Title of Invention

A PROCESS FOR PRODUCTION OF FRUCTOOLIGOSACCHARIDES (FOS)

Abstract

A process for the production of fructooligosaccharides The present invention relates to a process for the reduction of fructo oligosaccharides (FOS). The process in particular uses the extracellular fructosyl transferase (FTase) enzyme obtained from Aspergillus oryzae MTCC 5154 grown on by - products of coffee berries like coffee pulp, coffee husk, spent coffee and spent tea. The novelty of the process is that it uses coffee and tea processing by - products like coffee pulp, coffee husk, spent coffee and spent tea as substrates for solid state fermentative production of FTase This gives value addition to these by - products which are otherwise used as fuel and fertilizer. The final product contains up to 30 % FOS with functional properties like non-carciogenicity, low calorific value and prebiotic property. FOS also improves mineral absorption, reduces the total cholesterol and triglyceride levels in the body.

Full Text

The present invention relates to a process for the reduction of fructo oligosaccharides (FOS). The process in particular uses the extracellular fructosyl transferase (FTase) enzyme obtained from Aspergillus oryzae is soil isolate from the laboratory and the deposition number is MTCC 5154 grown on by - products of coffee berries like coffee pulp, coffee husk, spent coffee and spent tea. Fructooligosaccharides are food ingredients that are of increasing importance because of their favourable functional properties such as being low caloric, non-cariogenic and acting as a growth factor for beneficial microorganisms. They are used in beverages, infant milk powders, confectionery, bakery products, yoghurts and dairy desserts. FOS are usually extracted from plants like Chicory and Jerusalem artichoke or prepared by the action of microbial FTase enzyme on sucrose. There are many reports on the production of FTase by submerged fermentation (SmF). However, Solid State Fermentation (SSF) is an unexplored area in the production of FTase. SSF is an attractive process for enzyme production because it presents high productivity per reactor volume, lower capital and operating costs, lower space requirements, simpler equipment and easier downstream processing compared to SmF. It also permits the use of agricultural by - products as substrates, which can be converted into bulk chemicals and fine products. In the present study, coffee by - products and spent tea are used as substrates for the production of FTase. In India, the area planted with coffee is more than 2 lakh hectares and the annual production of coffee on an average comes to more than three lakh tonnes. Processing of coffee fruits often results in byproducts, which present serious disposal problems. This include coffee pulp, coffee husk, mucilage etc. These by - products are not environmental friendly and has a potential problem of disposal. Any type of value addition to these by - products is an important aspect of biotechnology. Coffee pulp being a potential source of pectin, tannins, reducing sugars, non-reducing sugars, caffeine, lignin, cellulose and amino acids, could be processed scientifically to produce more useful products. Coffee husk is a rich source of nitrogen, phosphorous and potassium. Spent coffee could be used for extracting oil and as a fertilizer. Spent tea is a waste in the manufacture of instant tea and thus is a cheap source. Reference may be made to Y. D. Hang, E. E. Woodams and K. Y. Jang, Biotechnology Letters, 17 (3), 295-298, 1995, wherein fructooligosaccharides were produced from sucrose using extracellular fructosyl transferase (FTase - EC. 2.4.1.9) from Aspergillus foetidus NRRL 337. The organism was grown in a medium consisting of 50 g of apple pomace in solid state culture. After 120 h of growth at 30 °C, the pomace was blended with water, centrifuged and the supernatant was used as a source of enzyme. The production of FOS was carried out at 40 °C with 0.3 M sucrose and 0.01 M sodium acetate buffer (pH 5.0) in a total volume of 2 mL. The maximum yield of FOS obtained was 224 g/L at the end of 59 h. This corresponds to a conversion yield of 47 % of the initial sucrose (476 g/L). The drawback of this process was the longer reaction period. In addition, the product contains only kestose, the trisaccharide. Reference