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ScienceDirect Novel enzymatic processes applied to the food industry Anil Kumar Patel1, Reeta Rani Singhania1 and Ashok Pandey2 There is a deep relation between enzymes and their application in food and feed industries. These ‘green’ biological catalysts have altered the way we process our food. The enzymes applied in the food industry have evolved in several ways during several past decades. The desired traits as thermostability, ability to act at wide pH range, non-metal ion dependency, fast reaction rate, wide substrates utilization, etc., have been developed by using single, or integrated approaches as screening, rDNA technology, protein engineering, etc. Immobilization of enzymes has also enabled them to be employed more economically as being able to reuse them with minor loss, or without any loss in the activity. These ‘green’ molecules have exerted a great impact on human life that expresses perspectives of enzymes in the area of food industry. Addresses 1 DBT-IOC Centre for Advanced Bio-Energy Research, Indian Oil Corporation Ltd, R&D Centre, Sector-13, Faridabad 121007, India 2 Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum 695019, India Corresponding author: Patel, Anil Kumar ([email protected])

Current Opinion in Food Science 2016, 7:64–72 This review comes from a themed issue on Food bioprocessing Edited by Carlos Ricardo Soccol For a complete overview see the Issue and the Editorial Available online 17th December 2015 http://dx.doi.org/10.1016/j.cofs.2015.12.002 2214-7993/# 2015 Elsevier Ltd. All rights reserved.

Introduction All living organisms are built-up and maintained by the biological catalysts, called as ‘enzymes’, which catalyze biochemical reactions that are necessary to support life [1]. Some enzymes have been designed by the nature to form complex molecules from simpler ones, while others have been designed for breaking up the complex molecules into simpler ones; also few are present for modifying the molecules. These reactions involve making and breaking of the chemical bonds existing in the components. Owing to their ‘specificity’, a property of enzyme that allows it to recognize a particular chemical compound, or substrate that they are designed to target; they are useful for industrial processes and are capable of catalyzing the reaction between particular chemicals even though present in mixtures with many chemicals. The application of enzymes in various processes for the Current Opinion in Food Science 2016, 7:64–72

production of industrial chemicals, often referred as ‘White biotechnology’ remains a challenge since new biocatalytic processes have to compete economically with the well-established chemical processes that have been optimized since long years [2–4]. Humans recognized the importance of enzymes thousands of years ago, where clarification and filtration of wines and beer are among the earliest examples of application of industrial enzymes. Microbial enzymes find great importance for the development of efficient bioprocesses in several industries, for example, chemical, pulp and paper, textiles, pharmaceuticals, leather, detergent, food and beverages, biofuels, animal feed, personal care, etc. These are exploited in several process based on their unique properties such as high specificity, high rate of catalysis, ability to perform with improved yield and reduced waste generation [5]. Enzymes are usually userfriendly for these processes as they catalyze reactions under mild conditions (e.g., temperature, pH and pressure), do not require protecting the functional groups of the substrate, exhibit longer half-life, higher stereo-selectivity, which yields stereo-selective and regio-selective reaction products at the rate of 105–108. Moreover, they work on unnatural substrates also [6]. Today, more than 500 products are made via enzymatic processes in several industries [7]. About 150 industrial processes exploit enzymes, or whole microbial cell as catalysts. Microbial enzymes find applications in numerous segments of industries as mentioned above [8]. However, the segment of food and beverage enzymes comprises the largest segment of industrial enzymes [9]. It created revenues of nearly $1.2 billion in 2011, which was expected to grow up to $1.8 billion by 2016, with average annual growth rate of 10.4% [10]. Within food and beverage enzymes segment, the milk and dairy market had the highest sales, with $401.8 million in 2009 [3]. Novozymes is the largest supplier of the industrial enzymes, followed by DSM and DuPont (based on holding stake in Danisco and Genencor Division). North America and Europe are the largest consumers of industrial enzymes; however, the Asia-Pacific region is predicted for a rapid increase in enzyme demand in China, Japan and India. Current industrial market is very competitive in terms of product cost; therefore, there is a demand for novel, durable and more versatile enzymes in order to develop sustainable and economically viable and competitive production processes. The companies generally compete due to their product quality, performance, use of intellectual property rights, and the ability to innovate among them. Most enzymes available in the market are in www.sciencedirect.com

Novel enzymatic processes applied to the food industry Patel, Singhania and Pandey 65

recombinant form, which is produced from bacteria, fungi and yeasts. In order to discover new potential enzymes, several strategies and evolution practices have been applied. Apart from the microbial diversity, modern molecular techniques such as metagenomics, protein engineering and genome shuffling are being used to discover novel microbial enzymes with improved catalytic properties [11].

