. As opposed to more conventional ones, these methods are more sensitive and selective.

## **2.1.5 Crop monitoring systems**

Nanotechnology is contributing very much in the development of sensors with applications in fields such as: agriculture, farming or food packaging. In agriculture, nano-sensors could make real-time detection of: humidity, nutrient status, temperature, pH or pesticides, pollutants and pathogens presence in air, water, soil or plants. All these collected data could help to save agro-chemicals and to reduce waste production (Baruah & Dutta, 2009; Joseph & Morrison, 2006).

## **2.1.6 Particle farming**

One of the cornerstones of nanotechnology is the synthesis of nanoparticles and their selfassembly (Gardea et al., 2002) since the methods used until now are very expensive and some of them involve the use of hazardous chemical reagents.

Alternative nanoparticles production processes are continually sought in order to make them more easily scalable and affordable. One of these routes of synthesis under study is known as "particle farming" and involves the usage of living plants or their extracts as factories of nanoparticles. This process opens up new opportunities in the recycling of wastes and could be useful in areas such as cosmetics, food or medicine.

The latest research in this field focus on the synthesis of gold and silver nanoparticles with various plants: *Medicago sativa* (Bali & Harris, 2010; Gardea et al., 2002), *Vigna radiata*, *Arachis hypogaea*, *Cyamopsis tetragonolobus*, *Zea mays* , *Pennisetum glaucum*, *Sorghum vulgare* (Rajani et al., 2010), *Brassica juncea* (Bali & Harris, 2010; Beattie & Haverkamp, 2011) or extracts from *B. juncea* and *M.sativa* (Bali & Harris, 2010), *Memecylon edule* (Elavazhagan & Arunachalam, 2011) or *Allium sativum* L. (Ahamed et al., 2011).

Depending on the nanoparticle's nature, specie of plant or tissue in which they are stored, metal nanoparticles of different shapes and sizes can be obtained. However, all these processes share the advantages of being simple, cost-effective and environmentally friendly.

Apart from the potential benefits of nanotechnology in agricultural sector (described throughout this section), it also involves some risks. Farmers' chronic exposure to nanomaterials, unknown life cycles, interactions with the biotic or abiotic environment and their possible amplified bioaccumulation effects, should be seriously considered before these applications move from laboratories to the field.

## **2.2 Nanotechnology and animal production**

Livestock contribute 40 percent of the global value of agricultural output and support the livelihoods and food security of almost a billion people (FAO, 2009). Rapidly rising incomes and urbanization, combined with underlying population growth, are driving demand for meat and other animal products in many developing countries, being the annual growth rate 0,9% in developed countries and 2,7% annual worldwide rate. In the last decade, per capita consumption in developing countries is nearly twice and this tendency keeps on

2010), electrodes coated with multiwalled carbon nanotubes (Sundari & Manisankar, 2011) or nano TiO2 (Kumaravel & Chandrasekaran, 2011) are some examples of nanomaterials which have being successfully used. They all act as sensors of pesticides. As opposed to

Nanotechnology is contributing very much in the development of sensors with applications in fields such as: agriculture, farming or food packaging. In agriculture, nano-sensors could make real-time detection of: humidity, nutrient status, temperature, pH or pesticides, pollutants and pathogens presence in air, water, soil or plants. All these collected data could help to save agro-chemicals and to reduce waste production (Baruah & Dutta, 2009; Joseph

One of the cornerstones of nanotechnology is the synthesis of nanoparticles and their selfassembly (Gardea et al., 2002) since the methods used until now are very expensive and

Alternative nanoparticles production processes are continually sought in order to make them more easily scalable and affordable. One of these routes of synthesis under study is known as "particle farming" and involves the usage of living plants or their extracts as factories of nanoparticles. This process opens up new opportunities in the recycling of

