Title of Invention

A PROCESS FOR OBTAINING XYLOOLIGOSACCHARIDES FROM CEREALS AND THEIR MALTS

Abstract

The present invention relates to a process for obtaining xylooligosaccharides from cereals. The invention is aimed at obtaining oligosaccharides from cereals using cereal/cereal malt enzymes. Xylans present in the cereal/cereal malt/ bran cell wall are hydrolyzed with the cereal/cereal malt enzyme extract, particularly rich with xylanase to obtain xylooligosaccharides. The present method exploits the cereal/cereal malt xylanase, which is induced during germination to hydrolyze cereal/cereal malt/cereal bran xylans leading to the liberation of xylooligosaccharides.

Full Text

The present invention relates to a process for obtaining xylooligosaccharides from cereals. The invention is aimed at obtaining oligosaccharides from cereals using cereal/cereal malt enzymes. Oligosaccharides of various types are found as natural components in many common foods, and in the recent years their popularity has grown rapidly as they are considered as functional food ingredients. This has largely been due to the possible health benefits associated with the consumption of these compounds. Oligosaccharides are used as low/non-cariogenic sugar substitutes, and as ingredients in prebiotic and low-calorie diet foods. As components of soluble dietary fibres, they prevent constipation, effect lipid/cholesterol metabolism, increase mineral absorption, influence immune modulation, and help prevention of colon cancer. Apart from these health benefits, they influence physico-chemical properties of food such as increased viscosity, water binding and gelling properties, improved body and mouth feel, and inhibition of starch retrogradation. They safeguard qualities of cold and heat processed foods and help in food preservation with high moisture retaining capacity, preventing excessive drying, but maintaining low water activity, which is convenient in controlling microbial contamination. Oligosaccharides can be used as bulking agents with reduced sweetness, suitable for consumption by diabetic individuals. They are mainly utilized in confectionary, chewing gums, yoghurts, drinks/beverages, desserts, bakery products and in livestock industry. Non-food applications include drug delivery, cosmetics and mouthwashes. Oligosaccharides are usually defined as glycosides that contain between two to ten sugar moieties. They can be produced either by enzymatic synthesis (step-up approach) from constituent monosaccharides or by enzymatic/chemical hydrolysis (step-down approach) of parent polysaccharides. Food grade oligosaccharides are not pure products, but mixtures containing oligosaccharides of different nature and degrees of polymerization, the parent polysaccharides and monosaccharides. Xylooligosaccharides are an important class of oligosaccharides, which have potential applications in such areas as pharmaceuticals, and prebiotic foods. They promote the growth of bifidobacteria in the colon, and are used predominantly in prebiotic drinks. Industrially xylooligosaccharides are produced by the hydrolysis of polysaccharide - xylan. Although enormous amount of raw material - xylan, as cheap cereal agro-wastes is available, xylooligosaccharide production has not been exploited commensurate with xylan. Therefore, xylooligosaccharides represent only a tiny fraction of more than hundred thousand tons of world's total oligosaccharide market. At the moment, there is tremendous scope for the production of xylooligosaccharides. Reference may be made to Zhao, Z., Egashira, Y. and Sanada, H. J. Agric. Food Chem. 2003, 51, 5534 - 5539; Saulnier, L. Vigouroux, J. and Thibault, J.-F. Carbohydr. Res. 1995, 272, 241 - 253 and Ohta, T., Semboku, N. Kuchii, A. Egashira, Y. and Sanada, H. J. Agric. Food Chem. 1997, 45, 1644 - 1648 wherein cereal brans are hydrolyzed with acids such as oxalic or trifloroacetic acid to obtain xylooligosaccharides. The drawbacks are that acid hydrolysis is nonspecific and uncontrolled, leading to non-desirable product quality. Reference may be made to Mueller-Harvey, I. and Hartley, R. D. Carbohydr. Res. 1986, 148, 71 - 85 and Lequart, C, Nuzillard, J.