Title of Invention	A PROCESS FOR THE MANUFACTURE OF LIPSTATIN BY SOLID SUBSTRATE
Abstract	A process for the manufacture of lipstatin by solid substrate fermentation comprising the steps of: preparing an inoculum of the microorganism Streptomyces toxytricini, inoculating a solid substrate matrix with the inoculum prepared, incubating the inoculated solid substrate matrix for 4-7 days at 25-30 deg.C and extracting the incubated solid substrate matrix to obtain lipstatin.

Full Text

FIELD OF THE INVENTION The current invention relates to a novel process for the manufacture of lipstatin by solid substrate fermentation of Streptomyces toxytricini optionally using fed-batch production technique in a self contained bioreactor. BACKGROUND OF THE INVENTION The compound Lipstatin, a pancreatic lipase inhibitor is produced by fermentation of Streptomyces toxytricini. Tetrahydrolipstatin the active substance of the anti-obesity drug is chemically reduced derivative of lipstatin and is obtained by hydrogenation of Lipstatin, The biosynthesis of the pancreatic lipase inhibitor lipstatin was investigated by fermentation experiment using cultures of Streptomyces toxytricini, which were supplied with soybean oil and a crude mixture of U-13C-lipids obtained from algal biomass cultured with 13C02 (Eisenreich, Wolfgang, etal J. Biol. Chem., 1997, 272(2), 867-874). KR 9709294 discloses novel active substance lipstatin, which is produced by a Streptomyces sp. (KCTC-9303) isolated from soil. EP 0 803 576, this patent discloses a process for the fermentative production of lipstatin, which comprises (a) aerobically cultivating a micro-organism of the order of actinomycetes which produces lipstatin, in an aqueous culture medium, substantially free of fats and oils, containing a suitable carbon and nitrogen sources and inorganic salts, (b) adding to the broth linoleic acid, optionally together with capryiic acid, their ester(s) or salt(s), stabilized by an antioxidant. Lipstatin can be isolated from the broth and hydrogenated to tetrahydrolipstatin. EP 0 129 748, discloses a process for the production of Lipstatin by fermentation of Streptomyces toxytncini, in a liquid nutrient media comprising potato starch, glucose, soybean meal, (NH4)2S04, ribose, glycerol, and peptone 0.2% and incubated at 28*C for 124 h with stirring and aeration, followed b}' extraction of the product. Existing prior art does not disclose the use of "solid state fermentation" or the use of "fed-batch technique" in the production of Lipstatin. SUMMARY OF THE INVENTION: The object of the present invention is to provide a novel commercially viable process for the production of Lipstatin by solid state fermentation of Streptomyces toxytncini optionally using fed-batch production technique in a contained bioreactor. Accordingly the present invention provides a process for the manufacture of lipstatin by solid substrate fermentation a comprising the steps of: preparing an inoculum of the microorganism of the genus Streptomyces, inoculating a solid substrate matrix with the inoculum prepared, incubating the inoculated solid substrate matrix for 4-7 days at 25-30 deg.C and extracting the incubated solid suostrate matrix to obtain lipstatin. The micro-organism is Streptomyces toxytndm. The solid substrate matrix for fermentation is selected from wheat bran, wheat rawa, broken wheat, boiled rice, rice bran, rice rawa, beaten rice, maize bran, maize grits, oat meal, bagasse, tapioca residue, soya grits, soya flakes, ceramic beads, glass beads, sponge or a mixture of one or more of these. The solid substrate fermentation is a fed-batch fermentation. The fed-batch fermentation is carried out by oil feeding. The oil used for feeding is selected from soyabean, safflower, groundnut, rapeseed, cottonseed. The oil used for feeding also contains lecithin. The feeding for fed-batch fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation. The incKnilum is prepared by peculating a seed medium with slant of the micro-organism of the genus Streptomyces. The.seed medium comprises defatted soy, glycerol and yeast. DEATAILED DESCRIPTION Definitions "Solid state fermentation" or "solid state cultivation" or Solid Substrate fermentation: The term "solid state fermentation" or "solid state cultivation or solid substrate fermention", sometimes referred to as "semi-solid state fermentation" as used hereiiv means the process of fermenting microorganisms on a solid medium that provides anchorage points for the microorganisms in the absence of any freely flowing ^ substance. The amount of water in the solid medium can vary to the extent till no free flowing of substances is noticed. For example/ the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the terms "solid state fermentation" and "semi-solid state fermentation" are interchangeable. "Fed-batch fermentation" or "fed-batch technique": The term fed-batch fermentaticm as used hereiiv means a fermentation process carried out where substrate or nutrients are added in small increments as the