Title of Invention

"A PROCESS FOR THE PREPARATION OF FRUCTOOLIGOSACCHARIDES"

Abstract

The invention relates to a process for the production of fructooligosaccharides (FOS). The novelty lies in use of extracchular fructosyl transferase frdm Aspergillus oryzae, a new source. The product are food ingreadients that are important for their functional properties, non-cariogenic, low calorific value. The process gives high field of the order of 54.7% compared to 47% by the existing process. Higher yield obtained using lower source concentration of 460 gm/l. Fermentation time is also less then the existing process.

Full Text

The present invention relates to a process for the preparation of fructooligosaccharides. The process in particular uses the extracellular fructosyl transferase from Aspergillus oryzae FT.SGP.98, a new source hitherto unreported. Fructooligosaccharides are food ingredients that are important for their functional properties and their mild sweetness very similar to sucrose. They are non-cariogenic, have low calorific value and promote the proliferation of bifidobacteria in the colon. These properties make them suitable for use in beverages, infant milk powders, confectionery, bakery products, yoghurts and dairy desserts. 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 (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. T le maximum yield of FOS obtained was 224 g/L at the end of 59 h. This corresponded to a conversion yield of 47 % of the initial sucrose (476 g/L). The drawbacks of this process were the lower yields and longer reaction periods. Reference may be made to Xiao-Dong Wang and S. K. Rakshit, Process Biochemistry, 35, 771-775, 2000, wherein production of FOS by multiple forms of transferase enzymes from Aspergillus foetidus was reported. Aspergillus foetidus was grown on liquid medium containing 1 % maltose, 3 % corn steep liquor, 0.05 % MgS04, 0.2 % K2HPO4 and 0.7 % KH2PO4 with a pH of 6.0. A portion (1 ml) of this suspension was added to 100 ml of the cultivation medium in 500 ml flasks and incubated at 30 °C for 7 days with agitation (200 rpm). After incubation, cultures were filtered through Whatman No.1 filter paper to remove mycelia. Filtrates were centrifuged at 6000 rpm for 20 min at 4 °C and supernatants were used as enzyme source. The production of FOS was carried out using 30 % sucrose and pH 5.0 with 12-h incubation. The maximum yield obtained was 29 % of the total products. The drawbacks of this process are the low yield obtained and long hours of fermentation time needed to produce the enzyme. The main object of the present invention is to provide a process for the production of fructooligosaccharides, which obviates the drawbacks as detailed above. Accordingly, the present invention provides a process for the preparation of fructooligosaccharides using extracellular fructosyl transferase which comprises; (a) growing the culture from Aspergillus oryzae sp. in a medium consisting of 1 % sucrose and 0.2 % yeast extract at pH ranging between 5-6 , at a temperature ranging between 25 - 35 °C at about 250 rpm for 24- 120 hrs to develop the inoculum, (b) transferring 5-15 % of the said inoculum to a medium consisting of 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNOa, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and growing for a period of ranging between 24-120 tirs at 250 rpm at 25-35°c, (c) harvesting the entire culture broth, (d) separating the pellets from culture both by known filtration method such as herein described to obtain culture fluid, (e) Incubating the culture fluid with the substrate as herein described for 1-36 h at 50-60 °C, pH 5 - 6, (f) stopping the reaction by keeping reaction mixture as obtained in step e) in boiling water bath, isolating the product from reaction mixture by known methods such as herein described. In another embodiment of the present invention, the inoculum used may be developed from 5 days old slant culture. In another embodiment of the present invention, the inoculum may be grown for 24-48 h under agitation at 250 rpm. In another embodiment of the present invention, the inoculum transferred to the femientation medium is preferably -10%. In another embodiment of the present invention, the culture may be grown in a known manner for a period of 48-120 hrs. In yet another embodiment of the present invention, the culture broth may be filtered to separate the pellets from the culture fluid. In yet another embodiment of the present invention, the culture fluid may be incubated with sucrose as substrate in a concentration of 400 g/L to 800 g/L. In yet another embodiment of the present invention, the incubation period may be from 1-36 h. In an another embodiment of the present invention the yield of fructooligosaccharides is up to 55 % of the initial sucrose concentration. The substrate solution (sucrose) was prepared by weighing sucrose (55 g or 80 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 sucrose concentration was measured using a refractometer and found to be 46 ° brix for 550 g/L and 66 ° brix for 800 g/L respectively. Hence, the concentration as indicated by the refractometer was taken as the exact sucrose concentration to calculate the percentage FOS yield. 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 FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNOa, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 48 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (460 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 249 g/L after 12 h of reaction, which corresponded to 54.12 % of the initial sucrose. EXAMPLE - 2 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNOa, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 48 h at 30 + 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (660 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 356.8 g/L after 12 h of reaction, which corresponded to 54.06 % of the initial sucrose. EXAMPLE - 3 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNOa, 0.25 % K2 HPO4. 