The latest research in this field focus on the synthesis of gold and silver nanoparticles with various plants: *Medicago sativa* (Bali & Harris, 2010; Gardea et al., 2002), *Vigna radiata*, *Arachis hypogaea*, *Cyamopsis tetragonolobus*, *Zea mays* , *Pennisetum glaucum*, *Sorghum vulgare* (Rajani et al., 2010), *Brassica juncea* (Bali & Harris, 2010; Beattie & Haverkamp, 2011) or extracts from *B. juncea* and *M.sativa* (Bali & Harris, 2010), *Memecylon edule* (Elavazhagan & Arunachalam,

Depending on the nanoparticle's nature, specie of plant or tissue in which they are stored, metal nanoparticles of different shapes and sizes can be obtained. However, all these processes share the advantages of being simple, cost-effective and environmentally

Apart from the potential benefits of nanotechnology in agricultural sector (described throughout this section), it also involves some risks. Farmers' chronic exposure to nanomaterials, unknown life cycles, interactions with the biotic or abiotic environment and their possible amplified bioaccumulation effects, should be seriously considered before

Livestock contribute 40 percent of the global value of agricultural output and support the livelihoods and food security of almost a billion people (FAO, 2009). Rapidly rising incomes and urbanization, combined with underlying population growth, are driving demand for meat and other animal products in many developing countries, being the annual growth rate 0,9% in developed countries and 2,7% annual worldwide rate. In the last decade, per capita consumption in developing countries is nearly twice and this tendency keeps on

more conventional ones, these methods are more sensitive and selective.

some of them involve the use of hazardous chemical reagents.

2011) or *Allium sativum* L. (Ahamed et al., 2011).

these applications move from laboratories to the field.

**2.2 Nanotechnology and animal production**

wastes and could be useful in areas such as cosmetics, food or medicine.

**2.1.5 Crop monitoring systems**

& Morrison, 2006).

friendly.

**2.1.6 Particle farming**

growing surpassing 100 million tonnes of produced meat in Asia in 2007 for instance (FAO, 2009).

Therefore, some of the challenges the animal production sector will have to deal with are:

In this way, nanotechnology will be able to solve these problems in the animal production sector. In order to explain this issue, we will speak of the different areas within the animal production where nanotechnology can give support and provide some important solutions. These fields can be classified within 5 categories (Kuzma, 2010) being currently all of them under research and development.

## **2.2.1 Pathogen detection and removal**

This first category includes the use of nanodetectors not only to detect pathogens but to bind and remove them. An example will be the case of *S. typhi* detection in chickens skins, which uses magnetic particles functionalised with antibodies that after binding the pathogen, removes them by means of the introduction of magnetic forces. Other case is the study against foot-and mouth disease (FMD) virus in chickens, with the development of surfaces based on nanostructured gold films with topography matched to that of the size of FMDV. These surfaces are functionalized with a single chain antibody specific for FMDV in such a way that liquid crystals will uniformly anchor on these surfaces. FMDV will bind to these surfaces in such a way that it will give rise to easily visualized changes in the appearance of liquid crystals anchored on these surfaces and so virus presence is detected (Platypus Technologies LLC, 2002).

Nanotechnology and Food Industry 103

Animal health and breeding would be improved as well as animal production. For instance, some kind of genetic diseases could be avoided and genetic configuration could be determined in a way that animals will be more fertile. However, before more studies about toxicity, safety and legislation must be made. Moreover, public perception and current legal framework related to genetic modification would have to change a lot, since society

Concerning traceability and identity preservation chips of DNA are being studied in order to detect genes in feed and food, likewise nanobarcodes to get feed (Jayarao et al., 2011) and animal traceability from farm to fork. This will aid to maintain animals healthy but also to improve public health, since feed traceability would help to prevent and avoid possible food related illnesses, being able to find quickly the source of such diseases and even to prevent

In conclusion, the potential benefit of these technologies would allow a diminution cost and an improvement in the animal production sector as much in quality as in quantity, the waste reduction, increasing efficiency in the use of resources and rising safety in animal derived

