Title of Invention	"AN IMPROVED PROCESS FOR THE PREPARATION OF ß--D-GLUCAN ENRICHED FRACTION FROM CEREAL FLOUR"
Abstract	An improved process for the preparation of ß-ß-glucan enriched fraction from cereal flour, which comprises refluxing cereal flour with alcohol for a period of 4-6 hours to obtain an extract, separating the alcohol-insoluble residue from the extract by conventional manner, subjecting the residue so obtained to simultaneous hot water gelatinisation and amyloiv.sis using heat stable amylase enzyme in the range of 0.4-0.6 ml per 100 g Hour to obtain a slurry, allowing the slurry to stand at boiling water bath for a period of 2 hours, separating the residue by conventional method as herein described, precipitating the supernatant with alcohol to obtain of (ß-D-glucan, the said process characterized in using heat stable amylase enzyme.

Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF ß--D-GLUCAN ENRICHED FRACTION FROM CEREAL FLOUR"

Abstract

An improved process for the preparation of ß-ß-glucan enriched fraction from cereal flour, which comprises refluxing cereal flour with alcohol for a period of 4-6 hours to obtain an extract, separating the alcohol-insoluble residue from the extract by conventional manner, subjecting the residue so obtained to simultaneous hot water gelatinisation and amyloiv.sis using heat stable amylase enzyme in the range of 0.4-0.6 ml per 100 g Hour to obtain a slurry, allowing the slurry to stand at boiling water bath for a period of 2 hours, separating the residue by conventional method as herein described, precipitating the supernatant with alcohol to obtain of (ß-D-glucan, the said process characterized in using heat stable amylase enzyme.