-M, Kurek, B. and Debeire, P. Carbohydr. Res. 1999, 319, 102 - 111 wherein cereal bran/straws are hydrolyzed with partially purified microbial enzymes such as cellulase or endoxylanase to obtain xylooligosaccharides. The drawbacks are that purification of microbial enzymes is cumbersome and costly. Reference may be made to Ralet, M.-C, Faulds, C. B., Williamson, G. and Thibault, J.-F. Carbohydr. Res. 1994, 263, 257 - 269; Wende, G, and Fry, S. C. Phytochem. 1997, 44 (6), 1019 - 1030 and Ohta, T., Yamasaki, S., Egashira, Y. and Sanada, H. J. Agric. Food Chem. 1994, 42, 653 - 656 wherein cereal brans are hydrolyzed both with acids such as oxalic or trifloroacetic acid and partially purified microbial enzymes such as driselase or hemicellulase to obtain xylooligosaccharides. The drawbacks are that acid hydrolysis is nonspecific and uncontrolled, and purification of microbial enzymes is cumbersome and costly. The main object of the present invention is to provide a process for obtaining xylooligosaccharides from cereals and their malts, which obviates the drawbacks as mentioned above. Another object of the present invention is to obtain feruloylated xylooligosaccharides from cereals and their malts. Still another object of the present invention is to obtain xylanase from cereals and their malts. Yet another object of the present invention is to obtain cell wall degrading enzymes from cereals and their malts. Statement of invention Accordingly the present invention provides a process for obtaining xylooligosaccharides from cereals and their malts, the said process comprising steps of: a) soaking cereals/cereal malts in water in the ratio of 1:4 for a period of 16-17 h at the temperature ranging from 20-27 degree C; b) spreading soaked cereals/cereal malts of step (a) in trays and incubating them at a temperature range of 25-27 degree C for a period of 96-98 h; c) drying cereals/cereal malts of step (b) at the temperature range of 50-52 degree C for a period of 48-50 h; d) grinding the product of step (c) for preparing the cereal/cereal malt flour; e) extracting xylanase from cereal or cereal malt flours of step d) by using acetate buffer, of pH 4.8-4.9,by incubating for a duration of 2-2.5 h at a temperature range of 4-6 degree C, and centrifuging the buffer enriched with xylanase at 4000-4200 x g and at 4-6 degree C for 20-25 min in order to separate out cereal or cereal malt residues to obtain xylanase; f) de-starching cereal/ cereal malt or cereal brans by treating with termamyl at a temperature range of 92-95 degree C for a period of 60-65 min, followed by glucoamylase at a temperature range of 37-39 degree C for a period of 24-26 h, centrifuging the contents, upon mixing with 3 volumes of ethanol, at the range of 4000-4200 x g for a period of 20-25 min at room temperature (25 degree C to 30 degree C), washing the residue with ethanol and drying with acetone to obtain xylans, g) characterized in incubating xylans obtained from step (f) and xylanase rich extract obtained in step (e) in acetate buffer, of pH 4.8-4.9 at a temperature range of 37-39 degree C for a period of 48-50 h; h) treating the above said reaction mixture with 3 volumes of ethanol, and centrifuging at a range of 4000-4200 x g for a period of 20-25 min at room temperature (25 degree C to 30 degree C) to obtain supernatant; i) concentrating the supernatant by using a rotary evaporator at a temperature range of 30-35 degree C under reduced pressure to get concentrated solution of xylooligosaccharides. j) partially purifying the said concentrated solution of xylooligosaccharides by passing through a column of Sephadex LH-20 using 1:3 methanol: water to obtain xylooligosaccharides. In an embodiment of the present invention cereals as such, without malting, are used as the source to obtain xylanase. In another embodiment of the present invention cereal/cereal malt xylanase rich extract is dialyzed to remove salts and free sugars. In still another embodiment of the present invention cereal/cereal malt bran used is from rice and ragi. In yet another embodiment of the present invention