Title of Invention	AN IMPROVED PROCESS FOR THE PRODUCTION OF GALACTOOLIGOSACCHARIDES
Abstract	The present invention deals with an improved process for the production of high yield of pure Galactooligosaccharides using immobilized microbial whole cells in a two-microbe system. The process is economical as it eliminated the need to carry out downstream processing for the removal of mono and disaccharides from the final product.

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THE PATENTS ACT, 1970 COMPLETE SPECIFICATION Section 10 "Novel Process for the Production of Galactooligosaccharides by Coimmobilization" Tata Chemicals Pvt. Limited, of Leela Business Park, Andheri Khuria Road, Andheri (E) Mumbai, India. The following specification particularly describes the nature of this invention and the manner in which it is to be performed: Novel Process for the Production of Galactooligosaccharides by Coimmobilization FIELD OF THE INVENTION The present invention relates to a process for the production of galactooligosaccharides/oligosaccharides using immobilized microbial whole cells in a two-microbe system. The microbial cells are immobilized in hydrocolloid matrices. The process leads to the production of galactooligosaccharides of high purity and yield without the downstream separation of mono and disaccharides. BACKGROUND OF THE INVENTION Galactooligosaccharides find widespread use in the industry as pro-biotic compounds. A number of processes have been developed for the production of galactooligosaccharides. Some processes involve the use of P-galactosidase enzyme obtained from different microbial sources, example Aspergillus oryzae, Bullera singular is, Candida, Kluveromyces sp., Bacillus circulans, Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium sp., (Akiyama et. al., 2001, Ribu et. al., 2001, Tzortiz et. al., 2005, Jorgensen etal., 2001, Shin et. al., 1998, US5032509, EP00272095A2). Use P-galactosidase enzyme or whole cells in immobilized matrices in place of free whole cells or enzymes has also been reported (Akiyama et. al., 2001, Ribu et. al., 2001, Tzortiz et. al., 2005, Jorgensen et al., 2001, Shin et. al., 1998, US5032509, EP00272095A2). Axelsson et al., (1991), Lewandoska et al., (2003) and Hideo et al., (1984) have coimmobilized P-galactosidase enzyme with Saccharomyces cerevisiae cells in calcium alginate beads to produce ethanol from whey. The objective was to cleave lactose to glucose and galactose and thereby yeast can produce more ethanol. The processes involving immobilized whole cells or enzyme over the free enzyme has certain advantages (1) catalytic power is stabilized (2) the immobilized matrices can be recycled which reduces cost and (3) products can be isolated in a simple manner. However, the use of immobilized enzymes depends upon the cost benefit and technical feasibility factors. In some processes, the extraction and purification of enzyme is costly and in some cases the enzyme denatures after extraction. Under such conditions, the use of immobilized or coimmobilized whole cells has added advantages over the immobilized enzymes (Zhang et al., 1992, Kiss et al., 1999). The most prevalent method of microbial immobilization is cell entrapment in. hydrocolloids like alginate, carrageenan, polyacrylamide, agarose, gelatin, gellan gum (US5175093, US5288632, US5093253, US4572897, US5070019, US5759578, US5939294 and US5034324, Birnbhaum et al., 1981). JP2005042037, JP5815243, and JP561132890 describe the use of polyvinyl alcohol with polyethylene glycol and boric acid as a successfully alternative to other hydrocolloids. In the industry, microbial cells are immobilized in calcium-alginate beads, which are produced by mixing microbial cells with sodium alginate (Na-alginate) solution and adding the mixture drop wise into calcium chloride solution. Chang et al., 1998 mixed microbial cells in calcium chloride solution containing small amount of xanthan gum and then dropped into sodium alginate. The capsule membrane formed by ionic bond between calcium and alginate prevented swelling of the membrane and resulted in a high concentration of microbes within the capsule. US5034324 discloses that polyvinyl alcohol has a high affinity for