Full Text	This invention relates to a process for the preparation of. is--D-glucan enriched fraction from cereal flour. Important cereal grains of the world include wheat, rice, ragi, barley, oat.b, maize, sorghum and a few minor m: J lets. Starch and non-starch carbohydrates are the major ons 11 tuent s of cereals. Horn i ce !. 1 u1os; s represent non-starchy and non-cellulosic polysaccharides, which are quite diverse in their chemical structure, varying from simple sugars to polymers containing pentoses, hexoses, proteins and phenolics. Hemicelluloses include fructans, (1->3)/(1->4) -S-D-qlucan, arabinoxylans and glucomannans ;soluble dietary fibres) . Reference may be made to Fleming,M. and Kawakami,K., Carbohydr. Res., 5 7 (1977) 15, for the information on fS-D-glucan linear molecules associated with peptide sequences especially in endosperm ceil walls. Aleurone and sub-aleurone leiyers are the major sites of location, of these mixed-linkage S-D-glucans in the cereal grains. Mixed-linkage S-D-glucans have been reported to contain 70% (1~>4) and. 30% (l->3) linkages. These linear molecules consist of blocks of (1 - >4) -fS-D-oligoglucosides joined by isolated 3) linkages. Thus runs of two or three contiguous (l->4) linkages are separated by (l->3) linkages which has been reported by Parrish,F.W., Perlin,A.S. and Reese, E.T., Can. J. Chem., 3 8 (1960) 2094. More than three upto ten contiguous (l->4) linkages have been observed in barley. It has been suggested that the number and length of the blocks of adjacent (l->4) linkages alter the properties, particularly viscoelasticity of (l->3, 1 - >4) -D-glucans as reported by Macgregor,A.W. and Fincher,G.B. in "Barley Chemistry and Technology", Macgregor,A.W. and Bhatty,R.S. ieds. , 1993, AACC, St. Paul, Mmnisota, pp.73. According to recent studies, oat E-D-glucan is mainly ft-(l->3) linked ceilotriosyl and cellotetraosyl units with 15% by weight consisting of regions containing more than three consecutive S(l->4) linkages. These mixed linkage fi-D-glucans have beneficial implications in nutrition and health of the consumer. Reference may be made to Newman,R.K., Lewis,S.E., Newman,C.W., Boik,R.J. and Ramage,R.T., Nutr. Rep. Int., 39 (1987) 749 wherein the hypocholesterolemic effect of barley foods on humans has been observed. Beneficiary effects of barley S-D-glucan on blood glucose, blood cholesterol, hormone responses, colonic cancer and micronutrient availability are also reported by Bhatty,R.S. in "Barley Chemistry and Technology, Macgregor,A.W, and Bhatty.R.S. feds,), 1993, AACC, St.Paul, Minn., pp. 355. Experiments in chicks and rats and clinical trials in humans have shown that dietary oat. gum to be instrumental in selectively lowering total cholesterol and LDL-cholesterol as reported by chen,W-J.L., Anderson,J.W. and Gould,M.R., Nutr. Rep. Int., 24 (1981) 1093. The actual mechanism of cholesterol lowering properties of these S-D-glucans is not completely understood. Bacterial fermentation of these "soluble dietary fibres" in colon, results in the formation of short chain fatty acids, which in turn will be utilised by the host for its own benefits. Reference may be made to .opt enste i a , C , F . , Cereal Foods Wot id, (1988 865, ROI-CI J qer , W , E . W . , Cast r oen - ero iogy , 8 ' I9H2) 424 wherein butyric: acid has been found to be involved m inhibition of. coi.oi. cancer'. In view of these beneficial properties there is a demand for soluble S-D-glucans in pharmaceutical industries. In this regard, "oatrim", a fat substitute, consisting of soluble S-D-glucans and amylodextrins from oat flour has gained much attention as reported by Inglett,G.E . , J. Food Chem., 47 (1993) 133. Reference may be made to Inglett,G.E., Warner,K. and Newman,R.K., Cereal Foods World, 39 (1994) 755 wherein applications of oatrin, a heat-stable gel, in processed meat, pasteurized cheeses and packed products including breads, cookies, muffins, and cakes have been documented. Similarly, barleytrim (soluble S-D-glucan and amylodextrins from barley flour) is getting attention of late. Hydrolysed S-D-glucan can also be used as a bulking agent for replacement of sucrose. Barley 6-D-glucan has application as a thickening agent in food industry and it has potential as an industrial hydrocolloid. The existing methods available for the extraction of S-D-glucans are mostly been used for barley or oats. Reference may be made to Preece,I.A. and Mackenzie, K. G. , J. Inst. Brewing, 58(1952) 353; and Bourme,D.T. and Pierce,J.S., J. Inst. Brewing, 76 (1976) 328 wherein ammonium sulphate has been used for precipitation of S-D-glucan from the aqueous extracts done at 40° and 60°C of the ethanol-refluxed barley flour, respectively. The drawbacks are low yield of S-D- gjucan and involvement of other steps such as dialysis and subsequent; freeze drying. Reference may be made to Bass, E.J. and Merethi,W.O.S., Cereal (hem. , 3 2 (1955) 374, wherein papain, trichloroacetic acid (54%) and ammonium sulphate-have been used for extraction, precipitation and fractionation. The drawbacks are involvement of series or steps such as use of enzyme, acid and salts which need to be removed from the final product. Reference may be made to Fleming,M. and Kawakami,K., Carbohydr. Res., 57(1977) 15 wherein the ethanol refluxed barley flour was extracted with water at 40°, 65° and 100°C followed by filtration, dialysis, ammonium sulphate precipitation and reprecipitation with acetone. Although high recovery values are reported, the extraction method seems to be tedious because of the involvement of so many steps. Reference may be made to Fleming,M., Manners,D.J., Jackson,R.M. and Cooke,S.C., J. Inst. Brewing, 80 (1974) 399 wherein aqueous extraction is done at 40°C and no prior amylolysis is done. The drawbacks are alpha-glucan (starch) contamination and also very low yield of the product. Reference may be made to Palmer,G.H., Proc. Amer. Soc. Brewing Chem., 33 (1975) 174, wherein sodium hydroxide (4%) is used at 20°C for extraction. The drawback is alkaline degradation, resulting in ': he decrease in molecular weight and solution viscosity which may reduce its bio-pharmacological activity. Due to involvement of various chemicals and steps in the above said procedures for the extraction of pure 6-D-glucan fraction, the final cost of the product escalates. Thus commercially available (to be imported) S-D-glucans are quite expensive. The main object of the present invention is to provide a process for the preparation of ß-D-glucan enriched fraction from cereal flour which obviates the drawbacks as detailed above. Another object of the present invention is to provide a process which is simple and economical. Accordingly the present invention provides an improved process for the preparation of P - D-glucan enriched fraction from cereal flour , which comprises refluxing cereal flour with alcohol for a period of 4 - 6 hours to obtain an extract , separating the alcohol-insoluble residue from the extract by conventional manner , subjecting the residue so obtained to simultaneous hot water gelatinisation and amylolysis using heat stable amylase enzyme in the range of 0.4 - 0.6 ml per 100 g flour to obtain a slurry , allowing the slurry to stand at boiling water bath for a period of 2 hours , separating the residue by conventional method as herein described , precipitating the supernatant with alcohol