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CN1607245A - High efficiency fermentation method for pristinamycin - Google Patents

High efficiency fermentation method for pristinamycin Download PDF

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Publication number
CN1607245A
CN1607245A CN 200310100179 CN200310100179A CN1607245A CN 1607245 A CN1607245 A CN 1607245A CN 200310100179 CN200310100179 CN 200310100179 CN 200310100179 A CN200310100179 A CN 200310100179A CN 1607245 A CN1607245 A CN 1607245A
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China
Prior art keywords
carbon source
organic nitrogen
stapyocine
nitrogen source
seed
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CN 200310100179
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CN1311069C (en
Inventor
金志华
李春波
岑沛霖
张国钧
金一平
陶正利
林建平
徐志南
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Zhejiang Pharmaceutical Co Ltd
Zhejiang University ZJU
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Zhejiang Pharmaceutical Co Ltd
Zhejiang University ZJU
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Abstract

本发明涉及始旋链霉菌CGMCC 0957，用其生产原始霉素。本发明还涉及生产原始霉素的方法，即在培养基中以适宜的条件培养始旋链霉菌CGMCC 0957，高效生产原始霉素的方法。The present invention relates to Streptomyces initiating spinus CGMCC 0957, which is used to produce protomycin. The present invention also relates to a method for producing protomycin, that is, a method for cultivating Streptomyces spirulina CGMCC 0957 in a culture medium under suitable conditions to efficiently produce protomycin.

Description

A kind of efficient fermentation process of Stapyocine
Technical field:
The present invention relates to revolve streptomycete CGMCC 0957 fermentation, the method for High-efficient Production Stapyocine with the beginning.
Background technology:
Stapyocine (pristinamycin) belongs to streptogramin class microbiotic, and Stapyocine I by about 30% and about 70% Stapyocine II form.Stapyocine I is a cyclic hexadepsipeptides, by Stapyocine I A, Stapyocine I BWith Stapyocine I CForm.Stapyocine I AMolecular weight be 866, molecular formula is C 45H 54O 10N 8Stapyocine I BMolecular weight be 852, molecular formula is C 44H 52O 10N 8Stapyocine I CMolecular weight be 852, molecular formula is C 44H 52O 10N 8Stapyocine II is a polyunsaturated macrolactone, by Stapyocine II AWith Stapyocine II BForm.Stapyocine II AMolecular weight 525, molecular formula is C 28H 35O 7N 3Stapyocine II BMolecular weight 527, molecular formula is C 28H 37O 7N 3Stapyocine I AWith Stapyocine II AIt is the main ingredient of Stapyocine.
Stapyocine mainly has better antibacterial activity to gram-positive microorganism especially golden Portugal bacterium and suis, and its mechanism of action suppresses the synthetic of bacterioprotein for acting on bacterial ribosome 50S subunit.Has synergy between Stapyocine I component and the II component.
In the prior art, traditional zymotic and the extracting method of producing Stapyocine have been described among U.S. Pat 3154475 (1964) and the English Patent GB998195 (nineteen sixty-five).Bacterial classification used in these two patents revolves streptomycete for the beginning, and particularly bacterial strain 5647, or NRRL 2958, and used fermention medium is for cultivating the nutritional medium of streptomycete, and the substratum that particularly contains analysis for soybean powder and glucose can produce result preferably.The seed culture medium and the fermention medium of Stapyocine fermentative production are provided in the example of these two patents: used seed culture medium contains enzymic hydrolysis casein 1200 grams, glucose 1200 grams, malt extract 360 grams, 100 liters in tap water, 60 milliliters in soya-bean oil in 170 liters of seeding tanks; Used fermention medium contains 17.75 kilograms of analysis for soybean powder, 2.25 kilograms of distiller ' s solubles, 2.25 kilograms in sodium-chlor, 1800 milliliters in soya-bean oil, 2.25 kilograms in lime carbonate, 360 liters in tap water in 800 liters of fermentor tanks.With this substratum fermentative production Stapyocine, the fermentation titer of Stapyocine is 1190u/ml.Because the fermentation titer of Stapyocine is lower, so production cost is higher.
Summary of the invention:
In order to overcome the existing shortcoming that the Stapyocine fermentation titer is low, production cost is high, the invention provides a kind of Streptomyces pristinaespiralis microorganism.
The invention provides a kind of efficient fermentation process of producing Stapyocine.
Fermentation process of the present invention can be brought up to 3000-5000u/ml with the fermentation titer of Stapyocine.
The microorganism of using
The streptomyces that belongs to Streptomyces pristinaespiralis begins to revolve streptomyces strain, is actinomycetes.
Streptomyces pristinaespiralis is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 25th, 2003, and it abbreviates CGMCC as, and deposit number is 0957.
The microorganism of using have following character:
The bacteria characteristic of bacterial classification
(a) morphological specificity: fibrillae of spores ripple song or loose spiral end shape, 1-3 circle.The spore ovum
Circle, surface band wart.At inorganic salt Starch Agar, glycerine asparagine fine jade
Aerial hyphae grey on fat and the yeast wheat toothpaste agar, the substrate mycelium reverse side
Colourless to sallow or pale yellow brown, but lysochrome does not have or a small amount of yellow, does not produce
Give birth to melanochrome.
(b) utilization of carbon source
Can utilize: wood sugar, pectinose, rhamnosyl, trehalose, glucose,
Semi-lactosi, fructose, seminose, lactose, maltose, sucrose, cellobiose,
Raffinose, dextrin, starch, glycerine, N.F,USP MANNITOL, inose.
Do not utilize or grow suspicious: sorbose, ethylene glycol, erythritol, oneself
Six alcohol, sorbyl alcohol, ethanol, sodium acetate, sodium oleate, sodium formiate, succsinic acid,
Calglucon.
(c) nitrogenous source utilization
Can utilize: SODIUMNITRATE, Sodium Nitrite, sulfate of ammoniac, adenosine, urea,
Arginine, glycine, L-Ala, Xie Ansuan, L-glutamic acid, N,
Serine, Threonine, phenylalanine, tyrosine, proline(Pro), oxyproline,
Histidine, peptone, yeast extract, soybean cake powder, groundnut meal, cotton
Sub-cake powder, fish meal.
Do not utilize or grow suspicious: ethanamide, succinate, benzamide,
Sarkosine, Creatinine, trimethyl-glycine.
Any bacterial classification that can produce Stapyocine all is applicable to the present invention.Preferred bacterial classification revolves streptomycete (Streptomyces pristinaespiralis) for the beginning, this bacterial classification all has preservation in USS type culture collection institute (ATCC) and USDA north development and use research departments (NRRL), and numbering is respectively ATCC 25486 and NRRL 2958.Preferred bacterial classification is Streptomyces pristinaespiralis CGMCC 0957.
The invention still further relates to the method for carrying out the fermentative production Stapyocine with the bacterial classification that produces Stapyocine, specifically comprise the preparation and the fermentative production of seed.In order to improve Stapyocine output, need to adopt improved seed culture medium and fermention medium.Substratum in above-mentioned two patents of improved substratum and prior art has very big difference, uses substratum of the present invention can improve the output of Stapyocine significantly.
The per-cent of all substratum is the quality of medium component and the ratio of the cumulative volume of seed culture medium among the application, and unit is grams per milliliter or kilogram/liter (being g/ml or kg/l).
Improved seed culture medium contains:
A) carbon source utilized of 2-5%, the suitable carbon source utilized comprise starch, dextrin, wheat
Bud sugar, sucrose, lactose, glucose, seminose and semi-lactosi etc.
B) organic nitrogen source of 1-4%, suitable organic nitrogen source comprises soybean cake powder, peanut cake
Powder, cotton seed powder cake, peptone, yeast powder, fish meal etc.
Improved fermention medium contains:
A) carbon source utilized of 4-8%, the suitable carbon source utilized comprise slowly utilizes carbon source
Utilize carbon source to add fast with 2: 1 to 6: 1 ratio.Slowly utilize
Carbon source comprises starch, dextrin, maltose, sucrose, lactose, raffinose, fibre
Dimension disaccharides etc.; The preferred carbon source of slowly utilizing comprises starch.Utilize fast
Carbon source comprises glucose, fructose, semi-lactosi, seminose, glycerine etc.; Preferably
The carbon source of utilizing fast be glucose.
B) 2-5% (W/V, the quality of medium component and the cumulative volume of fermention medium it
Than) organic nitrogen source, suitable organic nitrogen source comprises and slowly utilizes organic nitrogen source
Utilize organic nitrogen source to add fast with 3: 1 to 8: 1 ratio.Suitable
Slowly utilize organic nitrogen source comprise soybean cake powder, groundnut meal, cotton seed powder cake and
Imperial or royal seal powder etc.; The suitable organic nitrogen source that utilizes fast comprises peptone, yeast powder etc.
C) 0.1-1% (W/V, the quality of medium component and the cumulative volume of fermention medium
Ratio) inorganic nitrogen-sourced, suitable inorganic nitrogen-sourced be KNO 3, NaNO 3,
(NH 4) 2SO 4, NH 4Cl, NH 4NO 3Deng, preferred inorganic nitrogen-sourced is (NH 4) 2SO 4
The substratum that use has above-mentioned feature is the main aspect that realizes the present invention's high level fermentative production Stapyocine.
The spore culture that produces the bacterial classification (revolving streptomycete as the beginning) of Stapyocine is inoculated in the seed culture medium of the present invention prepares seed, seed is inoculated into ferments in the fermention medium again to produce Stapyocine.
The preparation of spore culture:
The bacterial classification (revolving streptomycete as the beginning) that produces Stapyocine is inoculated into to produce on the spore substratum and cultivates, produce the spore substratum and can adopt No. 1 substratum of Gao Shi, ISP2 substratum, more preferably No. 1 substratum of Gao Shi.Suitable culture temperature is 20-40 ℃, and preferred culture temperature is 25-37 ℃, and preferred culture temperature is 30 ℃.Incubation time is 2-15 days, is preferably 5-10 days, is more preferably 6 days.
The preparation of seed:
Suitable seed culture medium contains: the 1) carbon source utilized of 2-5%, the suitable carbon source utilized comprise starch, dextrin, maltose, sucrose, lactose, glucose, seminose and semi-lactosi etc.2) organic nitrogen source of 1-4%, suitable organic nitrogen source comprises soybean cake powder, groundnut meal, cotton seed powder cake, peptone, yeast powder, fish meal etc.
The preparation of seed culture medium: seed culture medium can prepare by conventional art (as at 120-140 ℃ of difference or sterilize simultaneously carbon source and nitrogenous source).
Inoculation: the spore culture made be inoculated into behind the spore suspension in the seed culture medium or cultivate in the seed culture medium and prepare seed with digging piece method direct inoculation.
Cultivate: seed culture adopts deep-layer liquid sinking culture method, carries out in shaking bottle or seeding tank according to scale.(shaking bottled liquid measure by control controls by bottleneck media filtration air when shake-flask culture, shake bottled liquid measure and be and shake the long-pending 10-20% of bottle), and the oxygen in the sterile air is fully contacted with bacterial classification with substratum by shaking table vibration (shaking speed is 150-250 rev/min); Provide oxygen (air flow is every liter of fermented liquid 0.5-1.5 of per minute litres of air, and stirring velocity is 100-600 rev/min) by feeding aseptic compressed air and stirring when in seeding tank, cultivating.Culture temperature suitable during seed culture is 20-40 ℃, is preferably 25-37 ℃, and suitable incubation time is 24-72 hour, is preferably 36-60 hour.Seed culture requires the mycelial concentration in the seed liquor to reach 10-30% when finishing, be preferably 15-25%, and more preferably 20%.
The Stapyocine fermentation:
Suitable fermention medium contains: the 1) carbon source utilized of 4-8%, the suitable carbon source utilized comprise a certain proportion ofly slowly to be utilized carbon source and utilizes carbon source fast.Slowly the carbon source of utilizing comprises starch, dextrin, maltose, sucrose, lactose, raffinose, cellobiose etc., and the carbon source of utilizing comprises glucose, fructose, semi-lactosi, seminose, glycerine etc. fast, preferably utilizes carbon source to be glucose fast.2) organic nitrogen source of 2-5%, suitable organic nitrogen source comprise and a certain proportion ofly slowly utilize organic nitrogen source and utilize organic nitrogen source fast.The suitable organic nitrogen source that slowly utilizes comprises soybean cake powder, groundnut meal, cotton seed powder cake and fish meal etc.; The suitable organic nitrogen source that utilizes fast comprises peptone, yeast powder etc.3) 0.1-1%'s is inorganic nitrogen-sourced, suitable inorganic nitrogen-sourced be KNO 3, NaNO 3, (NH 4) 2SO 4, NH 4Cl, NH 4NO 3Deng, preferred inorganic nitrogen-sourced is (NH 4) 2SO 4
Prepare fermention medium (as at 120-140 ℃ of difference or sterilize simultaneously carbon source and nitrogenous source) by conventional art.Seed is inoculated into the inoculum size of 1-20% (the seed liquor volume of access and the ratio of fermention medium volume) carries out fermentation culture in the fermention medium.Deep-layer liquid sinking culture method is also adopted in fermentation, carries out in shaking bottle or fermentor tank according to scale.(shaking bottled liquid measure by control controls by bottleneck media filtration air when shake flask fermentation, shake bottled liquid measure and be and shake the long-pending 10-20% of bottle), and the oxygen in the sterile air is fully contacted with bacterial classification with substratum by shaking table vibration (shaking speed is 150-250 rev/min); When fermentation cylinder for fermentation, provide oxygen by feeding aseptic compressed air (air flow is every liter of fermented liquid 0.5-1.5 of per minute litres of air) and stirring (mixing speed is 100-600 rev/min).Culture temperature suitable during fermentation culture is 25-37 ℃, is preferably 28-32 ℃, and suitable incubation time is 24-72 hour, is preferably 36-60 hour.
In the preferred embodiment of the invention, suitable breeding condition happiness oxygen condition, the aeration speed of substratum be the substratum, per minute volume of air of per unit volume greater than 0.5 (vvm), aeration speed generally all adopts 0.5-1.5 (vvm).
In the preferred embodiment of the invention, in seeding tank and fermentor tank,, can add defoamer such as soya-bean oil, rape oil, peanut oil, bubble enemy (polyoxyethylene oxypropylene glycerine) etc. for the generation of control foam, preferred defoamer is the bubble enemy.
In embodiments of the invention, by seed culture medium and the fermention medium that use has above-mentioned feature, can improve the output of Stapyocine significantly.
By an improved environment being provided for the bacterial classification that produces Stapyocine, can make the fermentation titer of Stapyocine bring up to 3000-5000u/ml.Have been found that by using improved seed culture medium and fermention medium, can improve Stapyocine output significantly, shorten the production time, reduce cost.
Embodiment:
Experimental example 1
The preparation of spore culture:
Spore substratum: adopt No. 1 substratum of Gao Shi (Zulkovsky starch 20 grams, KNO 31 gram, K 2HPO 40.5 gram, NaCl0.5 gram, MgSO 40.5 gram, FeSO 40.01 gram, agar 20 grams, distilled water adds to 1 liter, transfers pH7.0-7.2 with sodium hydroxide before the medium sterilization).Above-mentioned medium component is mixed the back branch install in the small test tube, 121 ℃ of sterilizations were paved into the inclined-plane with the substratum in the test tube after 30 minutes.
Beginning being revolved streptomycete be inoculated into to produce on the spore substratum and cultivate, is to cultivate 6 days under 30 ℃ the condition in temperature.
The preparation of seed:
Seed culture medium: glucose 30 grams, soybean cake powder 10 grams, peptone 5 grams, yeast powder 5 grams, KNO 33 grams, NaCl2 restrains %, CaCO 34 grams, tap water adds to 1 liter, transfers pH7.0 with sodium hydroxide before the sterilization.
The preparation of seed culture medium: after seed culture medium mixes, divide to install in 500 ml shake flasks (every is shaken bottled 100 milliliters of seed culture mediums), 121 ℃ of sterilizations 30 minutes.
Inoculation: the spore culture is inoculated into cultivation preparation seed in 100 milliliters of seed culture mediums that prepare with digging piece method (digging piece size is 1 * 1 square centimeter).
Cultivate: seed culture adopts deep-layer liquid sinking culture method, carries out in 500 ml shake flasks (adorning 100 milliliters of seed culture mediums).When shake-flask culture, pass through bottleneck medium (10 layers of gauze) filtrated air, and the oxygen in the sterile air is fully contacted with substratum with bacterial classification by shaking table vibration (shaking speed is 220 rev/mins).Keeping the seed culture temperature is 30 ℃, cultivates 36 hours, finishes to cultivate when the mycelial concentration of streptomycete in the seed liquor reaches 20%.
The Stapyocine fermentation:
The preparation of fermention medium: starch 30 grams, glucose 20 grams, soybean cake powder 25 grams, peptone 5 grams, (NH 4) 2SO 43 grams, MgSO 41 gram, KH 2PO 41 gram, CaCO 34 grams, tap water adds to 1 liter, transfers pH7.0-7.2 with sodium hydroxide before the sterilization.After fermention medium mixes, divide to install in 500 ml shake flasks (every is shaken bottled 50 milliliters of seed culture mediums), 121 ℃ of sterilizations 30 minutes.
3 milliliters of seeds are inoculated in 50 milliliters of fermention mediums, in 500 ml shake flasks (adorning 50 milliliters of seed culture mediums), carry out fermentation culture with deep-layer liquid sinking culture method.When shake-flask culture, pass through bottleneck medium (10 layers of gauze) filtrated air, and the oxygen in the sterile air is fully contacted with substratum with bacterial classification by shaking table vibration (shaking speed is 220 rev/mins).Culture temperature during fermentation culture is 30 ℃, and incubation time is 48 hours.
Detecting Stapyocine in the fermented liquid with bioassay method during fermentation ends tires and is 4000u/ml.
Experimental example 2
The preparation of seed in 110 liters of seeding tanks:
The 60 liters of seed culture mediums of packing in 110 liters of seeding tanks, medium component is identical with shake-flask seed substratum in the experimental example 1, but will add 26 milliliters of bubble enemies.Real jar sterilization was adopted in the sterilization of substratum, 121 ℃ of sterilizations 30 minutes.
100 ml shake flask seeds are inoculated into cultivation preparation seed in 110 liters of seeding tanks that contain 60 liters of seed culture mediums with flame method.In seeding tank, provide oxygen (air flow is every liter of seed liquor of per minute 0.8 litres of air, and mixing speed is 200 rev/mins) by feeding aseptic compressed air and stirring.Keeping the seed culture temperature is 30 ℃, cultivates 36 hours.When reaching 20%, finishes the mycelial concentration that revolves streptomycete when the beginning in the seed liquor to cultivate.
Stapyocine fermentation in 1000 liters of fermentor tanks:
The 700 liters of fermention mediums of packing in 1000 liters of fermentor tanks, medium component is identical with shake flask fermentation substratum in the experimental example 1, but need add 300 milliliters of bubble enemies.Real jar sterilization is adopted in the sterilization of substratum, and sterilization is 30 minutes under 121 ℃ condition.
60 liters of seeds are inoculated in 1000 liters of fermentor tanks that contain 700 liters of fermention mediums carry out fermentation culture.In fermentor tank, provide oxygen (stirring velocity is 250 rev/mins) by feeding aseptic compressed air (air flow is every liter of fermented liquid of per minute 1.2 litres of air) and stirring.Culture temperature during fermentation culture is 30 ℃, and incubation time is 48 hours.
Detecting Stapyocine in the fermented liquid with bioassay method during fermentation ends tires and is 3500u/ml.
Experimental example 3
700 liters of fermented liquids (3500u/ml) that experimental example 2 is obtained transfer to pH3 with 6N hydrochloric acid, filter, and wash with 100 premium on currency top again.Filtrate is transferred pH7 with dilute solution of sodium hydroxide, uses dichloromethane extraction then.Organic phase is evaporated to 4.8 liters.Add 5 liters of sherwood oils in the concentrated solution, obtain precipitation, obtain crude product 236 grams after the washing drying, potency of crude is 7560u/mg.
236 gram crude products (7560u/mg) are dissolved in 12 liters of methylene dichloride; filter; filtrate advancing contains the post of 3.6 liters of granulated active carbons, with 22 liters of dichloromethane rinse, collects middle body; be evaporated to 1.2 liters; concentrated solution filters, and filtrate is handled with sherwood oil, obtains precipitation; obtain pure product 141 grams after the washing drying, pure product are tired and are 8055u/mg.

Claims (11)

1, the beginning is revolved streptomycete (Streptomyces pristinaespiralis) CGMCC0957
2, a kind of method for preparing Stapyocine is characterized in that, the described bacterial strain Streptomyces of fermentation claim 1 pristinaespiralis CGMCC 0957.
3, the method for claim 2 is characterized in that, this method comprises the steps:
A) seed preparation
B) fermentation culture.
According to the method for claim 3, it is characterized in that 4, described seed prepares used substratum and contains the carbon source utilized of 2-5% and the organic nitrogen source of 1-4%.
According to the method for claim 3, it is characterized in that 5, the used substratum of described fermentation culture contains the inorganic nitrogen-sourced of the organic nitrogen source of the carbon source utilized, 2-5% of 4-8% and 0.1-1%.
According to the method for claim 5, it is characterized in that 6, described carbon source contains 2: 1 to 6: 1 slowly utilizing carbon source and utilizing carbon source fast.
According to the method for claim 6, it is characterized in that 7, the carbon source of described slow utilization comprises starch, dextrin, maltose, sucrose, lactose, raffinose, cellobiose etc.
According to the method for claim 6, it is characterized in that 8, the carbon source of described quick utilization comprises glucose, fructose, semi-lactosi, seminose, glycerine etc.
According to the method for claim 5, it is characterized in that 9, described organic nitrogen source contains 3: 1 to 8: 1 slowly utilizing organic nitrogen source and utilizing organic nitrogen source fast.
According to the method for claim 9, it is characterized in that 10, the described organic nitrogen source that slowly utilizes is selected from soybean cake powder, groundnut meal, cotton seed powder cake and fish meal.
According to the method for claim 9, it is characterized in that 11, the described organic nitrogen source that utilizes fast is selected from peptone, yeast powder.
CNB200310100179XA 2003-10-16 2003-10-16 High efficiency fermentation method for pristinamycin Expired - Fee Related CN1311069C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586370A (en) * 2012-01-18 2012-07-18 石家庄佰锐生物技术有限公司 Fermentation medium for pristinamycin and fermentation method of pristinamycin
CN114807275A (en) * 2022-05-26 2022-07-29 海正药业(杭州)有限公司 Fermentation method for increasing pristinamycin yield
CN118726202A (en) * 2024-08-14 2024-10-01 青岛海科生物技术有限公司 A marine fish meal culture medium and its application in antibiotic fermentation production and fermentation method

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3154475A (en) * 1961-06-27 1964-10-27 Rhone Poulenc Sa Process for the production of pristinamycin

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586370A (en) * 2012-01-18 2012-07-18 石家庄佰锐生物技术有限公司 Fermentation medium for pristinamycin and fermentation method of pristinamycin
CN114807275A (en) * 2022-05-26 2022-07-29 海正药业(杭州)有限公司 Fermentation method for increasing pristinamycin yield
CN118726202A (en) * 2024-08-14 2024-10-01 青岛海科生物技术有限公司 A marine fish meal culture medium and its application in antibiotic fermentation production and fermentation method

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