Title of Invention

A PROCESS FOR THE PREPARATION OF FRUCTOOLIGOSACCHARIDES (FOS) JAGGERY

Abstract

A process for the production of fructooligosaccharides The present invention relates to a process for the production of fructooligosaccharides (FOS) using jaggery. The process in particular uses the extracellular fructosyl transferase (FTase) from Aspergillus oryzae CFR 202. The novelty of the process is that it uses jaggery as carbon source in the fermentation medium and as substrate to prepare FOS. The final product contains 48.59 % 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. Another novelty of the process is the value addition to a lower value gur to a high value product with FOS

Full Text

The present invention relates to a process for the preparation of fructooligosaccharides (FOS) using jaggery. The process in particular uses the extracellular fructosyl transferase (FTase) from Aspergillus oryzae CFR 202, Fructooligosaccharides are food ingredients that are in great demand for their functional properties. Their non-cariogenicity, low calorific value and prebiotic properties make them suitable for use 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. Jaggery, a concentrated sugar cane juice with or without purification produced by cottage industries (also known as gur) containing 75 - 85 % sucrose is widely used in India as a substitute for white and refined cane sugar. In the present study, jaggery is used to prepare FOS using FTase from Aspergillus oryzae CFR 202. There are many reports regarding the improvement of the properties and keeping qualities of jaggery and the preparation of various sweet dishes using jaggery. However, there are only very few reports wherein jaggery has been used as an ingredient in the fermentation media. Reference may be made to S. V. N. Vijayendra, D. Bansal, M. S. Prasad and Krishna Nand, Process Biochemistry, 37, 359-364, 2001, wherein jaggery was used as a novel substrate for pullulan production by Aureobasidium pullulans CFR 77. A pullulans CFR 77 was grown in batch fermentation using jaggery as a carbon source. The maximum yield of pullulan was obtained using 5 % jaggery in the fermentation broth in 72 h. The process is different from the present process since it results in the production of a polysaccharide. Reference may be made to P. Ambati and C. Ayyanna, World Journal of Microbiology and Biotechnology, 17, 331-335, 2001 where in palmyra jaggery was used as carbon source for citric acid production using Aspergillus niger MTCC 281 by submerged fermentation. Maximum citric acid production was obtained after 136.8 h of fermentation with 221.66 g/L of jaggery in the medium. The process isdifferent form the present one in that the product is an acid. The drawbacks of the process are the longer fermentation time (136.8 h) and high substrate concentration used in the fermentation medium (221.66 g/L). Reference may be made to a process for the production of fructooligosaccharides (Prapulla. S. G, Sangeetha. P. T and Ramesh M. 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 uses only sucrose as the carbon source in the medium and as substrate for FTase to produce FOS. Reference may be made to an improved process for the production of fructooligosaccharides (Prapulla. S. G, Sangeetha. P. T and Ramesh M. N, 411/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 uses the sonicated culture broth as source of FTase and it involves additional steps like sonication and centrifugation to obtain the enzyme. Further, it uses only sucrose as the carbon source for the production of FTase and as substrate for the production of FOS. The main object of the present invention is to provide a process for the production of fructooligosaccharides using jaggery, which obviates the drawbacks as detailed above. Accordingly, the present invention provides a process for the production of FOS using jaggery, which comprises (a) growing the culture Aspergillus oryzae MTCC 5154 in a medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 25 - 30 °C at 200 - 250 rpm for 24 - 48 h to develop the inoculum, (b) transferring 20 % of the inoculum to a medium consisting of 10 % sucrose /jaggery, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaNO3, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCl and 1 % NH4CI and growing for a period of 48 - 120 h under submerged fermentation conditions, The process is different form the present one in that it uses only sucrose as the carbon source in the medium and as substrate for FTase to produce FOS. Reference