Title of Invention	PROCESS FOR PRODUCTION OF GALACTOOLIGOSACCHARIDES (GOS)
Abstract	Process for production of Galactooligosaccharides by the hydrolysis of lactose with β-galactosidase enzyme immobilized on the matrices involving effective separation of mono and disaccharides in column chromatography using resin to recover GOS of high purity and yield.

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THE PATENT ACT, 1970 COMPLETE SPECIFICATION Section 10 Process for Production of Galactooligosaccharides (GOS) Tata Chemical Limited, Leela Business Park, Andheri Khurla Road, Andheri (E) Mumbai, India The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed: Field of Invention The instant application relates to the field of oligosaccharides, more specifically to the field of galactooligosaccharides. More particularly the instant application describes the process for producing galactooligosaccharides by hydrolysis of lactose. Background of the Invention; Number of processes have been described for the production of galactooligosaccharide by the hydrolysis of lactose for galactosidase enzyme obtained from different microbial sources (Bacteria, Fungi, and Yeast). Currently, galactooligosaccharide production is by batch processing. The increasing demand for functional oligosaccharide in various application (Food and beverages) emphasize that the purity and yield needs to be improved by developing new process. The use of immobilized enzyme allows enzyme to reuse in continuous operation (Akiyama et. al, 2001, Ribu et. al., 2001, Tzortiz et. al., 2005, Jorgensen et al, 2001, EP00272095A2, US5032509, Carra et al., 1994, Torres et al, 2003, Sumgur and Akbulut, 1993). Enzyme ß-galactosidase was immobilized by entrapment (Mannarella and Rubiolo 2005, Rodriguez-Nogales and Delgadillo, 2005) and in membrane (Novalli et al, 2005). Shin et al, 1998 used chitosan beads for immobilization of ß-galalctosidase for continuous production of galactooligosaccharides. Beddows et al, use p-amino-carbonillated cellulose acetate derivative for immobilization of b-galactosidase. While, Torres et al, (2003) compared immobilization of B-galactosidase commercially available amino-epoxy sepabeads with conventional epoxy supports. The galactooligosaccharide solution obtained after hydrolysis of lactose with ß-galactosidase contains a mixture galactooligosaccharides, unreacted lactose, and secondary products such as glucose and galactose. To obtain galactooligosaccharides having high activity, mono and disaccharides needs to be separated Activated carbon is most commonly used in industry to purify the product. Sugar solution is passed through activated carbon to adsorb saccharides Adsorption of saccharides depends upon the molecular weight. Low molecular saccharides preferentially adsorb first. Low molecular weight saccharides (mono and disaccharides are eluted first with water or low concentration of ethanol and the galactooligosaccharides is eluted with high concentration of ethanol. However, this method has several disadvantages (1) separation based on the molecular weight is not efficient. (2)Yield of galactooligosaccharides is extremely poor. (3) Use of ethanol lead to increased expenses. Rastal et al., used Nanofiltration with 10KD NMWCO cut off membrane for the separation of sachharides. A gel filtration method has been used to separate sugars based on the molecular size using dextran, cellulose, and polyacrylamide based on three-dimensional linking. However, this method is used only in laboratory scale because the gels are stable for short time and not applicable for industrial scale due to poor separation in increased loading and expensive gel. Strong cation exchange polystyrene divinylbenzene resins of Na/Ca with SO3 group (UBK530) have been used for separation saccharides (EP00272095) Polymers of no ion exchange group can be used for separation of oligosaccharides (JP 130297/1986). In this method saccharides are eluted with water or by concentration gradient of water and alcohol based on the differences in the affinities of the saccharides and the adsorbent in order of molecular weight. However, this method is inefficient, expensive and impractical because (1) elution with water requires too much time for galactosaccharides (2) Purity and recovery yield is low. Splechenta et.al., studied the option of oxidizing the residual lactose to lactobionic acid by the cellobiose dehydrogenase produced from Sclerotium rolfsii fungi. Lactobionic acid and monosaccharides were separated using ion exchange chromatography. The final product contained 97% GOS, 1.2% lactose, 2.1% monosacharides while the yield of GOS was 25% of original lactose. To reduce the cost of galactooligosacchardes production, more than 50% of unreacted lactose needs to be recycled. However, the presence of mono-saccharides (glucose and Galactose) as 