Sephadex LH-20 is used for partial purification of xylooligosaccharides. Xylans present in the cereal/cereal malt/ bran cell wall are hydrolyzed with the cereal/cereal malt enzyme extract, particularly rich with xylanase to obtain xylooligosaccharides. The use of cereal/cereal malt xylanase to obtain xylooligosaccharides from cereal/cereal malt bran thus overcomes all the limitations. Unlike acid hydrolysis enzyme hydrolysis is specific and reaction can be controlled. While microbial enzymes are cumbersome to purify and involve costly processes, cereals/cereal malts are rich source of enzymes and thus concentrated enzymes can be obtained without much purification, but with very low cost. The following examples are given by way of illustration of the present invention and therefore should not be constructed to limit the scope of the present invention. EXAMPLE - 1 Stepl 2.5 g cereal/cereal malt (96 h) flour was dispersed in a beaker containing 10 ml of 0.05 M acetate buffer, pH 4.8 and stirred on a magnetic stirrer using a magnetic bar for 2 h at 4 ° C, followed by centrifugation at 4000 x g at 4 ° C for 20 min. Supernatant was collected and dialyzed overnight against 0.05 M acetate buffer, pH 4.8, at 4 ° C. This was used as the xylanase rich extract. The xylanase activity as determined by DNS method using xylan as substrate was high in 96 h malts compared to native (0 h) and varied in different cereals such as rice (native: 0.19 U/min/g flour, malt: 0.78 U/min/g flour), maize (native: 0.28 U/min/g flour, malt: 0.68 U/min/g flour), wheat (native: 0.85 U/min/g flour, malt: 2.66 U/min/g flour) and ragi (native: 0.23 U/min/g flour, malt: 0.98 U/min/g flour). Step 2 10 g of rice flour was dispersed in a conical flask containing 100 ml of 0.05 M acetate buffer, pH 4.8 and 1 ml of termamyl was added and incubated at 95 ° C for 60 min. Contents were cooled and 100 mg of glucoamylase was added and incubated at 37 ° C for 24 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 C to 30 ° C) and residue is washed thrice with ethanol and dried using acetone. The yield of rice bran was 8.2 %. Step 3 250 mg of rice bran is dispersed in a conical flask containing 20 ml of 0.05 M acetate buffer, pH 4.8 and 5 ml of dialyzed wheat malt xylanase rich extract was added and incubated at 37 ° C for 48 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and supernatant was collected. It was concentrated by using a rotary evaporator at ~ 30 ° C under reduced pressure to get concentrated solution of xylooligosaccharides. Step 4 Xylooligosaccharides thus obtained in step 3 were partially purified by passing through a column of Sephadex LH-20 using 1:3 methanol:water. The yield of partially purified xylooligosaccharides was 5.72 %. EXAMPLE - 2 Step 1 2.5 g cereal/cereal malt (96 h) flour was dispersed in a beaker containing 10 ml of 0.05 M acetate buffer, pH 4.8 and stirred on a magnetic stirrer using a magnetic bar for 2 h at 4 ° C, followed by centrifugation at 4000 x g at 4 ° C for 20 min. Supernatant was collected and dialyzed overnight against 0.05 M acetate buffer, pH 4.8, at 4 ° C. This was used as the xylanase rich extract. The xylanase activity as determined by DNS method using xylan as substrate was high in 96 h malts compared to native (0 h) and varied in different cereals such as rice (native: 0.19 U/min/g flour, malt: 0.78 U/min/g flour), maize (native: 0.28 U/min/g flour, malt: 0.68 U/min/g flour), wheat (native: 0.85 U/min/g flour, malt: 2.66 U/min/g flour) and ragi (native: 0.23 U/min/g flour, malt: 0.98 U/min/g flour). Step 2 10 g of rice malt (96 h) flour was dispersed in a conical flask containing 100 ml of 0.05 M acetate buffer, pH 4.8 and 1 ml of termamyl was added and incubated at 95 ° C for 60 min. Contents were cooled and 100 mg of glucoamylase was added and incubated at 37 ° C for 24 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and residue is washed thrice with ethanol and dried using acetone. The yield of rice malt (96 h) bran was 7.5 %. Step 3 250 mg of rice malt (96 h) bran is dispersed in a conical flask containing 20 ml of 0.05 M acetate buffer, pH 4.8 and 5 ml of dialyzed