Rational of enzyme’s application in food industry Enzymes have been used for food processing as long as man has processed food. Food industry represents one of the economic sectors where enzymes have found a wide variety of applications. The use of enzymes in food industry is based on three basic aspects, (i) to control the quality of foods (presence, or absence of some enzymatic activities has a great impact in the quality control of the final product), (ii) to modify the properties of some food additives and the food itself (to modify the physicochemical and rheological properties of the foods, for example, the use of enzymes, such as amylases, lipases pectinases, etc.), and (iii) to be used as food additives (enzymes with direct applications in the food industry) [12]. Most of the commercial chemical processes are driven by high temperatures and pressures, which lead to high energy cost and needs high volume of cooling water. These ultimately increase the capital investment. Harsh processes requiring high pressure, temperature, acidity, or alkalinity necessitate heavy investment for designing the specific equipment being able to handle extreme conditions. Most of these processes generate undesired byproducts, which are difficult to separate and exert harmful impact on the environment. To combat with the above drawbacks, enzymes have emerged as a powerful tool [11]. Microbial production of enzymes has gained much importance compared to the plant and animal sources because of being economical as well as versatile. The enzymes are produced by the microorganisms in shorter times without the production of toxic compound. Enzymes are natural products, which is the most important reason of their widespread application in food industry [13]. Furthermore, their unsurpassed specificity, ability to perform under mild conditions of temperature, pressure and pH along with high rate of reaction and turnover number make them suitable candidates for food industry. Life-cycle assessment (LCA) has confirmed that the implementation of enzyme-based technology has a positive impact on the environment [14]. Microbial food enzymes may be utilized to increase the productivity, efficiency and quality of food products without a costly investment and have the advantage of being produced with simple technology and economically viable downstream processing. A survey on world sales of enzymes www.sciencedirect.com

ascribed 31% for food enzymes, 6% for feed enzymes and the remaining for technical enzymes [15]. Food processing via biological agent is possibly having the deepest route among all other processes and is historically well-established. Enzymes are environmentally safe, natural and are applied very safely in food and even pharmaceutical industries; still, these are proteins, which, like any protein can cause and have caused in the past allergic reactions, hence, protective measures are necessary in their production and applications.

Applications in various sectors of food industry Food enzymes comprise enzymes used in baking, brewing, beverage, fruit juice and wine industries, as well as dairy industry and the oils & fats industry. Food enzymes are added to the food during various stages of manufacture, processing, preparation, treatment, packaging, transport or storage of the foods [16,17]. Methods of food processing such as fining (e.g., addition of adsorptive compounds, followed by settling, or precipitation) and clarification (e.g., sedimentation, racking, centrifugation, filtration, etc.) include the removal of excess amounts of certain components to achieve clarity and to ensure the physicochemical stability of the end product [18]. The fining and clarification of fermented beverages often include expensive and laborious work that generates large volumes of disposal, thereby causing a loss of product and the removal of important aroma and flavor compounds from the remaining product. In order to minimize the disadvantages of these harsh processes, an increasing use of enzyme preparations (e.g., proteases, pectinases, glucanases, xylanases, arabinofuranosidases, etc.) are often added to the fermentation media (e.g., grape, must and wine). Emphasis is also given on microbial production of aroma liberating enzymes (e.g., pectinases, glycosidases, glucanases, arabinofuranosidases, etc.) [19]. Improved yeast can be used in the production of better flavored alcoholic beverages [20]. Almost all classes of enzymes have a role in food and feed applications; however, hydrolases are the most prevalent ones. Representative examples of enzyme from each classes and their application in food industry is given in Table 1. Enzymes used in food processing have historically been considered non-toxic. Still as every coin has two faces, enzymes too have their limitations. Some characteristics arising from their chemical nature and source, such as allergenicity, activity-related toxicity, residual microbiological activity and chemical toxicity are of high concern. These attributes of concern must, however, be addressed in light of the growing complexity and sophistication of the methodologies used in the production of food-grade enzymes. Safety evaluation of all food grade enzymes, Current Opinion in Food Science 2016, 7:64–72