microorganisms and provides mechanical strength and durability sufficiently high for use in any reactor, and high resistance to water and chemicals. Jianlong et al., (2004) used acryl amide as polymerizing agent in the polyvinyl alcohol matrix with boric acid as cross-linking agent to overcome the swelling of polyvinyl alcohol gels in aqueous solution. Continuous production of high yields of pure galactooligosacchandes is needed to meet up with the increasing demands for functional oligosaccharides in various applications. In the process of galactooligosaccharide production using immobilized cells or enzymes, the kinetics of enzymatic hydrolysis of lactose is limited due to competitive and noncompetitive inhibition caused by the production of glucose. US 716451 describes mixing of the saccharide solution, obtained after hydrolysis, with ethanol and passing through activated carbon column to remove the mono and disaccharide components. The galactooligosaccharide component is eluted using pure ethanol. This downstream processing results in substantially pure galactooligosaccharide solution However, this process is not efficient due to the loss in the yield of galactooligosaccharides. It is also not economical due to the use of ethanol. To overcome the limitation of activated carbon column treatment, EP00272095A2 and US5032509 describe loading of the saccharide solution on a strong cation exchange resin followed by elution of galactooligosaccharides with water at 60 to 80 °C. This process can improve the yield of galalctooligosaccharide. However, it is not cost effective because of the use of costly strong cation exchange resin. Therefore, the need for a cost effective process that results in high yields of pure galactooligosaccharides continues to prevail. SUMMARY OF THE INVENTION The invention discloses a novel process for the production of galactooligosaccharides of high purity and yield using immobilized whole cells of yeast in a two-microbe system. In accordance with one embodiment of the invention, the immobilized whole cells of yeasts are used in a sequential reactor composed of a series of three reactors wherein the hydrolysis products from one reactor serve as the feed for the next reactor in the series. In accordance with another embodiment, the yeast cells are coimmobilized and used in a single reactor. The present invention eliminates the need for the down stream process for the separation of contaminating saccharides. This process of coimmobilization of whole cells can be applied to other products such as oligosaccharides, organic acids and other biotechnological process where more than one microorganisms are involved; BRIEF DECRIPTION OF DRAWINGS The process described in this invention will become apparent from the drawings given below: Fig.l: Overall process of production of galactooligosaccharides using immobilized microbial strains. Fig.2: Production of GOS by free cells of B. singularis and mixed cells of B. singularis and Saccharomyces sp. Fig.3: GOS production by reuse of immobilized matrix DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION A novel and improved process for the production of galactooligosaccharides involving a two-microbe system is disclosed. The process comprises the following steps: (1) Growth of microbial cells (2) Independent immobilization of microbial cells or coimmobilization (4) Hydrolysis of lactose by the independently immobilized microbial cells in sequential reactor or by the coimmobilized cells in a continuous stir tank reactor (5) Concentration of galactooligosaccharide solution. A schematic representation of the process is depicted in figure 1. The two-microbe system comprises B. singularis TCL-IC/NUT-1 and Saccharomyces sp. TCL-IC/NUT-2, the wild strain of lac-glc+ Saccharomyces sp, the wild strain of lac-glc+ is used along with B. singularis having ß-galactosidase enzyme. Saccharomyces sp utilizes glucose. Thus competitive inhibition of ß-galactosidase enzyme by glucose is substantially reduced which results in galactooligosaccharides with increased yield and purity. Since glucose is consumed by Saccharomyces sp the need of complicated downstream process for separation of saccharides is also eliminated. This