8) Concentration of the product in Rota vapor 9) Reuse of recovered lactose to further increase galactooligosaccharide production. The different stages would be discussed in detail in the following section along with examples of the preferred embodiments. The microorganisms from the dairy waste were isolated on YEM medium of the following composition; Yeast extract, malt extract, dextrose and peptone. The isolated microorganisms were screened for the lactose hydrolysis leading to the production of galactooligosaccharides. The yeast culture showing highest galactooligosaccharide producer was selected. To optimize the growth medium, the medium was supplemented with Mg++ and Fe++ ions. The concentration of Mg++ and Fe++ varied between 0.01-0.1% and 0.001-0.05% respectively. The effect of pH & temperature on the hydrolysis by the free cells was investigated. The galactooligosaccharide production is not affected with the change in pH from 4,5 and 6.0. While galactooligosaccharide production increased with increase in temperature from 30 °C to 50 °C the increase in temperature. Microbial cell biomass was harvested from shaker flask and fermentor by centrifugation when the ß-galactosidase enzyme activity reached maximum. The harvested cell biomass was sonicated to extract crude enzym. Crude enzyme was further subjected to ammonium sulfate (60-80%) precipitation for purification of enzyme. The precipiated enzyme was dissolved and dialyzed to remove salts. Purified enzyme was concentrated to dry powder by lyophilization. Shin et al., observed 55% galactooligosaccharides during continuous production process where ß-galactosidase was immobilized in chitosan beads. Albayrak and Yang (2002) immobilized the ß-galactosidase enzyme on the fibrous matrix using polyethylenimine and glutarldehyde as cross linking agents. They claimed that formation of two layers of PEI-enzyme aggregates prevent enzyme leaching unlike in the monolayer (Bahulekar et al.,) . Also, they claimed that use of fibrous cotton as immobilizing matrix is safest, fastest and cheapest. The use of PEI and GDA may not be absolutely safe as they may leach out in the industrial scale production and may be a serious problem as they are carcinogenic. Because of the forthcoming strict regulation, it is necessary to develop the process where the carcinogenic cross linking agents are not used in the immobilization of enzyme, particularly where they are used in the food industry. The immobilization of the enzyme is on a polymer resin matrices with functional groups. As a preferred embodiment the crude/purified ß-galactosidase enzyme is immobilized on NR 2 resin, Thermax India Ltd.; wherein PEI and GDA are not used as cross linking agents. In the present process GOS production is not affected with the changes in the pH.5 -7.0. In accordance with the fourth embodiment, lactose was dissolved in the distilled water instead of buffers. This eliminates the process of removal of salts of inorganic and organic acids used in the buffer. The immobilized enzyme resin on NR 2 resin is used in the shaker flask stirred tank reactor or packed in the jacketed glass column for continuous production. Chockchaisawasdee et al., (2004) used batch and continuous ultrafiltration membrane fixed bioreactors for the production of galactooligosaccharides. The hydrolysis of lactose is carried out on aqueous solution of lactose. In a preferred embodiment the aqueous lactose solution (10-90% by weight) is passed through a column (45 ml/min) packed with immobilized B-galactosidase enzyme (mg/g of resin). Column temperature is maintained at 45 °C by circulating hot water (45 °C) in the outer jacket of the column. The sugar solution coming out of column typically contains the saacharides of the following compositions; 30-50% galactooligosaccharides, 10-15 % glucose, 30-50% lactose and 5-10% galactose. NR 2 resins has strong affinity to the enzymes and hence the enzyme do not leach out. Therefore, in accordance with seventh embodiment of the present invention, the immobilized resin is used in continuous production of GOS in the CSTR or columns for more than 100 days. Thus, this makes the process cost effective. The resin of Na, K, and Ca form composed of styrene divinyl benzene copolymer and SO3 as exchange group. Saccharides are eluted with hot water (60 °C). Sacchardies of larger molecular size, which are not cross linked, are eluted first, using above strong cation resin bases separation of the components of the saccharide solution by the molecular size exclusion effect. To separate saccharides efficiently the saccharide solution applied to the column should be preferably 25 to 80% total sugar concentration. Hot water is passed through the column to elute the saccharides. The strong acid cation exchange resin, TULSION from Thermax India Ltd., used in the separation of saccharides has excellent