wheat malt xylanase rich extract was added and incubated at 37 ° C for 48 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and supernatant was collected. It was concentrated by using a rotary evaporator at ~ 30 ° C under reduced pressure to get concentrated solution of xylooligosaccharides. Step 4 Xylooligosaccharides thus obtained in step 3 were partially purified by passing through a column of Sephadex LH-20 using 1:3 methanol:water. The yield of partially purified xylooligosaccharides was 5.01 %. EXAMPLE - 3 Step 1 2.5 g cereal/cereal malt (96 h) flour was dispersed in a beaker containing 10 ml of 0.05 M acetate buffer, pH 4.8 and stirred on a magnetic stirrer using a magnetic bar for 2 h at 4 ° C, followed by centrifugation at 4000 x g at 4 ° C for 20 min. Supernatant was collected and dialyzed overnight against 0.05 M acetate buffer, pH 4.8, at 4 ° C. This was used as the xylanase rich extract. The xylanase activity as determined by DNS method using xylan as substrate was high in 96 h malts compared to native (0 h) and varied in different cereals such as rice (native: 0.19 U/min/g flour, malt: 0.78 U/min/g flour), maize (native: 0.28 U/min/g flour, malt: 0.68 U/min/g flour), wheat (native: 0.85 U/min/g flour, malt: 2.66 U/min/g flour) andragi (native: 0.23 U/min/g flour, malt: 0.98 U/min/g flour). Step 2 10 g of ragi flour was dispersed in a conical flask containing 100 ml of 0.05 M acetate buffer, pH 4.8 and 1 ml of termamyl was added and incubated at 95 ° C for 60 min. Contents were cooled and 100 mg of glucoamylase was added and incubated at 37 ° C for 24 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and residue is washed thrice with ethanol and dried using acetone. The yield of ragi bran was 20.3 %. Step 3 250 mg of ragi bran is dispersed in a conical flask containing 20 ml of 0.05 M acetate buffer, pH 4.8 and 5 ml of dialyzed wheat malt xylanase rich extract was added and incubated at 37 ° C for 48 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and supernatant was collected. It was concentrated by using a rotary evaporator at ~ 30 ° C under reduced pressure to get concentrated solution of xylooligosaccharides. Step 4 Xylooligosaccharides thus obtained in step 3 were partially purified by passing through a column of Sephadex LH-20 using 1:3 methanol:water. The yield of partially purified xylooligosaccharides was 4.22 %. EXAMPLE -4 Stepl 2.5 g cereal/cereal malt (96 h) flour was dispersed in a beaker containing 10 ml of 0.05 M acetate buffer, pH 4.8 and stirred on a magnetic stirrer using a magnetic bar for 2 h at 4 ° C, followed by centrifugation at 4000 x g at 4 ° C for 20 min. Supernatant was collected and dialyzed overnight against 0.05 M acetate buffer, pH 4.8, at 4 ° C. This was used as the xylanase rich extract. The xylanase activity as determined by DNS method using xylan as substrate was high in 96 h malts compared to native (0 h) and varied in different cereals such as rice (native: 0.19 U/min/g flour, malt: 0.78 U/min/g flour), maize (native: 0.28 U/min/g flour, malt: 0.68 U/min/g flour), wheat (native: 0.85 U/min/g flour, malt: 2.66 U/min/g flour) and ragi (native: 0.23 U/min/g flour, malt: 0.98 U/min/g flour). Step 2 10 g of ragi malt (96 h) flour was dispersed in a conical flask containing 100 ml of 0.05 M acetate buffer, pH 4.8 and 1 ml of termamyl was added and incubated at 95 ° C for 60 min. Contents were cooled and 100 mg of glucoamylase was added and incubated at 37 ° C for 24 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and residue is washed thrice with ethanol and dried using acetone. The yield of ragi malt (96 h) bran was 20.3 %. Step 3 250 mg of ragi malt (96 h) bran is dispersed in a conical flask containing 20 ml of 0.05 M acetate buffer, pH 4.8 and 5 ml of dialyzed wheat malt xylanase rich extract was added and incubated at 37 ° C for 48 h. The contents were mixed with 3 volumes of ethanol, centrifuged at 4000 x g for 20 min at room temperature (25 ° C to 30 ° C) and supernatant was collected. It was concentrated by using a rotary evaporator at ~ 30 ° C under reduced pressure to get concentrated solution of xylooligosaccharides. Step 4 Xylooligosaccharides thus obtained in step 3 were partially purified by passing through a column of Sephadex LH-20 using 1:3 methanol:water. The yield of partially purified xylooligosaccharides was 3.98 %. The present method exploits the cereal/cereal malt xylanase, which is induced during germination to hydrolyze cereal/cereal malt bran xylans leading to the liberation of xylooligosaccharides. The main advantages of the present invention are: 1. using xylanase rich extract from cereal malts wherein malting can be easily performed. 