makes the process cost effective. B. singularis is isolated from dairy effluents and screened for the growth at different conditions (temperature and pH) and production of galactooligosaccharides. The isolate B. singularis showing highest galactooligosaccharide production in free cell is immobilized in a hydrocolloid matrix. The immobilized culture, B. singularis, produced 30-35% galactooligosaccharides in 24 h hydrolysis. Saccharomyces sp. (lac-, glc+) isolated from contaminated glucose syrup is used to selectively utilize glucose produced during the hydrolysis of lactose by ß-galactosidase. The mixed microbial cultures of B. singularis and Saccharomuces sp. (1:1 Ratio dry wt. basis) produced 70-90% galactooligosaccharides after 2 - 6 h hydrolysis at 30°C with 30% lactose. The isolated microbial cells are entrapped together in hydrocolloids of unique combination and supplemented with polymers for good stability of immobilized beads. Cells of B. singularis and Saccharomyces sp. were coimmobilized in the hydrocolloid comprising alginate, polyvinyl alcohol and gum arabica. For immobilization, the microbial cell pellet is mixed with sodium alginate, polyvinyl alcohol and gum arabica and then dropped into calcium chloride and saturated boric acid to form beads or noodles. In accordance with one embodiment of the invention, the coimmobilized beads or noodles are used in two reactors for hydrolysis of lactose solution. In the first reactor, the coimmobilized beads or noodles are packed in porous, tubular nylon sac and placed in stir tank reactor in spiral configuration. Lactose solution in the stir tank reactor is gently agitated at 50-100 rpm. In the second reactor, coimmobilized beads are packed in a column and lactose solution is recirculated. In both the reactors the temperature is maintained at 30°C and the reaction is carried out until galactooligosaccharide content exceeds 70%. In accordance with the second embodiment of the invention the coimmobilized beads or noodles are used in a single reactor. Lactose solution is recirculated in the column. Hydrolysis is carried out at 30 °C until the GOS of maximum purity and yield is obtained. In accordance with the third embodiment of the invention, the yeast whole cells are immobilized independently and used in a sequential stir tank reactor comprising three sequentially arranged reactors. The hydrolysis product of one reactor serves as the feed for the next reactor in series. Immobilized cells of B. singularis are present in the first and third reactors whereas immobilized Saccharomyces sp. cells are present in the second reactor. The galactooligosaccharide solution of more than 70% purity is concentrated in Rota vapor to obtain total solid content is 75-80%. The present invention is further illustrated in the following examples: Example 1: Isolation of lactose hydrolyzing B. singularis culture B. singularis, TCL-IC/NUT-1, was isolated from dairy effluents by serial dilution method on Yeast-Malt-Peptone (YMP) medium of the following composition; Yeast extract 0.3%, Malt extract 0.3%, Peptone 0.5%, Dextrose 1% and Lactose 1%, and agar 2.0 %, pH 6.5. Working stock culture: The cultures were streaked on YMP slants and incubated for 48 h at 27 °C. These slant cultures were used as working stock culture. Preparation of inoculum: 50 ml YMP medium in 250 ml flask was inoculated with a loop full of culture from the slants and incubated in a shaker for 48 h at 27 °C at 180 rpm. Shake flask trials: 150 ml of YMP medium in 500 ml flasks was inoculated with 5% inoculum and incubated on the shaker for 48 h at 27 °C at 180 rpm The samples from the shake flask were taken aseptically and assayed for GOS production as given in Example 2. Fermentation trials: 3 1 of YMP medium was charged in 5 1 fermentor and sterilized. The fermentor was inoculated with 5% inoculum from the shaker flask. Fermentation was carried out at 27 °C, pH 4.5, 600 rpm agitation, and with 1 vvm of air to maintain 40-60% dissolved oxygen. The samples from fermentor were taken at 24, 48 and 67 h to determine maximum GOS production as described in Example 2. Example 2: Assay for galactooligosaccharide production 25 ml of the cell suspension from the shaker flask or fermentor was centrifuged to obtain cell pellet. The cell pellet was suspended in 15 ml of 40% lactose solution and kept on shaker at 50°C and 280 rpm agitation. 