Purification of Fructosyl Transferase (FTase)

shade of Fructosyl Transferase (FTase)IntroductionFood harvests atomic number 18 develop for taste, appearance, speak to and convenience of the consumer. The composition of nourishment products that confer a intimatelyness benefit is a relatively new trend, and recognizes the growing acceptance of the fibre of diet in disease saloon, treatment and well-being. It is thus becoming more and more clear that in that location is a strong relationship between the sustenance we consume and our health. Scientific knowledge of the beneficial role of various nourishment component parts (nutrients) for the prevention of particularised diseases is rapidly advancing. us competent pabulums, pharma foods and nutraceuticals argon synonyms for foods that tush prevent and treat diseases.Generally, a functional food pot be defined as any food that has a positive advert on an individuals health, physical performance or fix of mind in addition to its nutritional content. Functional fo ods in addition to their basic nutritional content and natural being, forget verify the proper balance of ingredients which will help to emend many aspects of tender-hearted lives, including the prevention and treatment of illness and disease (Goldberg, 1994). So far, a tumidr number of functional foods in various forms buzz off already been introduced into the market. many of them contain a number of characteristic functional ingredients. They embroil dietetical fiber, oligosaccharides, sugar alcohols, peptides and proteins, prebiotics and probiotics, phytochemicals, antioxidants and polyunsaturated fatty acids (Stark and Madar, 1994).Oligosaccharides argon very well recognised as functional food ingredients beca hold of their positive military units on forgiving health. This look work focuses on the microbial product and purification of Fructosyl Transferase (FTase) and the achievement of Fructooligosaccharides (FOS) by transfructosylation using this enzyme.Oligosac charidesOligosaccharides argon usually defined as carbohydrates with a low degree of polymerization (DP) and consequently low molecular weight. They are composed of 3-10 unre military serviced sugar units linked together.The primary(prenominal) categories of nondigestible oligosaccharides embarrass carbohydrates in which the monosaccharide unit is fructose, galactose, glucose and xylose. Chemical resistences among moneymaking(prenominal)ly available oligosaccharides include compass length, monosaccharide composition, degree of branching and purity. They are found of course (small amounts) in many defines.. Plants with large amounts of oligosaccharides includechicory root, from which virtually commercial inulin is extracted, and so-called Jerusalem. They are similarly found in onions,leeks, garlic, legumes, wheat, asparagus, jicama, and other plant foods.Oligosaccharides are functional food ingredients that drop great potential to improve the quality of many foods. In add ition to providing useful modifications to food expression and physicochemical characteristics, they possess properties that are beneficial to the health of the consumers. In general, food mug oligosaccharides are not pure products, only if are mixtures containing oligosaccharides of distinguishable degrees of polymerization, the parent polysaccharide or disaccharide and the monomer sugars (Roberfroid and Slavin, 2000).The production and application of food grade oligosaccharides are increasing and their major uses are in beverages, infant milk powders, confectioneries, bakery products, yoghurts and dairy farm desserts.FRUCTOOLIGOSACCHARIDESFOS are chain polymers of the sugar fructose that are found in a variety of foods. The sugar units send packing be linked in a single straight chain or can be branched. In many cases small amounts of glucose are also contained in the chain. Chemically, FOS is difficult to define, because the length of the fructose chains can set forth from consultation to source. Therefore a FOS analysis of a food may report the total of several closely related compounds. Inulin is an example of a longer chained compound that is considered as FOS. The misfortunateer ( discredit molecular weight) compounds tend to have a sweet taste.The size and complexity of the FOS molecule desirable characteristics. Although the simple sugars fructose and glucose are quickly absorbed into the body by the intestines, FOS for the most part is indigestible and therefore acts as a non-digestible fiber in the diet. This is because humans does not have the enzymes to take out pop out FOS as it travels down the digestive tract. When the FOS reaches the large intestine and the