WO2004003212A1 - Novel process for the production of pancreatic lipase inhibitor - Google Patents

Novel process for the production of pancreatic lipase inhibitor Download PDF

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Publication number
WO2004003212A1
WO2004003212A1 PCT/IN2002/000139 IN0200139W WO2004003212A1 WO 2004003212 A1 WO2004003212 A1 WO 2004003212A1 IN 0200139 W IN0200139 W IN 0200139W WO 2004003212 A1 WO2004003212 A1 WO 2004003212A1
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Prior art keywords
fermentation
solid substrate
feeding
lipstatin
fed
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PCT/IN2002/000139
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French (fr)
Inventor
Ramavana Gururaja
Ramakrishnan Melarkode
Shrikumar Suryanarayan
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Biocon Limited
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Priority to PCT/IN2002/000139 priority Critical patent/WO2004003212A1/en
Priority to AU2002321819A priority patent/AU2002321819A1/en
Publication of WO2004003212A1 publication Critical patent/WO2004003212A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
    • C07D305/02—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D305/10—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms not condensed with other rings having one or more double bonds between ring members or between ring members and non-ring members
    • C07D305/12—Beta-lactones

Definitions

  • the current invention relates to a novel process for the manufacture of lipstatin by solid state fermentation of Streptomyces toxyt ⁇ cini optionally using fed-batch production technique in a self contained bioreactor.
  • the compound Lipstatin a pancreatic lipase inhibitor is produced by fermentation of Streptomyces toxyt ⁇ cini. 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 toxyt ⁇ cini, which were supplied with soybean oil and a crude mixtLire of U- 13 C-lipids obtained from algal biomass cultured with 13 C ⁇ 2 (Eisenreich, Wolfgang, et.al. J . Biol.
  • KR 9709294 discloses novel active substance lipstatin, which is produced by a Streptomyces sp. (KCTC-9303) isolated from soil.
  • I fermentative production of lipstatin which comprises (a) aerobically cultivating a micro-organism of the order of actinomycetes which produces lipstatin, in an aqueous c ulture 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 caprylic acid, their ester(s) or salt(s), stabilized by an antioxidant.
  • Lipstatin can be isolated from the broth and hydrogenated to tetrahydrolip statin.
  • EP O 129 748 discloses a process for the production of Lipstatin by fermentation of Streptomyces toxyt ⁇ cini, in a liquid nutrient media comprising potato starch, glucose, soybean meal, (NH4) 2 S0 4 , ribose, glycerol, and peptone 0.2% and incubated at 28°C for 124 h with stirring and aeration, followed by extraction of the product.
  • 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 toxyt ⁇ cini optionally using fed-batch production techniqLie in a contained bioreactor.
  • the present invention provides a process for the manufacture of lipstatin by solid substrate fermentation a comprising the steps of: preparing an inoc lum 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 substrate matrix to obtain lipstatin.
  • the micro-organism is Streptomyces toxyt ⁇ cini.
  • 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 inoculum is prepared by inoculating a seed medium with slant of the micro-organism of the genus Streptomyces.
  • the seed medium comprises defatted soy, glycerol and yeast.
  • Solid state fermentation or solid state cultivation:
  • solid state fermentation or “solid state CLiltivation”, sometimes referred to as “semi-solid state fermentation” as used herein, 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 be any amount of water.
  • the solid medium could be almost dry, or it could be slushy.
  • solid state fermentation and “semi-solid state fermentation” are interchangeable.
  • Feed-batch fermentation or “fed-batch technique”: The term fed-batch fermentation as used herein, 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 0 help in providing more energy to the microorganism which in turn increases the overall production of the secondar ⁇ ' metabolites.
  • Bioreactor means a device capable of holding fermentation 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, yeast 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.
  • a well grown slant of Streptomyces toxyt ⁇ cini was taken ( i and 5ml of distilled water w as 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:
  • 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 inoc ilum 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. Adequate amount of water was added and sterilized at 121 deg C for 30 minutes.
  • the inoculum from seed medium as obtained from Example 1 was used to prepare another production medium, lml 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.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention provides a novel method for producing lipstatin, by solid substrate fermentation by culturing microorganisms.