to obtain P -D-glucan , the said process is characterized in using heat stable amylase enzyme. In an embodiment of the present invention the cereal flour used may be such as barley, jowar, ragi , wheat. In another embodiment of the present invention the alcohol may be such as ethanol, methanol, propanol. In yet another embodiment of the present invention the separation may be effected using known methods such as filtration, centrifugation. In still. .1. another embodi rnent of the present invention, the Hinyly:;is may be effected using heat stable amylase surf; as aipha amylase, glucoamylase. In another embodiment ot the present Invention the duration of the extraction period may be in the range of 4-b hours. The principles underlying the process of the present-invention are : a. Preliminary refluxing of the cereal flour with alocohol such as ethanol, methanol, propanol in order to remove invisible fats and pigments and also inactivate any i.n situ S-D-glucanase in the resting seeds. b. Separating the alcohol-insoluble residue from the extract by known methods such as filtration, centrifugation. c. The residue obtained in step (b) is subjected to simultaneous gelatinisation in hot water and amylolysis of stairch by heat stable amylase such as alpha-amylase, glucoamylase, allowing the resultant to stand for at least 2 hours, preferably 4-6 hours at boiling water bath temperature to effect aqueous extraction of S-D-glucan. d. Separating the residue as obtained in step (c) by known methods such as filtration, centrifugation. e. Supernatant from step (d) is further subjected to precipitation with alcohol such as ethanol, methanol, propanol to preciptiate S-D-glucan enriched fraction. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present 'Invention. Examp'l e - 1 Barley (market variety) flour, LOO q, was subjected to hanol (400 ml) refluxing (2 hr . The extrad. was discarded and the residue was extracted with water ( i . 5 L containing thermostable alpha - amylase (0,4 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml; at boiling water temperature for 6.0 hr, The solution was centrifuged (10 min, 4000 rpm) to separate the supernatant from the insoluble residue, which, was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 3.0). Example-2 Barley (market variety) flour, 100 g, was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (1.5 L containing thermostable alpha-amyiase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 6.0 hr. The solution was centrifuged (10 min, 4000 rpm) to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in B-D-glucan content ("s recovery, 4.0) . Example-3 Barley (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (l.5 L containing thermostable alpha-amylase (0.6 ml) from an enzyme concentrate of 20 g enzyme protein/100 m i .'• at boiling water temperature for 6.0 hr. The solution was centrifuged (, 10 m Ln, 4 0 0 0 rpm) to separate the Hupe rnat ant: 11 om the insoluble lesidue, which was discarded. Addition of 3 volumes of: ethanol to Che clear supernatant gave a precipitate enriched i; ß L) glucan content (% recovery, .3 . L ; . Example-4 Barley (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (1.5 L containing thermostable alpha amylase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 4.0 hr duration. The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 3.1). Example-5 Barley (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water 1.5 L containing thermostable alpha - amylase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 5.0 hr duration. The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 4.4). Example-b Barley (market variety) flour, 100 a was subjected to hanoi (400 ml) refluxing ;2 hrj . The extract was discarded and the residue was extracted with water 1. .5 L containing thermostable alpha - amylase (0.5 ml) ttorn an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 6.0 hr. The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 3.0). Example-7 Jowar (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded .and the residue was extracted with water >1. 5 L containing thermostable alpha - amy1 ase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 5 hr . The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the former gave a precipitate enriched in S-D-glucan content % recovery, 4.0). Example-8 Ragi (Indaf variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (1.5 L containing thermostable alpha-amylase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml: at boiling water temperature for 5 hr . The solution was centrifuged co separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the cleai supenidtant. gave a precipitate enriched in ß-D glucan content . recovery, 0.05). The isolated glucan enriched preparations were readily soluble in water giving a slightly viscous solution. The extraction methodology reported in this invention was reproducible. Table 1 shows yield and properties of the barley S-D-glucan enriched fractions prepared in four batches as per the schedule shown in Example-5. Table 1: Yield (%) and composition (%) of barley ft-D-glucan preparations (Table Removed) The main advantages of the present invention are: 1. It uses water for extraction. The use of alkali is totally avoided as it may result in alkaline degradation of the polysaccharide and reduced viscosity/molecular weight. 2. Commercial enzyme preparations are being used to remove the starch from the flour prior to £-D-glucan extraction. No buffer salts/costly reagents are employed in this p r o c e d u v e , t h u s c i r c u in v e n t i n g d i a 1 y s i s and other expensive purification ;step. '['he entire extraction procedure upto t lie final drying is comp1ete in just 8 hours. No extraordinary technical competence is required to carry out this extraction procedure. The present process does not involve any costly pretreatments and subsequent purification steps and therefore highly cost effective. We Claim: 1. An improved process for the preparation of p - D-glucan enriched fraction from cereal flour, which comprises refluxing cereal flour with alcohol for a period of 4 - 6 hours to obtain an extract, separating the alcohol-insoluble residue from the extract by conventional manner , subjecting the residue so obtained to simultaneous hot water gelatinisation and amylolysis using heat stable amylase enzyme in the range of 0.4 - 0.6 ml per 100 g flour to obtain a slurry , allowing the slurry to stand at boiling water bath for a period of 2 hours , separating the residue by conventional method as herein described , precipitating the supernatant with alcohol to obtain P -D-glucan , the said process characterized in using heat stable amylase enzyme. 2. An improved process as claimed in claim 1 wherein the cereal flour used is selected from barley , ragi and jowar. 3. An improved process as claimed in claim 1 and 2 wherein the alcohol used is selected from ethanol, methanol, propanol. 4. An improved process as claimed in claim 1 to 3 , wherein the residues separated by conventional manner such as filtration , centrifugation . 5. An improved process as claimed in claim 1 to 4 , wherein the heat stable amylase enzyme is selected from alpha-amylase , glucomylase. 6. An improved process for the preparation of p - D-glucan enriched fraction from cereal flour substantially as herein described with reference to the examples.