may be made to an improved process for the production of fructooligosaccharides (Prapulla. S. G, Sangeetha. P. T and Ramesh M. N, 411/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 uses the sonicated culture broth as source of FTase and it involves additional steps like sonication and centrifugation to obtain the enzyme. Further, it uses only sucrose as the carbon source for the production of FTase and as substrate for the production of FOS. The main object of the present invention is to provide a process for the for the preparation of fructooligosaccharides (FOS) using jaggery, which obviates the drawbacks as detailed above. Accordingly, the present invention provides a process for the preparation of fructooligosaccharides (FOS) using jaggery, which comprises (a) growing the culture Aspergillus-oryzae CFR 202 in a medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 25 - 30 °C at 200 - 250 rpm for 24 - 48 h to develop the inoculum (b) transferring 20 % of the inoculum to a medium consisting of 10 % sucrose / jaggery, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaNO3, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCI and 1 % NH4CI and growing for a period of 48-120 h under submerged fermentation conditions (c) separating the pellets from culture fluid by filtration using filter paper (d) incubating the culture fluid with the substrate for 18-24 h at 50 - 55 °C, pH 5.0 - 5.5 and (e) analyzing the reaction mixture for the content of FOS, which yielded up to 48 %. 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 yet another embodiment of the present invention, the culture fluid may be incubated with sucrose / jaggery as substrate in a concentration of 400 g/L to 800 g/L. The substrate solution was prepared by weighing sucrose / jaggery (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 preparation of fructooligosaccahrides using jaggery is illustrated in the following flow chart Five -Eight days old slant of Aspergillus oryzae CFR 202 One loop full of spores transferred to medium containing 1 % sucrose / jaggery and 0.2 % yeast extract at pH ranging from 5-6 and incubated for 24-48 h at temperature ranging from 25-30 °C at about 200-250 rpm to develop inoculum 20 % inoculum transferred Fermentation medium consisting of 10 % sucrose / jaggery, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaNO3, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCI and 1 % NH4CI and incubated for 48-120 h at temperature ranging from 25-30 °C Filtered the culture broth - Pellets discarded Fructosyl Transferase (FTase) FTase (0.25 ml) mixed with 600 g/l sucrose / jaggery(1 .75 ml) at pH 5.0-5.5 for18-24hat50-55°C Fructooligosaccharides The novelty of the process is that it uses jaggery as carbon source in the fermentation medium and as substrate to prepare FOS. The final product contains 48.59 % 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. Another novelty of the process is the value addition to a lower value gur to a high value product with FOS 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 was grown in 50 ml medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 ± 1 °C for 24 h at 250 rpm to develop inoculum. 20 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 10 % sucrose, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaNO3, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCI and 1 % NH4CI and incubated at 250 rpm for 90 h at 30 + 1 °C. The pellets were separated from the culture fluid by filtration using filter paper (Whatman No. 2). The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 ml of the culture fluid was mixed with 1.75 ml of the substrate (600 g/L jaggery) 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 FOS yield obtained was 41.98 % of the initial sucrose which consisted of 32.07 % Kestose and 9.91 % Nystose. EXAMPLE - 2 Aspergillus oryzae was grown in 50 ml medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 ± 1 °C for 24 h at 250 rpm to develop inoculum. 20 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 10 % jaggery, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaNO3, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCI and 1 % NH4CI and incubated at 250 rpm for 90 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using filter paper (Whatman No. 2). The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 ml of the culture fluid was mixed with 1.75 ml of the substrate (600 g/L jaggery) 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 FOS yield obtained was 40.03 % of the initial sucrose which consisted of 32.75 % Kestose and 7.28 % Nystose. EXAMPLE - 3 Aspergillus oryzae was grown in 50 ml medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 ± 1 °C for 24 h at 250 rpm to develop inoculum. 