Page | 1AMITY INSTITUTE OF BIOTECHNOLOGY ENZYMOLOGY ASSIGNMENT-I ENZYMES USED IN DETERGENTS DATE OF SUBMISSION- 22.O4.08 SUBMITTED TO: DR. S.M. BHATT FACULTY,ENZYMOLOGY AIB SUBMITTED BY: MAYANK JAIN ROLL NO.-77 SECTION-U AIB CONTENTS: HISTORY................................................................................. INTRODUCTION................................................................... DETERGENT ENZYMES PROTEASES....................................................................... AMYLASES......................................................................... LIPASE................................................................................ CELLULASE...................................................................... PAGE NO. 3 3 5 7 8 9 Page | 2 MISCELLANEOUS DETERGENTS PEROXIDASES................................................................. PULLULANASE................................................................ ENZYME FORMULATION................................................... PRODUCTION OF ENZYME BASED DETERGENT........ ENZYME STABILITY............................................................. APPLICATION OF ENZYME BASED DETERGENT........ BENEFIT OF USING ENZYME IN DETERGENT.............. CONCLUSION .......................................................................... BIBLIOGRAPHY....................................................................... 10 10 11 13 14 16 19 20 22 History The original idea of using enzyme as detergents was described in 1913 by Dr Otto Rohm, who patented the use of crude pancreatic extracts in laundry presoak compositions to improve the removal of biological stains. In the same year, the first enzymatic detergent named Burnus was launched, but was not popular because of its own limitations. Subsequently, Bio- 40 - a detergent containing a bacterial protease was produced in Switzerland and launched in the market in 1959 and it gradually became popular. In the period from 1965 to 1970, use and sale of detergent enzymes grew very fast. In 1970, the use was distorted due to dust production by formulations leading to allergies to some workers. This problem was overcome in 1975 by encapsulating the granules of enzyme. From 1980s to the 1990s, several changes took place in the detergent industry like development of softening through the wash, development of concentrated heavy-duty power detergents, development of concentrated or structured or nonaqueous liquid detergent. Introduction Enzymes have been used to improve the cleaning efficiency of detergents for more than 35 years, and are now well accepted as ingredients in powder and liquid detergents, stain removers/laundry pre-spotters, automatic dishwashing detergents and industrial/institutional cleaning products. Detergent enzymes account for about 30% of the total worldwide enzyme production and represent Page | 3 one of the largest and most successful applications of modern industrial biotechnology. The largest segment within the global industrial enzyme market is the market for technical enzymes, estimated at around uss 980 million in 2002. In the technical enzymes category, detergent additives make up for nearly two-thirds of the market. These enzymes are used as functional ingredients in laundry detergents and automated dishwashing detergents. This article gives an overview of the detergent enzymes industry and discusses its manufacturing and downstream processing. Enzymes used in detergents are protein catalysts that consist of long chains of amino acids. They are similar to protein catalysts present in all living cells where they control metabolic processes, convert food nutrients to simple molecules, convert these molecules to energy and to new cell material. As catalysts; enzymes speed up specific chemical reactions, in mild conditions of temperature and pH, without being altered or consumed in the process. Consequently, small quantities of enzyme can repeatedly catalyze the breakdown of millions of molecules in minutes. Enzymes function optimally in detergents at temperatures of 20 - 60C and within a pH range of pH 7.5 - 10.5. The performance of enzymes in detergents depends on number of factors, including the detergent’s composition, type of stains to be removed, wash temperature, washing procedure and wash-water hardness. To help formulators optimize enzymatic detergent washing efficiency, Specialty Enzymes provides wash laboratory technical services. In our wash laboratory, customer, base detergents are evaluated on standard soils in both a model wash system (Terg-OTometer) and in full-scale household washing machines. Table 1 Compositions of an enzyme detergent Constituent Sodium tripolyphosphate (water softener, loosens dirt)a Sodium alkane sulphonate (surfactant) Sodium perborate tetrahydrate (oxidising agent) Soap (sodium alkane carboxylates) Sodium sulphate (filler, water softener) Sodium carboxymethyl cellulose (dirt-suspending agent) Sodium metasilicate (binder, loosens dirt) Bacillus protease (3% active) Fluorescent