fermentation progresses. The substrate or nutrient is added in small increment that would encourage the production of secondary metabolites because some secondary metabolite production is inhibited by high concentrations of substrate or substrates, so this method would encourage the production of such metabolites. Supplement of nutrients at a time when the initially fed nutrient are consumed by the microorganisms or culture also help in providing more energy to the microorganism which in turn increases the overall production of the secondary metabolites. "Bioreactor": The term "bioreactor* as used herein, means a device capable of holding f ermentation media inoculated with microorganism and carrying out the process of solid state fermentation in a contained manner. A bioreactor can be used to grow any microorganism capable of growing under specified conditions in a contained environment Some examples of microorganisms capable of growing in a bioreactor are fungi, veast and bacteria. Fed batch fermentation systems are generally defined as batch culture systems wherein fresh nutrients and/or other additives (such as precursors to products) are added but no medium is withdrawn. The advantages of the present invention over the other reported methods are: (i) Commercial viability making it cost effective. (ii) Self-contained bioreactor decreases nazardous o exposure and risk of contamination. The following Examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein. EXAMPLES EXAMPLE 1 A well grown slant of Streptomyces toxytricini was taken and 5ml of distilled water was added. It was shaken thoroughly and 100 micro litres of this suspension was used for the inoculation of seed medium. 30ml taken in a 250 ml conical flask. The composition of seed medium is as follows: Defatted soy flour = ]0g/L Glycerol = lOg/L Yeast extract = 5g/ L pH of this medium is adjusted to 7.0 by making up the volume with water. The culture is grown at 28 deg C for a day and used as an inoculum for solid state fermentation. Solid state fermentation: lOgm each of wheat bran, maize flakes, wheat rawa, rice rawa, oat meal/ maize grits, maize bran, soy grits were taken in separate petri plates. 35 ml of water was added in each petri plate arid sterilized at 121 deg. C for 30 minutes. Adequate amount of water was added and sterilized at 121 deg C for 30 minutes. 10 ml inoculum from 24 hr seed medium was added along with lgm of oil; lecithin [ratio of oil to soya lecithin - 4.6: l(w/w)] emulsion. The entire substrate was mixed properly and incubated at 28 deg C for 7 days. Following results were obtained: EXAMPLE2 The inoculum from seed medium as obtained from Example 1, was used to prepare another production medium. 1ml of the culture grown in the seed medium was used for the inoculation of 35 ml production medium taken in 250ml conical flasks. Production medium composition has the seed medium composition with additional oil and lecithin emulsion in the ratio of 4.6 : 1. The quantity of this emulsion in the medium is between 50 and lOOg/L. These flasks were grown for a da}' at - 28 deg C and used as an inoculum for solid state fermentation. Solid state fermentation was carried out similar to Example 1 using the production medium as inoculum. Following results were obtained: EXAMPLE 3 Solid state fermentation was conducted as in Example 2 using 75 g ceramic beads as the solid support in a petri-plate, 15 mL of Streptomyces toxytncini inoculum grown in production medium was added. The results obtained are given in the table below. EXAMPLE 6 15 kg of substrate mixture consisting of maize bran, wheat rawa and oat meal was loaded into a bioreactor having 22600 cm2 surface area. The bioreactor was sterilised at 121 deg C for 1 to 2 hours using steam. After the sterilization the temperature of the solid substrate was brought down to 28 deg C. 0.75 kg of oil and lecithin emulsion was sterilized separately and added to the solid substrate along with 15 L of Streptomyces toxytricini inoculum grown for 24 hours, in a fermenter using production medium. Then solid substrate, oil and inoculum are mixed. This was incubated for 3 days at 27 to 29 deg C. On the 3rd day, 0.75 kg of oil and lecithin emulsion was added to the solid substrate matrix and incubated at the same temperature for another 4 days. The entire biomass along with the solid substrate was harvested. A total of 17.25 g of lip statin was present at the end of fermentation. FIELD OF THE INVENTION The current invention relates to a novel process for the manufacture of lipstatin by solid substrate fermentation of Streptomyces toxytricini optionally using fed-batch production technique in a self contained bioreactor. BACKGROUND OF THE INVENTION The compound Lipstatin, a pancreatic lipase inhibitor is produced by fermentation of Streptomyces toxytricini. Tetrahydrolipstatin the active substance of the anti-obesity drug is chemically reduced derivative of lipstatin and is obtained by hydrogenation of Lipstatin, The biosynthesis of the pancreatic lipase