may be made to Antier P, Minjares A, Roussos S, Raimbaults M and Viniegra-Gonzalez G, Enzyme Microbial Technology, 15, 254-260, 1993, wherein pectinase was produced by Aspergillus niger mutants by solid state fermentation of coffee pulp. The strain attained a peak production of 21.1 U/ml (138 U/g dry pulp) of pectinase in 72 h. The process is different from the present process since it produces pectinase instead of FTase. Reference may be made to Soares M, Christen P, Pandey A and Soccol CR, Process Biochemistry, 35, 857-861, 2000 where in Ceratocystis fimbriata was grown on coffee husk for fruity flavour production. This media supplemented with glucose produced up to 6.58 mmol/1 per gram total volatiles. The process is different from the present one in that it describes the production of flavours and not an enzyme like FTase. Reference may be made to a process for the manufacture of natural antioxidant products from tea and spent tea (Chang, Stephen S, Bao and Yongde, US Patent 5043100 dated August 27, 1991) wherein superior oil - soluble antioxidants are produced by the vacuum steam distillation of alcohol extracts of spent black tea or spent green tea or even the tea itself. The process is different in that it does not involve fermentation. Reference may be made to a process for the production of fructooligosaccharides (Prapulla. S. G, Sangeetha. P. T and RameshM. N, 439/DEL/2001, March 2001) wherein FOS was prepared using extracellular fructosyl transferase enzyme obtained by growing Aspergillus oryzae under submerged fermentation conditions. The process is different form the present one in that it deals with the production of FTase by SmF. Reference may be made to an improved process for the production of fructooligosaccharides (Prapulla. S. G, Sangeetha. P. T and Ramesh M. N, 41 l/DEL/2001, March 2001) wherein FOS was prepared using FTase obtained from the sonicated culture broth of Aureobasidium pullulans CFR 77. The process is different from the present one in that it discusses submerged fermentation for FTase production. The main object of the present invention is to provide a process for the utilization of coffee and tea processing by - products for the production of fructooligosaccharides which obviates the drawbacks as detailed above. Accordingly, the present invention provides a process for the utilization of coffee and tea processing by - products for the production of fructooligosaccharides, which comprises (a) growing the culture Aspergillus oryzae MTCC 5154 in a medium consisting of 1 % sucrose and 0.2 % yeast extract at pH ranging between 5 and 6, at temperature ranging between 25 - 35 °C (b) shaking the medium at 200 - 250 rpm for 24 - 48 h to develop the inoculum (c) transferring 10 - 25 % (v/w) of the inoculum to 5-20 g of sterilized solid substrate selected from coffee and tea processing by - products such as coffee pulp, coffee husk, spent coffee and spent tea with a moisture content of 40 % - 60 % (d) mixing the substrate and incubating for a period of 48 - 120 h at a temperature of 25-35 °C. (e) adding water to the moldy substrate in a manner that the substrate is suspended in water (f) incubating the flasks containing the suspended moldy substrate on a rotary shaker at 200-220 rpm for a period of 1 - 2 h (g) separating the substrate by filtration (h) incubating the extract with substrate for a period of 18-20 h at a temperature of 50-60 °C at pH 5 -5.5 In an embodiment of the present invention, the culture used may be from Aspergillus oryzae. In another embodiment of the present invention, the inoculum used may be developed from 5-8 days old slant culture. In another embodiment of the present invention the coffee and tea processing by - products used may be coffee pulp, coffee husk, spent coffee or spent tea. In another embodiment of the present invention the coffee and tea processing by - products such as coffee pulp, coffee husk, spent coffee and spent tea may be supplemented with yeast extract solution to give 1 % nitrogen. In yet another embodiment of the present invention the coffee and tea processing by -products such as coffee pulp, coffee husk, spent coffee and spent tea may be supplemented with complete synthetic media with sucrose, yeast extract and mineral salts such as sodium nitrate, magnesium sulfate, potassium dihydrogen phosphate, di potassium