brighteners Foam-controlling agents Composition (%) 38.0 25.0 25.0 3.0 2.5 1.6 1.0 0.8 0.3 Trace Page | 4 Perfume Water Trace to 100% Detergent Enzymes Presently, detergent enzyme has become an integral part of detergent formulation. A look at the market share of detergent enzyme indicates it to be very high in comparison with other enzyme applications. Enzymes that have to be used as detergent composite must possess the following characters: Stability at temperature over a broad range of 20C to 50C and even above The optimum pH should be in alkaline or higher alkaline range It should be detergent compatible It should have specificity towards different proteins Major detergent enzymes include proteases, amylases, lipases, cellulases, miscellaneous enzymes such as peroxidases and pullulanase. A recent trend is to reduce this phosphate content for environmental reasons. It may be replaced by sodium carbonate plus extra protease. Proteases Proteases were introduced in the market in 1959 in the detergent Bio-40, produced by Schnyder Ltd in Switzerland. Most powder and liquid laundry detergents in the market, today, contain proteases. Proteases are of two types: Alkaline protease from Bacillus licheniformis, having optimum pH 8, for egg, liquid laundry product, (pH 7- 8.5), commercially known as Alcalase -Novonordisk Optimase- Genencor Inter . Page | 5 High alkaline protease from Bacillus alkalophilus and Bacillus lentus, having an optimum pH 10. For e.g., powder laundry products, automatic dish washing formulations, known by trade names of Savinase-Novo Nordisk, Purafet- Genencor Inter. Proteases enhance the cleaning of protein-based soils, such as grass and blood by catalyzing the breakdown of the constituent proteins in these soils through hydrolysis of the amide bonds between individual amino acids. In the case of serine endopeptidase, it contains a catalytic triad of amino acids at the active site; • An aspartyl residue containing ß-COO¯ • A histidine containing the imidazole group • A serine residue with p-OH as the functional group The serine hydroxyl group functions as a potential nucleophile, where as both the aspartyl and histidine functional groups behave as general base catalysts facilitating the hydrolysis process. . The serine group initiates the nucleophilic attack on the peptide bond to form a tetrahedral intermediate, which undergoes an active hydrogen transfer, facilitated by both the histidine and aspartyl residues. The net effect of the addition of water across the bond generates the original protein. The protease hydrolysis involves the transfer of electrons between the amino acids at the active site and substrate. For proteases the three-dimensional arrangement of the catalytic triad is required for the enzyme to be active. Disturbances in the confirmation are likely to affect enzyme efficacy and therefore cleaning performance. These were susceptible to oxygen bleaches and calcium sequestrates. But now, stable protease can be obtained . Oxidative attack by peroxides or per acids on the methionine residue adjacent to the catalytic serine results in nearly 90% loss of enzyme activity. However, replacing methionine with oxidatively stable amino acids like alanine improves stability of enzyme towards oxygen bleach (Boguslawski et al, 1992) Protease substilisin requires at least one calcium ion, which maintains three- dimensional structure of enzyme. However, calcium- sequestering agents used in many laundry procedures to control water hardness can remove this calcium resulting in the decreased thermal and autolytic Page | 6 stability. This can be corrected by the introduction of negatively charged residues near the calcium-binding site, which increases the binding affinity of enzyme for calcium and results in improved stability towards calcium sequestrants (Krawczyk et at, 1997) Protease has limited applications towards the detergency of wool and Constituent silk, because of the proteinaceous nature of these fibres. Proteases are added in an encapsulated or granulated form, which protects them from other detergent ingredient and eliminates the problem of autolysis or proteolysis of other enzymes. In aqueous detergent formulations, protease inhibitors show a preventive effect of avoiding contact of the protease molecules with each other as well as other enzyme molecules. This effect gets nullified on dilution and enzyme molecules are free to act on stains (Krawczyk et al, 1997) Amylases Amylases facilitate the removal of starch-based food soils, by catalyzing the hydrolysis of glycosidic linkages in starch polymers. Generally, starchcontaining stains are of chocolate, gravy, spaghetti, cocoa, pudding, etc. Amylases can be classified as: : a-amylases: These enzymes catalyze the hydrolysis of the amylose fractions of the starch under hydrolysis of the glycosidic bonds in the interior of the starch chain. The first step in the reaction is called as endoreaction & leads to oligosaccharides, where short chain water- soluble dextrins are produced. . ß-amylases: These enzymes acton dextrins from reducing end and forms maltose units. Pullulanases or isoamylases: These degrade starch directly into linear dextrins for they also attack ci-1,6 glycosidic bonds. . . Amyloglycosidases: These enzymes act on the dextrin or maltose units and forms glucose units. . a-amylases are mostly used for detergents, although recently other carbohydrate cleaving enzymes such as pullulanases or isoamylases have also been described Page | 7 for this application. a-amylases bring about the primary hydrolysis of starch into the oligosaccharides and dextrins. Currently, these enzymes are produced from bacteria. Bacillus subtilis. Bacillus amyloliquefaciens, and Bacillus licheniformis. These are available under the trade names Maxamyl- Genencor Int or Termamyl -Novo Nordisk Lipases Tomato-based sauces, butter, edible oils, chocolate and cosmetic stains are very difficult to remove as they form due to greasy food stains. Body soils, sebum and sweat on collars, cuffs and underarms, are generally composed of a mixture of proteins starch pigments and lipids. Lipases hydrolyze the water insoluble triglycerides components into the more water-soluble products as monoglycerides, diglycerides, free fatty acids and glycerol. The Novo Nordisk launched the first lipase product in 1987. They transferred the lipolase gene into the fungus Asper6yillus oryzae for industrial production, Genencor followed in 1993 with lumafast (Pseudomonas menocina) and Gist-Brocades in 1995 with Lupomax. . Currently, the known sources of lipases include mammalian lipases (human pancreases/colipases), fungal (Rhizomucor mehei, Humicola lanuginose, etc), yeast (Candida rugosa, Candida antartica), bacterial lipase (Pseudomonas glumae, Pseudomonas aeroginosa, Chrobacterium viscosum) (Ishida et al, 1995) . . Lipases possess a catalytic triad that is similar to the serine proteases of trypsin and subtilisin type. Hence, these are also called as serine hydrolysate lysate. Lipases can decompose a fatty stain up to 25%, which then can be removed very easily because of the hydrophilic character (Dorrit et al, 1991). It is generally thought that lipases get adsorbed on to the hydrophobic stain during the washing period. And, during the drying cycle when the water content is decreased, the enzyme is activated and can hydrolyze triglycerides in the stain. This facilitates the removal of stain in the next wash cycle (Dorrit et al 1991). The enzyme also has stability over a broad range of temperature 30C to 60C. These novel alkaline lipases also retained 100% activity in the presence of strong oxidants. Efforts are on to manufacture enzymes that can work below the normal temperature range of 30C to 40C to save energy. It has been observed that energy consumption per wash in household washing machine (3 kg clothes) at low temperature (30C) is less than 1 % of the energy used at a higher temperature (60C) (Edvardetal, 1991). . Page | 8 Cellulases These enzymes introduced in the late 1980s were described for the first time in a Japanese patent filed by Murata. These enzymes are used in UK and US since 1991. Cellulases remove microfibrils from cotton and cotton-blended fabrics. These microfibrils stick out from the main fibre of cotton and are formed during use and repeated washing condition of the tissue. This makes garments and household textiles unusable. The cellulases can be used as softening agents, to remove soil particles and to revive colours. . . The overall cellulose structure has two types of region; one that has a higher order of crystallinity is called crystalline region. The other type has less structured order, and hence is called as amorphous regions. The activating but not hydrolytically acting enzyme was named as C i-activity. According to this concept, microorganisms that are able to degrade crystalline cellulose have C iactivity. This enzyme is not present in that microorganism that attack only substituted cellulose like carboxymethyl cellulose as they have Cx-activity. According to a recent research, biodegradation of cellulose requires the interaction of three different hydrolytes or at least the first two enzymes to attack simultaneously. These include: · Cellobiohydrolase is also called as exocellulase (C i-activity) . . · Endoglucanases is also called as Endocellulases (C i-activity) . . · ß-Glucosidase is also called as cellobiase . . It was observed that sebum in the interior of cotton fibres cannot be removed by ordinary detergents satisfactorily, although they readily remove sebum on the exterior of the fibres. Alkaline cellulase interacts selectively with cellulose in interfiber spaces in the interior of fibre, and selectively removes the sebum soil. The removal of the soil is by the hydrolysis of amorphous regions (Murata et al, 1991). Cellulases can be chemically modified to have greater stability and efficiency in alkaline medium. It can be done by treating the acid cellulases with reagents like maleic anhydride (Bund and Singhal et al, 2002). Page | 9 . Currently, the