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 72 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (460 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 192.79 g/L after 24 h of reaction, which corresponded to 41.94 % of the initial sucrose. EXAMPLE - 4 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNO3, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 72 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (660 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 318.3 g/L after 12 h of reaction, which corresponded to 48 % of the initial sucrose. EXAMPLE - 5 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNO3, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 96 h at 30 + 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (460 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 219.3 g/L after 12 h of reaction, which corresponded to 47.7 % of the initial sucrose. EXAMPLE - 6 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNOa, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 96 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained wais used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (660 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 346.31 g/L after 12 h of reaction, which corresponded to 52.47 % of the initial sucrose. EXAMPLE - 7 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNO3, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 120 h at 30 ± 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (460 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 251.5 g/L after 12 h of reaction, which corresponded to 54.7 % of the initial sucrose. EXAMPLE- 8 Aspergillus oryzae FT.SGP.98 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. 10 % v/v of the inoculum was transferred to 50 ml fermentation medium in a 250 ml conical flask containing 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04. 7H2O, 1 % NaNO3. 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and incubated at 250 rpm for 120 h at 30 + 1 °C. The pellets were separated from the culture fluid by filtration using Whatman Number 2 filterpaper. The culture fluid obtained was used as the source of extracellular enzyme for the production of FOS. 0.5 ml of the culture fluid was mixed with 1.5 ml of the substrate (660 g/L sucrose) and incubated for 12 h at 55 °C at pH 5.0. 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 was 337.9 g/L after 24 h of reaction, which corresponded to 51 % of the initial sucrose. (Table Removed) The inferences from the above examples are detailed below. 1. Aspergillus oryzae FT.SGP.98can be identified as a new source of the enzyme fructosyl transferase for the production fructooligosaccharides. 2. High yield of FOS can be obtained at lower substrate concentration using extracellular enzyme from A oryzae FT.SGP.98. 3. High yield of FOS can be obtained in shorter reaction time using extracellular enzyme from A oryzae FT.SGP.98. 4. Fermentation time required for producing the enzyme, fructosyl transferase can be 48-72 h. The advantages of the present invention are: 1. Higher yield of the order 54.7 % obtained as against 47 % obtained by the existing process using extracellular enzyme from Aspergillus foetidus NRRL 337. 2. Higher yield of FOS obtained using lower sucrose concentration of 460 g/L. 3. A minimum of 72 h of fermentation is only needed to produce higher titres of fructosyl transferase as against 120-168 h iri the existing processes. We claim 1. A process for the preparation of fructooligosaccharides using extracellular fructosyl transferase which comprises; (a) growing the culture from Aspergillus oryzae sp. in a medium consisting of 1 % sucrose and 0.2 % yeast extract atpH ranging between 5-6 , at a temperature ranging between 25 - 35 °C at about 250 rpm for 24- 120 hrs to develop the inoculum, (b) transferring 5-15 % of the said inoculum to a medium consisting of 20 % sucrose, 0.5 % yeast extract, 0.05 % MgS04.7H2O, 1 % NaNO3, 0.25 % K2 HPO4, 0.25 % KH2PO4, 0.25 % NaCI and 0.5 % NH4CI and growing for a period of ranging between 24-120 hrs at 250 rpm at 25-35°c, (c) harvesting the entire culture broth, (d) separating the pellets from culture both by known filtration method such as herein described to obtain culture fluid, (e) Incubating the culture fluid with the substrate as herein described for 1-36 h at 50-60 °C, pH 5 - 6, (f) stopping the reaction by keeping reaction mixture as obtained in step e) in boiling water bath, isolating the product from reaction mixture by known methods such as herein described. 2. A process as claimed in claim 1 wherein the inoculum used is developed from 5 days old slant culture 3. A process as claimed in claims 1 to 2 wherein the inoculum transferred to the fermentation medium is preferably -10%. 4. A process as claimed in claims 1 to 3 wherein the culture is grown preferably for a period 48-120 hrs. 5. A process fas claimed in claims 1 to 4 wherein the culture fluid used is incubated with sucrose as substrate in a concentration of 400 g/L to 800 6. A process for the preparation of fructooligosaccharides using extracellular fructosyl transferase substantially as herein described with reference to the examples accompanying this specification.

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439-del-2001-abstract.pdf

439-del-2001-claims.pdf

439-del-2001-complete specification (granted).pdf

439-del-2001-correspondence-others.pdf

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439-del-2001-description (complete).pdf

439-del-2001-form-1.pdf

439-del-2001-form-2.pdf

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