0.5 ml of the samples was taken out and centrifuged to remove the cell biomass. The supernatant was diluted 50 times with milli Q water. 5 µ,l of diluted samples was injected in to the HPLC system. HPLC analysis: The concentration of sugars (glucose, galactose, lactose, and Galactooligosaccharides) was determined by HPLC. The HPLC (Waters 717) system consisted of refractive index detector (waters W2467) and carbohydrate column Phenomenex (RNM 00h-0316, REZEX 300 mm L X 7.5 mm, pore size 8 u) ID column. The column temperature was maintained at 80°C. Water was used as mobile solvent with flow rate of 0.5 ml/min. Galactooligosaccharides and other sugars were determined as weight percentage of total sugars based on the area of peak. Experiment 3 : Production of galactooligosaccharides by free cells of B. singularis 25 ml of cell suspension from the shaker flask or fermentor was centrifuged to obtain cell pellet The cell pellet was suspended in 15 ml of 30% lactose solution and kept on shaker at 50 °C and 180-rpm agitation. Table: 1 Galactooligosaccharide production by free cell of B. singularis. Reaction time (h) % GOS 1 16.00 3 25.40 6 35.20 The results in Table 1 indicate the galactooligosaccharides production is higher than reported by Yang et al., while it is same as reported by Shin et al.1998 Experiment 4: Immobilization whole cells of B. singularis The results of Table 3 indicate that lower the concentration of lactose, higher the galactooligosaccharide production. Low galactooligosaccharide production at high lactose concentration may be due to substrate inhibition and/or glucose produced during hydrolysis of lactose. Experiment 7: Isolation of Saccharomyces sp. This strain was isolated from contaminated dextrose syrup on MYGP medium composed of Malt extract 0.3%, Yeast extract 0.3%, Glucose 1.0%, Peptone 0.5% and agar 2.0 %, pH 6.4. The strain was characterized for the utilization of glucose and lactose. The strain, TCL-IC/NUT-2, growing only in presence of glucose and not in the presence of lactose was selected (lac-, glc+). Production of cell biomass from this strain in shaker flask and fermentor was similar to B. singularis except for the growth medium. Experiment 8: Hydrolysis of lactose and glucose by Saccharomyces sp. 25 ml of the cell suspension from the shaker flask or fermentor was centrifuged to obtain cell pellet. The cell pellet was suspended in 15 ml of 20% lactose and 2% glucose solution and kept on shaker at 50°C and 180 rpm agitation. The samples were taken at regular intervals of time and assayed for residual lactose and glucose concentration by HPLC as described in Example 2. Table 4: Hydrolysis of lactose and glucose by Saccharomyces sp. Hydrolysis time (h) Residual sugar (%) Lactose Glucose 0 100 100 3 86.44 5.21 5 92.61 4.75 The results of Table 4 show that Saccharomyces sp. utilize glucose preferably compared to lactose. Thus, the culture has been characterized as lac-, glc+ Experiment 9: Effect of mixed free cells of B. singularis and Saccharomyces sp. 25 ml of the cell suspension of B. singularis and 25 ml suspension of Saccharomyces sp from the shaker flask or fermentor was mixed and centrifuged to obtain cell pellet. The cell pellet was suspended in 15 ml of 30% lactose solution and kept on shaker at 30 °C and 180 rpm agitation. Haif a milliliter sample was taken at different time intervals and processed for galactooligosaccharide by HPLC. Table 5: Production of Galactooligosaccharide by mixed free cells of B. singularis and Saccharomyces Sp. Hydrolysis time (h) % GOS production 1 50.80 2 76.04 4 77.95 5 74.85 7 70.26 The results of Table 5 and Fig.2 indicate that there is two times (> 100%) increase in galactooligosaccharide production by the addition of Saccharomyces sp. The increase in galactooligosaccharide production may be due to the consumption of glucose, produced during hydrolysis, by Saccharomyces sp. The consumption of glucose eliminates the competitive inhibition of the enzyme, there by leading to increased galactooligosaccharide production. Experiment 