CN102080047A - Culture medium for producing antifungal substance by streptomyces fermentation and preparation method thereof - Google Patents
Culture medium for producing antifungal substance by streptomyces fermentation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a culture medium for producing an antifungal substance by streptomyces fermentation and a preparation method thereof. The culture medium comprises the following components in concentration by mass: 0.3 to 1.0 percent of corn flour, 1.5 to 2.0 percent of glucose, 2.5 to 3.5 percent of soy bean cake flour, 0.3 to 0.6 percent of potassium dihydrogen phosphate, 0.5 to 1.0 percent of magnesium sulfate and the balance of water. By adopting the fermentation culture medium, the yield of the streptomyces hygroscopicus BS-112 antifungal substance is greatly improved; and the industrially common raw materials such as the corn flour, the soy bean cake flour and the like are used for production, so the production cost is reduced and the culture medium is suitable for large-scale production. The produced antifungal substance has broad-spectrum antifungal activity on main mildewing fungi such as aspergillus flavus in grains and feed and multiple domestic silkworm and plant pathogenic fungi, has low toxicity, and has high practical application value and broad market prospect on mildew-proof agents for the grains and the feed and biological prevention and control of domestic silkworm fungi diseases and plant fungi diseases.
Description
Technical field
The present invention relates to a kind of fermentation culture medium for microbe and preparation method thereof, be specifically related to substratum of the antimycotic material of a kind of streptomyces hygroscopicus fermentative production and preparation method thereof.
Background technology
The polyene macrolide microbiotic is a chemical monoid in the microbiotic, and its structure is formed big lactonic ring by 25～37 carbon atoms, in a part of encircling the conjugated polyene structure is arranged, and corresponding position has hydroxyl, and connects one or more aminosugars by glycosidic link.Be divided into three, four, five, six, seven alkene owing to conjugated double bond number in the structure is different, every class all has special uv-absorbing peak value.The ultra-violet absorption spectrum of the macrolide of conjugation tetraene has charateristic avsorption band at 291 ± 2nm, 302 ± 2nm, 320 ± 3nm, all has antifungic action, kind surplus in the of about 50.The tetraene macrolide antibiotic is widely used in medical treatment, food and agriculture production, can suppress yeast and mould in obligate ground as tennecetin, and have good physical and chemical stability, become more than 30 widely used a kind of natural biological food preservatives of country and antimicrobial additive; Tetramycin has the better prevention effect to forest canker, Peptic Ulcers, black spot etc., and the healing of obvious promotion injured tissue, flourishing, the aging root regeneration of weak shoot root system are arranged, improve characteristics such as crop disease-resistant ability, and toxicity is low, to the person poultry safety, nuisanceless, free from environmental pollution, be a kind of novel agricultural antibiotic.In the microbiotic of having reported with anti-mycotic activity, great majority have the structure of polyene macrolide, and polyene antibiotics is that a class least is easy to generate drug-fast antifungal antibiotic in the clinical medicine commonly used at present.Therefore, the polyenoid class antifungal antibiotic of exploitation high-efficiency low-toxicity is still one of effective way of antifungal drug research and development.
This research department separates acquisition one plant height from the timbered soil of Mount Taishan imitates antagonism bacterium BS-112, through being accredited as streptomyces hygroscopicus (Streptomyces hygroscopicus), its meta-bolites has broad-spectrum antifungal activity, and common mould in grains such as flavus, Aspergillus ochraceus, aspergillus niger and the feed is had the obvious suppression effect.The antimycotic material that this laboratory produces this bacterial strain has carried out separation and purification and structure is differentiated, find the tetraene macrolide antibiotic that antimycotic material that this bacterial strain produces is made up of 4 active ingredients, their chemical structure is respectively Tetrins A and B, Tetramycins A and B.This strain fermentation proterties excellence, antimycotic physical property is stable, has the potentiality as grain and Midew preventive for feed.In addition, this antimycotic material also has good inhibitory effect to the pathogenic fungi of multiple silkworm and farm crop, also has potential using value on sericulture and agricultural.
Summary of the invention
The object of the present invention is to provide substratum of the antimycotic material of a kind of streptomyces hygroscopicus fermentative production and preparation method thereof, substratum to the antimycotic material of streptomyces hygroscopicus BS-112 fermentative production is optimized, with the output that improves antimycotic material and reduce production costs, significant to the suitability for industrialized production of the antimycotic material of this bacterial strain.
