Application of a-Amylases

Biotechnological approaches have been applied for the production of industrial enzymes, pharmaceutical agents, food additives, etc. Amylases are being used for various purposes like brewing, ethanol and biofuel production, textiles, paper, detergent, food and pharmaceutical industry, animal feed preparation, paper recycling, and bioconversion of wastes (Figure 2.5) (Yan and Wu 2016). The annual demand of amylases in the world market is estimated to increase by 4%, and the enzymes are significantly used in detergents (37%), textiles (12%), starch processing (11%), baking (8%) and animal feed production (6%) (Deb et al. 2013).

Liquefaction of Starch

Liquefaction is the initial essential step for production of glucose syrup, maltose, high fructose containing syrup (HFCS), oligosaccharide mixture, branched dextrin, etc. Starch is the unequal mixture of amylose and amylopectin and forms viscous suspension in water. Commercially available thermostable a-amylases, mostly from Bacillus amyloliquefaciens and Bacillus licheniformis, are used extensively in starch processing technology for initiation of liquefaction, which produces short-chain dextrin and reduces the viscosity of starch suspension. Saccharification and production of several useful products are made by using (3-amylase, isoamylase, pullulanase, glucoamylase, a-glucosidase, maltotetraose forming amylase (G4 amylase).

Applications of amylase in various purposes

FIGURE 2.5 Applications of amylase in various purposes.

2.10.1.1 Manufacture of Glucose Syrup and Glucose

Production of glucose syrup is the primary step for manufacturing of crystalline glucose, HFCS, fructose and maltose. Starch slurry, mostly corn starch, is subjected to liquefaction by thermostable a-amylase followed by saccharification through fungal glucoamylase and debranching enzyme. The efficacy of the saccharification process depends on the degree of dextrinization during liquefaction, as the affinity of glucoamylase decreases with high levels of dextrinized products. After conversion to glucose, the solution is purified through ion exchange treatment (cationic, anionic and mixed resin beds) followed by decolourization. Glucose syrup is obtained through subsequent increase of its concentration by evaporation technique. Production of glucose powder and anhydrous dextrose starts after solidification of whole content or crystallization of glucose syrup (Figure 2.6). Glucose has several applications (Table 2.4).

2.10.1.2 Manufacture of Maltose

Maltose is basically used as sweetener in food industries for its low tendency to crystallization and relatively non-hygroscopic nature. In medical fields, it is applied as intravenous sugar supplement. Several natural food items such as corn, potato, sweet potato, and cassava starches are used for the purpose of maltose production. The concentration of starch slurry is not fixed; 10%-20% is required for production of medical grade maltose while more concentration (20%—40%) is adjusted for food grade production.

Schematic representation of steps of manufacturing of glucose syrup, glucose, fructose, high fructose containing syrups (HFCS), maltose and dextrin

FIGURE 2.6 Schematic representation of steps of manufacturing of glucose syrup, glucose, fructose, high fructose containing syrups (HFCS), maltose and dextrin.

The initial liquefaction process is similar to glucose syrup production. Later, saccharification is done with isoamylase, pullulanase and p-amylase to breakdown of a-1,6 gly- cosidic bond and exo-amylase activity. Finally, decolourization and desalting is done.

Different Uses of Glucose, High Fructose Containing Syrups (HFCS), Fructose, Maltose and Dextrin.

TABLE 2.4

Products

Glucose

HFCS

Fructose

Maltose

Dextrin

Used for production of bread, confectionary products (jams, jelly, chewing gum), soft drinks, canned foods, ice cream, sake.

Used as instant powder food.

Medical uses.

Used for production of beverages, dairy products, bakery and confectionary products, canned foods, frozen candies

Fructose is used in food and

beverage industry as sweetener, flavour enhancer, colour developer, freezing point depressant, and osmotic stabilizer.

Medical grade maltose is used for

intravenous injection.

Food grade maltose is used as sweetener, quality improver in several bakery and confectionary products, infant and geriatric foods.