Possible risks related to human and animal health and also the environmental impact have to be overcome before applying of these technologies. Other matters such as exploitation rights, intellectual property and legal issues derived from the use of these inventions and

In this decade, the demand of water supplies is rapidly growing and the competition for water resources is being every day a bigger concern. Currently, 70% of the obtained fresh water in the entire world is used in agriculture, while 20% is used in the industry and 10%

The access to inexpensive and clean water sources is an overriding global challenge mainly

are devoted to municipal uses (FAO, Agotamiento de los Recursos de Agua dulce).

opposition is still rather difficult to save.

them.

due to:

food products.

patents must be considered too.

security and poverty.)

**2.3 Nanotechnology and water supplies**

**2.2.5 Identity preservation and supply-chain tracking**

The benefit of this application would allow improving human health reducing the risk of diseases derived from food consumption and allow great benets for animal health, likewise an economic advantage when producing. However, this technology is not still very developed and it is necessary studies to guarantee the lack of toxicity of these nanoparticles. Also, nanotechnology is used in the diagnosis of animal diseases control based on microfluidic, microarray, electronic and photo-electronic, integrated on-chip and nanotechnology together with analytical systems, which enable the development of pointof-care analysers (Bollo, 2007).

## **2.2.2 Veterinary medicine**

In the veterinary medicine field, nanoparticles to deliver animal drugs like growth hormones (somatotropine) in pigs through PGLA nanocapsules and the improvement in animal vaccination are two of the application examples. Among vaccination products, some polystyrene nanoparticles bound to antigens are experimentally being proved in sheep (Scheerlinck et al., 2005) and vaccines against *Salmonella enteriditis* based on an immunogenic subcellular extract obtained from whole bacteria encapsulated in nanoparticles made with the polymer Gantrez (Ochoa et al., 2007).

Animal production and food safety will be increased and this will lead to cheaper and greater availability of meat products. However, some of these measures could not be successfully applied in some countries, as it would be the case of hormones supply to animals, which is not accepted as good practice by consumers or even banned in some countries.

#### **2.2.3 Feed Improvement and waste remediation**

In the category of feed improvement and waste remediation are being studied the use of nanoparticles in feed for pathogen detection. These particles would bind pathogens in the gut of poultry preventing its colonization and growth and avoiding its presence in waste.

Other example would be the use of nanoparticles to detect chemical and microbiological contamination in feed production. Advantages of both applications are clear: Farms would be more environmentally friendly and alternatives for using of antibiotics to face pathogens will be proposed. The former option could be very interesting due to the growth of antibiotic resistance found in animals and humans. However, life cycle of these particles must be verified in order to warrant that these particles are not harmful for the animal and that they don't end up on animal-derived food products or food chain.

Toxicology must be studied and environmental exposure, likewise the effect that animal wastes containing these particles would have. In feed production case, workers safety must be studied and the effect of leftovers remaining in farms and fields.

Within this part, we can also mention the use of polymeric nanocapsules to carry bioactive compounds (such as proteins, peptides, vitamins, etc.) in such a way that they will be able to pass through the gastrointestinal mucus layer and/or epithelial tissue providing or increasing the absorption of these bioactive compounds (Luppi et al., 2008; Plapied et al., 2011), improving the availability of these nutrients and therefore, the quality of animal production.

#### **2.2.4 Animal breeding and genetics**

Genetic animal therapy is one of the possible applications in a near future. Currently, studies of genes delivery in animal cells for selection and temporal expression are being carried out in order to determine specific features of livestock (Mc Knight et al., 2003).