resistance to compression, good durability, and superior operating properties and low price suitable for industrial applications. The hot water used as elutant in place of organic solvents used in activated carbon column negates the requirement of equilibration with water after each cycle. Therefore, continuous purification can be easily performed with good efficiency resulting in increased purity and yield of galactooligosaccharides. It is a preferred embodiment of the invention that lactose rich fraction be recycled again in cation exchange column for increased purity of galactooligosaccharides. Galactooligosaccharide fraction more than 50% was passed through ultrafiltration to remove any suspended particles and concentrated in Rota vapor to obtain the syrup with 75-80% total sugar concentration and fraction rich in mono and disaccharide solution can be recycled as carbon source in the growth of cell biomass to produce the enzyme. The process disclosed in the instant application produces galactooligosaccharides of high purity and yield. The various stages of the process for production of oligosaccharides as disclosed in the instant application shall now be described with the help of some examples. A person skilled in the art would appreciate that the process followed in the examples may be varied without deviating from the spirit of the invention and hence examples only broadly represent the invention's embodiments. samples was taken out, centrifuged to remove the cell biomass. The supernatant was diluted to 50 times with milliq water. 5 ul-diluted samples was injected in to the HPLC Galactooligosaccharides and other sugars HPLC analysis: The concentration of sugars (glucose, galactose, lactose, and Galactooligosaccharides) was determined by HPLC. The HPLC system consists of (Waters 717) with refractive index detector (waters W2467).and carbohydrate column. Phenomenex (RNM 00h-0316, REZEX 300 mm L X 7.5 mm, pore size 8 u) IDcolumn. The column temperature was 80 C with Refractive index detector. Water was used as mobile solvent with flow rat 0.5 ml/min. Galactooligosaccahrides and other sugars were determined as weight percentage of total sugars based on the area of peak. Assay for Protein estimation: The samples for protein estimation are diluted 200 times. 150 ul of diluted sample was used for protein estimation by Bradford method. Absorbancy was recorded 595 nm in spectrophotometer (Eppendorf Biophotometer). Bovine serum albumin of 5-25 ug/ml was used standard. Example 3: Optimization of growth medium: The YMP medium was supplemented with MgS04 0.05% and FeS04 0.001%. Modified medium in the shaker flask was inoculated and incubated as given in Example 1. Samples were taken at different time intervals assayed for GOS production as shown in Example 2. The results tabulated in Table-1 shows 42-80 % increase in GOS production. The modified medium was used in the growth of cell biomass either in shaker flasks or fermentor. Table: 1 Effect of addition of Mg and Fe in the growth medium on the GOS Production by B. singularis Medium supplementation % GOS 2h 3h Control 17.89 28.71 MgSo4 30.32 41.36 Fe 18.34 30.70 sodium phosphate buffer of pH 7.0. and sonicated in ultrasonicator under chilled conditions for 10 min (15.sec. ON/OFF cycle) at 40 amplitude. Sonication was carried out twice. Both the extraction showed galactooligosaccharide production. The sonicated sample was centrifuged at 10000 rpm and 4 °C for 5 min. to remove the cell debris. The crude extract was pooled for purification of enzyme. Purification : The supernatant was precipitated with 20-50% ammonium sulfate and the resulting supernatant was further precipitated with 50-80% ammonium sulphate. The precipitate from 60-80 Ammonium sulfate was dissolved in a buffer and dialyzed against distilled water for overnight. Dialysate was concentrated to dryness with 10% PEG followed by lyophilization in lyophilizer (Virtis , Benchtop K). The lyophilized powder was assayed for galactooligosaccharide production and then used for immobilization. The results of the Table-4 indicate that the purified enzyme reduced reaction time from 46 h to 2.5 h. GOS production by crude and purified enzyme : 0.4 ml of the crude or purified enzyme (1.4 mg protein/ml) was taken in 0.6 ml of 20% lactose in eppendorf tube. The reaction was carried out on shaker at 50 °C and 180 rpm agitation. 10 ul of sample was taken for GOS analysis using HPLC as described in Example 2. Table 4: Comparison of GOS production by the purified enzyme extracted from B. singularis. Reaction time Crude enzyme Purified Enzyme (h) %GOS %Lac %GLC %GOS %LAC %GLC 2.5 4.29 95.71 0.00 36.22 58.85 18.25 5 - - - 39.33 48.25 19.32 22 16.91 77.26 5.83 35.65 34.53 5.83 30 20.33 71.78 7.89 - - - 46 25.76 66.12 8.73 - - - The results of Table-4 indicate that specific activity of purified B-galactosidase enzyme is higher than the crude enzyme. Example: 7 Immobilization of B-galactosidase enzyme from B. singularis. On NR2 resin. 