colon, the bacterium that are found there start to break down the FOS. These bacteria have the enzymes needed to break down FOS. Bifidobacteria have been reported to use FOS. It is believed that foods that promote bifidobacterial growth are well for the health.Of all the olig osaccharides known so far, FOS has attracted special attention. FOS is a public name for fructose oligomers that are mainly composed of 1- Kestose (GF2), 1 Nystose (GF3) and 1F Fructofuranosyl nystose (GF4) in which fructosyl units (F) are bound at the 2, 1 position of sucrose (GF). social structure of FructooligosaccharidesOccurrenceFOS derived from sucrose occur in many higher plants as reserve carbohydrates. They are found in a variety of nourishment plants, including banana, barley, garlic, honey, onion, rye, brown sugar, tomato, asparagus root, Jerusalem artichoke plant, wheat and triticale (Fishbein et al, 1988). The concentration of FOS in these foods is diagrammatically represented in Figure 1.2 (derived using data from the Environmental defense Agencys Dietary Risk Evaluation system EPA, 1984). FOS uniform 1- kestose, neokestose, 6- kestose and their derivatives have also been isolated from the plant Agave vera cruz (Satyanarayana, 1976).FTase producing organisms FTase by transfructosylation body process helps in the production of FOS. The enzyme source of FOS deduction can be dual-lane into two classes one is plants such as asparagus, sugar beet, onion, Jerusalem artichoke etc the other consists of bacteria and fungi such as Arthrobacter sp., Fusarium sp., Aspergillus sp., Aureobasidium sp.Functional properties of FOSLow calorific valueThe - configuration of the anomeric carbon, C2 in their fructose monomers sop up FOS nondigestible by human digestive enzymes which are mostly specific for -osidic linkages and hence they are not use as an energy source in the body. However, overdue to colonic fermentation, they have an energy contribution to food of about 1.5 kcal/ g. This property makes them suitable for use in sweet, low-calorie diet foods and risk-free for consumption by individuals with diabetes. In the case of very sweet foods, they may be utilize as bulking agents in conjunction with intense conventionalized sweeteners such as aspartame, phenylalanine or sucralose, thereby masking the aftertaste contractd by just about of these intense sweeteners (Crittenden and Playne, 1996).Non cariogenicityUnlike starch and simple sugars, FOSs are not utilized by oral microflora like Streptococcus mutans to form acids and insoluble -glucans, that serve as a matrix for plaque formation and are the main culprits in causing dental caries (Oku, 1994). Hence, FOSs are presently used as non cariogenic sugar substitutes in confectionery, chewing gums, yoghurts and drinks.Dietary fibre outcomeDietary fibre is the edible part of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with fulfil or partial fermentation in the large intestine (AACC Report, 2001). The nondigestible quality of FOS makes them act similar to dietary fibre, and thus prevent constipation. However, extravagant consumption of FOS may cause flatulence or dissolution, the minimum acid being 4 0-50 g/day. The advantages FOS has over dietary fibre are that they have a smaller daily requirement, do not cause diarrhea in recommended doses, are slightly sweet, have neither bad food grain nor bad taste, are completely water soluble, do not manakin viscosity, do not bind minerals, are physically stable, and are easier to stop into processed foods and drinks (Tomomatsu H, 1994).Prebiotic effectIn recent years, the ability of FOS to promote the proliferation of bifidobacteria in the colon has been recognized. These intestinal bacteria metabolize FOS readily and produce large amounts of short chain fatty acids mattering in an acidic pH in the lumen of the large intestine. The beneficial bacteria such as Bifidobacterium sp. and Lactobacillus sp. are resistant to the acidic pH, whereas the harmful bacteria such as clostridium sp. are sensitive to the acidic conditions. Therefore the proliferation of useful bacteria is aroused and that of harmful bacteria is suppressed. Subsequ ently, FOS has been described as one of several prebiotics, which can be defined as a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and / or the act of one or a limited number of bacteria in the colon and thus improves host health (Gibson and Roberfroid, 1995). FOS have been demo to increase bifidobacteria numbers in the colon at doses of Lipid wakeless effectFOS in diet has been found to decrease the levels of triglycerides, serum cholesterin and lipids. Hypotriglyceridemia is due to decrease in the hepatic synthesis