The invention provides the substratum of the antimycotic material of a kind of streptomyces hygroscopicus fermentative production, each component and mass concentration thereof are: Semen Maydis powder 0.3%%～1.0%, glucose 1.5%～2.0%, soybean cake powder 2.5%～3.5%, potassium primary phosphate 0.3%～0.6%, sal epsom 0.5%～1.0%, all the other are water.
The preferred mass concentration of each component of the present invention is: Semen Maydis powder 0.5%, glucose 1.8%, soybean cake powder 3.1%, potassium primary phosphate 0.38%, sal epsom 0.85%.
The present invention also provides the preparation method of the antimycotic material culture medium of a kind of streptomyces hygroscopicus fermentative production, it is characterized in that mainly comprising the steps:
(1) mass concentration according to glucose 1.5%～2.0%, potassium primary phosphate 0.3%～0.6% and sal epsom 0.5%～1.0% takes by weighing each component, adds an amount of water it is dissolved fully;
(2) mass concentration according to Semen Maydis powder 0.3%～1.0% and soybean cake powder 2.5%～3.5% takes by weighing each component, adds a spot of water and stirs into pasty state;
(3) lysate of step (1) is poured in the pasty state solution of step (2), added the water constant volume again, obtain antimycotic fermentation of materials substratum.
The present invention adopts following technical scheme to realize: a kind of streptomyces hygroscopicus, this bacterial strain are expressed as streptomyces hygroscopicus (Streptomyces hygroscopicus) BS-112.It is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 13rd, 2009 and (abbreviates CGMCC as, the address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.Postcode: 100101), preserving number is CGMCC No.3504, and class categories is: streptomyces hygroscopicus (Streptomyces hygroscopicus).
BS-112 bacterial strain of the present invention screens from the timbered soil of Mount Taishan and obtains, this bacterial strain all has the common fungi that goes mouldy (flavus, Aspergillus ochraceus, aspergillus niger, Aspergillus fumigatus, aspergillus oryzae), silkworm pathogenic fungi (muscardine, green muscardine fungus, flavus) and plant disease fungi (botrytis cinerea, Colletotricum destructivum bacterium, dry thread Pyrenomycetes, ring rot of apple bacterium, willow rotten pathogenic bacteria) etc. in grain and the feed and suppresses effect preferably, and this bacterial strain has the good antibacterial stability that goes down to posterity.
The tetraene macrolide antibiotic that the antimycotic material that the BS-112 bacterial strain produces is made up of 4 active ingredients, their chemical structure is respectively Tetrins A and B, Tetramycins A and B.This antimycotic material physico-chemical property is stable, and toxicity is low.In the biological control of grain and Midew preventive for feed, silkworm epiphyte disease and fungal diseases of plants, all have higher actual application value and vast market prospect.
The present invention is applied to single single-factor, Plackett-Burman Design and Response Surface Methodology in the antimycotic fermentation of materials Optimum of culture medium of the BS-112 bacterial strain process, comprises the steps:
At first filter out optimum carbon source (glucose) and nitrogenous source (soybean cake powder), secondly select 3 key factors of the antimycotic material output of influence: glucose, soybean cake powder and potassium primary phosphate with Plackett-Burman Design testing sieve according to the single one-factor experiment.Adopt the method for Response Surface Methodology at last, obtained the best in quality concentration of glucose, soybean cake powder and potassium primary phosphate.
Fermention medium after the optimization is applied in the fermentation culture process of the antimycotic material of BS-112 bacterial strain, the raising in various degree of antimycotic material output 3～4 times.And reduced the higher carbon source of cost---the content of glucose, replenish slow carbon source---Semen Maydis powder, removed the higher nitrogenous source of cost---peptone, replenished cheap nitrogenous source---soybean cake powder, production cost is reduced significantly, for the suitability for industrialized production of this antimycotic material is laid a good foundation.
The calculating of the antimycotic material concentration of streptomyces hygroscopicus BS-112 fermented liquid:
There is stronger characteristic peak to absorb at the 305nm place according to antimycotic material, adopt its concentration of determined by ultraviolet spectrophotometry: with testing sample or fermented liquid dilution suitable multiple, its light absorption value at the 305nm place is between 0.2～1.0, measure and the record light absorption value, each is handled and repeats 2 times, with numerical value substitution equation: y=[(x-0.037)/0.0011] * the n[formula in, y: the concentration of antimycotic material (μ g/mL), x: light absorption value (0.2＜x＜1.0), n: extension rate], calculate the concentration of antimycotic material.