66 Food bioprocessing

Table 1 Application of enzymes representing different class in food industry. Class

Sub food industry

Enzymes

Purpose

Oxidoreductase

Starch industry

Glucose oxidase

Enhance the storability of food by removing oxygen and glucose from the food stuff Improves dough quality and conditioning Loaf volume, shelf life Dough strengthening and bread whitening Clarification of juices and flavor enhancer (beer)

Baking industry Lipoxygenase Laccase Transferase

Transglutaminase

Cyclodextrin Glucosyltrasferase Fructosyltransferase Hydrolase

Baking industry

Brewing industry

Dairy

Alpha amylase Alpha and b amylase, pullulanase and invertase Phospholipase Cellulase, xylanase and pectinase Glucanase Papain Renin Lactase Lipase and protease Peptidase

Texturing agent in the processing of yoghurt, sausages and noodles where cross-linking of proteins provides improved viscoelastic properties of the products Cyclodextrin production Synthesis of fructose oligomers Loaf volume, shelf life Production of various types of syrups from starch and sucrose In situ emulsification for dough conditioning Juice clarification Filter aid Haze control Protein coagulation Lactose hydrolysis Ripening of cheese Hydrolysis of proteins (namely, soy, gluten) for savory flavors, cheese ripening Release of phosphate from phytate, enhanced digestibility

Animal feed

Phytase

Lyase

Brewing industry

Acetolactactate decarboxylase

Beer maturation

Isomerase

Starch industry

Glucose isomerase

Production of various types of syrups from starch and sucrose Glucose isomerization to fructose

including those produced by the genetically modified (GM) microorganisms is essential to assure consumer safety [21]. Enzymes produced using GM microorganisms wherein the enzyme is not part of the final food product have specifically been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) [22]. Safety evaluations have been conducted using the general specifications and considerations for enzyme preparations used in food processing. Also, some of the broad generalizations on the limitations of enzymes for application as biocatalysts in commercial scale, namely, their high cost, low productivity and stability, and narrow range of substrates, have been rebutted [23].

grains. In the processing of sausages, noodles and yoghurt, transglutaminase plays major role as texturing agent where cross-linking of proteins provides improved viscoelastic properties of the products [24].

Starch industry

Xylanase (hemicellulases) are of great value in baking as they improve the bread volume, crumb structure and reduce stickiness [25]. There is evidence that the use of xylanases decreases the water absorption, and thus reduces the amount of added water needed in baking. This leads to more stable dough. Especially xylanases are used in whole meal rye baking and dry crisps common in Scandinavia. Enzymes such as proteases, xylanases, and cellulases directly or indirectly increase the strength of the gluten network, and therefore, improve the quality of bread [26]. Glucose oxidase has been used to replace the

The use of starch degrading enzymes was the first largescale application of microbial enzymes in food industry. Alpha amylase, beta amylase, glucoamylase, pullulanase, transglutaminase are the enzymes involved in starch industry. Mainly two enzymes carry out the conversion of starch to glucose: alpha-amylase and glucoamylase. Sometimes additional debranching enzymes such as pullulanase are added to improve the glucose yield. Betaamylase is used for the production of the disaccharide maltose which is commercially produced from barley Current Opinion in Food Science 2016, 7:64–72

Baking industry

Enzymes have gained real importance in bread making, where they improve the dough and bread quality, leading to improved dough flexibility, stability, loaf volume and crumb structure. Bread making involves the use of microbial enzymes such as amylases, amyloglucosidases, cellulases, glucanases, glucose oxidase, hemicellulases, lipases, pentosanases, and proteinases.