cellulases used in detergents are manufactured from bacteria and fungi. Bacterial cellulases have been in use since 1987, for example, Biotex. Some genetically engineered strains, which are widely used include Streptomyces sp. KSM-2, Bacillus KSM-635. The fungal cellulase from Humicola isolens DSM1800 is active under mild alkaline conditions. Miscellaneous detergent enzymes Peroxidases: These are one of the newest classes of enzymes that have been included in detergent formulations. Peroxidases are subclass of general oxidoreductases and are very popular and commercially available for manufacturing detergents. Novo Nordisk produces this under the brand name Guardzyme obtained from mushroom Corprinus cinereus. It is a heme containing protein, which in the presence of H2O2 can mediate the oxidation of fugitive dyes in solution and inhibits the dye transfer. Pullulanases: In recent years, pullulanases (Pullulan 6- glucanohydrolase) a debranching enzyme has been gaining importance due to its efficiency of starch hydrolysis by cleaving a-1, 6 linkages. Pullulanases with other amylolytic enzymes are used in detergents for improved stain removal and enhanced overall cleaning performance. This enzyme was first isolated in Klebsiella pnuemoniae (Shaw et al, 1995). Manufacturing and downstream processing Nearly all-detergent enzymes, which are used and marketed today, are produced through large-scale fermentation of microorganisms. Most of enzymes "are obtained from the bacterial or fungal strains. As low cost enzymes are needed to support the requirements of the global detergents business, enzyme manufacturers should consider the following points to ensure lower costs: P a g e | 10 The enzyme-producing micro-organism must be capable of secreting the enzyme extra-cellularly in the bulk fermentation broth, as the cost in terms of both money and time to recover enzyme from the fermentation broth is very high The production organism should be able to produce highest possible yields. Strain optimisation can be accomplished either through classical mutagenesis and screening methods or using genetic engineering · The number of steps in the downstream processing should be kept to a minimum to be economical and also to avoid yield losses The production organism should produce the desired enzyme in a highly pure state without any contaminating side activities or proteins. This can be done by deleting the genes, which codes for unwanted enzymes and proteins Enzyme formulations . Enzymes are formulated mainly in two forms, as a liquid product or as a granular product . 1. Liquid product formulation . The highly concentrated liquids of the evaporator or the ultra filtration unit can be used for the manufacturing of the liquid formulations. The liquids, which are to be incorporated into the formulation, must be sterilized against microbial growth. Stabilizing agents like borax, organic boric acid derivatives, alkali salts, etc should be added along with preservatives like urea, propel glycol, diglycol, and sorbitol. The current trend is to formulate these liquid formulations as structured liquids with the help of salts and polymers, so that all surfactant remains in the structured liquid and enzymes remain in the aqueous phase. (Hermann et al, 1997) 2. Granular enzyme products . Highly ultra filtered and dialysed enzyme solution is subjected to adjustment of pH, turbulences, and temperature in the suitable range, when the enzyme crystallises out. It can be precipitated at high salt concentrations. The following four types of granulation process are employed: i. Enzyme pulling: : The enzyme (dry) is dispersed into a molten wax, non-ionic surfactant, or polymer matrix, and then sprayed in a cooling tower to form solid, spherical, molten water-soluble or water dispersible material with a P a g e | 11 melting point above 50C. This technique offers the advantages of high throughput and ability to recycle particles that fall outside the desired size range but has a drawback i.e., the particle has relatively poor physical strength, leading to break- up and high dust generation in subsequent processing. Polyethylene glycols can be used to improve physical strength and thus lesser dust formation, but as particle breakup cannot be completely ruled out, it is not used widely at present. ii. Granulation by extrusion process: : In this technique, all the ingredients like enzyme powder or liquid concentrate with binders, such as clay sugar, starch, some anticlogging agents like cellulose fibres and solubility enhancers like sodium sulphate are mixed together and an extrudable dough is produced, which is then pressed through the perforated metal plate. The extruded noodles are cut into small cylinders and then given a round shape by a spheroniser. After sieving, the particles are coated with pigments such as titanium dioxide, and protective outer layers to achieve desired appearance and to improve granulate integrity. The drawback of this method is the high capital investment in a multi-step process