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In this article we will discuss about:- 1. Introduction to Utilization of Enzymes in Food Industry 2. Techniques for Immobilized Enzymes in Food Industry 3. Application of Immobilized Enzyme.
Introduction to Utilization of Enzymes in Food Industry:
Enzyme technology determines the future perspectives of food industry, enzymes are biocatalyst in food industry. Enzymes have found widespread applications in processing and production of all kinds of food products.
In food production enzymes have number of advantages:
(i) Enzymes can modify and improve the functional, sensory properties of ingredients and products.
(ii) Protein engineering may also be used to alter the substrate specificity of an enzyme. These alterations may confer highly desirable features for enzymes used in food processing applications.
(iii) Enzymes are used as alternatives to traditional chemical based technology. Enzymes can thus replace synthetic chemicals in a wide range of processes.
(iv) Enzymes mediated processes achieved lowering of energy consumption and contribute biodegradability of products.
(v) Enzymes are highly specific in their action than chemical reactants. Specificity of reaction including substrate specificity, positional specificity and stereo-specificity.
(vi) Enzyme catalyzed processes have fewer side reaction and waste products; hence it results high quality products and less pollution.
(vii) Eliminates the need to use organic solvents in processing.
(viii) Reduces number of process steps required.
(ix) Enzymes can catalyze reactions under very mild conditions, e.g., temperature, pH, sterility, etc., hence it preserves valuable attributes of food and food components.
(x) Enzymes can be modified according to the specific need for food industries. Introduction of a single disulfide bond or substitution of a specific amino acid can increase stability of an enzyme to elevated temperature, pH, oxidizing agents or proteolytic degradation.
(xi) Enzymes allows process to be carried out which would be impossible by other means.
(xii) Enzymes can be immobilized to a surface of a membrane or other inert object in contact with the food being processed.
The first commercial food product produced by biotechnology was an enzyme used in cheese making. Prior to Biotech techniques enzymes had to be extracted from the stomach of calves, lambs and baby goats. But now it is produced by microorganisms that were given the gene for the enzymes. The food industry uses more than 55 different enzymes products in food processing.
Some of the most important food industries which utilizing the benefits of enzymes are given below:
The proteases ‘papain’ (from papaya) or ‘bromelain’ (from pineapple), are used to tenderize meat by hydrolyzing peptide bonds. It is also used to stabilize chill proof beer.
2. Enzymes Used in the Dairy Industry:
In the dairy industry, some enzymes are required for the production of cheeses, yogurt and other dairy products, while others are used in a more specialized fashion to improve texture or flavor.
Six of the most common types of enzymes and their role in the dairy industry are described below:
Milk contains proteins, specifically caseins, which maintain its liquid form. Proteases are enzymes that are added to milk during cheese production, to hydrolyze caseins. Rennet and rennin are general terms for any enzyme used to coagulate milk. The most common enzyme isolated from rennet is chymosin.
Chymosin can also be obtained from several other animal, microbial or vegetable sources, but indigenous microbial chymosin (from fungi or bacteria) is ineffective for making cheddar and other hard cheeses. ‘Renin’ obtained from the calf stomach is used in the manufacture of cheese, since it converts calcium-casein of the milk to calcium- paracaseinate, which is curd like in appearance.
The curd is solidified and processed as cheese after inoculation with an appropriate mixture of microorganisms. Limited supplies of calf rennet have prompted genetic engineering of microbial chymosin by cloning calf prochymosin genes into bacteria. Bioengineered chymosin may be involved in production of up to 70% of cheese products.
Milk contains a number of different types of proteins, in addition to the caseins. Cow milk also contains whey proteins such as lactalbumin and lactoglobulin. The denaturing of these whey proteins, using proteases, results in a creamier yogurt product. Destruction of whey proteins is also essential for cheese production. During production of soft cheeses, whey is separated from the milk after curdling and may be sold as a nutrient supplement for body building, weight loss and lowing blood pressure, among other things.
There have even been reports of dietary whey for cancer therapies and having a role in the induction of insulin production for those with Type 2 diabetes. Proteases are used to produce hydrolyzed whey protein, which is whey protein broken down into shorter polypeptide sequences. Hydrolyzed whey is less likely to cause allergic reactions and is used to prepare supplements for infant formulas and medical uses.
Lactase is milk sugar lactose degrading enzyme.
The enzyme catalase has found limited use in one particular area of cheese production. Hydrogen peroxide is a potent oxidizer and toxic to cells. It is used instead of pasteurization, when making certain cheeses such as Swiss, in order to preserve natural milk enzymes that are beneficial to the end product and flavor development of the cheese. These enzymes would be destroyed by the high heat of pasteurization.
