MAJOR ENZYME APPLICATIONS IN FOOD INDUSTRY
Enzymes are produced by all living cells as catalysts for specific chemical reactions. Not surprisingly enzymes are present in all foods at some time, and play an increasingly important role in food processing techniques. Enzymes, although not recognized as such, have played an essential part in some food processes, notably the making of cheese, leavened bread, wine and beer, for thousands of years (Dewdney, 1973).
II. Major Enzyme Applications in Food Industry
In food industry, enzyme has been used to produce and to increase the quality and the diversity of food. Some examples of products that use enzyme are cheese, yoghurt, bread syrup etc. Ancient traditional arts such as brewing, cheese making, meat tenderization with papaya leaves and condiment preparation (e.g., soy sauce and fish sauce) rely on proteolysis, albeit the methods were developed prior to our knowledge of enzymes. Early food processes involving proteolysis were normally the inadvertent consequence of endogenous or microbial enzyme activity in the foodstuff. Some major applications by types of enzymes are:
The use of rennet in cheese manufacture was among the earliest applications of exogenous enzymes in food processing, dating back to approximately 6000 B C. In 1994, the total production of cheese was 8000 metric tons against a total demand of 9000 metric tons. The projected demand by 2000 A D is around 30,000 metric tons. The use of rennet, as an exogenous enzyme, in cheese manufacture is perhaps the largest single application of enzymes in food processing. In recent years, proteinases have found additional applications in dairy technology, for example in acceleration of cheese ripening, modification of functional properties and preparation of dietic products (IDF, 1990).
Picture 1. Chymosin crystal structure (www.fst.rdg.ac.uk., 2002)
Animal rennet (bovine chymosin) is conventionally used as a milk-clotting agent in dairy industry for the manufacture of quality cheeses with good flavor and texture. Owing to an increase in demand for cheese production world wide – i.e. 4% per annum over the past 20 years, approximating 13.533 million tons (ref. 3) – coupled with reduced supply of calf rennet, has therefore led to a search for rennet substitutes, such as microbial rennet. At present, microbial rennet is used for one-third of all the cheese produced worldwide. Rennin acts on the milk protein in two stages, by enzymatic and by nonenzymatic action, resulting in coagulation of milk. In the enzymatic phase, the resultant milk becomes a gel due to the influence of calcium ions and the temperature used in the process (Bhoopathy, 1994). Many microorganisms are known to produce rennet-like proteinases which can substitute the calf rennet. Microorganisms like Rhizomucor pusillus, R. miehei, Endothia parasitica, Aspergillus oryzae, and Irpex lactis are used extensively for rennet production in cheese manufacture. Extensive research that has been carried out so far on rennet substitutes has been reviewed by several authors (Green, M. L., 1977 Fox, P. F.1993; Farkye, N. Y., 1995).
Different strains of species of Mucor are often used for the production of microbial rennets. Whereas best yields of the milk-clotting protease from Rhizomucor pusillus are obtained from semisolid cultures containing 50% wheat bran, R. miehei and Endothia parasitica are well suited for submerged cultivation. Using the former, good yields of milk-clotting protease may be obtained in a medium containing 4% potato starch, 3% soybean meal, and 10% barley. During growth, lipase is secreted together with the protease. Therefore, the lipase activity has to be destroyed by reducing the pH, before the preparation can be used as cheese rennet.
Lactose, the sugar found in milk and whey, and its corresponding hydrolase, lactase or b-galactosidase, have been extensively researched during the past decade (Mehaiya, 1987). This is because of the enzyme immobilization technique which has given new and interesting possibilities for the utilization of this sugar. Because of intestinal enzyme insufficiency, some individuals, and even a population, show lactose intolerance and difficulty in consuming milk and dairy products. Hence, low-lactose or lactose-free food aid programme is essential for lactose-intolerant people to prevent severe tissue dehydration, diarrhoea, and, at times, even death. Another advantage of lactase-treated milk is the increased sweetness of the resultant milk, thereby avoiding the requirement for addition of sugars in the manufacture of flavored milk drinks. Manufacturers of ice cream, yoghurt and frozen desserts use lactase to improve scoop and creaminess, sweetness, and digestibility, and to reduce sandiness due to crystallization of lactose in concentrated preparations. Cheese manufactured from hydrolyzed milk ripens more quickly than the cheese manufactured from normal milk.