and the sensitivity of the process variation in feedstock moisture and composition. : In this process the enzyme is mixed with controlled amounts of water, binders such as polyethylene glycol, ethoxylated fatty alcohols, fatty acids, bentonite, waxes having low melting temperature and other granulating agents so as to form a low-moisture agglomerate. This agglomerate is then passed on to the high-shear mixer in which it is broken up into smaller particles. The particles are then dried in a fluidised bed and coated with a final protective layer and pigments like titanium dioxide. . : In this process, on the inert support or core material like sodium chloride, calcium alginate, urea, or saccharose beads, liquid enzyme is sprayed and the coating material is transferred to the drying zone with the help of heated air stream. Then once the enzyme layer has dried, additional coatings of stabilizers, chelating agents, antioxidants and pigments are applied. The outer coating consists of film forming polymer such as titanium dioxide. The volume of flow for proper fluidization is dependent on the surface area and the shape & density of the core material. The proper fluidization should flow sufficient core material through the spray zone to coat all the atomized liquids iii. High shear granulation: iv. Fluid bed coating: P a g e | 12 on . to the core material to prevent spray drying. Production of enzyme-based detergents The manufacture of an enzyme detergent is not very different from that of the conventional synthetic one. Modern industrial cultivation of enzymes begins with fermentation of a vial of dried or frozen micro-organisms called a production strain. This production strain is selected to produce large amounts of the enzymes of interest. The production strain is first cultivated in a small flask containing nutrients and agar. The flask is placed in an incubator which provides the optimal temperature for the previously frozen or dried cells to germinate. Once the flask is ready, the cells are transferred to a seed fermenter, which is a large tank containing previously sterilized raw materials and water, known as the medium. Seed fermentation allows the cells to reproduce and adapt to the environment and nutrients that they will encounter later on. The cells are then transferred to a larger tank, the main fermenter, where temperature, pH and dissolved oxygen are carefully controlled to optimize enzyme production. Additional nutrients may be added to enhance productivity. When main fermentation is complete, the mixture of cells, nutrients and enzymes, referred to as the broth, is ready for filtration and purification. The prime step for the formulation of an enzyme-based detergent is the compatibility of the enzyme(s) with various detergent ingredients. In general, the suitability of an enzyme preparation mainly depends on its compatibility with the detergents at moderately higher temperatures. An ideal enzyme for detergent preparation should be effective at low levels (0.4–0.8%) in the detergent solution. It should also be compatible with various detergent components along with oxidizing and sequestering agents and possess adequate temperature stability to be active in a wide range of cleaning temperatures. It must also have a long shelf life 11. Moreover, the very low use concentration is due to the fact that the enzymes added to the product are biocatalysts. In this context, the term biocatalyzation is implied wherein the enzymes themselves are not being consumed during the cleaning process and a single enzyme triggers numerous chemical reactions. As a result, the disadvantages of the conventional detergents are eliminated. The early use of enzyme powders in detergents led to dust problems in the production process. In addition, the reduced stability of the enzymes due to autolysis and detrimental effects by the other detergent ingredients in the presence of moisture were encountered. These problems led to the use of granulation techniques and enzyme prilling with enzymes being encapsulated in an inert water soluble waxy substance. In powder detergents, the enzymes are mixed with the finished powders as granulates or prills. Currently, wax-coated enzyme detergent granules are being offered in colours identical to the non- P a g e | 13 coloured detergent granules. The coloured granules are termed as signal granules by the detergent manufacturers which symbolize the presence of an extra-added active ingredient in the detergent preparations. In case of enzyme-based automatic dishwashing detergents, citrate and other polyacrylate builders are added. Moreover, perborates and percarbonates are also used. These peroxybleach generating systems are not too harmful for enzymes and through the action of activators such as tetraacetylethylene diamine (TAED), enable the acceptable bleaching action at low temperature. Enzyme stabilization Most early enzyme products such as detergent proteases were just powders. Almost all of them were granulated and further protected by