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Novel enzymatic processes applied to the food industry Patel, Singhania and Pandey 67

chemical oxidants and lipases to strengthen gluten, which leads to more stable dough and better bread quality [27]. Brewing industry

Brewing industry employs the fermentation process to produce the wine, beer, sake and soy sauce. Many glycosidic enzymes such as cellulase, xylanase, amylases, amyloglucosidase, glucanase, acetolactase, and also other enzymes such as decarboxylase, lipase, pentosanase, proteinase, etc. are used in this industry. In the wine industry, enzymes such as amylase, amyloglucosidase, cellulase, glucanases, hemicellulases, pectinases, proteases, glucose oxidase and catalase are needed [16]. Wines are produced by fermenting the red and white grapes. Some other alcoholic beverages such as cider and perry are produced using fruit juices. Saccharomyces carlsbergensis is used for the production of light colored and less cloudy beers. S. cerevisiae is used in top fermenters, as the cells are carried to the surface of the fermentation by carbon dioxide bubbles, while S. carlsbergensis cells form the sediment at the bottom and is used in bottom fermenters. Enzymes can be used to help the starch hydrolysis (typically alpha-amylases), solve filtration problems caused by beta-glucans present in malt (beta-glucanases), hydrolyse proteins (neutral proteinase), and control haze during maturation, filtration and storage (papain, alphaamylase and beta-glucanase). Tea and coffee are among the main beverages used worldwide. In coffee industry, enzymes from microbial sources such as cellulases, hemicellulases, galactomannanase, pectinases are used from Leuconostoc mesenteroides, Saccharomyces marscianus, Flavobacterium spp., Fusarium spp. [28]. Tea processing requires cellulases, glucanases, pectinases and tannase. Most familiar Asian food products such as soy sauce, koji, moromi, etc. are also made through fermentation of Aspergillus oryzae and later inoculated with a bacterium, Peiococcus soyae, and yeasts such as Saccharomyces rouxii and Torulopsis sp., which ferment the mixture for approximately six months. Dairy industry

Milk is the chief dairy product and is processed and converted in to yoghurt, buttermilk, cheeses, butter, cream, soured cream, etc. through fermentation. The enzymes proteases, lactases and lipases are chiefly used in the dairy industry to develop the flavor compounds. Lipases from Mucor miehei, or Aspergillus niger are used to give stronger flavors in Italian cheeses from milk before adding the rennet, by a modest lipolysis, increasing the amount of free butyric acid. Rennin acts on the milk protein in two stages, by enzymatic and by nonenzymatic action, resulting in coagulation of milk. Other enzymes used for dairy food application include proteases to reduce the allergic properties of cow milk products for infants, and lipases for the development of lipolytic flavors in speciality cheeses. Chymosin is used in cheese www.sciencedirect.com

making to coagulate milk protein. Another enzyme used in milk industry is b-galactosidase, or lactase, which splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers. The functional properties of milk proteins may be improved by limited proteolysis through the enzymatic modification of milk proteins. The antigenicity of casein is destroyed by proteolysis, and the hydrolysate is suitable for use in milk-protein-based foods for infants allergic to cow milk. Lipolysis makes an important contribution to Swiss cheese flavors, due to the lipolytic enzymes of the starter cultures. The characteristic peppery flavor of Blue cheese is due to short-chain fatty acids and methyl ketones catalyzed by Penicillium roqueforti lipase [29]. Other minor enzymes having limited applications in dairy processing include glucose oxidase, catalase, superoxide dismutase, sulphydryl oxidase, lactoperoxidase and lysozymes [30]. Vegetable and fruit processing industry