Technologically, lactose crystallizes easily which sets limits to certain processes in the dairy industry, and the use of lactase to overcome this problem has not reached its fullest potential because of the associated high costs. Moreover, the main problem associated with discharging large quantities of cheese whey is that it pollutes the environment. But, the discharged whey could be exploited as an alternate cheap source of lactose for the production of lactic acid by fermentation. The whey permeate, which is a by-product in the manufacture of whey protein concentrates, by ultrafiltration could be fermented efficiently by Lactobacillus bulgaricus (Mehaiya, 1987)
Picture 3. Lactase crystal structure (Pitman, S. D., 2004)
Lactose can be obtained from various sources like plants, animal organs, bacteria, yeasts (intracellular enzyme), or molds. Some of these sources are used for commercial enzyme preparations. Lactase preparations from A. niger, A. oryzae, and Kluyveromyces lactis are considered safe because these sources already have a history of safe use and have been subjected to numerous safety tests. The most investigated E. coli lactase is not used in food processing because of its cost and toxicity problems.
The properties of the enzyme depend on its source. Temperature and pH optima differ from source to source and with the type of particular commercial preparation. Immobilization of the enzymes, method of immobilization, and type of carrier can also influence these optima values. In general, fungal lactase have pH optima in the acidic range 2.5–4.5, and yeast and bacterial lactases in the neutral region 6–7 and 6.5–7.5, respectively. The variation in pH optima of lactases makes them suitable for specific applications, for example fungal lactases are used for acid whey hydrolysis, while yeast and bacterial lactases are suitable for milk (pH 6.6) and sweet whey (pH 6.1) hydrolysis. Product inhibition, e.g. inhibition by galactose, is another property which also depends on the source of lactase. The enzyme from A. niger is more strongly inhibited by galactose than that from A. oryzae. This inhibition can be overcome by hydrolyzing lactose at low concentrations by using immobilized enzyme systems or by recovering the enzyme using ultrafiltration after batch hydrolysis. Lactases from Bacillus species are superior with respect to thermostability, pH operation range, product inhibition, and sensitivity against high-substrate concentration. Thermostable enzymes, able to retain their activity at 60°C or above for prolonged periods, have two distinct advantages viz. they give higher conversion rate or shorter residence time for a given conversion rate, and the process is less prone to microbial contamination due to higher operating temperature. Bacillus species have a pH optima of 6.8 and temperature optima of 65°C. Its high activity for skim milk and less inhibition by galactose has made it suitable for use as a production organism for lactase (Gekas, V. and Lopez-Levia, M., 1985).
The enzymatic hydrolysis of lactose can be achieved either by free enzymes, usually in batch fermentation process, or by immobilized enzymes or even by immobilized whole cells producing intracellular enzyme. Although numerous hydrolysis systems have been investigated, only few of them have been scaled up with success and even fewer have been applied at an industrial or semi-industrial level. Several acid hydrolysis systems have been developed to industry-scale level. Large-scale systems which use free enzyme process have been developed for processing of UHT-milk and processing of whey, using K. lactis lactase (Maxilact, Lactozyme).
Several commercial immobilized systems have been developed for commercial exploitation. Snamprogretti process of industrial-scale milk processing technology in Italy is one such working system. They make use of fibre-entrapped yeast lactase in a batch process, and the milk used is previously sterilized by UHT. For pilot plants, there are three other processes designed and developed to handle milk; (i) by Gist-Brocades, Rohm GmbH (Germany), and (ii) by Sumitomo, Japan. These are continuous processes with short residence times. Processing of whey UF-permeate is accomplished by the system developed by Corning Glass, Connecticut, Lehigh, Valio and Amerace corp. The process by Corning Glass is being applied at commercial scale in the bakers yeast production using hydrolyzed- whey (Gekas, V. and Lopez-Levia, M., 1985).
Catalase is an enzyme that can be produced from bovine livers or microbial sources. It is used to change hydrogen peroxide to become water and oxygen molecules. This enzyme can be used in a limited amount in cheese production. Catalase is the enzyme that breaks down hydrogen peroxide to water and molecular oxygen. Catalase effectively removes the residual hydrogen peroxide, ensuring that the fabric is peroxide-free and mainly used in food industry and also in egg processing with other enzymes. Catalase is a common enzyme found in nearly all living organisms which are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen (Chelikani P, Fita I, Loewen PC, 2004).