The benefit of this application would allow improving human health reducing the risk of diseases derived from food consumption and allow great benets for animal health, likewise an economic advantage when producing. However, this technology is not still very developed and it is necessary studies to guarantee the lack of toxicity of these nanoparticles. Also, nanotechnology is used in the diagnosis of animal diseases control based on microfluidic, microarray, electronic and photo-electronic, integrated on-chip and nanotechnology together with analytical systems, which enable the development of point-

In the veterinary medicine field, nanoparticles to deliver animal drugs like growth hormones (somatotropine) in pigs through PGLA nanocapsules and the improvement in animal vaccination are two of the application examples. Among vaccination products, some polystyrene nanoparticles bound to antigens are experimentally being proved in sheep (Scheerlinck et al., 2005) and vaccines against *Salmonella enteriditis* based on an immunogenic subcellular extract obtained from whole bacteria encapsulated in nanoparticles made with

Animal production and food safety will be increased and this will lead to cheaper and greater availability of meat products. However, some of these measures could not be successfully applied in some countries, as it would be the case of hormones supply to animals, which is not

In the category of feed improvement and waste remediation are being studied the use of nanoparticles in feed for pathogen detection. These particles would bind pathogens in the gut of poultry preventing its colonization and growth and avoiding its presence in waste. Other example would be the use of nanoparticles to detect chemical and microbiological contamination in feed production. Advantages of both applications are clear: Farms would be more environmentally friendly and alternatives for using of antibiotics to face pathogens will be proposed. The former option could be very interesting due to the growth of antibiotic resistance found in animals and humans. However, life cycle of these particles must be verified in order to warrant that these particles are not harmful for the animal and

Toxicology must be studied and environmental exposure, likewise the effect that animal wastes containing these particles would have. In feed production case, workers safety must

Within this part, we can also mention the use of polymeric nanocapsules to carry bioactive compounds (such as proteins, peptides, vitamins, etc.) in such a way that they will be able to pass through the gastrointestinal mucus layer and/or epithelial tissue providing or increasing the absorption of these bioactive compounds (Luppi et al., 2008; Plapied et al., 2011), improving the availability of these nutrients and therefore, the quality of animal production.

Genetic animal therapy is one of the possible applications in a near future. Currently, studies of genes delivery in animal cells for selection and temporal expression are being carried out in order to determine specific features of livestock (Mc Knight et al., 2003).

accepted as good practice by consumers or even banned in some countries.

that they don't end up on animal-derived food products or food chain.

be studied and the effect of leftovers remaining in farms and fields.

of-care analysers (Bollo, 2007).

the polymer Gantrez (Ochoa et al., 2007).

**2.2.4 Animal breeding and genetics**

**2.2.3 Feed Improvement and waste remediation**

**2.2.2 Veterinary medicine**

Animal health and breeding would be improved as well as animal production. For instance, some kind of genetic diseases could be avoided and genetic configuration could be determined in a way that animals will be more fertile. However, before more studies about toxicity, safety and legislation must be made. Moreover, public perception and current legal framework related to genetic modification would have to change a lot, since society opposition is still rather difficult to save.

## **2.2.5 Identity preservation and supply-chain tracking**

Concerning traceability and identity preservation chips of DNA are being studied in order to detect genes in feed and food, likewise nanobarcodes to get feed (Jayarao et al., 2011) and animal traceability from farm to fork. This will aid to maintain animals healthy but also to improve public health, since feed traceability would help to prevent and avoid possible food related illnesses, being able to find quickly the source of such diseases and even to prevent them.

In conclusion, the potential benefit of these technologies would allow a diminution cost and an improvement in the animal production sector as much in quality as in quantity, the waste reduction, increasing efficiency in the use of resources and rising safety in animal derived food products.

Possible risks related to human and animal health and also the environmental impact have to be overcome before applying of these technologies. Other matters such as exploitation rights, intellectual property and legal issues derived from the use of these inventions and patents must be considered too.

## **2.3 Nanotechnology and water supplies**

In this decade, the demand of water supplies is rapidly growing and the competition for water resources is being every day a bigger concern. Currently, 70% of the obtained fresh water in the entire world is used in agriculture, while 20% is used in the industry and 10% are devoted to municipal uses (FAO, Agotamiento de los Recursos de Agua dulce).