of triglycerides and hypocholesterolemia is likely to result from the antagonistic effect of short chain fatty acids, especially propionate on cholesterol metabolism. Propionate has been found to be an inhibitor of HMG CoA reductase, an important enzyme in cholesterol synthesis (Roberfroid and Slavin, 2000).Effect on mineral absorptionFOS have been found to rear the absorption of Ca2+ and Mg2+ ions and mainta in balance of iron and Zn2+ ions. This has been found to be due to the osmotic effect, acidification of the colonic content due to fermentation and production of short chain carboxylic acids, formation of calcium and magnesium salts of these acids and hypertrophy of the colon wall (Roberfroid and Slavin, 2000).Anticancer effectFOS has an indirect effect on prevention of cancer in human beings due to its prebiotic properties. This is due to immunity enhancements by the cells, cell wall components and extracellular components of bifidobacteria (Tomomatsu H, 1994).Production of nutrientsThe presence of a good colonic environment with high bifidobacterial count take aims to the production of Vitamins B-1, B-2, B-6, B-12, nicotinic acid and folic acid. Bifidobacteria fermented dairy products also improve lactose tolerance, calcium absorbability and digestibility (Tomomatsu H, 1994).Antidiabetic effectFOS have been claimed to have no effect on blood glucose levels in patients with type 2 diabetes, possibly due to effects of short chain fatty acids produced during fermentation. (Luo et al, 2000).PROBLEM STATEMENTNormally, pure sucrose is expensive and the cost to produce FTase by using sucrose is high. There are more or less spin-off like cereal bran, corn-products, sugarcane molasses and by-products of coffee and tea process industries were used as a substrate to produce FTase from microorganism (Sangeetha et al., 2003). This by-product can lower the cost of production of FTase. Before FOS can be produced, the enzyme FTase must be undergone a series of purification. The purification of FTase must be done in order to get high yield of FOS. many another(prenominal) researchers have reported the purification and portraying of FTase from various sources and FTase has been found to differ in their molecular weight and properties from one source to another (Lateef et al., 2006, Sangeetha et al., 2003).FTase exhibits hydrolytic activity which can dominate the process (Delphine et al., 2007). This fact will lead to lower production yields and to a contamination of the final product with glucose and fructose (Delphine et al., 2007). Purification of FTase is important for batch production of FOS. By doing so, the nature of its hydrolytic activity can be studied, improve the understanding its mode of operation and be able to classified which type of enzyme should it belong to (LHocine et al., 2000).RESEARCH OBJECTIVESThe main objective of this research is to mitigate FTase enzyme from microfungi Aspergillus oryzae isolated from CFTRI. To enhance the production of FOS as a food additive.SIGNIFICANCE OF STUDYIn Malaysia, the FTase enzyme used to be merchandise from other country such as Japan, India and United States (Sangeetha et al., 2005). Normally, the cost for production of FOS from commercialized enzyme is high, so by producing and purifying the enzyme, we can reduce the cost.Normally, bare-ass enzyme will produce lower yield of FOS than pur ified enzyme. In order to do high customer demand on the functional food such as FOS, the production of FOS must be increased. In order to produce high yield of FOS, the enzyme need to be purified. By doing this, the activity of enzyme will be increased and so do the production of FOS.Purification and characterization of the enzyme (FTase) are necessary steps to obtain the product (FOS) rapidly, with high purity, to improve our understanding of its mode of action and the nature of activity. Efforts have been made to purify the FTase enzyme to get high specific activity and fold of purification but the reaction mechanism involved in FOS production does not lead to higher yields than the theoretical maxima of 56 58 % due to inhibition caused by accumulation of glucose. Nevertheless, the time involved in obtaining maximum FOS yield has been easily reduced by using the purified FTase in comparison to the crude FTase. The product FOS formed has also been characterized for its physicoc hemical properties and structure using HPLC or LC-MS. Studies on the prebiotic effects of FOS are required to establish its functional properties and to demonstrate its use in product development. The present research work has also focus on the scaling up of both the production of FTase as well as FOS.