10: Galactooligosaccharide production by immobilized whole cells in sequential reactor B. singularis and Saccharomyces sp cultures were grown in the fermentor as described in Example 1. The cell biomass of B. singularis and Saccharomyces sp. was immobilized separately as described in Example 4. Galactooligosaccharide production by immobilized B. singularis in shaker flask was carried out using 20% lactose solution as described in Example 5 (Reactor 1). Galactooligosaccharide produced in Reactor 1 was used as feed for Reactor-2 where immobilized Saccharomyces sp. was used. The reaction was carried out for 12 h at 50 °C as described in Example 5. GOS produced from Reactor-2 was used as feed for Reactor -3 where immobilized B. singularis was used. The reaction was carried out for 6 h at 50 °C for 6 h as described in Example 5. The results of Table 7 show that galactooligosaccharide production increased by 24% over the control when galactooligosaccharide produced by B. singularis is used as feed for the Saccharomyces sp. This could be due to utilization of glucose alone. In Reactor 3, galactooligosaccharide production increased by 43.37% over the control. The increase in galactooligosaccharide production in Reactor 3 is due to removal of glucose, the competitive inhibitor of ß-galactosidase enzyme, by Saccharomyces sp. in Reactor 2. Thus, galactooligosaccharide production in a sequential reactor is the best option to increase the purity of galactooligosaccharide. . Table 6: Galactooligosaccharide production in Sequential Reactor Enzyme source Feed Reaction % % time (h) GOS increase Immobilized cells of BS 20 % lactose 18 40.70 - (Reactor 1) Immobilized cells of GOS feed from Reactor 1 12 5.0.57 24.25. Saccharomyces sp. (Reactor 2) Immobilized cells of BS GOS feed from Reactor 2 6 58.35 43.37 (Reactor 3) We claim: 1. An improved process for the production of galactooligosaccharides of high yield and purity comprising the steps of: obtaining microbial cell mass of the B. singularis and Saccharomyces sp. lac-glu+; characterized in that said microbial cell mass is used for the hydrolysis of lactose, wherein the Saccharomyces sp is used for removal of glucose, hydrolysis of lactose by the said microbial cells, such that lactose is converted to GOS and the glucose byproduct obtained by reaction with B. singularis is further utilized by Saccharomyces sp.; said reaction being carried out until galactooligosaccharides content being at least 65 % . 2. The process as claimed in claim 1 wherein said B. singularis is isolated from whey. 3. The process as claimed in claim 1 wherein said Saccharomyces sp. is isolated from contaminated sugar solution. 4. The process as claimed in claim 1 wherein the ratio of microbial cell is 1-2: 1-2 dry weight basis. 5. The process as claimed in claim 1 wherein the step of hydrolysis of lactose is carried out in sequential reactors consisting of series of at least three reactors. 6. The process as claimed in claim 5 wherein said first and third reactors contain B. singularis and the second reactor contains Saccharomyces sp. 7. The process as claimed in claim 5 wherein, galactooligosaccharide production is carried out in the first reactor containing B. singularis using 20% lactose solution for 12-48 h hours at 10-60°C, pH 3-10 and 50-200 rpm agitation. 8. The process as claimed in claim 5 wherein, galactooligosaccharide production is carried out in the second reactor containing Saccharomyces sp. for 12-48 hours at 20-40°C, pH 3-10 and 50-200 rpm agitation. 9. The process as claimed in claim 5 wherein, galactooligosaccharide production is carried out in the third reactor containing B. singularis for 1-10 hours at 10-60°C, pH 3-10 and 50-200 rpm agitation. 10. The process as claimed in claim 1 wherein, galactooligosaccharide production is carried out using 10-40% lactose solution at 25-60 °C. 11. The process as claimed in claim 1 wherein, optionally the galactooligosaccharide solution is concentrated, preferably said concentration is in a rota vapor at 60-80 °C. 12. The process as claimed in claim 1 wherein the concentrated galactooligosaccharide syrup comprises 70-80% total solids.

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