Used for production glucose, maltose in industrial sector.

2.10.1.3 Manufacture of High Fructose Containing Syrups

Glucose syrup (glucose content >90%) is initially produced from starch as raw material, which is converted to HFCS 42F (fructose content, 42%) by enzymatic isomerization. 55F HFCS is prepared by mixture of 42F HFCS and purified fructose. The glucose syrup is passed through a step of conditioning with pH 8-8.5 at 60°C in presence of Mg2+ ion followed by isomerization with commercially available immobilized glucose isomerase. Ion exchanger and activated carbon are used for desaltation and decolourization respectively. 42F HFCS is formed after maintaining the appropriate concentration. The separation of fructose fraction is made through cation exchanger resin, which is blended with 42F HFCS for preparation of 55F HFCS (Figure 2.6). The composition of 42F HFCS and 55F HFCS are like that: fructose 42%, glucose 50%, oligosaccharide 8% and fructose 55%, glucose 39%, oligosaccharide 6% respectively. HFCS are used for preparation of beverages, dairy products, baking products, frozen candies, confectioneries, etc.

2.10.1.4 Manufacture of Fructose

Fructose is the naturally sweetest sugar present commonly in fruits. It has several applications. Fructose fraction of 42F HFCS has been separated through affinity chromatography followed by crystallization. Crystallization starts in the presence of crystalline fructose powder (l%-4% w/w) at 60°C and slowly cooled for crystal formation. The newly formed crystal is very small (100-200 pm) in size and to increase the size, double stage crystallization method has been applied.

2.10.1.5 Manufacture of Maltotriose-Rich Maltooligosaccharides Mixture (Maltooligomer Mix)

Maltooligomer mix has some unique properties and several applications in food industry. The initial step of liquefaction of starch is very similar to glucose syrup production followed by treatment with p-amylase and pullulanase. Maltooligomer mix is separated from maltose through chromatographic technique and subsequent treatment of decolourization and deionization with active carbon and ion exchanger respectively. This process gives liquid product which is converted to powder form after spray drying.

2.10.1.6 Manufacture of Maltotetraose Syrup (G4 Syrup)

G4 syrup production is initiated by the same process of liquefaction of starch, but the later stage of saccharification is mediated by G4 amylase and isoamylase. Final product is prepared after purification (desaltation by ion exchanger and decolourization by activated carbon). The general properties and applications of G4 syrup are very similar to maltooligosaccharides mixture.

2.10.1.7 Production of Anomalously Linked Oligosaccharides Mixture (Alo Mixture)

Alo mixture is a mixture of isomaltose, panose, isomaltotriose and branched oligosaccharide. The initial step is similar to other oligosaccharide preparation. Dextrinized product is subjected to saccharification and transglucosidation by using (3-amylase and fungal a-glucosidase followed by desaltation and decolourization through cation and anion exchanger mixed bed, and active carbon respectively. Table 2.5 shows the properties and applications of Alo mixture.

2.10.1.8 Manufacture of High Molecular Weight (MW) Branched Dextrins

High MW branched dextrins are manufactured mostly from corn starch and waxy corn starch which contains exceptionally high amount of amylopectin. Liquefaction of starch by bacterial a-amylase produces high MW branched dextrins which is separated from lower oligosaccharides (Figure 2.7). Branched dextrins are used in food and pharmaceutical industry.

Applications in Food Industry

Alpha amylase is used for production of chocolate syrup from cocoa slurries. Chocolate syrup is a low viscous stable compound with cocoa flavour, used for manufacturing of frozen chocolate, flavoured confectioneries, etc. Bakery industry uses a-amylase for hydrolysis starch in dough. This process improves the quality of bread by increasing texture, loaf volume, flavour, better colour, a softer crumb, taste, toasting qualities and shelf life. The fermentable products of a-amylase act as anti-staling agents and they prevent the staling effects like crumb firmness, loss of crispness of the crust and decrease in moisture content. Amylases are also used for the clarification of beer, fruit juices and animal feed production to improve the digestibility of fiber (Kumari et al. 2012; Dey and Banerjee 2014).