Microbial pectic enzymes are used for the extraction and clarification of fruit juices and grape musts, citrus fruit juice and wine technology, maceration of vegetables and fruits and in the extraction of olive oil. Amylase, amyloglucosidase, cellulase, pectinase, pentosanase, dehydrogenase, naringinase, etc. are also used in the extraction of fruit juice and fruit processing. Pectins contribute to fruit juice viscosity and turbidity. A mixture of pectinases and amylases is used to clarify the fruit juices. Pectinases improve the production yields and result in crystal clear juice [31]. The treatment of fruit pulps with pectinases also shows an increase in fruit juice volume from banana, grapes and apples. Pectinases in combination with other enzymes, viz., cellulases, arabinases and xylanases have been used to increase the pressing efficiency of the fruits for juice extraction [32]. The pectolytic enzymes complex specifically is added to apple mash during the milling. Infusion of free stone peaches with pectin methyl esterase and calcium resulted in four times firmer fruits. This could be applied to pickle processing where excessive softening may occur during the fermentation and storage, where enzyme would solubilize the protopectin, the bonding agent of vegetable cells. Carbohydrases such as amylase, amyloglucosidase and glucose isomerase have found usage in the starch and syrup industry for the production of high dextrose, fructose and maltose syrup production. Other enzymes such as glucose oxidase, pectinase, and naringinase are of value to the wine and fruit juice industries [33]. Fat and oil industry

In the fat and oil industries, several enzyme-based processes such as use of immobilized lipases in the interesterification of triglycerides for the production of margarine, removal of phospholipids in vegetable oils (de-gumming), etc. using a highly selective microbial lipases, phospholipase, etc. are applied [25]. The immobilization of lipases on Current Opinion in Food Science 2016, 7:64–72

68 Food bioprocessing

the granulated silica was utilized for the production of commodity fats and oils with no content of trans-fatty acids. The interesterification and hydrogenation are techniques, which have been useful in the preparation of glyceride products for use in the manufacture of butter and margarine. An interesterification process conducted in the presence of lipase, unlike the chemical one, is highly selective and requires the presence of water to activate the lipase [20].

Considerable progress has been made in recent times toward the improvement of microbial strains used in the production of enzymes. Microbial host strains developed for enzyme production have been engineered to increase enzyme yields by deleting native genes encoding extracellular proteases. Certain fungal producing strains have also been modified to reduce or eliminate their potential for producing toxic metabolites [1].

Fish and meat industry

Enzymes from plant, or animal cells, such as transglutaminase or even slow-growing microorganisms were naturally produced in the conditions that prevented large-scale application. By allowing gene cloning in the microorganisms compatible with industrial requirements, this methodology enabled cost feasible production of those enzymes. When successfully implemented, the undertaken approaches allow continuous operations at relatively high temperatures given the reduced need for processing the reaction media (pH adjustments; metal ion removal/addition) throughout the intermediate steps of a multistep biotransformation (namely, starch to high fructose syrup); and (c) the use of raw substrates, preferably as high-concentrated solutions, hence cutting back in costs related to upstream processing and increasing productivity [36,37]. Methodologies with a high level of parallelization, anchored in computer-monitored microtiter plates equipped with optic fibers and temperature control have also been developed, which provide high-throughput capability for a quick and detailed characterization of enzymes performance [38]. Particular focus was given to the prediction of the long-term stability of enzymes under moderate conditions using short-term runs (up to 3 h). Several enzymes (namely, a-amylases; pullulanases) currently used in food processing, namely, in starch hydrolysis, are actually produced by recombinant microorganisms. Despite some complexity in the implementation of their use in large-scale applications, partly resulting from lack of uniformity in the US and EU legislation, quite a few enzyme preparations have been accepted for industrial use [1,21].

In meat industry, proteases are used to tenderize the muscle and to obtain the flavor precursors. In the preparation of cured meat products such as sausages, lipases and proteases from bacterial cultures are utilized [34]. Much work has been carried out on the application of trans-glutaminase as a texturing agent in the processing of sea foods, sausages, meat products as well as noodles and yoghurt, where cross-linking of proteins provides improved viscoelastic properties of the products. The enzyme improves the firmness, elasticity, viscosity, heat stability, and water-holding capacity of prepared foods through the mild reaction. At present, only the transglutaminase from Streptoverticillium sp. and in recombinant Escherichia coli is used [24].