coatings. Another method to prevent enzyme dust in the air is liquid formulations. Today a lot of research work is being done in the different formulations and stabilization techniques in many enzyme detergent production facilities. The enzymes used in various detergent formulations are subject to proteolytic and autolytic degradation on storage and sudden exposure to harsh operating conditions results in rapid inactivation of enzyme activity. Loss of enzyme activity is also encountered during storage in the factory, shipment to client(s) and/or storage in client(s) facilities. Hence, storage stability is of prime concern to enzyme manufactures. The rate of enzyme inactivation is largely dependent on temperature, pH and other detergent components such as surface active agents, sequestrants and bleaching agents. Moreover, the higher the temperature and alkalinity, the less stable is the enzyme. The loss of the enzyme activity is mainly due to the partial unfolding of the polypeptide chain, since the inactivating agent breaks down the delicate balance of noncovalent bonds which maintain the native conformation. The ideal approach to stabilize the enzyme would be to identify the mechanism of inactivation and then design a procedure which would prevent that mechanism. In order to protect the enzyme against denaturation, addition of stabilizers like calcium salts, sodium formate, borate, polyhydric alcohols and protein preparations have proved successful. To prevent contamination of the final commercial crude preparation during storage, addition of sodium chloride at 18–20% concentration has been suggested. These processes maintain the enzyme activity and improve storage stability. In certain cases, for the purpose of convenience in handling and storage, liquid enzyme preparations are often brought to powder form by vacuum or air drying which are milder and less expensive than lyophilization. The stabilization of enzymes has also been made possible through use of protein engineering to design tailor-made enzymes with specific enzyme properties and stability and this technique is leading new insights into the process of biocatalysis. Protein engineering is rapidly emerging today as a new science and P a g e | 14 is basically an art of modifying an existing protein or creating de novo, a protein of pre-specified properties. From a commercial viewpoint, this technology is inherently complex, costly and time consuming. Despite these inherent drawbacks, commercial detergent enzyme producers adopt this technology for producing novel and/or superior enzymes with stable, new and/or improved properties like stain removing ability, improved stability due to resistance to oxidizing agents (oxygen-based bleaching), etc.. Applications of enzyme-based detergents Data published up to now indicate that the enzyme detergents are being mainly explored for their application in laundry, dishwashing, textile and other such industries. Of late, in view of their advantages and increased potentiality, some researchers have tried to use them in the food and dairy industries. The different applications wherein the enzyme detergents are being currently used are: In laundry The microbial enzymes which have found application so far in laundry are the proteases, amylases and lipases. More recently, the cellulases have also been employed in the detergent industry with an added dimension. The proteases hydrolyse the proteinaceous residues of blood, egg, grass and sweat to form soluble peptides which are subsequently easily removed by detergent suds. The amylases degrade the residues of starchy foods like porridge, potatoes, gravies, custard, chocolate, etc. to dextrins, while the lipases catalyse the hydrolysis of salad oil, sauces, lipstick, etc. The cellulases in the detergents degrade mainly the microfibrils which are generated during continuous use and repeated washings of the garment and also help in restoring the original shine and colour of the garment. The washing performance of the enzyme detergent depends on many factors to achieve better results. These are detergent composition and dosage, pH and buffer capacity, water hardness, washing time and temperature, mechanical handling, soiling agents, textile types to name a few. In addition, the specificity of the enzyme is another most important parameter. As a general opinion, it is considered that a detergent enzyme should have as wide a specificity as possible. For example, a protease should be capable of degrading as many proteins as possible. However, a reasonably good wash performance can be achieved by a specific protease, in comparison to a non-specific protease . As the hydro-lysis proceeds, small peptide fragments are formed by the action of an unspecific protease, which are rather difficult to remove as they are not very much soluble in detergent solutions. On the other hand, larger protein or peptide fragments are formed on hydrolysis with a specific protease due to the P a g e | 15 breakdown of very few peptide bonds which can be easily removed during the washing process. Presently, use of dual enzymes in detergent formulations is practised, wherein the enzymatic hydrolysis and degradation can be broadened considerably in comparison to a single enzyme approach. Recently, workers of the Genencor International Inc., USA have developed enzymes called endoglycosidases which deglycosylate biopolymers like glycoproteins which are widely distributed in living organisms. They employed rDNA technology to develop Endo-b -N-acetyl glucosaminidase H (Endo H) as a cleaning agent. Endo H has a unique property to remove bacteria (Staphylococci and E. coli) from glass and cloth surfaces in buffer and detergent solutions. At present, most of the advanced countries like Japan, United States and some European countries almost invariably use the detergents incorporated with enzymes. Interestingly, in Japan, all detergent brands contain enzymes. In India, a few premium detergent brands presently available in the market like Ariel (Procter and Gamble (India) Ltd.), Surf Ultra, Rin Biolites, Revel Plus (Hindustan Lever Ltd.) and Zymo (Henkel) contain enzymes in their formulations. Recently, Procter and Gamble (P&G) has introduced a new cellulase enzyme in the detergent powder, Ariel, presently marketed in India, that eliminates the fuzz formed during washing and tumble-drying, particularly of the cotton fabrics. The manufacturers claim that use of this product retains the colour and improves the texture of the fabric on repeated washings. In dishwashing Enzymes have been successfully used in laundry detergents for many years as an aid to remove tough stains. However, the interest in using enzymes in automatic dishwashing detergents (ADDs) has increased recently. Both laundry and dishwashing detergents share similar functions such as removal of stains from egg, milk and starch-based soilings, etc. The performance of the enzymes in the ADDs is strongly influenced by the ADD formulation and the conditions of the automatic dishwashing. At present, proteases and amylases are the only two enzymes which have found major application in dishwashing detergents. In particular, enzyme-based dishwashing detergents are less abrasive in function and thus are suitable for use on delicate chinaware; they prevent the erosion of designs and colours. This application was first exploited in Japan where the use of richly decorated chinaware and wooden kitchen utensils is widespread. Enzymatic ADDs have gained widespread usage since the last decade. In the past 2–3 years, ADDs with enzymes were launched in several European countries, viz. Austria, Germany, Switzerland, Denmark and the United Kingdom. In Japan, all major ADD brands contain enzymes, whereas only one brand in the US market currently contains enzymes. However, at present, there are no enzymatic dishwashing detergents available in India. P a g e | 16 In the textile industry Currently, in the textile industry, there is a widespread demand for faded jeans. This involves subjecting such clothes to amylases – a process commonly referred to as biowashing or biobleaching, an alternative to the term, enzymefade. This allows elegant softness and unique shades to be given to the cloth which overcomes the traditional methods of bleaching by sodium hypochlorite or tumbling with pumice stones, and also offers better safety as well as economy. In food and dairy industries With the better understanding of such enzymes, more and more areas of their application are emerging, such as in dairy, food and beverage industries. The use of enzymes in these industries in the cleaning operations helps in creating the required hygienic conditions in such plants. Probably, the use of enzymebased detergents in the in-place cleaning of membranes of ultrafiltration (UF) and reverse osmosis (RO) equipments proves promising and forms one of the most important aspects of modern dairy and food industries. The UF and RO membranes are put to a variety of uses including concentration, clarification and/or sterilization of liquid foods like skim milk, whey, egg white, fruit juices and beverages. Despite their diverse applications, these two membrane processes have some inherent disadvantages. The membrane filters come in contact with the feed stock during use. Even a small degree of adsorption causes pore blockage resulting in clogging of filters, a phenomenon called fouling, and thereby cause a reduction in the permeate flux rate and loss in the product quality with increase in production costs. In general, the proteins, inorganic salts and fat residues along with bacteria constitute the common and important fouling agents responsible for lowering the flux and affecting the product quality. Depending on the type of application, the precise formulations are made; for instance, proteases are used for fouled dairy filters, a -amylases and b -glucanases in yeast and cereal, and cellulases and pectinases for wines and fruit juices. The enzyme detergent preparations presently marketed for cleaning of membrane systems are