However, residues of hydrogen peroxide in the milk will inhibit the bacterial cultures that are required for the actual cheese production, so all traces of it must be removed. Catalase enzymes are typically obtained from bovine livers or microbial sources and are added to convert the hydrogen peroxide to water and molecular oxygen.
(e) Lipases:
Stronger flavored cheeses, for example, the Italian cheese, Romano, are prepared using lipases. Hydrolysis of the shorter fats by lipases is preferred because it results in the desirable taste of many cheeses.
(f) Glycoside Hydrolase:
Glycoside hydrolases (also called glycosidases) catalyze the hydrolysis of the glycosidic linkage present in sugars.
Enzymes from yeast are used for alcoholic fermentation in beverage industry, since they convert sugars to alcohol and CO2.
‘Pectinases’ are often added in canned fruit juices and in wine, since they hydrolyze the pectin making the juice or wine clear.
‘Invertase’ is used in the manufacture of chocolate covered berries and other such candies.
‘Glucose isomerase’ is used for the production of fructose and high fructose syrups from hydrolysed maize starch, to be used in soft drinks.
‘Lactase’ is used for the prevention of lactose crystals in ice-cream.
8. Lipases and Hydrolases:
i. Lipases:
One of the most important enzymes used in food industry is lipases. It is a water soluble enzymes which participating the hydrolysis of long chain fatty acid into simpler subunits. Lipases are used to break down milk fats and give characteristic flavors to cheeses. The flavor comes from the free fatty acids produced when milk fats are hydrolyzed.
Under appropriate condition, alkaline pH lipases will hydrolyses long chain fatty acid and triglycerides into disaccharides and short chain fatty acids. Disaccharides are then converted to monglycerides and fatty acid, later it hydrolyzed to form free glycerol and fatty acid chains.
Types of Lipases:
a. Animal lipases:
Animal lipases are obtained from kid, calf and lamb.
b. Microbial lipases:
Microbial lipases produced from bacteria and fungi.
(i) Bacterial lipase – It is produced by Arthrobacter sps, Chromobacterium sp. Pseudomonas sps, etc.
(ii) Fungal lipase – Fungal lipases are the products of Mucor meihei, Aspergillus niger, Rhizopus arrhizus and Rhizopus japonicus.
ii. Hydrolases:
The most important hydrolases are used in food industries are referred as glycoside hydrolases (also called glycosidases). This enzyme catalyzes the hydrolysis of the glycosidic linkage to generate two smaller sugars. They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose and hemicellulose, in anti-bacterial defense strategies (e.g., lysozyme), in pathogenesis mechanisms (e.g., viral neuraminidases) and in normal cellular function. Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.
The important types of glycoside hydrolases are:
(a) Amylases:
Amylases are starch processing enzymes.
Types of Amylases:
Three classes of starch-degrading enzymes are used commercially in large amounts:
(i) α-amylase (α-1, 4, glucan-4-glucano hydrolase):
This enzyme is one of the most important enzymes of industrial use. α-amylase enzyme hydrolyses internal α-1, 4 linkage and by pass α-1, 6 linkages to produce glucose, maltose and maltotriose.
(ii) Glucoamylase (amyloglucosidase or α-1, 4 glucan glycohydrolase):
Glucoamylase hydrolyses α-1, 4 and α-1, 6 bonds to produce glucose as the major product of hydrolysis.
(iii) Pullulanase:
This enzyme hydrolyses the α -1, 6 linkages of starch polymer.
(b) Lactases:
Lactase is a glycoside hydrolase enzyme that cuts lactose into its constituent sugars, galactose and glucose. Without sufficient production of lactase enzyme in the small intestine, human become lactose intolerant, resulting in discomfort (cramps, gas and diarrhea) in the digestive tract upon ingestion of milk products.
Lactase is used commercially to prepare lactose-free products, particularly milk, for such individuals. It is also used in preparation of ice cream, to make a creamier and sweeter-tasting product. Lactase is usually prepared from Kluyveromyces sp. of yeast and Aspergillus sp. of fungi.
(c) Invertase (Sucrase):
Invertase (systematic name – β-fructofuranosidase) is a sucrase enzyme. It catalyzes the hydrolysis (breakdown) of sucrose (table sugar) to fructose and glucose, usually in the form of inverted sugar syrup. Invertase is a yeast-derived enzyme. Invertase splits sucrose into glucose and fructose (invert syrup) and can be applied for any inversion of sucrose especially liquefied cherry centers, creams, mints, truffles, marshmallow, invert syrup and other fondants. Invertase is used to improve shelf life of confections.