Biotechnological tools for improving enzymes Recombinant DNA technology

Recombinant DNA technology, which is considered as important biotechnological tool, is widely employed in research and development for microbial strain improvement. The availability of genetic manipulation tools and the opportunities that exist to improve the microbial cultures associated with food fermentations are tempered by the concerns over regulatory issues and consumer perceptions. Genetically modified (GM) microbial cultures are, however, used in the production of enzymes and various food-processing ingredients such as monosodium glutamate, polyunsaturated fatty acids and amino acids. The use of recombinant DNA technology has made it possible to manufacture novel enzymes that are tailored to specific food processing conditions. Alpha amylases with increased heat stability have, for example, been engineered for use in the production of high-fructose corn syrups. These improvements were accomplished by introducing the changes in the a-amylase amino acid sequences through DNA sequence modifications of the a-amylase genes [1]. Bovine chymosin used in cheese manufacture was the first recombinant enzyme approved for used in food by the US Food and Drug Administration [35]. The Phospholipase A1 gene from Fusarium venenatum was expressed in GM A. oryzae to produce the phospholipase A1 enzyme used in the dairy industry for cheese manufacture to improve process efficiencies and cheese yields [1]. Current Opinion in Food Science 2016, 7:64–72

Screening

Another approach for obtaining improved enzyme relies on screening efforts, which has been consistently undertaken, as summarized by several workers [39–41]. Focus was on extremophiles, particularly thermophiles, since operation at high temperatures (roughly above 45–50 8C) minimizes the risk of microbial contamination, a particularly delicate matter under continuous operation. Furthermore, the extension of some reactions in relevant food applications is favored at relatively high temperatures (namely, isomerization of glucose to fructose), although care should be taken to avoid an operational environment that may lead by-product formation (namely, Maillard reactions). www.sciencedirect.com

Novel enzymatic processes applied to the food industry Patel, Singhania and Pandey 69

Examples of screened enzymes include the isolation of amylases which are calcium independent, amylopullulanases, fructosyltransferases, glucoamylases, glucose (xylose) isomerases, glucosidases, inulinases, levansucrases, pullulanases and xylanases. Gomes and Steiner [42] have reviewed several enzymes, with some of which being able to retain stability even at 90 8C or higher. The majority of enzymes used in food and feed processing is of terrestrial microbial origin, and screening-efforts for isolation of promising enzyme producing strains have accordingly been performed in such background. Since few years now, marine environment has also been explored as source for useful enzymes from either microbial or higher organisms origin. This latter environment has allowed the isolation of some promising biocatalysts, such as the heatstable invertase/inulinase from Thermotoga neapolitana DSM 4359 or inulinase from Cryptococcus aureus [43,44], amylolytic enzymes, glucosidases and proteases from several genera [45], esterase from Vibrio fischeri [46], and glycosyl hydrolases [47,48]. Protein engineering

With recent advances in PCR technology, site-specific and random mutageneses are readily available to improve enzyme stability in a wide range of pH and temperature and tolerance to a variety of organic solvents. Since a large quantity of enzyme can be obtained by recombinant expression, X-ray crystallography can facilitate the understanding of the tertiary structure of an enzyme and its substrate binding/recognition sites. This information may assist a rational design of the enzyme, predicting amino acid changes for altering substrate specificity, catalytic rate and enantioselectivity (in the case of chiral compound synthesis). To engineer a commercially available enzyme to be a better industrial catalyst, two different approaches are presently available: a random method called directed evolution and a protein engineering method called rational design. The protein engineering as well as random mutagenesis are capable of changing a protein sequence to achieve a desired result, such as a change in the substrate specificity, or increased stability to the temperature, organic solvents, and/or extremes of pH. Many specific methods for the protein engineering exist, but they can be grouped into two major categories: those involving the rational design of the protein changes, and the combinatorial methods which make changes in a more random fashion. Protein engineering or rational methods, such as the sitedirected mutagenesis, require targeted amino acid substitutions, and therefore, require a large body of the knowledge about the biocatalyst being improved, including the three-dimensional structure and the chemical mechanism of the reaction. The main advantage of the rational design is that a very small number of protein variants are created, meaning that very little effort is necessary to screen for the improved properties. One www.sciencedirect.com