Full Text

This invention relates to a process for the preparation of. is--D-glucan enriched fraction from cereal flour.
Important cereal grains of the world include wheat, rice, ragi, barley, oat.b, maize, sorghum and a few minor m: J lets. Starch and non-starch carbohydrates are the major ons 11 tuent s of cereals. Horn i ce !. 1 u1os; s represent non-starchy and non-cellulosic polysaccharides, which are quite diverse in their chemical structure, varying from simple sugars to polymers containing pentoses, hexoses, proteins and phenolics. Hemicelluloses include fructans, (1->3)/(1->4) -S-D-qlucan, arabinoxylans and glucomannans ;soluble dietary fibres) . Reference may be made to Fleming,M. and Kawakami,K., Carbohydr. Res., 5 7 (1977) 15, for the information on fS-D-glucan linear molecules associated with peptide sequences especially in endosperm ceil walls. Aleurone and sub-aleurone leiyers are the major sites of location, of these mixed-linkage S-D-glucans in the cereal grains.
Mixed-linkage S-D-glucans have been reported to contain 70% (1~>4) and. 30% (l->3) linkages. These linear molecules consist of blocks of (1 - >4) -fS-D-oligoglucosides joined by isolated 3) linkages. Thus runs of two or three contiguous (l->4) linkages are separated by (l->3) linkages which has been reported by Parrish,F.W., Perlin,A.S. and Reese, E.T., Can. J. Chem., 3 8 (1960) 2094. More than three upto ten contiguous (l->4) linkages have been observed in barley. It has been suggested that the number and length of the blocks of adjacent (l->4) linkages alter the properties,