Invertase is mainly used in the food (confectionary) industry where fructose is preferred over sucrose because it is sweeter and does not crystallize as easily. However, the use of invertase is rather limited because another enzyme, glucose isomerase, can be used to convert glucose to fructose more inexpensively. For health and taste reasons, its use in food industry requires that invertase be highly purified. Optimum temperature at which the rate of reaction is at its greatest is 60°C and an optimum pH of 4. 5.
Techniques for Immobilized Enzymes in Food Industry:
The immobilization of enzymes is an emerging technology that involves the fixing of an enzyme within or onto an insoluble matrix. The preparation of an immobilized enzyme involves an enzyme, a support material and a method of immobilization. Immobilizations often stabilize structure of the enzymes, thereby allowing their applications even under harsh environmental conditions of pH, temperature and organic solvents and thus enable their uses at high temperature in no aqueous enzymology.
Large number of techniques and supports are now available for the immobilization of enzymes on a variety of natural and synthetic supports. The choice of the support as well as the technique depends on the nature of the enzyme, nature of the substrate and its ultimate application.
Techniques for immobilization have been broadly classified into four categories, namely:
1. Entrapment:
The entrapment technique is the entrapment in a gel matrix made from polyacrylamide, sephadox or agar. Biocatalysts have been entrapped in natural polymers like agar, agarose and gelatin through thermo reversal polymerization. A number of synthetic polymers such as the photo-cross linkable resins, polyurethane prepolymers and acrylic polymers like polyacrylamide.
Among these, the most widely used one is the polyacrylamide gel. Polyacrylamide may not be a useful support for use in food industry because of its toxicity, but can have potentials in the treatment of waste and in the fabrication of analytical devices containing biocatalysts. Recently, the development of hydrogels and thermoreactive water-soluble polymers, like the albumin- poly (ethylene glycol) hydrogel, has attracted attention in the field of biotechnology.
It is the covalent attachment of enzyme to insoluble support material such as metals glass, ceramics, nylon, cellulose, sephardose or sephadex. Enzymes are covalently linked to the support through the functional groups in the enzymes (amino, carboxyl and the phenolic groups), which are not essential for the catalytic activity.
The enzyme could then be covalently linked to a support containing an alkyl amine group through Schiffs base reaction. Enzymes like glucose oxidase, peroxidase, invertase, etc. have been immobilized using this technique. Enzymes have also been bound to synthetic membranes are used in the biconversion, downstream processing and food technology.
3. Cross-Linking:
Biocatalysts can also be immobilized through chemical cross- linking using homo as well as hetero bifunctional cross-linking agents. Among these, glutaraldehyde which interacts with the amino groups through a base reaction has been extensively used in view of its GRAS status, low cost, high efficiency and stability. The enzymes have been normally cross-linked in the presence of an inert protein like gelatine, albumin and collagen.
Adsorption of enzyme on an insoluble matrix by hydrophobic, electrostatic or other non-covalent affinity methods. This is the simplest technique of immobilization. In case of enzymes immobilized through ionic interactions, adsorption and desorption of the enzyme depends on the basicity of the ion exchanger.
A dynamic equilibrium is normally observed between the adsorbed enzyme and the support which is often affected by pH as well as the ionic strength of the surrounding medium. Enzymes with low pi like invertase, urease, glucose oxidase, catalase and other enzymes have been bound through adsorption followed by cross-linking on polyethylenimine-coated supports.
Another method for adsorption is by hydrophobic interaction. Hydrophobic interactions are usually stabilized by high ionic concentrations, thus enabling the use of high concentrations of substrates as desired in an industrial process.
Factors Influencing Immobilization Techniques:
The most important factors influencing immobilization techniques are:
(1) Stability of the enzyme during repeated use,
(2) Retention activity of the enzyme following immobilization,
(3) Stability of the matrix, flow, pressure, temperature and other conditions,
(4) Accessibility of the substrate to the enzyme,
(5) Efficiency of converting substrate to product,
(6) Compatibility of the system with subsequent steps in conversion and recovery of products,
(7) Need for cofactors,
(8) Compatibility of the pure enzyme, crude enzyme to be immobilized (9) susceptibility to microbial contamination,
(10) Renewal efficiency and,
(11) Cost.
Application of Immobilized Enzyme in Food Industry:
The most important application of immobilized enzymes in food industry is the conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase. Another important application of immobilized enzyme is the production of aspartic used acid. L-aspartic acid is widely used in medicines and as a food additive.