5 g of NR2 resin (Thermax India Ltd.,) was suspended in 20 ml crude enzyme solution (lmg protein/ ml). The enzyme - resin solution was kept on shaker for 12 h at 30 °C and 180 rpm agitation. Samples were taken different time interval to estimate the percentage protein binding on the resin. The agitation was stopped when binding reached more than 75% (Table 5) and the solution was drained and the immobilized resin was washed thrice with 50 ml of 50 mM NaCl solution. The washed resin was treated with 20 ml of 3M glycine at 27 °C for 16 h and then washed thrice with 50ml of 50mM NaCl. Table 5: Percentage of binding of enzyme Resin Protein (mg/ml) Binding (%) Before immobilization After immobilization NR2 1.88 0.22 79.0 The immobilized resin was tested for GOS production using 1 g immobilized resin in 10 ml of 20% lactose solution. The reaction was carried out in shaker flask at 50 °C , 180 rpm agitation. 0.5 ml sample were taken at different time interval and assayed for GOS by HPLC as described in Example 2. Table; 6 Extraction of crude enzyme from B. singularis immobilized in NR2 Experiment % GOS production 7h 10 h 24 h Experiment 1 25.36 31.22 41.70 Immobilized crude enzyme showed maximum GOS production after 24 h (Table 6) Example 8: Hydrolysis of lactose for the production of Galactooligosaccharide. Thirty five g of immobilized resin was packed in to a jacketed glass column(300mm L x 25 mm ID). Resin in glass column was contained in between two sintered filter (G1). 40% aqueous lactose maintained at 50 °C was recirculated in the above column.The column was maintained at 50 °C by passing hot water through the jacket of glass column. The lactose solution coming out of column showed 30-35% GOS. The results of Table-8 indicate that GOS produced in each cycle of reuse of immobilized resin is stable. Example: 10 Down stream separation of galactooligosaccharides. Forty five ml of TULSION resin from Thermax India Ltd. packed in glass column of 1.1 cm ID and 60 cm height. Resin was flushed with 4 bead volume of distilled water. 6 ml of GOS solution from the enzyme reactor was loaded on the top of resin bed in glass column. Saccharide solution was eluted with hot water (60 °C) with flow rate 0.2 ml/min. The initial fractions having no saccharides were discarded. The collected fraction shows the purity of galactooligosaccharide in the range of 4-86% (Table-9). The fraction having more than 50% GOS were pooled. While fractions rich in lactose were pooled. Fractions more than 50% glalactooligosaccharides were pooled. Fractions less than 50% galactooligosaccharides were pooled and passed through downstream column 2 for recovering galactooligosaccharides of high purity and yield. The fractions rich in glucose and Galactose were pooled and used as constituents of YMP medium for the growth B. singularis. Table: 9 Separations of Saccharides Fraction No % GOS % Lactose % Glucose % Galactose Fl 86.70 13.30 0.00 0.00 F2 65.62 34.38 0.00 0.00 F3 52.39 47.11 0.56 0.00 F4 43.12 54.34 2.53 0.00 F5 35.88 59.56 4.56 0.01 F6 24.02 66.56 8.92 0.50 F7 18.29 64.26 15.32 2.13 F8 10.43 50.12 32.52 6.93 F9 4.36 19.34 60.33 15.97 The results of Table-9 clearly shows that there is good separation of GOS, lactose, glucose and galactose. The high molecular GOS is eluted first followed by lactose, glucose and galactose Example: 11 Recycling of lactose and monosaccharide rich fractions; GOS produced from the enzyme reactor (Table 10) was passed through the Downs tream column for the separation of saccharide fractions as described in the Example 10. The results of the separation is given in Table 11. The fractions F1-F6 were pooled (PF 1) to get 52.13% GOS while the fractions F7-F16 were pooled (PF 2) of 37.89 % GOS. The pooled fraction PF-1 and PF-2 were further subjected to separation of saccharides in DSP-2 for further enrichment of GOS. The results of Table 12a and 12b showed GOS and PF-1 and PF-2 was increased to 66.19 and 51.85 respectively. (a) Recycling of lactose rich fraction; this fraction was either recycled in another DSP column for further separation of galactooligosaccharides. (b) Recycling of Monosaccharide fraction: This fraction is used as medium constituents for the production cellbiomss to produce ß-galactosidase enzyme. Table 10: GOS production from B. singularis immobilized in NR 2 Resin Immobilized Resin % GOS % Lactose % Glucose NR2 30.24 58.29 11.14 Table 11 :Downstream separation I: Primary purification Fraction No % GOS % Lactose % Glucose % Galactose Fl 61.65 38.35 0.00 0.00 F2 56.98 43.02 0.00 0.00 F3 53.09 46.17 0.74 0.00 F4 50.02 48.29 1.69 0.00 F5 46.83 50.36 2.81 0.00 F6 44.24 51.56 4.20 0.00 F7 43.02 51.31 5.67 0.00 F8 40.92 51.36 7.73 0.00 F9 39.54 50.66 9.80 0.00 F10 40.46 