The present invention has the following advantages:
1. broad-spectrum antibacterial property is good, and the main fungi (as flavus, Aspergillus ochraceus, aspergillus niger, Aspergillus fumigatus, aspergillus oryzae) that causes grain and feed mold is had stronger restraining effect.Antimycotic material also all has good inhibitory effect to silkworm pathogenic fungi (muscardine, green muscardine fungus, flavus) and plant pathogenic fungi (botrytis cinerea, Colletotricum destructivum bacterium, dry thread Pyrenomycetes, ring rot of apple bacterium, willow rotten pathogenic bacteria) etc.
2. the fermentation proterties is good, and the antibacterial proterties that goes down to posterity is stable, and antimycotic material is stable to heat and alkali, storage endurance.
3. the fermention medium after optimizing and optimize before fermention medium compare, the employing Semen Maydis powder is as the slow carbon source, with soybean cake powder as nitrogenous source, greatly reduce composition, shortened fermentation period, and the output of antimycotic material improves 3～4 times, be beneficial to suitability for industrialized production.
Embodiment
Embodiment 1:
(1) preparation fermention medium: according to glucose 1.8%, potassium primary phosphate 0.38%, the mass concentration of sal epsom 0.5% takes by weighing each component, adds an amount of water it is dissolved fully, obtains lysate; Take by weighing Semen Maydis powder 0.5% and soybean cake powder 3.1% places beaker according to mass concentration, add a spot of water and stir into pasty state; Above-mentioned lysate is poured in the beaker, mixed, add the water constant volume again with pasty state Semen Maydis powder and soybean cake powder;
(2) bottling sterilization: 60 capacity are cleaned for the 250mL triangular flask, and the amount of every bottled fermention medium is 50mL, in the process of dress liquid, otherwise the time stir with glass stick, prevent Semen Maydis powder and soybean cake powder precipitation.After the triangular flask that installs fermention medium sealed with tampon, autoclaving;
(3) inoculation: after treating the fermention medium cooling, the inoculum size by 2% under aseptic condition inserts BS-112 bacterial strain seed liquor;
(4) fermentation: the fermention medium that will connect the BS-112 bacterial classification is put into shaking table and is fermented, and fermentation time is 84 hours, and temperature is 28 ℃, and rotating speed is 200 rev/mins;
(5) concentration of the antimycotic material of mensuration: the concentration that records antimycotic material according to ultraviolet spectrophotometry is 5.94g/L.
Embodiment 2:
(1) preparation fermention medium: according to glucose 1.5%, potassium primary phosphate 0.3%, the mass concentration of sal epsom 0.85% takes by weighing each component, adds an amount of water it is dissolved fully, obtains lysate; Take by weighing Semen Maydis powder 0.3% and soybean cake powder 2.5% places beaker according to mass concentration, add a spot of water and stir into pasty state; Above-mentioned lysate is poured in the beaker, mixed, add the water constant volume again with pasty state Semen Maydis powder and soybean cake powder;
(2) bottling sterilization: 60 capacity are cleaned for the 250mL triangular flask, and the amount of every bottled fermention medium is 50mL, in the process of dress liquid, otherwise the time stir with glass stick, prevent Semen Maydis powder and soybean cake powder precipitation.After the triangular flask that installs fermention medium sealed with tampon, autoclaving;
(3) inoculation: after treating the fermention medium cooling, every bottle of inoculum size by 2% inserts BS-112 bacterial strain seed liquor under aseptic condition;
(4) fermentation: the fermention medium that will connect the BS-112 bacterial classification is put into shaking table and is fermented, and fermentation time is 84 hours, and temperature is 28 ℃, and rotating speed is 200 rev/mins;
(5) concentration of the antimycotic material of mensuration: the concentration that records antimycotic material according to ultraviolet spectrophotometry is 5.36g/L.