The enzyme aspartase catalyses the addition of ammonia to the double bond of fumaric acid to produce aspartic acid. Enzymes are immobilized biocatalysts in dairy industry is used in the preparation of lactose-hydrolyzed milk and whey, using beta galactosidase. A large population of lactose intolerants can consume lactose-hydrolyzed milk.
Lactose-hydrolyzed whey may be used as a component of whey-based beverages, leavening agents, feed stuffs, or may be fermented to produce ethanol and yeast, thus converting an inexpensive byproduct into a highly nutritious, good quality food ingredient.
Another application of immobilized enzymes in pharmaceutical industry is the production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either penicillin G or V, using penicillin acylase (penicillin amidase). Immobilized oxidoreductases are important in biotechnology to carry out synthetic transformations.
Immobilized glucose oxidase can find application in the production of gluconic acid, removal of oxygen from beverages and in the removal of glucose from eggs prior to dehydration in order to prevent Maillard reaction. A variety of biologically active peptides are gaining importance in various fields including in pharmaceutical industries and in food industries as sweeteners, flavorings, antioxidants and nutritional supplements.
Proteolytic enzymes, such as subtilisin, a-chymotrypsin, papain, ficin or bromelain, which have been immobilized by covalent binding, adsorption or cross-linking to polymeric supports are used for the production of various flavoring compounds.
Plasteins are insoluble polypeptide formed through the random condensation of amino acids or peptides under the catalytic influence of a proteinase like chymotrypsin. The plastein technology provides possible future use of immobilized enzymes in food technology.
Since the plastein reaction can be used to covalently bind aminoacid esters into proteins, food proteins could be nutritionally improved by the incorporation of selected essential amino acids, e.g., soyabean protein fortified with methionine had a protein efficiency ratio (PER) greater than casien, milk protein. Similarly the functionality of food proteins can be modified by increasing the solubility of denatured soy protein by the incorporation of glutamic acid.
Immobilized enzymes can be used as effective antioxidants. The important antioxidants are superoxide dismutase (SOD), catalase and glutathione peroxidase.
Other Applications in Food Industry:
Immobilized enzymes permits repeated use of the same enzyme, thus reducing the cost per analysis. The immobilized enzyme can make the assay faster and easier. There are at least four possible arrangements that work well. These are immobilized enzyme column, enzyme electrodes, enzyme containing chips and enzyme immunosorbent assay (ELISA) type systems.
Generation of Flavor and Aroma Compounds by Enzymes:
Enzymes Used as Flavanoids:
Flavor is usually the result of the presence of many volatile and nonvolatile components possessing diverse chemical and physicochemical properties. The nonvolatile compounds contribute mainly to the taste while the volatile compounds influence both taste and aroma.
There are lots of compounds responsible for the aroma of the food products including as alcohols, aldehydes, esters, dicarbonyls, short to medium-chain free fatty acids, methyl ketones, lactones, phenolic compounds and sulphur compounds. Although flavors may be produced by chemical transformation of natural substances, the resulting products cannot be legally labeled as natural substance.
Also, chemical synthesis often results in environmentally unfriendly production processes and lack substrate selectivity, which may cause the formation of undesirable racemic mixtures, thus reducing process efficiency and increasing downstream costs. But the same time flavor substances have been extracted from plant sources. The production of natural flavors by direct extraction from plants is also subject to various problems.
These raw materials contain low concentrations of the desired flavor compounds, making the extraction expensive. Moreover, their use depends on factors difficult to control such as weather conditions and plant diseases. The disadvantages of both methods are replaced with the use of microbial enzymes by fermentation reaction.
Microorganisms can synthesize flavors as secondary metabolites during fermentation on nutrients such as sugars and amino acids. The use of enzyme-catalyzed reactions solves many problems of the chemical synthesis of enzymes, due to the substrate specificity, regio and enantioselectivity of these biocatalysts, which can be utilized at mild reaction conditions. By choosing the suitable enzymes, enantiomerically pure flavor compounds can be obtained, thus increasing process efficiency and lowering downstream costs. Moreover, flavors obtained through biocatalysis can be considered as natural products.
The most commonly used enzyme applications for the generation of flavor and aroma compounds are described below:
One of the most important applications of enzyme technology in the food aroma field is the use of reversed lipolysis in low water-content systems in order to carry out esterification or transesterification reactions for the production of esters from inexpensive raw materials (i.e. fatty acids and alcohols).