Removal of Starch Sizer from Textile (Desizing)

In textile industry, starch paste is applied as sizer agent during weaving. The starch sizer protects the string from friction, cutting and generation of static electricity on the string at the time of warping. After making the cloth, starch is removed (desizing) before scouring and dyeing. There are several processes of desizing such as enzymatic, oxidative, acid wash, and fermentative. Thermostable a-amylase is used for enzymatic desizing process which is most effective for increasing the quality of cloth. The following steps are essential to continue the process: gelatinized starch used as sizer to the cloth —> conditioning at high temperature (90°C) -»treatment by thermostable a-amylase -* hot water washing -> cold water washing -> drying.

Fermentation of Starch to Ethanol for Production of Biofuel

Ethanol is used as biofuel in several countries to fight against environmental pollution and global warming. In this purpose, starch is used as substrate due its low cost and maximum availability. There are three main stages for production of ethanol from starch. Stage 1: liquefaction of starch is mediated by thermostable a-amylase as described by Pervez et al. (2014). Stage 2: saccharification of liquefied starch is performed by other sets of enzymes like (3-amylase, glucoamylase, pullulanase, etc.

Properties and Uses of Different Oligosaccharides

Products

Composition (%)

Properties

Uses

Maltotriose-rich

maltooligosaccharides

mixture

G-2.0%, G2-37.5%, G3-46.5%, G4 and maltotetraose and larger maltooligosaccharides 14.0%

Sweetness is only 30% than sucrose, low viscosity, low' freezing point, less colour formation than com syrup, highly hygroscopic.

Used as moisture regulator of foods, substitute of sucrose, lowers the hardness and maintain the texture of the foods during storage.

Mostly used for manufacturing of confectionary products (jams, jelly, cake, chewing gum, butter cream, custard cream), bakery products, frozen foods, canned coffee, cocoa, fruit drinks, alcoholic beverages.

G4 syrup

G-1.0%, G2-6.5%, G3-10.0%, G4-50% dextrin s-30%

Less sweeter (20% of sucrose) and used as sucrose substituent, high moisture retaining capacity, less colouration due to low glucose and maltose, viscosity is 2.5 times than sucrose, low freezing point, low osmotic pressure and gloss imparter.

Used to improve the texture of the foods, controls the freezing point of frozen foods, regulates the moisture of the foods.

Used in preparation of bakery products, confectionary products and soft drinks.

Alo mixture

G-40.5%, G2-6.5%, isomaltose-17%, G3-l%, anomalously linked tetraoligosaccharides-10%

Mildly sweet, low viscosity, high moisture retaining capacity, low water activity—controls microbial contamination.

Used in bakery products, confectionary products, soft drinks, sake making.

High molecular weight branched dextrins

Maximum amount of branched dextrins (85%-90%), minimum amount of G5, G4 and negligible amount of G3, G2, G

No retrogradation after long standing in low temperature, low viscous than starch, hygroscopic property.

Used as extender and glazing agents for preparation of powdery food products

High molecular weight branched dextrins from waxy starch

Oligosaccharides higher than maltohexaose is 96%

No retrogradation and maintenance clarity after long standing, no deliquescent property

Used as dilution powder in pharmaceutical products; Used as binding, stabilizing, texturing and glazing agent for preparation of custard powder, cake mixture, bread improvers, canned foods.

G = glucose, G2 = maltose, G3 = maltotriose, G4 = maltotetraose, G5 = maltopentaose

Schematic representation of steps of manufacturing of Maltotriose-rich maltooligo- saccharides mixture. G4 syrup. Alo mixture and high molecular weight branched dextrins

FIGURE 2.7 Schematic representation of steps of manufacturing of Maltotriose-rich maltooligo- saccharides mixture. G4 syrup. Alo mixture and high molecular weight branched dextrins.