49.59 9.95 0.00 Fll 40.65 49.83 9.52 0.00 F12 40.71 50.04 9.24 0.00 F13 40.92 50.80 8.28 0.00 F14 39.38 52.95 7.67 0.00 F15 35.64 54.98 9.37 0.00 F16 31.87 57.51 10.62 0.00 F17 23.69 62.50 13.81 0.00 FI8 10.95 53.21 34.84 1.00 F19 2.08 14.60 77.55 5.77 F20 0.00 0.00 78.20 21.80 % GOS of pooled fractions (Fl-F6)= 52.13 % GOS of pooled fractions (F7-F16) = = 37.89 We claim: 1. Process for production of galactooligosaccharides comprising: a) growing Bullera singularis producing an enzyme that hydrolyses lactose to oligosaccharide in optimum medium and condition to obtain cell biomass, wherein said medium contains Mg++ cation, b) extracting said enzyme, c) immobilizing said enzyme on polymer resin matrices with functional groups, characterized in that said immobilization is in a resin allowing up to 15 recycling without the use of polyethyleneimine and glutarldehyde as cross linking agents, wherein said immobilization of the purified enzyme is on the resin, using buffer (5mm to 1M) of pH 7, at 20°C for 24h and washing non-covalently attached protein to matrix, d) hydrolyzing aqueous lactose by said immobilized enzyme wherein said hydrolysis is at a temperature of 10 to 60 degree Celsius and at a pH of 3 to 10 to obtain galactooligosaccharides, and e) separating said oligosaccharides using strong cation exchange resins. 2. The process as claimed in claim 1 optionally comprising down stream processing for concentration of oligosaccharides. 3. The process as claimed in claim 1 wherein said enzyme that hydrolyses lactose to oligosaccharide is ß-galactosidase. 4. The process as claimed in claim 1 wherein said growth of cell biomass is by shake flask culture and fermentation. 5. The process as claimed in claim 1 wherein said cation in the cation exchange resin is in the concentration of 0.001 to 0.100%. 6. The process as claimed in claim 1 wherein said temperature is 15 to 55°C. 7. The process as claimed in claim 1 wherein said growth medium has a pH of 4 to 10. 8. The process as claimed in claim 1 wherein said growth period is 12 to 96 h. 9. The process as claimed in claim 1 wherein said cell biomass is harvested when the ß-galactosidase enzyme activity in the free cell is maximum. 10. The process as claimed in claim 9 wherein said 6-galactosidase enzyme activity in the free cell is determined in the shaker flask using 5-30% lactose at 45°C. 1 J. The process as claimed in claim 1 wherein said extraction of enzyme is in a buffer of P04and salts of organic acids of divalent cation. 12. The process as claimed in claim 1 wherein said extraction of enzyme is by ultrasonication, osmolysis, and ball mill with glass beads. 13. The process as claimed in claim 12 wherein the extracted enzyme is precipitated with Ammonium sulphate 60-80% followed by optionally dissolving in buffer and dialyzed. 14. The process as claimed in claim 13 wherein said dialyzed enzyme solution is concentrated to dryness by lyophilization. 15. The process as claimed in claim 1 wherein said enzyme resin matrix composite is further incubated in 1-10 M glycine for 10-24h at 10-30°C. 16. The process as claimed in claim 1 wherein said aqueous lactose solution has 10-50% concentration. 17. The process as claimed in claim 1 wherein said hydrolysis of lactose is carried out with immobilized matrix packed in jacketed glass column, shaker flask or CSTR (continuous stir tank reactor) at 45°C. 18. The process as claimed in claim 17 wherein said enzymatic hydrolysis of lactose is carried out for l-50h. 19. The process as claimed in claim 2 wherein said oligosaccharide solution obtained by hydrolysis of lactose is loaded on the resin, packed in the glass column for said separation of galactooligosaccharides, mono and disaccharides. 20. The process as claimed in claim 19 wherein said different fractions of saccharides are eluted with hot water at 40-80°C. 21. The process as claimed in claim 19 wherein said fractions are pooled to give 55% -90% GOS. 22. The process as claimed in claim 21 wherein said pooled fraction rich in lactose is recycled in the second column packed with an immobilized matrix to obtain 55% GOS. 23. The process as claimed in claimed in claims 19 or 22 wherein said fraction rich in mono and disaccharides were pooled and used in the medium for the growth cell biomass. 24. The process as claimed in claims 21 or 22 wherein said GOS rich fraction is ultra filtered and concentrated to syrup in Rota vapor to obtain GOS more than 55% and 75% total sugar content. Abstract Process for the Production of Galactooligosaccharides Process for production of Galactooligosaccharides by the hydrolysis of lactose with B-galactosidase enzyme immobilized on the matrices involving effective separation of mono-and disaccharides in column chromatography using resin to recover GOS of high purity and yield.

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