example of this is the mutant glucose isomerase from Actinoplanes missouriensis displayed an enhanced thermal stability, alongside with improved stability at different pH, as compared with the original enzyme, with no changes in catalytic properties [49]. The double mutant isomerase (G138P, G247D) displayed a 2.5-fold increase in half-life, and additionally a 45% increase in the specific activity, when compared to the wild type. Such features were ascribed to increased molecular rigidity due to the introduction of a proline in the turn of a random coil [50]. The work by Lin and coworkers on amylase mutants from Bacillus sp. strain TS-23 highlighted the relevance of E219 for the thermal stability of the enzyme [51]. The combinatorial methods, on the other hand, create a large number of variants that must be assayed; however, they have the advantage of not requiring such extensive knowledge about the protein. In addition, often nonobvious changes in the protein sequence lead to large improvements in their properties, which are extremely hard to predict rationally, and thus, can only be identified by the combinatorial methods. Table 2 gives an account of enzymes modified by protein engineering and other methods. Several enzymes have already been engineered to function better in the industrial processes. These include the proteinases, lipases, cellulases, a-amylases and glucoamylases. Xylanase is a good example of an industrial enzyme, which needs to be stable at high temperature and active at the physiological temperature and pH when used as the feed additive. One of the industrial production organisms of the xylanases is Trichoderma sp. Its xylanase has been purified and crystallized. By the designed mutagenesis, its thermal stability has been increased by about 15 8C. The mutational changes increased the half-life in the thermal inactivation of this enzyme from approximately 40 s to approximately 20 min at 65 8C, and from less than 10 s to approximately 6 min at 70 8C [59]. By designed mutagenesis its thermal stability has been increased about 2000 times at 70 8C and its pH optimum shifted toward alkaline region by one pH-unit. The most successful strategies to improve the stability of the Trichoderma xylanase include the stabilization of the alpha-helix region and the N-terminus. Immobilization

Enzyme immobilization is a powerful industrial technique to increase life line of any enzyme. It has the capacity to turn the process economically feasible by allowing highenzyme loading with high activity within the bioreactor, hence leading to high-volumetric productivities; it enables the control of the extension of the reaction; downstream process is simplified, since biocatalyst is easily recovered and reused; the product stream is clear from biocatalyst; continuous operation (or batch operation on a drain-and-fill basis) and process automation is possible; and substrate inhibition can be minimized. Along with this, adequately Current Opinion in Food Science 2016, 7:64–72

70 Food bioprocessing

Table 2 Improvement in enzyme properties and strategies applied. Enzyme

Microorganism

Attributed properties

Role

Modification type

Reference

Glucose isomerase

Actinoplanes missouriensis

Isomerization of hexoses

Site directed mutagenesis

[52]

Amylosucrase

Neisseria polysaccharea Not applicable, as the variant was from library

Enhanced thermostability (85 8C) with stability at various pH with no effect in activity 5-Fold increase in activity and 2-fold increase in overall catalytic activity 10-Fold increase in half life time at 50 8C

Synthesis of amylose type polymers from sucrose Twice in length amylose synthesis at high sucrose concentration (600 mM) as that of wild-type enzyme Could tolerate pH inside stomach when used in animal feed Starch liquefaction

Mutation via error prone PCR Combinatorial engineering

[53]

Phytase

Aspergillus niger

Active at low pH 3.5 and 1.5-fold higher activity at 100 8C

Alpha amylase

Bacillus stearothermophilus US100 Peas –

Thermostability, mutant displayed enhanced half life of 70 min at 100 8C Thermostable Enhanced activity