hydrogen phosphate, sodium chloride and ammonium chloride. In yet another embodiment of the present invention, the FTase may be incubated with sucrose as substrate in a concentration of 400 g/L to 800 g/L. The substrate solution was prepared by weighing sucrose (60 g) and dissolving it slowly in 0.1 M citrate buffer (pH 5.0) and making up the total volume to 100 ml using citrate buffer. The process for the utilization of coffee and tea processing by - products for the production of fructooligosaccharides is illustrated in the following flow chart (Chart Removed) The novelty of the process is that it uses coffee and tea processing by - products like coffee pulp, coffee husk, spent coffee and spent tea as substrates for solid state fermentative production of FTase This gives value addition to these by - products which are otherwise used as fuel and fertilizer. The final product contains up to 30 % FOS with functional properties like non-cariogenicity, low calorific value and prebiotic property. FOS also improves mineral absorption, reduces the total cholesterol and triglyceride levels in the body. 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 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g dried coffee pulp moistened with 10 mL complete synthetic media consisting of 10 % sucrose, 0.8 % yeast extract, 0.03 % MgS04. 7H20, 2 % NaN03, 0.4 % K2 HP04, 0.9 % KH2P04, 0.6 % NaCl and 1 % NH4C1 and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 °C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 15 % of the initial sucrose. EXAMPLE - 2 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g dried coffee husk moistened with 10 mL complete synthetic media consisting of 10 % sucrose, 0.8 % yeast extract, 0.03 % MgS04. 7H20, 2 % NaN03, 0.4 % K2 HP04, 0.9 % KH2P04, 0.6 % NaCl and 1 % NH4C1 and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 °C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 8 % of the initial sucrose. EXAMPLE - 3 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g dried coffee husk moistened with 10 mL yeast extract solution containing 1 % nitrogen and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 °C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 20 % of the initial sucrose. EXAMPLE - 4 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g spent coffee moistened with 10 mL complete synthetic media consisting of 10 % sucrose, 0.8 % yeast extract, 0.03 % MgSC^. 7H20, 2 % NaN03, 0.4 % K2 HP04, 0.9 % KH2P04, 0.6 % NaCI and 1 % NH4C1 and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 21.25 % of the initial sucrose. EXAMPLE - 5 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g spent coffee moistened with yeast extract solution to provide 1 % nitrogen and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 °C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 33 % of the initial sucrose. EXAMPLE - 6 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g spent tea moistened with 10 mL complete synthetic media consisting of 10 % sucrose, 0.8 % yeast extract, 0.03 % MgSO.}. 7H2O, 2 % NaN03, 0.4 % K2 HPO4, 0.9 % KH2P04, 0.6 % NaCl and 1 % NH4CI and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 °C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 30 % of the initial sucrose. EXAMPLE - 7 Aspergillus oryzae CFR 202 was grown in 50 mL medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 °C for 24 h at 250 rpm to develop inoculum. 20 % v/w of the inoculum was transferred to a 250 mL conical flask containing 10 g spent tea moistened with 10 mL yeast extract solution to provide 1 % nitrogen and incubated for 72 h at 30 °C. The moldy substrate was mixed with 50 mL water and subjected to shaking at 220 rpm for one and a half hours. The extract is then filtered using filter paper (Whatman number 2). The extract obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 mL of the extract was mixed with 1.75 mL of the substrate (600 g/L sucrose) and incubated for 18 h at 55 °C at pH 5.15. The reaction was stopped by keeping the reaction mixture in boiling water bath. The reaction products were analyzed by HPLC using refractive index detector. The maximum yield obtained corresponded to 28 % of the initial sucrose. The summary of the results given in the examples is presented in Table 1 Table 1 (Table Removed) The inferences from the above examples are detailed below. 