Description

TITLE OF THE INVENTION NOVEL PROCESS FOR THE PRODUCTION OF PANCREATIC
LIPASE INHIBITOR FIELD OF THE INVENTION
- The current invention relates to a novel process for the manufacture of lipstatin by solid state fermentation of Streptomyces toxytήcini optionally using fed-batch production technique in a self contained bioreactor. BACKGROUND OF THE INVENTION
HI The compound Lipstatin, a pancreatic lipase inhibitor is produced by fermentation of Streptomyces toxytήcini. 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 toxytήcini, which were supplied with soybean oil and a crude mixtLire of U- 13C-lipids obtained from algal biomass cultured with 13Cθ2 (Eisenreich, Wolfgang, et.al. J . Biol.
2n 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 parent discloses a process for the
I fermentative production of lipstatin, which comprises (a) aerobically cultivating a micro-organism of the order of actinomycetes which produces lipstatin, in an aqueous c ulture 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 caprylic acid, their ester(s) or salt(s), stabilized by an antioxidant. Lipstatin can be isolated from the broth and hydrogenated to tetrahydrolip statin.
EP O 129 748, discloses a process for the production of Lipstatin by fermentation of Streptomyces toxytήcini, 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 by 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 toxytήcini optionally using fed-batch production techniqLie 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 inoc lum 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 substrate matrix to obtain lipstatin.
The micro-organism is Streptomyces toxytήcini.
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 inoculum is prepared by inoculating 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" :
The term "solid state fermentation" or "solid state CLiltivation", sometimes referred to as "semi-solid state fermentation" as used herein, 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 be any amount of water. 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 fermentation as used herein, 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 0 help in providing more energy to the microorganism which in turn increases the overall production of the secondar}' metabolites.
"Bioreactor" : The term "bioreactor" as used herein, means a device capable of holding fermentation 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, yeast 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 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 toxytήcini was taken ( i and 5ml of distilled water w as 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 = 1 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 inoc ilum 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. 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 Igm 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:
Figure imgf000007_0001
EXAMPLE 2
The inoculum from seed medium as obtained from Example 1 , was used to prepare another production medium, lml 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}r 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:
Figure imgf000008_0001
Soy grits 143
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 Streptoinyces toxytncini inoculum grown in production medium was added. The results obtained are given in the table below. Solid substrate Lipstatin mg/kg substrate
Ceramic beads 126
EXAMPLE 4
Solid state fermentation was conducted as in Example 2, using different solid supports in combination. Streptomyces toxytήcini inoculum grown in production medium was added. The results obtained are given in the table below.
1 Solid substrates Lipstatin
| mg/kg substrate
| Maize bran + wheat rawa 1020
1
Maize bran + oat meal 1081
Wheat rawa + oat meal 1 122
Maize bran + wheat rawa + oat 1256 meal
EXAMPLE 5
Solid state fermentation was conducted as in Example 4 w ith feeding of oil : lecithin mixture carried out initially with 0.5 gm/ plate and on the 3l d da\ . mixed and incubated for another days. Following results are obtained:
Solid substrates Lipstatin , mg/kg substrate ι
Maize bran + wheat rawa 1240
Maize bran + oat meal 1218 ! !
Figure imgf000010_0001
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 toxytήcini 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 1 7.25 g of lipstatin was present at the end of fermentation.

Claims

We claim:
1. 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 substrate matrix to obtain lipstatin.
2. A process as claimed in claim 1 , wherein the said microorganism is Streptomyces toxytήcini.
3. 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.
4. A process as claimed in claim 1 , wherein the solid substrate fermentation is a fed-batch fermentation.
5. A process as claimed in claim 4, wherein the fed-batch fermentation is carried out by oil feeding.
6. A process as claimed in claim 5, wherein the oil used for feeding is selected from soyabean, safflower, groundnut, rapeseed, cottonseed.
7. A process as claimed in claim 5, wherein the oil used for feeding also contains lecithin .
8. A process as claimed in claim 4-7, wherein the feeding for fed-batch fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation.
9. A process as claimed in claim 1 , wherein inoculum is prepared bw inoculating a seed medium with slant of the micro-' organism of the genus Streptomyces.
1 0. A process as claimed in claim 9, wherein said seed medium comprises defatted soy, glycerol and yeast.
PCT/IN2002/000139 2002-06-26 2002-06-26 Novel process for the production of pancreatic lipase inhibitor WO2004003212A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009118743A1 (en) * 2008-03-26 2009-10-01 Biocon Limited Improved fermentation process for higher yield coefficient of lipase-inhibitor with respect to consumed fatty acid
EP2141236A1 (en) 2008-07-03 2010-01-06 KRKA, D.D., Novo Mesto Process for production of lipstatin and microorganisms therefore
CN108753861A (en) * 2018-06-08 2018-11-06 福建省微生物研究所 A kind of culture medium and method of Streptomyces toxytricini fermentation high yield Lipstatin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EISENREICH W. ET AL.: "Tracer studies with Crude U-13 C-lipid mixtures biosynthesis of the lipase inhibitor lipstatin", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 2, 10 January 1997 (1997-01-10), pages 867 - 874 *
LEUENBERGER H.G. ET AL.: "Synthesis and metabolism of drugs by means of enzyme-catalysed reactions", CHIMIA, vol. 53, 1999, pages 536 - 540 *
WEIBEL E.K. ET AL.: "Lipstatin, an inhibitor of pancreatic lipase produced by streptomyces toxytricini part I", THE JOURNAL OF ANTIBIOTICS, vol. XL, no. 8, pages 1081 - 1085 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009118743A1 (en) * 2008-03-26 2009-10-01 Biocon Limited Improved fermentation process for higher yield coefficient of lipase-inhibitor with respect to consumed fatty acid
JP2011515100A (en) * 2008-03-26 2011-05-19 バイオコン リミテッド Improved fermentation process to obtain high yield factor lipase inhibitors for consumed fatty acids
US8501444B2 (en) 2008-03-26 2013-08-06 Biocon Limited Fermentation process for higher yield coefficient of lipase-inhibitor with respect to consumed fatty acid
EP2141236A1 (en) 2008-07-03 2010-01-06 KRKA, D.D., Novo Mesto Process for production of lipstatin and microorganisms therefore
CN108753861A (en) * 2018-06-08 2018-11-06 福建省微生物研究所 A kind of culture medium and method of Streptomyces toxytricini fermentation high yield Lipstatin
CN108753861B (en) * 2018-06-08 2022-02-01 福建省微生物研究所 Culture medium and method for producing lipstatin by fermenting streptomyces toxytricini

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