Embodiment 3:
(1) preparation fermention medium: according to glucose 2.0%, potassium primary phosphate 0.6%, the mass concentration of sal epsom 1.0% takes by weighing each component, adds an amount of water it is dissolved fully, obtains lysate; Take by weighing Semen Maydis powder 1.0% and soybean cake powder 3.5% places beaker according to mass concentration, add a spot of water and stir into pasty state; Above-mentioned lysate is poured in the beaker, mixed, add the water constant volume again with pasty state Semen Maydis powder and soybean cake powder;
(2) bottling sterilization: 30 capacity are cleaned for the 500mL triangular flask, and the amount of every bottled fermention medium is 100mL, in the process of dress liquid, otherwise the time stir with glass stick, prevent Semen Maydis powder and soybean cake powder precipitation.After the triangular flask that installs fermention medium sealed with tampon, autoclaving;
(3) inoculation: after treating the fermention medium cooling, every bottle of inoculum size by 2% inserts BS-112 bacterial strain seed liquor under aseptic condition;
(4) fermentation: the fermention medium that will connect the BS-112 bacterial classification is put into shaking table and is fermented, and fermentation time is 84 hours, and temperature is 28 ℃, and rotating speed is 200 rev/mins;
(5) concentration of the antimycotic material of mensuration: the concentration that records antimycotic material according to ultraviolet spectrophotometry is 5.46g/L.
Embodiment 4:(simultaneous test)
(1) prepare original fermention medium: according to glucose 2.5%, peptone 3.0%, potassium primary phosphate 1.5%, the mass concentration of sal epsom 1.5% takes by weighing each component, adds an amount of water it is dissolved fully, obtains lysate; Mass concentration according to 2% takes by weighing potato and boiled 15 minutes, through 4 layers of filtered through gauze, collects supernatant liquor; Above-mentioned lysate is poured in the supernatant liquor of collection, added the water constant volume again;
(2) bottling sterilization: 60 capacity are cleaned for the 250mL triangular flask, and the amount of every bottled fermention medium is 50mL, after the triangular flask that installs fermention medium is sealed with tampon, and autoclaving;
(3) inoculation: after treating the fermention medium cooling, every bottle of inoculum size by 2% inserts BS-112 bacterial strain seed liquor under aseptic condition;
(4) fermentation: the fermention medium that will connect the BS-112 bacterial classification is put into shaking table and is fermented, and fermentation time is 84 hours, and temperature is 28 ℃, and rotating speed is 200 rev/mins;
(5) concentration of the antimycotic material of mensuration: the concentration that records antimycotic material according to ultraviolet spectrophotometry is 1.63g/L;
(6) concentration of antimycotic material in the fermention medium before and after contrast is optimized: among the embodiment 1 in the fermention medium concentration of antimycotic material be start 3.64 times of ferment substratum of present embodiment Central Plains.
Claims (3)
1. a streptomycete fermentation is produced the substratum of antimycotic material, it is characterized in that its each component and mass concentration thereof are: Semen Maydis powder 0.3%～1.0%, glucose 1.5%～2.0%, soybean cake powder 2.5%～3.5%, potassium primary phosphate 0.3%～0.6%, sal epsom 0.5%～1.0%, all the other are water.
2. the preparation method that streptomycete fermentation is produced the substratum of antimycotic material is characterized in that comprising the steps:
(1) mass concentration according to glucose 1.5%～2.0%, potassium primary phosphate 0.3%～0.6% and sal epsom 0.5%～1.0% takes by weighing each component, adds an amount of water it is dissolved fully;
(2) mass concentration according to Semen Maydis powder 0.3%～1.0% and soybean cake powder 2.5%～3.5% takes by weighing each component, adds a spot of water and stirs into pasty state;
(3) lysate of step (1) is poured in the pasty state solution of step (2), added the water constant volume again.
3. a kind of streptomycete fermentation according to claim 1 is produced the substratum of antimycotic material, it is characterized in that the preferred mass concentration of each component is: Semen Maydis powder 0.5%, glucose 1.8%, soybean cake powder 3.1%, potassium primary phosphate 0.38%, sal epsom 0.85%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108300677A (en) * | 2018-02-22 | 2018-07-20 | 广东省微生物研究所（广东省微生物分析检测中心） | One plant of streptomyces albus and its application in preparing microbialpreservatives |
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Patent Citations (8)
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| CN108300677A (en) * | 2018-02-22 | 2018-07-20 | 广东省微生物研究所（广东省微生物分析检测中心） | One plant of streptomyces albus and its application in preparing microbialpreservatives |
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