The access to inexpensive and clean water sources is an overriding global challenge mainly due to:

Nanotechnology and Food Industry 105

The key point is that these technologies would allow to treat processing and residual water from food industry and recycle and reuse them, contributing to the economy, environmental

This is essential in areas where water scarcity is a problem, drinkable water is difficult to

Although the operation cost of some of these installations and facilities would be higher than the operation costs of the traditional facilities, the initial investment would be cheaper (construction costs), moreover life cycle of some of these materials is unknown and these are only some of issues that must be studied before trying to implement anything, so there is

Up to now in this chapter, the nanotechnology applications have been referred to water use and commodity production but, nanotechnology can be a way to get better manufacturing food process (or even a completely new and different one). Actually, food sector could be facing a paradigm shift. As commented by Pray & Yaktine, 2009, nowadays foods are structured using a recipe (o formulation) whereas two simultaneous

water sources and if the water is polluted or not (Walter et al., 2005).

improvement and water security of local communities and industries.

still a long way to cover before some of these applications can be real facts.

obtain and water is considered as a precious resource.

**2.4 Nanotechnology in food processing**

development of new knowledge and techniques.

processes occur:

transformation …)

and there could be a more efficient water mist catching (Grimshaw, 2009).

Zodrow et al., 2009).

Universidad de Pretoria).

performance and could inactivate virus and eliminate organic matter. An example would be reverse osmosis membranes and its spacers with nanosilver coating, which would improve bacteria removal and antifouling performance (Yang at al., 2009;

rate than the population is increasing (by 2050, population will reach 9.000 million people), the planet will lose every year around 18.000000 km3 of water resources (UNESCO, 2007).

These are some of the reasons why alternatives should be thought for obtaining and recycling water and in this point is where nanotechnology could provide innovative solutions.

Currently, there are several nanotechnology research lines concerning water treatment. Some of these examples are mentioned next:

These are some of the reasons why alternatives should be thought for obtaining and recycling water and in this point is where nanotechnology could provide innovative

Currently, there are several nanotechnology research lines concerning water treatment.

industrial animal production systems connected to cities.

can adsorb ionic components from water (Oren, 2008).

distribution, connexion and storage systems. (Brame et al., 2011).

Some of these examples are mentioned next:

magnet (Mayo et al., 2007; Wolfe, 2006).

al., 2005; Senthilnathan & Ligy, 2010).

(UNESCO, 2007).

traditional sources.

solutions.

rate than the population is increasing (by 2050, population will reach 9.000 million people), the planet will lose every year around 18.000000 km3 of water resources performance and could inactivate virus and eliminate organic matter. An example would be reverse osmosis membranes and its spacers with nanosilver coating, which would improve bacteria removal and antifouling performance (Yang at al., 2009; Zodrow et al., 2009).

The key point is that these technologies would allow to treat processing and residual water from food industry and recycle and reuse them, contributing to the economy, environmental improvement and water security of local communities and industries.

This is essential in areas where water scarcity is a problem, drinkable water is difficult to obtain and water is considered as a precious resource.

Although the operation cost of some of these installations and facilities would be higher than the operation costs of the traditional facilities, the initial investment would be cheaper (construction costs), moreover life cycle of some of these materials is unknown and these are only some of issues that must be studied before trying to implement anything, so there is still a long way to cover before some of these applications can be real facts.

## **2.4 Nanotechnology in food processing**

Up to now in this chapter, the nanotechnology applications have been referred to water use and commodity production but, nanotechnology can be a way to get better manufacturing food process (or even a completely new and different one). Actually, food sector could be facing a paradigm shift. As commented by Pray & Yaktine, 2009, nowadays foods are structured using a recipe (o formulation) whereas two simultaneous processes occur: