CN106868070A - A kind of microbial fermentation produces the culture medium of Lipstatin - Google Patents
A kind of microbial fermentation produces the culture medium of Lipstatin Download PDFInfo
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- CN106868070A CN106868070A CN201710242727.4A CN201710242727A CN106868070A CN 106868070 A CN106868070 A CN 106868070A CN 201710242727 A CN201710242727 A CN 201710242727A CN 106868070 A CN106868070 A CN 106868070A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses the culture medium that a kind of microbial fermentation produces Lipstatin, the culture medium composition is as follows：The 50g/L of soybean cake powder 40, the 30g/L of agar 10, the 70g/L of vegetable oil 50, the 25g/L of soybean lecithin 15, the 20g/L of whole milk 10, amino acid/11 5g/L, the 7g/L of vitamin 2, the g/L of inorganic salts 38, solvent are water, pH7.0 7.3；The culture medium also enteromorpha extract including 20 40g/ L.Culture medium of the present invention increased enteromorpha extract compared with existing culture medium, containing abundant polysaccharide component and small molecule functional polypeptide in Enteromorpha, can improve the stability expressed in fermentation process, improve fermentation titer.
Description
Technical field
The present invention relates to a kind of microbial fermentation production Lipstatin culture medium and its method, belong to microbial fermentation neck
Domain.
Background technology
Lipstatin（Lipstatin）It is by Streptomyces toxytricini（Streptomyces toxytricini）It is fermented
The metabolite of generation, its four hydroperoxide derivative is orlistat（olistat）, it is Roche Holding Ag（Roche）What success was developed subtracts
Fertile medicine（Xenical）, orlistat is first gastrointestinal lipases inhibitor, it by with gastric lipase in stomach and small intestinal lumen
Forming covalent bond with the active ser position of pancreatic lipase inactivates enzyme, the decomposition and absorption of fat is reduced, so as to reach control
The effect of body weight processed.Orlistat is currently the only not enter blood, do not act on the slimming drugs of central nervous system.
Orlistat can by full chemistry synthetic method or first pass through fermentation synthetic method produce Lipstatin, after carry out hydrogen
It is combined to.Because complete synthesizing process production cost is higher, mainly produced by microbial fermentation synthetic method at present, the method cost compared with
It is low, it is environment-friendly, it is adapted to large-scale production.
In fermentation method production, mainly pass through Streptomyces toxytricini fermenting and producing.Therefore, by the improvement to the bacteria strain,
The fermentation titer for optimizing to improve Lipstatin of culture medium and technological condition for fermentation is all the target of research.
Enteromorpha is rich in carbohydrate, protein, crude fibre, amino acid, aliphatic acid, vitamin and several mineral materials, its
Middle iron content be recited as on Chinese food nutrient component meter China's food most, while also containing fat and vitamin.In waterside
In tongue protein, amino acid classes are complete, it is necessary to which amino acid content is higher, and wherein the limiting amino acid of edge pipe Enteromorpha is to rely ammonia
Acid, AAS is 79；The limiting amino acid of bar Enteromorpha is methionine, and AAS is 80.The aliphatic acid composition of Enteromorpha
In, the content of polyunsaturated fatty acid, monounsaturated fatty acids and saturated fatty acid is respectively 50.5%, 12.7% and 36.8%,
Including nearly 4% odd carbon fatty acid.Therefore Enteromorpha is high protein, high dietary-fiber, low fat, low energy, and rich
The raw material of the natural ideal nutritional food containing minerals and vitamins.
The content of the invention
A kind of culture of Streptomyces toxytricini fermenting and producing Lipstatin is provided in the technical problem to be solved in the present invention
Base.
The culture medium composition that the present invention is provided is as follows：Soybean cake powder 40-50g/L, agar 10-30g/L, vegetable oil 50-
70g/L, soybean lecithin 15-25g/L, whole milk 10-20g/L, amino acid/11-5g/L, vitamin 2-7g/L, inorganic salts
3-8 g/L, solvent is water, pH7.0-7.3.
Preferably, the difference with disclosed culture medium in the prior art is that culture medium of the invention also includes 20-40g/
The enteromorpha extract of L.
Applicants have found that, enteromorpha extract can promote cell growth, stimulate microbial reproduction, in enteromorpha extract
The multiple biological activities composition for containing, can be greatly facilitated growing for microorganism.
The preparation method of enteromorpha extract of the present invention is as follows：
1）The pretreatment of Enteromorpha：Fresh Enteromorpha is cleaned, drying, grinds to obtain enteromorpha powder.
2）Enteromorpha extract：Above-mentioned enteromorpha powder is taken, the water of 3-5 times of enteromorpha powder quality is added, 70-80 DEG C is heated to, so
Homogenizer homogeneous is used afterwards, after homogeneous is complete, 90-100 DEG C is heated to, and keeps 30-50min；30 DEG C are cooled to, complex enzyme 400- is added
500U/g；35-45 DEG C is warmed up to, 50-90min is kept,
Complex enzyme is selected from one or more in cellulase, lysozyme, protease.
3）Extract solution is filtered, and takes filtrate, adds glycerine, and the weight ratio of filtrate and glycerine is 10:2-3.
Preferably, in culture medium of the present invention, vegetable oil includes peanut oil, soybean oil, corn oil, sesame
One or more in oil, castor oil.Further, the present invention provides the combination of a vegetable oil, including peanut oil, sesame oil
And castor oil, three's weight ratio is 3:2:1.
Preferably, in culture medium of the present invention, amino acid includes following ingredients：Glycine, L- asparagines,
Pidolidone, L-Histidine, L-Leu, L-phenylalanine, L-Trp.The various amino acid part by weight are according to reality
Need rationally to determine ratio.For example, the weight of the every kind of amino acid for adding is identical.
Preferably, in culture medium of the present invention, vitamin includes following ingredients：Biotin, Choline Chloride,
Folic acid, niacinamide, vitamin B6, vitamin B12, i- inositols.The various vitamin part by weight are reasonable according to being actually needed
Determine ratio.For example, the weight of the every kind of vitamin for adding is identical.
Preferably, in culture medium of the present invention, described inorganic salts include following ingredients：Anhydrous magnesium sulfate,
Sodium acid carbonate, potassium chloride, sodium chloride.The various inorganic salts part by weight are according to being actually needed reasonable determination ratio.For example, plus
The weight of the every kind of inorganic salts for entering is identical.
Sterilizing methods when basal culture medium is used：Basal culture medium uses filtration sterilization method, uses nuclepore membrane filter.Make
With filter membrane is loaded, when solution injects filter from syringe, various microorganisms are delayed above miillpore filter, and liquid and small point
Sub- material passes through filter membrane, so as to reach degerming purpose.Its filter membrane is made up of nitrocellulose, cellulose acetate etc., uses
0.22 μm or 0.1 μm of aperture, every filter membrane are only used 1 time.
Beneficial effects of the present invention：
Culture medium of the present invention increased enteromorpha extract compared with existing culture medium, in Enteromorpha containing abundant polysaccharide component and
Small molecule functional polypeptide, can improve the stability expressed in fermentation process, fermentation titer be improved, using Streptomyces toxytricini
Production Lipstatin, may be such that the accumulation of product potency reaches 16.3g/L.
Specific embodiment
Embodiment 1
The culture medium composition of the fermenting and producing Lipstatin that the present embodiment is provided is as follows：Soybean cake powder 50g/L, agar 30g/L,
Vegetable oil 70g/L, soybean lecithin 25g/L, whole milk 20g/L, amino acid 5g/L, vitamin 7g/L, the g/L of inorganic salts 8,
Enteromorpha extract 40g/ L solvents are water, pH7.2.
The preparation method of described enteromorpha extract is as follows：
1）The pretreatment of Enteromorpha：Fresh Enteromorpha is cleaned, drying, grinds to obtain enteromorpha powder.
2）Enteromorpha extract：Take above-mentioned enteromorpha powder, add the water of 5 times of enteromorpha powder quality, be heated to 80 DEG C, then with equal
Matter machine homogeneous, after homogeneous is complete, is heated to 100 DEG C, keeps 50min；30 DEG C are cooled to, complex enzyme 500U/g is added；It is warmed up to 45
DEG C, keep 90min.
Complex enzyme is selected from cellulase, protease, and both enzyme activity unit ratios are 3:2.
3）Extract solution is filtered, and takes filtrate, adds glycerine, and the weight ratio of filtrate and glycerine is 10: 3.
Vegetable oil includes peanut oil, sesame oil and castor oil, and three's weight ratio is 3:2:1.
Amino acid includes following ingredients：Glycine, L- asparagines, Pidolidone, L-Histidine, L-Leu,
L-phenylalanine, L-Trp.The various amino acid part by weight are identical.
Vitamin includes following ingredients：Biotin, Choline Chloride, folic acid, niacinamide, vitamin B6, vitamin
B12, i- inositol.The various vitamin part by weight are identical.
Described in inorganic salts include following ingredients：Anhydrous magnesium sulfate, sodium acid carbonate, potassium chloride, sodium chloride.It is described each
Plant inorganic salts part by weight identical.
Embodiment 2
The culture medium composition of the fermenting and producing Lipstatin that the present embodiment is provided is as follows：Soybean cake powder 40g/L, agar 10g/L,
Vegetable oil 50g/L, soybean lecithin 15g/L, whole milk 10g/L, amino acid/11 g/L, vitamin 2g/L, the g/L of inorganic salts 3,
Enteromorpha extract 20g/ L, solvent is water, pH7.0.
The preparation method of described enteromorpha extract is as follows：
1）The pretreatment of Enteromorpha：Fresh Enteromorpha is cleaned, drying, grinds to obtain enteromorpha powder.
2）Enteromorpha extract：Take above-mentioned enteromorpha powder, add the water of 3 times of enteromorpha powder quality, be heated to 70 DEG C, then with equal
Matter machine homogeneous, after homogeneous is complete, is heated to 90 DEG C, keeps 30min；30 DEG C are cooled to, complex enzyme 400U/g is added；It is warmed up to 35
DEG C, 50min is kept,
Complex enzyme is selected from cellulase, lysozyme, protease, and the enzyme activity unit ratio of three is 3：2：3.