20 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 10 % jaggery, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaN03, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCI and 1 % NH4CI and incubated at 250 rpm for 90 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using filter paper (Whatman No. 2). The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 ml of the culture fluid 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 FOS yield obtained was 48.59 % of the initial sucrose which consisted of 38.47 % Kestose and 10.12 % Nystose. EXAMPLE - 4 Aspergillus oryzae was grown in 50 ml medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 30 ± 1 °C for 24 h at 250 rpm to develop inoculum. 20 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 10 % sucrose, 0.8 % yeast extract, 0.03 % MgSO4. 7H2O, 2 % NaNO3, 0.4 % K2 HPO4, 0.9 % KH2PO4, 0.6 % NaCI and 1 % NH4CI and incubated at 250 rpm for 90 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using filter paper (Whatman No. 2). The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.25 ml of the culture fluid was mixed with 1.75 ml of the substrate (600 g/L sucrose) and incubated for 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 FO3 yield obtained was 58 % of the initial sucrose which consisted of 32.62 % Kestose, 22.47 % Nystose and 3.06 % Fructofuranosyl nystose. A summary of the results given in the examples are presented in Table 1 The inferences from the above examples are detailed below. 1. FTase can be obtained from A oryzae CFR 202 using jaggery / sucrose as carbon source in the fermentation medium. 2. FOS can be obtained by incubating extracellular FTase from A oryzae CFR 202 with jaggery / sucrose. 3. Fermentation time required for producing the enzyme, fructosyl transferase can be 48-96 h. 4. The yield of FOS obtained is 40 % using jaggery as carbon source in fermentation media and substrate, 41.98 % using sucrose in fermentation media and jaggery as substrate and 48.59 % using jaggery in fermentation media and sucrose as substrate where as the FOS yields obtained using sucrose in fermentation media and substrate is 58 %. The advantages of the present invention are: 1. FTase obtained by growing A oryzae CFR 202 on a cheaper carbon source like jaggery. 2. FOS can be obtained using jaggery / sucrose as substrate at a concentration of 600 g/L 3. A minimum of 90 h of fermentation is only needed to produce higher litres of fructosyl transferase. We claim 1. A process for the production of fructooligosaccharides using jaggery, which comprises (a) growing the culture Aspergillus oryzae MTCC 5154 in a medium consisting of 1 % sucrose and 0.2 % yeast extract (pH 5.5) at 25-30 °C at 200-250 rpm for 24-48 h to develop the inoculum (b) transferring 20 % of the inoculum to a medium consisting of 10 % sucrose / jaggery, 0.5 % yeast extract, 0.05 % MgSO4. 7H2O, 1 % NaNO3, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCl and 0.5 % NH4CI and growing for a period of 48 - 120 h under submerged fermentation conditions (c) separating the pellets from culture fluid by filtration using filter paper (Whatman no. 2) (d) incubating the culture fluid with the substrate for 18-24 h at 50-55 °C, pH 5-5.5 and (e) analyzing the reaction mixture for the content of FOS, which yielded up to 48.59 % of the initial sucrose concentration. 2. 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 3. A process for the production of fructooligosaccharides as claimed in claims 1 to 3 wherein the culture fluid is incubated with sucrose / jaggery as substrate in a concentration of 400 g/L to 800 g/L. 4. A process for the production of fructooligosaccharides substantially as herein described with reference to the examples accompanying this specification.

Documents:

487-DEL-2003-Abstract-(06-01-2009).pdf

487-del-2003-abstract.pdf

487-DEL-2003-Claims-(06-01-2009).pdf

487-del-2003-claims.pdf

487-DEL-2003-Correspondence-Others-(06-01-2009).pdf

487-del-2003-correspondence-others.pdf

487-del-2003-correspondence-po.pdf

487-DEL-2003-Description (Complete)-(06-01-2009).pdf

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

487-del-2003-form-1.pdf

487-del-2003-form-18.pdf

487-DEL-2003-Form-2-(06-01-2009).pdf

487-del-2003-form-2.pdf

487-DEL-2003-Form-3-(06-01-2009).pdf

487-del-2003-form-3.pdf

487-del-2003-gpa.pdf