A number of lipases have been isolated from the organisms such as Candida cylindracea, Pseudomonas fluorescens, Mucor miehei, Aspergillus sp., Rhizopus arrhizus and Candida rugosa. These lipases are highly active and the influence of variables such as reagent or enzyme characteristics and concentrations, temperature, water content or solvent properties have been thoroughly studied in many cases.
Lipases are usually highly specific, which makes esterification between carboxylic acids and alcohols dependent on alcohol and/or acid chain length. Also the enantiomeric selectivity of the enzymes can be an important feature for the synthesis of food aromas. For example – only the (S)-form of 2-methylbutanoic acid methyl ester (main flavor component of apple or strawberry) has the characteristic fruit flavor.
The ability of a number of lipases to catalyze enantiomeric selective synthesis of (S)-2-methylbutanoic acid methyl ester, are found good results for lipases from Rhizomucor miehei, Aspergillus niger and Aspergillus javanicus. Also, Candida rugosa lipase is catalyzed the esterification of L-menthol with long-chain unsaturated fatty acids in a solvent-free system create strong flavor.
Ester synthesis reactions by lipases are carried out in low-water content media. The most commonly used systems are organic solvents such as n-hexane, n-heptane, cyclohexane or isooctane. Generally highly hydrophobic solvents are preferred because they do not penetrate the water layer surrounding the enzyme surface so favors maintenance of its active stereoconfiguration.
2. Proteases:
Protease in the form of short oligopeptides contributes significant role in the sensory appreciation of food especially the four basic taste sensations (sweet, bitter, sour and salty). Peptides can be obtained by protease-mediated hydrolysis of proteins. Protease-catalysed hydrolysis in some food items increase in the concentration of benzaldehyde and pyrazines, which contribute some kinds of desirable aroma and flavors.
Peptides can be synthesized by recombinant DNA technology, chemical synthesis and enzymatic synthesis, depending on the desired size (large, intermediate and short sequences, respectively). The majority of commercially available proteases (metallo-, endo-, exo-, serine and aspartate) may be used for various kinds of aroma generation. Immobilized and chemically modified enzymes have also been proposed for the flavor generation.
Glucosidases can also be used to synthesize glycosides as aroma compounds. Glucosides are adequate derivatives as flavors, due to their very low vapor pressures and the possibility of obtaining them as natural compounds. Glucosidases can be used to enhance the aroma of some wines by freeing glycosidically bound volatile terpenes and flavor precursors. Terpenols in grapes are mostly found in glycosidically bound forms which are odourless.
The addition of exogenous enzymes like beta-glucosidase during the fermentation has been found to be the most effective way to improve the hydrolysis of the aroma precursor compounds and achieve an increase in wine flavor; β-glucosidases have been isolated from numerous sources including Saccharomyces, Aspergillus and Candida.
4. Other Enzymes:
Some other enzymes have been mentioned for their potential applicability in the production of flavor compounds.
They are listed below:
(i) Immobilised Alcohol Dehydrogenase:
The use of an immobilised alcohol dehydrogenase from Lactobacillus to synthesize phenylethanol from acetophenone in an organic solvent (hexane).
(ii) Hydroxylase:
Hydroxylation of sesquiterpenes by hydroxylase, an enzyme of the cytochrome monooxygenase generates a grapefruit flavor compound.
(iii) Amine Oxidase:
Enzymatic synthesis of vanillin from vanillylamine using amine oxidase from Aspergillus niger has been reported. Vanillylamine can be isolated from capsaicin, a natural ingredient of peppers and capsicums.
(iv) Glutaminase:
The use of microbial glutaminases in food industry leads to the synthesis of L-glutamic acid, the main compound responsible for the unique flavor of fermented soy sauce.
Esters are common in organic chemistry and biological materials and often have a characteristic pleasant, fruity odor. This leads to their extensive use in the fragrance and flavor industry. Transesterification is the general term used to describe the important class of organic reactions where an ester is transformed into another through interchange of the alkoxy moiety, i.e. Transesterification is the process of exchanging the organic group R” of an ester with the organic group R’ of an alcohol. These reactions are often catalyzed by the addition of an acid or base.
In the transesterification of vegetable oils, a triglyceride reacts with an alcohol in the presence of a strong acid or base, producing a mixture of fatty acids alkyl esters and glycerol. The overall process is a sequence of three consecutive and reversible reactions, in which di and monoglycerides are formed as intermediates. When the original ester is reacted with an alcohol, the transesterification process is called alcoholysis.
In food industry transesterification reaction modifies the lipids in many ways and contributing new flavor and aroma to many food items. The formation of new ester directly influences the concentration of more volatile compounds responsible for many kinds of flavors in fruits and vegetable oil.