3）Extract solution is filtered, and takes filtrate, adds glycerine, and the weight ratio of filtrate and glycerine is 10:2.
Vegetable oil includes peanut oil, sesame oil and castor oil, and three's weight ratio is 3:2:1.
Amino acid includes following ingredients：Glycine, L- asparagines, Pidolidone, L-Histidine, L-Leu,
L-phenylalanine, L-Trp.The various amino acid part by weight are identical.
Vitamin includes following ingredients：Biotin, Choline Chloride, folic acid, niacinamide, vitamin B6, vitamin
B12, i- inositol.The various vitamin part by weight are identical.
Described in inorganic salts include following ingredients：Anhydrous magnesium sulfate, sodium acid carbonate, potassium chloride, sodium chloride.It is described each
Plant inorganic salts part by weight identical.
Embodiment 3
The ferment effect of culture medium of the invention is determined.
According to fermentation Streptomyces toxytricini, the fermentation side of Lipstatin disclosed in Chinese patent 201310434885.1
Method and titer detection method evaluate the effect of culture medium of the invention.
Culture medium disclosed in embodiment 1 is applied in culture amplification test.The streptomyces that will have been activated
The fresh slant pores of toxytricini YKY-20, through seed flask culture 24h, 28 DEG C of condition of culture, shaking speed
240rpm.Seed liquor is accessed according to 5% inoculum concentration is equipped with the 5L fermentation tanks of 2L fermentation mediums.Fermentation condition is：It is empty
Throughput 1:1（vvm）, speed of agitator 500rpm, 28 DEG C of fermentation temperature.With the increase of fermentation time, Lipstatin is continuous
Accumulation, ferments during to 110h, and the potency accumulation of Lipstatin reaches 16.3g/L.
Claims (7)
1. a kind of microbial fermentation produces the culture medium of Lipstatin, it is characterised in that the culture medium composition is as follows：Soya bean
Cake powder 40-50g/L, agar 10-30g/L, vegetable oil 50-70g/L, soybean lecithin 15-25g/L, whole milk 10-20g/
L, amino acid/11-5g/L, vitamin 2-7g/L, inorganic salts 3-8 g/L, solvent is water, pH7.0-7.3.
2. culture medium according to claim 1, it is characterised in that the culture medium also Enteromorpha including 20-40g/ L is carried
Take liquid.
3. culture medium according to claim 1, it is characterised in that vegetable oil includes peanut oil, soybean oil, corn oil, sesame
One or more in sesame oil, castor oil.
4. culture medium according to claim 1, it is characterised in that vegetable oil includes peanut oil, sesame oil and castor oil.
5. culture medium according to claim 1, it is characterised in that described amino acid includes following ingredients：
Glycine, L- asparagines, Pidolidone, L-Histidine, L-Leu, L-phenylalanine, L-Trp.
6. culture medium according to claim 1, it is characterised in that described vitamin includes following ingredients：
Biotin, Choline Chloride, folic acid, niacinamide, vitamin B6, vitamin B12, i- inositols.
7. culture medium according to claim 1, it is characterised in that described inorganic salts include following ingredients：
Anhydrous magnesium sulfate, sodium acid carbonate, potassium chloride, sodium chloride.
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| CN201710242727.4A CN106868070A (en) | 2017-04-14 | 2017-04-14 | A kind of microbial fermentation produces the culture medium of Lipstatin |
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| CN201710242727.4A CN106868070A (en) | 2017-04-14 | 2017-04-14 | A kind of microbial fermentation produces the culture medium of Lipstatin |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007134836A1 (en) * | 2006-05-22 | 2007-11-29 | Krka | Fermentative production of lipstatin |
| CN103820510A (en) * | 2014-01-17 | 2014-05-28 | 浙江工业大学 | Method for producing lipstatin through microbial fermentation and culture medium |
| CN104341535A (en) * | 2013-08-07 | 2015-02-11 | 青岛博研达工业技术研究所(普通合伙) | Enteromorpha prolifera high-valued extraction method |
-
2017
- 2017-04-14 CN CN201710242727.4A patent/CN106868070A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007134836A1 (en) * | 2006-05-22 | 2007-11-29 | Krka | Fermentative production of lipstatin |
| CN104341535A (en) * | 2013-08-07 | 2015-02-11 | 青岛博研达工业技术研究所(普通合伙) | Enteromorpha prolifera high-valued extraction method |
| CN103820510A (en) * | 2014-01-17 | 2014-05-28 | 浙江工业大学 | Method for producing lipstatin through microbial fermentation and culture medium |
Non-Patent Citations (3)
| Title |
|---|
| PUNIT KUMAR,ET AL.: "Current trends and future prospects of lipstatin:a lipase inhibitor and pro-drug for obesity", 《RSC ADVANCES》 * |
| 禹邦超等: "《酶工程 第3版》", 31 January 2014, 华中师范大学出版社 * |
| 蒋新龙: "《发酵工程》", 28 February 2011, 浙江大学出版社 * |
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