This step gives simple sugars and smaller oligosaccharides for alcoholic fermentation. Stage 3: fermentable sugars are then fermented by yeast to produce ethanol. To avoid these three steps, a new genetically engineered strain of yeast is applied, which is constructed by protoplast fusion between the amylolytic yeast Saccharomycesfibu- ligera and S. cerevisiae. This strain is able to ferment starch directly to ethanol (Chi et al. 2009).

Treatment of Starch Processing Waste Water (SPW)

Starch is present in the effluent of various industries like textile, paper, sugar, bakery confectionary, brewing, etc. The removal of waste from industrial effluent is an expensive process and sometimes produces biochemical hazards in the environment. Enzymatic treatment of waste is an eco-friendly process and economically beneficial. Starchy waste has been treated with a-amylase for degradation followed by production of microbial biomass or biofuel. Eco-biotechnological strategies can also be acceptable for bioconversion of wastes into hydrogen and methane (Kumar et al. 2014).

Preparation of Starch Hydrolysate as Sizer of Paper

Starch paste is applied as sizing agent during papermaking, which protects paper against mechanical damage during processing and reduces liquid absorption tendency at the time of drying. Thermostable a-amylase treated low viscous starchy solution is used as sizer. This process enhances the quality, texture, strength, smoothness, writing and erasability capacity of the finished paper (Sundarram and Murthy 2014).

Use in Detergent Preparation

Enzymes are the major constituent of 90% of liquid detergents. Application of enzyme- based detergent is environmentally safe. Among the different enzymes, a-amylase is the major ingredient to formulate the enzyme-based detergents. Starch holds the dust particle on the surface of the cloth, a-amylase degrades the starchy ingredients to increase the whiteness and brightness of the cloth. Similarly, the residues of starchy food ingredients are present as remnants on the used food dishes. These residual starchy materials are removed by a-amylase. Generally, there is no fixed temperature to use the detergent and the manufacturers have tried to formulate the products to be effective at a broad range of temperatures, especially at the lower scale. For this purpose, psychrophilic organism Pseudoalteromonas arctica GS230 a-amylase gene has been cloned to express in E. coli for production of cold sensitive efficient enzyme (Lu et al. 2010). Moreover, a-amylase active at alkaline pH is mostly preferable for detergent preparation. Protein engineering is also applied for production of alkalophilic, chelator insensitive, and oxidant resistant a-amylase (Kumari et al. 2012, Sundarram and Murthy 2014).

Clinical Applications

The fungal a-amylase from Aspergillus oryzae (Takadiastase) is used as digestive aids for the treatment of indigestion. The liquid and capsule formulations of digestive medicines are available in the market. Ultrasensitive amylase biosensor has been developed by using a film of polysaccharides like glycogen and amylopectin through spin-coated onto gold-coated quartz crystals (Gibbs et al. 2015) or immobilized maltopentaose along with a-glucosidase on a membrane (Wang et al. 2015). This biosensor can determine the changes in serum or urine a-amylase concentration for the diagnosis of acute pancreatitis, which is associated with hyperamylasemia as well as urinary amylase. The obstruction of biliopancreatic duct is another source of serum amylase (Fishman et al. 2014). a-Amylase also serves as a biomarker for pancreatic fistula, acute stress, gastric aspiration, etc. (Yan and Wu 2016). Thus, biosensor- mediated amylase detection is the diagnostic tool for hyperamylasemia. Therapeutic inhibitors (flavonoids or alkaloids or acarbose in nature) of a-amylase are commonly extracted from wheat seed, white kidney bean, Phaseolus vulgaris, Sophora japonica, Varthemia iphionoides and Ginkgo biloba, which are used for treatment of post-pran- dial hyperglycemia, obesity and type 2 diabetes (Wulan et al. 2014; Gupta et al. 2014).

 
Source
< Prev   CONTENTS   Source   Next >