Terg-a-zyme (Alconox, Inc, New York, USA) and Ultrasil 53 (Henkel KGaA, Dusseldorf, Germany). These enzyme-based cleaners that have been marketed rely very much on the proteases to cleave and solubilize the protein foulant. The use of alkaline proteases from Bacillus sp. strain MK5-6 has also proved successful in our laboratory. Pilot scale evaluation of the enzymes at plant level operations for UF membrane cleaning indicated the enzyme preparation to be highly effective and restored 100% flux in comparison to Terg-a-zyme, a commercial preparation which resulted in only 80% restoration of the flux. The use of proteases and lipases to degrade and solubilize protein and fat foulants has also proved beneficial. P a g e | 17 Other uses The application of enzyme-cleaners in the optical industry is important, enabling one to give 100% safe and efficient cleaning to lenses. In India, presently one such enzyme-based optical cleaner in the form of tablets containing Subtilopeptidase A is being marketed by M/s Bausch and Lomb (India) Ltd. Enzyme detergents have also found application in hospitals. Promod 153L, a protease enzyme-based cleaner, has been used to clean surgical instruments fouled by blood proteins. Benefits of enzymes The past decades with a growing number of enzyme applications in consumer detergents have led to major improvements in terms of benefits for consumers. Low temperature efficiency . Enzymes catalyze the breakdown of soils and stain materials at lower temperatures. This allowed washing at lower temperatures and using less water throughout Europe whilst washing performance has improved. The energysaving in the home from the temperature reduction and consequent reduction in environmental emissions (such as carbon dioxide) is considerable as a washing machine operated at 40°C consumes only one third of the energy it would use at 95°C. Weight-efficiency Because enzymes act as catalysts (which can be used repeatedly to speed up chemical reactions without themselves being depleted) they are very weight efficient and cost effective. In other words, they can potentially replace a larger usage of conventional chemicals in the detergent. From an eco-toxicological viewpoint, enzymes can be considered as highly optimized laundry products ingredients which contribute positively to the overall environmental profile of detergents. Other Technical and consumer research has demonstrated that the formulation of P&G detergents with enzyme has led to significant consumer benefits in terms of performance. The benefits of enzymes are related to both the laundry process and the wash results, and include the abilities to: P a g e | 18 Wash at varying pH levels, from mild to high alkalinity; Use different wash temperatures, from 60°C to as low as the "30-40°C range"; Retain laundering performance in the presence of chemicals such as bleach; builder, surfactant, etc…. Soften fabrics; Brighten their colors; Improve whiteness Remove fatty stains at low wash temperatures; Conclusions Thus to conclude, cleaning forms an important aspect for the maintenance of hygiene and safety of foods in the food processing industry. Improperly cleaned food-contact surfaces lead to the accumulation of food particulates which favour the formation of biofilms, i.e. attachment of microorganisms. These cause post contamination and spoilage of foods. It is, therefore, necessary to understand the interactions of the biotic and abiotic entities in the food-processing operations and further effectively analyse the impacts of cleaning and sanitation from a microbiological viewpoint. The use of enzyme-based detergents as biocleaners can also serve as a viable option to overcome the biofilm problem in the food industry. Further, the technology and production of these enzymes and the enzyme-based detergents is mostly patent-protected. As such most of the enzymes used in the detergent industry in India are being imported. Even the large scale detergent manufacture seems highly technical requiring specific know-how and infrastructure. Work has been going on in the recent past in order to develop an indigenous technology on different enzyme systems in certain well reputed laboratories, viz. National Dairy Research Institute, Karnal, National Chemical Laboratory, Pune and Institute of Microbial Technology, Chandigarh. Due to their high efficiency and safety, it is assumed that the enzyme detergents will eventually capture a bulk of the Indian detergent market. P a g e | 19 As in the other sectors of the chemical process industry, where enzymes are increasingly playing a crucial role in making conventional processes more environment-friendly, the detergents industry has also benefited from the introduction of enzymes. The enzyme detergents are proving to be better than the traditional detergents with respect to washing performance, but there are still few constraints like the inability of the enzymes to withstand high alkalinity and variable temperatures. 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