2. cereals as such, without malting, can be used as the source to obtain xylanase. 3. obtaining xylanase rich extract from cereal/cereal malts using easy extraction methods. 4. obviating purification of xylanase to get high activity as the concentration of the xylanase can be increased by taking more cereal/cereal malt flour. 5. cheap and abundant supply of cereals. We claim: 1. A process for obtaining xylooligosaccharides from cereals and their malts, the said process comprising steps of; a) soaking cereals/cereal malts in water in the ratio of 1:4 for a period of 16-17 h at the temperature ranging from 20-27 degree C; b) spreading soaked cereals/cereal malts of step (a) in trays and incubating them at a temperature range of 25-27 degree C for a period of 96-98 h; c) drying cereals/cereal malts of step (b) at the temperature range of 50-52 degree C for a period of 48-50 h; d) grinding the product of step (c) for preparing the cereal/cereal malt flour; e) extracting xylanase from cereal or cereal malt flours of step d) by using acetate buffer, of pH 4.8-4.9,by incubating for a duration of 2-2.5 h at a temperature range of 4-6 degree C, and centrifuging the buffer enriched with xylanase at 4000-4200 x g and at 4-6 degree C for 20-25 min in order to separate out cereal or cereal malt residues to obtain xylanase; f) de-starching cereal/ cereal malt or cereal brans by treating with termamyl at a temperature range of 92-95 degree C for a period of 60-65 min, followed by glucoamylase at a temperature range of 37-39 degree C for a period of 24-26 h, centrifuging the contents, upon mixing with 3 volumes of ethanol, at the range of 4000-4200 x g for a period of 20-25 min at room temperature (25 degree C to 30 degree C), washing the residue with ethanol and drying with acetone to obtain xylans, g) characterized in incubating xylans obtained from step (f) and xylanase rich extract obtained in step (e) in acetate buffer, of pH 4.8-4.9 at a temperature range of 37-39 degree C for a period of 48-50 h; h) treating the above said reaction mixture with 3 volumes of ethanol, and centrifuging at a range of 4000-4200 x g for a period of 20-25 min at room temperature (25 degree C to 30 degree C) to obtain supernatant; i) concentrating the supernatant by using a rotary evaporator at a temperature range of 30-35 degree C under reduced pressure to get concentrated solution of xylooligosaccharides. j) partially purifying the said concentrated solution of xylooligosaccharides by passing through a column of Sephadex LH-20 using 1:3 methanol: water to obtain xylooligosaccharides. 2. A process as claimed in claim 1, wherein cereals without malting are used as the source to obtain xylanase. 3. A process as claimed in claims 1 to 2, wherein acetate buffer used is of 0.05 M; 4. A process as claimed in claims 1 to 4, wherein cereal or cereal malt bran used is selected from a group consisting of rice, maize, wheat and ragi; 5. A process for obtaining xylooligosaccharides from cereals and their malts substantially as herein described with reference to the examples.

Documents:

732-DEL-2005-Abstract-(30-03-2011).pdf

732-del-2005-abstract.pdf

732-DEL-2005-Claims-(30-03-2011).pdf

732-del-2005-claims.pdf

732-DEL-2005-Correspondence Other-(07-06-2011).pdf

732-DEL-2005-Correspondence Others-(30-03-2011).pdf

732-del-2005-correspondence-others.pdf

732-del-2005-description (complete).pdf

732-DEL-2005-Description Complete-(30-03-2011).pdf

732-DEL-2005-Form-1-(30-03-2011).pdf

732-del-2005-form-1.pdf

732-del-2005-form-18.pdf

732-del-2005-form-2.pdf

732-DEL-2005-Form-3-(30-03-2011).pdf