Picture 2. Catalase crystal structure in beef (green) (Mary Maj, 2009)
Glucose oxidase and catalase are often used together in selected foods for preservation. Superoxide dismutase is an antioxidant for foods and generates H2O2, but is more effective when catalase is present. Thermally induced generation of volatile sulphydryl groups is thought to be responsible for the cooked off-flavour in ultra high temperature (UHT) processed milk. Use of sulphydryl oxidase under aseptic conditions can eliminate this defect. The natural inhibitory mechanism in raw milk is due to the presence of low levels of lactoperoxidase (LP), which can be activated by the external addition of traces of H2O2 and thiocyanate. It has been reported that the potential of LP-system and its activation enhances the keeping quality of milk, (Muir, D. D., 1996)
A lipase is a water –soluble enzyme that catalyzes the hydrolysis of ester bonds in water-insoluble, lipid substrates (Svendsen, 2000). Lipases (triacylglycerol acylhydrolases) are produced by microorganism in individual or together with esterase. Microorganisms that produce lipases are Pseudomonas aeruginosa, Serratia marcescens, Staphylocococcus aureus dan Bacillus subtilis. Lipase is used as biocatalyst to produce free fatty acid, glycerol and various esters, part of glycerides and fat that is modified or esterified from cheap substrate i.e. palm oil. Those products are extensively used in pharmacy, chemical and food industry.
Picture 4. Lipase crystal structure (Berman, H. M., J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, and P. E. Bourne, 2000)
Various animal or microbial lipases gave pronounced cheese flavor, low bitterness and strong rancidity, while lipases in combination with proteinases and/or peptidases give good cheese flavor with low levels of bitterness. In a more balanced approach to the acceleration of cheese ripening using mixtures of proteinases and peptidases, attenuated starter cells or cell-free extracts (CFE) are being favored (Wilkinson, M. G., 1995)
The proteolytic system of lactic acid bacteria is essential for their growth in milk, and contributes significantly to flavor development in fermented milk products. The proteolytic system is composed of proteinases which initially cleaves the milk protein to peptides; peptidases which cleave the peptides to small peptides and amino acids; and transport system responsible for cellular uptake of small peptides and amino acids. Lactic acid bacteria have a complex proteolytic system capable of converting milk casein to the free amino acids and peptides necessary for their growth. These proteinases include extracellular proteinases, endopeptidases, aminopeptidases, tripeptidases, and proline-specific peptidases, which are all serine proteases. Apart from lactic streptococcal proteinases, several other proteinases from nonlactostreptococcal origin have been reported. There are also serine type of proteinases, e.g. proteinases from Lactobacillus acidophillus, L. plantarum, L. delbrueckii sp. bulgaricus, L. lactis, and L. helveticus. Aminopeptidases are important for the development of flavor in fermented milk products, since they are capable of releasing single amino acid residues from oligopeptides formed by extracellular proteinase activity (Law, J., And Haandrikman, A., 1997)
Picture 5. Protease crystal structure (www.proteases.org, 2009)
They can be derived from bacteria and fungi. They play a major role in the food and beverages (baking), brewing, starch, sugar industries. Amylase is used to hydrolyze amilum into a product that is water soluble and has low molecular weight: glucose. This enzyme is used extensively in drink industry for example the production of High Fructose Syrup (HFS) or in textile industry. Amylases can be made from various microorganisms especially from Bacillus, Pseudomonas and Clostridium family. Potential bacteria that are recently used to produce amylases in industrial scale are Bacillus licheniformis and B.stearothermophillus. It is preferable to use B.stearothermophillus because it can produce enzyme that is thermo stable so that can reduce production cost.
Alpha amylases have significant effects on baked goods. If the content is low, this leads to low dextrin production and poor gas production. This in turn results in inferior quality bread with reduced size and poor crust color. To compensate for the deficiencies of the grain, it is necessary to add either sugar or alpha amylase. The addition of enzymes offers certain advantages over sugar. At a flour mill, it is possible to standardize the enzyme content of the flour so that a uniform commodity can be supplied. Furthermore, enzymes bring about a gradual formation of sugar, which matches the needs of the yeast. When the dough is placed in the oven, the steadily increasing temperature leads to an increase in the enzymes’ rate of reaction and more sugar is produced. Malt flour and malt extract can be used as enzyme supplements as malt is rich in alpha amylases. However, it is better to use a fungal alpha amylase.
Picture 6. Alpha-amylase crystal structure (www.polyenzyme.com, 2009)
The alpha-amylases degrade the damaged starch in wheat flour into small dextrins, thus allowing yeast to work continuously during dough fermentation, proofing and the early stage of baking. This results in improved bread volume and crumb texture. In addition, the small oligosaccharides and sugars such as glucose and maltose produced by these enzymes enhance the reactions for the browning of the crust and baked flavour. Cereal beta-amylases are perhaps best known in terms of the vital role they play in releasing easily fermentable sugars from cereal grain starch to fuel the production of alcohol by yeast in brewing. The extent to which they have been investigated is indeed largely due to their significance in this economically important industry. However, cereal beta-amylases are also, or could be, employed in many other aspects of the food industry and the analysis of starch, and they constitute valuable markers in cereal assessment and breeding studies (Ziegler, 1999).
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