Lactase Pullulanases

designed immobilization prevents denaturation by autolysis or organic solvents, and can bring along thermal, operational and storage stabilization [60]. Along with the above advantages, immobilization has some intrinsic drawbacks also, namely, mass transfer limitations, loss of activity during immobilization procedures, particularly due to chemical interaction or steric blocking of the active site; the possibility of enzyme leakage during operation; risk of support deterioration under operational conditions, due to mechanical or chemical stress. Economical issues are furthermore to be taken into consideration when commercial processes are envisaged, although immobilization can prove critical for economic viability if costly enzymes are used. Still, the cost of the support, immobilization procedure and processing the biocatalyst once exhausted, upstream and downstream processing of the bioconversion systems, and sanitation requirements have to be taken into consideration. In the overall, the enhanced stability allowing for consecutive reuse leads to high specific productivity, which influences biocatalyst-related production costs [60]. A typical example is the output of immobilized glucose isomerase, allowing for 12,000–15,000 kg of dryproduct high-fructose corn syrup (containing 42% fructose) per kilogram of biocatalyst, throughout the operational lifetime of the biocatalyst [61]. Increased thermal stability, allowing for routine reactor operation above 60 8C minimizes the risks of microbial growth, hence leading to lower risks of microbial growth and to less demanding sanitation requirements, since cleaning needs of the reactor are less frequent [61]. There several methods for immobilization and advanced ones have been discussed here. Carrier-free macroparticles, where a bifunctional reagent (namely, glutaraldehyde), is used to cross-link enzyme aggregates (CLEAs) or crystals (CLECs), leading to a biocatalyst displaying Current Opinion in Food Science 2016, 7:64–72

Lactose hydrolysis Starch debranching

[54]

Site directed mutagenesis

[55]

Protein engineering through site directed mutagenesis Immobilization Protein engineering through directed evolution

[56]

[57] [58]

highly concentrated enzyme activity, high stability and low production costs [60,62]. The use of CLEAs is favored given the lower complexity of the process. This approach is recent, as compared with entrapment and binding to a solid carrier, and there are still relatively few examples of its application to enzymes used in the area of food processing. Among those are following. (1) First is the immobilization of Pectinex Ultra SPL, a commercial enzyme preparation containing pectinase, xylanase, and cellulase activities [63]. The CLEA biocatalyst displayed a slight (30%) in the Vmax, maximal reaction rate/KM ratio, but a significant enhancement in thermal stability (a roughly 10-fold increase in half-life), when the pectinase activity of the immobilized biocatalyst was compared with the free form. (2) Second is the immobilization of lactase for the hydrolysis of lactose, where, under similar operational conditions as for the free enzyme, the CLEA yielded 78% monosaccharides in 12 h as compared to 3.9% of the free form [64].

There are several other examples of improved enzymatic properties or enhancement in reaction rate by applying various technologies as above; and are nicely compiled by Fernandes [65]. A great deal of research has been carried out in food products by using microbial enzymes. Unlike western cultures, in which fermented food is usually processed by the yeasts, eastern cultures utilize different mycelial fungi.

Conclusion and future perspective The integration of enzymes in food and feed processes is a well-established approach; however there are clear www.sciencedirect.com

Novel enzymatic processes applied to the food industry Patel, Singhania and Pandey 71

evidences that dedicated research efforts are consistently being made to make the applications of biological agents more effective as well as diversified. Various techniques have been employed such as rDNA technology and protein engineering (site-directed mutagenesis and random mutation) for the design of new/improved biocatalysts, especially thermostable having wide pH stability, less dependent on metal ions and less susceptible to inhibitory agents and to aggressive environmental conditions, while maintaining the targeted activity, or evolving novel activities. These properties result enhanced performance of enzymes under operational conditions in food industry. Advances in molecular biology, evolutionary protein engineering expertise, the (bio) computational tools, and the implementation of high-throughput methodologies enabling the efficient and timely screening/ characterization of the biocatalysts has enabled the versatility of the enzymes for its wide application in various sectors of food industry. Alongside with these strategies, the immobilization of enzymes has also been a key supporting tool for rendering these proteins fit for industrial application, while simultaneously enabling the improvement of their catalytic features. This is obvious that we do not have any universal rules, which can be applied to all the enzymes for improved properties, so there needs to be a proper understanding as well as integration among different tools and methodologies to develop a desired trait in enzyme. Though enzyme technology has gone a long way through, there needs to be continuing efforts in the direction to have more diverse, versatile and robust enzymes to be applied in food technology. We are moving toward greener era and enzymes are the key molecules, which have the capacity to drive human population through. Definitely enzymes have changed the way we process our food.

Acknowledgement AKP and RRS acknowledge Department of Biotechnology, Govt. of India for DBT-Energy Bioscience Overseas Fellowship and IOCL R & D for providing working space.

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