particularly viscoelasticity of (l->3, 1 - >4) -D-glucans as reported by Macgregor,A.W. and Fincher,G.B. in "Barley Chemistry and Technology", Macgregor,A.W. and Bhatty,R.S. ieds. , 1993, AACC, St. Paul, Mmnisota, pp.73. According to recent studies, oat E-D-glucan is mainly ft-(l->3) linked ceilotriosyl and cellotetraosyl units with 15% by weight consisting of regions containing more than three consecutive S(l->4) linkages.
These mixed linkage fi-D-glucans have beneficial implications in nutrition and health of the consumer. Reference may be made to Newman,R.K., Lewis,S.E., Newman,C.W., Boik,R.J. and Ramage,R.T., Nutr. Rep. Int., 39 (1987) 749 wherein the hypocholesterolemic effect of barley foods on humans has been observed. Beneficiary effects of barley S-D-glucan on blood glucose, blood cholesterol, hormone responses, colonic cancer and micronutrient availability are also reported by Bhatty,R.S. in "Barley Chemistry and Technology, Macgregor,A.W, and Bhatty.R.S. feds,), 1993, AACC, St.Paul, Minn., pp. 355. Experiments in chicks and rats and clinical trials in humans have shown that dietary oat. gum to be instrumental in selectively lowering total cholesterol and LDL-cholesterol as reported by chen,W-J.L., Anderson,J.W. and Gould,M.R., Nutr. Rep. Int., 24 (1981) 1093. The actual mechanism of cholesterol lowering properties of these S-D-glucans is not completely understood.
Bacterial fermentation of these "soluble dietary fibres" in colon, results in the formation of short chain fatty acids, which in turn will be utilised by the host for
its own benefits. Reference may be made to .opt enste i a , C , F . , Cereal Foods Wot id, (1988 865, ROI-CI J qer , W , E . W . , Cast r oen - ero iogy , 8 ' I9H2) 424 wherein butyric: acid has been found to be involved m inhibition of. coi.oi. cancer'. In view of these beneficial properties there is a demand for soluble S-D-glucans in pharmaceutical industries. In this regard, "oatrim", a fat substitute, consisting of soluble S-D-glucans and amylodextrins from oat flour has gained much attention as reported by Inglett,G.E . , J. Food Chem., 47 (1993) 133. Reference may be made to Inglett,G.E., Warner,K. and Newman,R.K., Cereal Foods World, 39 (1994) 755 wherein applications of oatrin, a heat-stable gel, in processed meat, pasteurized cheeses and packed products including breads, cookies, muffins, and cakes have been documented. Similarly, barleytrim (soluble S-D-glucan and amylodextrins from barley flour) is getting attention of late. Hydrolysed S-D-glucan can also be used as a bulking agent for replacement of sucrose. Barley 6-D-glucan has application as a thickening agent in food industry and it has potential as an industrial hydrocolloid.
The existing methods available for the extraction of S-D-glucans are mostly been used for barley or oats. Reference may be made to Preece,I.A. and Mackenzie, K. G. , J. Inst. Brewing, 58(1952) 353; and Bourme,D.T. and Pierce,J.S., J. Inst. Brewing, 76 (1976) 328 wherein ammonium sulphate has been used for precipitation of S-D-glucan from the aqueous extracts done at 40° and 60°C of the ethanol-refluxed barley flour, respectively. The drawbacks are low yield of S-D-
gjucan and involvement of other steps such as dialysis and subsequent; freeze drying. Reference may be made to Bass, E.J. and Merethi,W.O.S., Cereal (hem. , 3 2 (1955) 374, wherein papain, trichloroacetic acid (54%) and ammonium sulphate-have been used for extraction, precipitation and fractionation. The drawbacks are involvement of series or steps such as use of enzyme, acid and salts which need to be removed from the final product. Reference may be made to Fleming,M. and Kawakami,K., Carbohydr. Res., 57(1977) 15 wherein the ethanol refluxed barley flour was extracted with water at 40°, 65° and 100°C followed by filtration, dialysis, ammonium sulphate precipitation and reprecipitation with acetone. Although high recovery values are reported, the extraction method seems to be tedious because of the involvement of so many steps. Reference may be made to Fleming,M., Manners,D.J., Jackson,R.M. and Cooke,S.C., J. Inst. Brewing, 80 (1974) 399 wherein aqueous extraction is done at 40°C and no prior amylolysis is done. The drawbacks are alpha-glucan (starch) contamination and also very low yield of the product. Reference may be made to Palmer,G.H., Proc. Amer. Soc. Brewing Chem., 33 (1975) 174, wherein sodium hydroxide (4%) is used at 20°C for extraction. The drawback is alkaline degradation, resulting in ': he decrease in molecular weight and solution viscosity which may reduce its bio-pharmacological activity. Due to involvement of various chemicals and steps in the above said procedures for the extraction of pure 6-D-glucan fraction, the final cost of the
product escalates. Thus commercially available (to be imported) S-D-glucans are quite expensive.
The main object of the present invention is to provide a process for the preparation of ß-D-glucan enriched fraction from cereal flour which obviates the drawbacks as detailed above.
Another object of the present invention is to provide a process which is simple and economical.
Accordingly the present invention provides an improved process for the preparation of P - D-glucan enriched fraction from cereal flour , which comprises refluxing cereal flour with alcohol for a period of 4 - 6 hours to obtain an extract , separating the alcohol-insoluble residue from the extract by conventional manner , subjecting the residue so obtained to simultaneous hot water gelatinisation and amylolysis using heat stable amylase enzyme in the range of 0.4 - 0.6 ml per 100 g flour to obtain a slurry , allowing the slurry to stand at boiling water bath for a period of 2 hours , separating the residue by conventional method as herein described , precipitating the supernatant with alcohol to obtain P -D-glucan , the said process is characterized in using heat stable amylase enzyme.
In an embodiment of the present invention the cereal flour used may be such as barley, jowar, ragi , wheat.