1. FTase can be obtained from A oryzae CFR 202 grown on coffee and tea by - products such as coffee pulp, coffee husk, spent coffee and spent tea by solid state fermentation. 2. FOS can be obtained by incubating extracellular FTase from A oryzae CFR 202 with sucrose. 3. Fermentation time required for producing the enzyme, FTase can be 48-96 h. 4. The yield of FOS obtained is up to 10 % when the solid substrates were moistened with water. However, the yields could be increased up to 33 % up on supplementing the substrates with yeast extract solution or complete synthetic media containing sucrose, yeast extract and mineral salts. The advantages of the present invention are: 1. FTase can be obtained by growing A oryzae CFR 202 on a cheaper substrate like coffee pulp, coffee husk, spent coffee and spent tea. 2. FOS can be obtained by incubating the FTase with sucrose as substrate at a concentration of 600 g/L. 3. A minimum of 90 h of fermentation is only needed to produce higher titres of fructosyl transferase. 4. Value addition to coffee and tea processing by - products such as coffee pulp, coffee husk, spent coffee and spent tea. We claim: 1. A process for the production of fructooligosaccharides(FOS), which comprises: (a) growing the culture Aspergillus oryzae MTCC 5154 in a medium consisting of 1 % sucrose and 0.2 % yeast extract at pH ranging between 5 and 6, at temperature ranging between 25-35 °C, (b) shaking the medium at 200 - 250 rpm for 24 - 48 h to develop the inoculum, (c) transferring 10 - 25 % (v/w) of the inoculum to 5-20 g of sterilized solid substrate selected from coffee and tea processing by-products such as coffee pulp, coffee husk, spent coffee and spent tea supplemented with yeast extract solution or synthetic media, with a moisture content of 40 % to 60 %, (d) mixing the substrate and incubating for a period of 48 - 120 h at a temperature of 25-35 °C, (e) adding water to the moldy substrate in a manner that the bran is suspended in water, (f) incubating the flasks containing the suspended moldy substrate on a rotary shaker at 200-220 rpm for a period of 1 - 2 h , (g) separating the substrate by filtration (h) incubating the extract with substrate for a period of 18-20 h at a temperature of 50-60 °C at pH 5 -5.5 to obtain fructooligosaccharides. 2. A process for production of fructooligosaccharides as claimed in claim 1 wherein the culture used is Aspergillus oryzae. 3. A process for the production of fructooligosaccharides as claimed in claims 1 & 2 wherein the inoculum used is developed from 5 -8 days old slant culture. 4. A process for production of fructooligosaccharides as claimed in claims 1-3, wherein the coffee by - products such as coffee pulp, coffee husk, spent coffee and spent tea is supplemented with yeast extract solution to give 1 % nitrogen. 5. A process for the production of fructooligosaccharides as claimed in claims 1 - 4, wherein the coffee by - products such as coffee pulp, coffee husk, spent coffee and spent tea is supplemented with synthetic media consisting of sucrose, yeast extract and mineral salts such as sodium nitrate, magnesium sulfate, potassium dihydrogen phosphate, di potassium hydrogen phosphate, sodium chloride and ammonium chloride. 6. A process for the production of fructooligosaccharides as claimed in claims 1 to 5, wherein the FTase is incubated with sucrose as substrate in a concentration of 400 g/L to 800 g/L. 7. A process for the production of fructooligosaccharides substantially as herein described with reference to the examples accompanying this specification.

Documents:

521-DEL-2003-Abstract-(29-12-2008).pdf

521-DEL-2003-Abstract-05-12-2008.pdf

521-del-2003-abstract.pdf

521-DEL-2003-Claims-(29-12-2008).pdf

521-DEL-2003-Claims-05-12-2008.pdf

521-del-2003-claims.pdf

521-del-2003-corresondence-others.pdf

521-del-2003-corresondence-po.pdf

521-DEL-2003-Correspondence-Others-(29-12-2008).pdf

521-DEL-2003-Correspondence-Others-05-12-2008.pdf

521-DEL-2003-Description (Complete)-(29-12-2008).pdf

521-DEL-2003-Description (Complete)-05-12-2008.pdf

521-del-2003-description (complete).pdf

521-del-2003-form-1.pdf

521-del-2003-form-18.pdf

521-DEL-2003-Form-2-(29-12-2008).pdf

521-DEL-2003-Form-2-05-12-2008.pdf

521-del-2003-form-2.pdf

521-DEL-2003-Form-3-05-12-2008.pdf

521-del-2003-form-3.pdf