inhibitor lipstatin was investigated by fermentation experiment using cultures of Streptomyces toxytricini, which were supplied with soybean oil and a crude mixture of U-13C-lipids obtained from algal biomass cultured with 13C02 (Eisenreich, Wolfgang, etal J. Biol. Chem., 1997, 272(2), 867-874). KR 9709294 discloses novel active substance lipstatin, which is produced by a Streptomyces sp. (KCTC-9303) isolated from soil. EP 0 803 576, this patent discloses a process for the fermentative production of lipstatin, which comprises (a) aerobically cultivating a micro-organism of the order of actinomycetes which produces lipstatin, in an aqueous culture medium, substantially free of fats and oils, containing a suitable carbon and nitrogen sources and inorganic salts, (b) adding to the broth linoleic acid, optionally together with capryiic acid, their ester(s) or salt(s), stabilized by an antioxidant. Lipstatin can be isolated from the broth and hydrogenated to tetrahydrolipstatin. EP 0 129 748, discloses a process for the production of Lipstatin by fermentation of Streptomyces toxytncini, in a liquid nutrient media comprising potato starch, glucose, soybean meal, (NH4)2S04, ribose, glycerol, and peptone 0.2% and incubated at 28*C for 124 h with stirring and aeration, followed b}' extraction of the product. Existing prior art does not disclose the use of "solid state fermentation" or the use of "fed-batch technique" in the production of Lipstatin. SUMMARY OF THE INVENTION: The object of the present invention is to provide a novel commercially viable process for the production of Lipstatin by solid state fermentation of Streptomyces toxytncini optionally using fed-batch production technique in a contained bioreactor. Accordingly the present invention provides a process for the manufacture of lipstatin by solid substrate fermentation a comprising the steps of: preparing an inoculum of the microorganism of the genus Streptomyces, inoculating a solid substrate matrix with the inoculum prepared, incubating the inoculated solid substrate matrix for 4-7 days at 25-30 deg.C and extracting the incubated solid suostrate matrix to obtain lipstatin. The micro-organism is Streptomyces toxytndm. The solid substrate matrix for fermentation is selected from wheat bran, wheat rawa, broken wheat, boiled rice, rice bran, rice rawa, beaten rice, maize bran, maize grits, oat meal, bagasse, tapioca residue, soya grits, soya flakes, ceramic beads, glass beads, sponge or a mixture of one or more of these. The solid substrate fermentation is a fed-batch fermentation. The fed-batch fermentation is carried out by oil feeding. The oil used for feeding is selected from soyabean, safflower, groundnut, rapeseed, cottonseed. The oil used for feeding also contains lecithin. The feeding for fed-batch fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation. The incKnilum is prepared by peculating a seed medium with slant of the micro-organism of the genus Streptomyces. The.seed medium comprises defatted soy, glycerol and yeast. DEATAILED DESCRIPTION Definitions "Solid state fermentation" or "solid state cultivation" or Solid Substrate fermentation: The term "solid state fermentation" or "solid state cultivation or solid substrate fermention", sometimes referred to as "semi-solid state fermentation" as used hereiiv means the process of fermenting microorganisms on a solid medium that provides anchorage points for the microorganisms in the absence of any freely flowing ^ substance. The amount of water in the solid medium can vary to the extent till no free flowing of substances is noticed. For example/ the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the terms "solid state fermentation" and "semi-solid state fermentation" are interchangeable. "Fed-batch fermentation" or "fed-batch technique": The term fed-batch fermentaticm as used hereiiv means a fermentation process carried out where substrate or nutrients are added in small increments as the fermentation progresses. The substrate or nutrient is added in small increment that would encourage the production of secondary metabolites because some secondary metabolite production is inhibited by high concentrations of substrate or substrates, so this method would encourage the production of such metabolites. Supplement of nutrients at a time when the initially fed nutrient are consumed by the microorganisms or culture also help in providing more energy to the microorganism which in turn increases the overall production of the secondary metabolites. "Bioreactor": The term "bioreactor* as used herein, means a device capable of holding f ermentation media inoculated with microorganism and carrying out the process of solid state fermentation in a contained manner. A bioreactor can be used to grow any microorganism capable of growing under specified conditions in a contained environment Some examples of microorganisms capable of growing in a bioreactor are fungi, veast and bacteria. Fed batch fermentation systems are generally defined as batch culture systems wherein fresh nutrients and/or other additives (such as precursors to products) are added but no medium is withdrawn. The advantages of the present invention over the other reported methods are: (i) Commercial viability making it cost effective. (ii) Self-contained bioreactor decreases nazardous o exposure and risk of contamination. The following Examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein. EXAMPLES EXAMPLE 1 A well grown slant of Streptomyces toxytricini was taken and 5ml of distilled water was added. It was shaken thoroughly and 100 micro litres of this suspension was used for the inoculation of seed medium. 