In another embodiment of the present invention the alcohol may be such as ethanol, methanol, propanol.
In yet another embodiment of the present invention the separation may be effected using known methods such as filtration, centrifugation.
In still. .1. another embodi rnent of the present invention, the Hinyly:;is may be effected using heat stable amylase surf; as aipha amylase, glucoamylase.
In another embodiment ot the present Invention the duration of the extraction period may be in the range of 4-b hours.
The principles underlying the process of the present-invention are :
a. Preliminary refluxing of the cereal flour with alocohol
such as ethanol, methanol, propanol in order to remove
invisible fats and pigments and also inactivate any i.n
situ S-D-glucanase in the resting seeds.
b. Separating the alcohol-insoluble residue from the extract
by known methods such as filtration, centrifugation.
c. The residue obtained in step (b) is subjected to
simultaneous gelatinisation in hot water and amylolysis
of stairch by heat stable amylase such as alpha-amylase,
glucoamylase, allowing the resultant to stand for at
least 2 hours, preferably 4-6 hours at boiling water bath
temperature to effect aqueous extraction of S-D-glucan.
d. Separating the residue as obtained in step (c) by known
methods such as filtration, centrifugation.
e. Supernatant from step (d) is further subjected to
precipitation with alcohol such as ethanol, methanol,
propanol to preciptiate S-D-glucan enriched fraction.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present 'Invention.
Examp'l e - 1 Barley (market variety) flour, LOO q, was subjected to hanol (400 ml) refluxing (2 hr . The extrad. was discarded and the residue was extracted with water ( i . 5 L containing thermostable alpha - amylase (0,4 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml; at boiling water temperature for 6.0 hr, The solution was centrifuged (10 min, 4000 rpm) to separate the supernatant from the insoluble residue, which, was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 3.0).
Example-2 Barley (market variety) flour, 100 g, was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (1.5 L containing thermostable alpha-amyiase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 6.0 hr. The solution was centrifuged (10 min, 4000 rpm) to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in B-D-glucan content ("s recovery, 4.0) .
Example-3 Barley (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (l.5 L containing thermostable alpha-amylase (0.6 ml) from an enzyme concentrate of 20 g enzyme protein/100 m i .'• at boiling water
temperature for 6.0 hr. The solution was centrifuged (, 10 m Ln, 4 0 0 0 rpm) to separate the Hupe rnat ant: 11 om the insoluble lesidue, which was discarded. Addition of 3 volumes of: ethanol to Che clear supernatant gave a precipitate enriched i; ß L) glucan content (% recovery, .3 . L ; .
Example-4 Barley (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (1.5 L containing thermostable alpha amylase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 4.0 hr duration. The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 3.1).
Example-5 Barley (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water 1.5 L containing thermostable alpha - amylase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 5.0 hr duration. The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 4.4).
Example-b Barley (market variety) flour, 100 a was subjected to hanoi (400 ml) refluxing ;2 hrj . The extract was discarded and the residue was extracted with water 1. .5 L containing thermostable alpha - amylase (0.5 ml) ttorn an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 6.0 hr. The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the clear supernatant gave a precipitate enriched in S-D-glucan content (% recovery, 3.0).
Example-7 Jowar (market variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded .and the residue was extracted with water >1. 5 L containing thermostable alpha - amy1 ase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml) at boiling water temperature for 5 hr . The solution was centrifuged to separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the former gave a precipitate enriched in S-D-glucan content % recovery, 4.0).
Example-8 Ragi (Indaf variety) flour, 100 g was subjected to ethanol (400 ml) refluxing (2 hr). The extract was discarded and the residue was extracted with water (1.5 L containing thermostable alpha-amylase (0.5 ml) from an enzyme concentrate of 20 g enzyme protein/100 ml: at boiling water temperature for 5 hr . The solution was centrifuged co
separate the supernatant from the insoluble residue, which was discarded. Addition of 3 volumes of ethanol to the cleai supenidtant. gave a precipitate enriched in ß-D glucan content . recovery, 0.05).
The isolated glucan enriched preparations were readily soluble in water giving a slightly viscous solution. The extraction methodology reported in this invention was reproducible. Table 1 shows yield and properties of the barley S-D-glucan enriched fractions prepared in four batches as per the schedule shown in Example-5.
Table 1: Yield (%) and composition (%) of barley ft-D-glucan preparations
(Table Removed)
The main advantages of the present invention are:
1. It uses water for extraction. The use of alkali is totally avoided as it may result in alkaline degradation of the polysaccharide and reduced viscosity/molecular weight.
2. Commercial enzyme preparations are being used to remove the starch from the flour prior to £-D-glucan extraction.
No buffer salts/costly reagents are employed in this
p r o c e d u v e , t h u s c i r c u in v e n t i n g d i a 1 y s i s and other
expensive purification ;step.
'['he entire extraction procedure upto t lie final drying is
comp1ete in just 8 hours.
No extraordinary technical competence is required to
carry out this extraction procedure.
The present process does not involve any costly
pretreatments and subsequent purification steps and
therefore highly cost effective.