30ml taken in a 250 ml conical flask. The composition of seed medium is as follows: Defatted soy flour = ]0g/L Glycerol = lOg/L Yeast extract = 5g/ L pH of this medium is adjusted to 7.0 by making up the volume with water. The culture is grown at 28 deg C for a day and used as an inoculum for solid state fermentation. Solid state fermentation: lOgm each of wheat bran, maize flakes, wheat rawa, rice rawa, oat meal/ maize grits, maize bran, soy grits were taken in separate petri plates. 35 ml of water was added in each petri plate arid sterilized at 121 deg. C for 30 minutes. Adequate amount of water was added and sterilized at 121 deg C for 30 minutes. 10 ml inoculum from 24 hr seed medium was added along with lgm of oil; lecithin [ratio of oil to soya lecithin - 4.6: l(w/w)] emulsion. The entire substrate was mixed properly and incubated at 28 deg C for 7 days. Following results were obtained: EXAMPLE2 The inoculum from seed medium as obtained from Example 1, was used to prepare another production medium. 1ml of the culture grown in the seed medium was used for the inoculation of 35 ml production medium taken in 250ml conical flasks. Production medium composition has the seed medium composition with additional oil and lecithin emulsion in the ratio of 4.6 : 1. The quantity of this emulsion in the medium is between 50 and lOOg/L. These flasks were grown for a da}' at - 28 deg C and used as an inoculum for solid state fermentation. Solid state fermentation was carried out similar to Example 1 using the production medium as inoculum. Following results were obtained: EXAMPLE 3 Solid state fermentation was conducted as in Example 2 using 75 g ceramic beads as the solid support in a petri-plate, 15 mL of Streptomyces toxytncini inoculum grown in production medium was added. The results obtained are given in the table below. EXAMPLE 6 15 kg of substrate mixture consisting of maize bran, wheat rawa and oat meal was loaded into a bioreactor having 22600 cm2 surface area. The bioreactor was sterilised at 121 deg C for 1 to 2 hours using steam. After the sterilization the temperature of the solid substrate was brought down to 28 deg C. 0.75 kg of oil and lecithin emulsion was sterilized separately and added to the solid substrate along with 15 L of Streptomyces toxytricini inoculum grown for 24 hours, in a fermenter using production medium. Then solid substrate, oil and inoculum are mixed. This was incubated for 3 days at 27 to 29 deg C. On the 3rd day, 0.75 kg of oil and lecithin emulsion was added to the solid substrate matrix and incubated at the same temperature for another 4 days. The entire biomass along with the solid substrate was harvested. A total of 17.25 g of lip statin was present at the end of fermentation. We Claim: 1. A process for the manufacture of lipstatin by solid substrate fermentation comprising the steps of: preparing an inoculum of the microorganism Streptomyces toxytricini, inoculating a solid substrate matrix with the inoculum prepared, incubating the inoculated solid substrate matrix for 4-7 days at 25-30 deg.C and extracting the incubated solid substrate matrix to obtain lipstatin. 2. A process as claimed in claim 1, wherein said solid substrate matrix for fermentation is selected from wheat bran, wheat rawa, broken wheat, boiled rice, rice bran, rice rawa, beaten rice, maize bran, maize grits, oat meal, bagasse, tapioca residue, soya grits, soya flakes, ceramic beads, glass beads, sponge or a mixture of one or more of these. 3. A process as claimed in claim 1, wherein the solid substrate fermentation is fed-batch fermentation. 4. A process as claimed in claim 3, wherein the fed-batch fermentation is carried out bv oil feeding. 5. A process as claimed in claim 4, wherein the oil used for feeding is selected from soyabean, safflower, groundnut, rapeseed, cottonseed. 6. A process as claimed in claim 4, wherein the oil used for feeding also contains lecithin. 7. A process as claimed in claim 3-6, wherein the feeding for fed-batch fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation. 8. A process as claimed in claim 1, wherein inoculum is prepared by inoculating a seed medium with slant of Streptomyces toxytricini. 9. A process as claimed in claim 8, wherein said seed medium comprises defatted soy, glycerol and yeast extract. Dated this 23rd day of December 2004

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