We Claim:
1. An improved process for the preparation of p - D-glucan enriched fraction from cereal flour, which comprises refluxing cereal flour with alcohol for a period of 4 - 6 hours to obtain an extract, separating the alcohol-insoluble residue from the extract by conventional manner , subjecting the residue so obtained to simultaneous hot water gelatinisation and amylolysis using heat stable amylase enzyme in the range of 0.4 - 0.6 ml per 100 g flour to obtain a slurry , allowing the slurry to stand at boiling water bath for a period of 2 hours , separating the residue by conventional method as herein described , precipitating the supernatant with alcohol to obtain P -D-glucan , the said process characterized in using heat stable amylase enzyme.
2. An improved process as claimed in claim 1 wherein the cereal flour used is selected from barley , ragi and jowar.
3. An improved process as claimed in claim 1 and 2 wherein the alcohol used is selected from ethanol, methanol, propanol.
4. An improved process as claimed in claim 1 to 3 , wherein the residues separated by conventional manner such as filtration , centrifugation .
5. An improved process as claimed in claim 1 to 4 , wherein the heat stable amylase enzyme is selected from alpha-amylase , glucomylase.
6. An improved process for the preparation of p - D-glucan enriched fraction from cereal flour substantially as herein described with reference to the examples.

Documents:

2151-del-1998-abstract.pdf

2151-del-1998-claims.pdf

2151-del-1998-complete-spacition(granted).pdf

2151-del-1998-correspondence-others.pdf

2151-del-1998-correspondence-po.pdf

2151-del-1998-description (complete).pdf

2151-del-1998-form-1.pdf

2151-del-1998-form-2.pdf

2151-del-1998-form-3.pdf

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Patent Number

189037

Indian Patent Application Number

2151/DEL/1998

PG Journal Number

49/2002

Publication Date

07-Dec-2002

Grant Date

17-Oct-2003

Date of Filing

24-Jul-1998

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	RUDRAPATNAM NARAYANASWAMI THARANATHAN	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE,MYSORE- 570 013, INDIA
2	HONNAVALLY PUTTASWAMI RAMESH	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE,MYSORE- 570 013, INDIA

PCT International Classification Number

C07K 1/14

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA