CN109182170A - One plant of Surfactin superior strain and its application - Google Patents

One plant of Surfactin superior strain and its application Download PDF

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CN109182170A
CN109182170A CN201811026114.8A CN201811026114A CN109182170A CN 109182170 A CN109182170 A CN 109182170A CN 201811026114 A CN201811026114 A CN 201811026114A CN 109182170 A CN109182170 A CN 109182170A
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surfactin
lm34s07
bacillus subtilis
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bacterial strain
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CN109182170B (en
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陈亮
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Henan University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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Abstract

The invention belongs to microorganisms technical field, bacterial strain and its application of a kind of high yield Surfactin are disclosed.The bacterial strain is bacillus subtilis LM34S07, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 18th, 2018, and deposit number is CGMCC 15620.There are the outstanding advantages that synthesis rate is fast, yield is high using bacterial strain production Surfactin, it can be applied to the fields such as fresh-keeping microbiological feed, feed addictive, microbial manure, control of plant disease, food antiseptic, cosmetics, medicine, environmental protection, there is huge application potential and economic value.

Description

One plant of Surfactin superior strain and its application
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of bacillus subtilis strain of high yield Surfactin And its application.
Background technique
Surfactin, Chinese Surfactin are times synthesized by bacillus through non-ribosomal approach Grade metabolite, basic structure is the ring being formed by connecting by 1 beta-hydroxy fatty acid and the polypeptide containing 7 amino acid through lactone bond Shape lipopeptid, because of the difference of carbon chain lengths in fatty acid, it may appear that one or several-CH are differed on molecular weight2Homologue.
Surfactin has big surface-active, emulsification and the good prominent characteristic of foam performance, can significantly reduce the table of water Face tension and other liquid-liquid interface tension；In addition, Surfactin is also proven to have antibacterium, antimycotic, antiviral, anti-branch The antimicrobial acivities such as substance, antitumor；Simultaneously as Surfactin is the cyclic annular rouge being formed by connecting by fatty acid, polypeptide chain Peptide, Surfactin also have the excellent characteristics such as easily biological-degradable, no pollution to the environment.
Just because of cyclic lipopeptide Surfactin has many prominent characteristics, so that micro- life of Surfactin, production Surfactin Object bacterial strain is by development and application in microbiological feed, feed addictive, microbial manure, control of plant disease, food processing, doctor The multiple fields such as medicine, cosmetics, environmental protection, oil exploitation, by the favor of researchers at home and abroad, industrial development personnel etc..
But since the Surfactin yield of existing microbial strains is lower, cause Surfactin production cost higher, Surfactin is expensive, greatly limits Surfactin application range, large-scale production and commercial development application.
Summary of the invention
In order to solve the problems, such as current Surfactin low yield, be unable to satisfy industrialized production, the present invention provides a kind of high Produce bacterial strain, production method and its application of Surfactin.
The present invention provides one plant of Surfactin superior strain, which is bacillus subtilis LM34S07 (Bacillus subtilis LM34S07), Chinese microorganism strain preservation conservator is preserved on April 18th, 2018 Meeting common micro-organisms center, deposit number are CGMCC 15620, and address is section of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Institute of microbiology of institute.
Surfactin superior strain bacillus subtilis LM34S07 provided by the present invention is by starting strain withered grass bud Spore bacillus obtains through nitrosoguanidine, He-Ne laser irradiation complex mutation breeding.
Bacillus subtilis LM34S07 well-grown on nutrient agar panel, bacterium colony milky, opaque, bacterium colony circle Shape, edge are irregular；Cell is rod-shaped, Gram-positive, raw in gemma, ellipse.
It is safe strain generally acknowledged in the world that bacterial strain provided by the invention, which belongs to, and being classified as by U.S. FDA and Environmental Protection Administration can quotient Exempt to do one of first class inoculum of secure authentication as defined in one of microorganism fungus kind applied in industry and the Ministry of Agriculture of China.
The present invention provides the methods using bacillus subtilis LM34S07 production Surfactin, comprising: picking bacterial strain Mono- ring of LM34S07, access is equipped in the 250mL triangular flask of 30-100mL beef-protein medium, in 30-35 DEG C, 150- Shaking table culture 16-24h under 200rpm, obtains seed liquor；Seed liquor is equipped with 100-200mL by the inoculum concentration access of 3-10% (v/v) In the 500mL triangular flask of fermentation medium, shaking table culture 28-48h, obtains fermentation liquid under 30-35 DEG C, 150-200rpm；Fermentation Culture medium group becomes glucose 15-35g, glutamic acid 3-8g, yeast extract 1-4g, potassium dihydrogen phosphate 0.7-1.1g, potassium chloride 0.3- 0.6g, epsom salt 0.3-0.6g, copper sulphate 0.1-0.2mg, ferrous sulfate heptahydrate 0.1-0.2mg, manganese sulfate 3-10mg, water 1L, pH7.0,115 DEG C of sterilizing 30min.
The present invention provides with bacillus subtilis LM34S07 or its tunning or contain bacillus subtilis The microbial inoculum of LM34S07 is in field of fodder, the application of agriculture field.
The present invention provides with bacillus subtilis LM34S07 or its tunning or contain bacillus subtilis Application of the microbial inoculum of LM34S07 in food, medicine, cosmetic field.
The present invention provides with bacillus subtilis LM34S07 or its tunning or contain bacillus subtilis Application of the microbial inoculum of LM34S07 in field of environment protection.
Beneficial effects of the present invention:
The present invention provides the bacillus subtilis LM34S07 that a plant height produces Surfactin, which synthesizes Surfactin speed Rate is fast, yield is high, and fermentation 40h fermentation liquid Surfactin content is up to 9.78g/L；It is produced using bacterial strain LM34S07 Surfactin can substantially reduce production cost, improve production efficiency, have huge industrial applications prospect.
Detailed description of the invention
Fig. 1 Surfactin standard items HPLC map；
Fig. 2 methanol extract sample HPLC map.
Specific embodiment
In order to keep the present invention easier to understand, the present invention will be further explained by examples below.It should be understood that These embodiments are merely to illustrate the present invention rather than limit the scope of the invention.
Embodiment 1:Surfactin superior strain complex mutation breeding
The access of one ring of picking starting strain is equipped in the 50mL triangular flask of 10mL beef-protein medium, in 33 DEG C, 175r/ After min shaking table culture 16h, it is centrifuged 15min under 4 DEG C, 8000r/min, collects thallus, thallus is washed with pH7.0 phosphate buffer It washs 3 times, a certain amount of pH7.0 phosphate buffer suspension thalline is added, obtain the bacteria suspension (10 for complex mutation8cfu/mL)； The nitroso guanidine solution of 1mL bacteria suspension, 1mL concentration 180ug/mL are uniformly mixed in small test tube, are placed in 33 DEG C of water-baths, during which Using He-Ne laser illumination (the quasi- single mode He-Ne laser of external cavity type, wavelength 632.8nm, irradiation power 12mW), shine every time Lasting 2min is penetrated, is irradiated 3 times, after water bath processing 12min, above-mentioned mixed liquor is poured into 48mL sterile saline, is terminated anti- It answers；Then, each dilution is coated on blood plate using gradient dilution method, the constant temperature incubation 36h at 33 DEG C, picking haemolysis circle Big bacterium colony, switching save, and detect mutant strain fermentation liquid Surfactin yield using HPLC method, finally screen to obtain Surfactin high productive mutant LM34S07.
Embodiment 2:Surfactin content detection
Fermentation liquid is centrifuged 20min under 4 DEG C, 12000rpm, collects supernatant, adjusts supernatant pH to 2.0,4 with concentrated hydrochloric acid 20min is centrifuged after DEG C standing overnight under 4 DEG C, 12000rpm, collects precipitating；5mL methanol is added into precipitating, in magnetic agitation It after stirring 30min on device, is put into 4 DEG C of refrigerators and stands extracting 12h, be centrifuged 20min in 4 DEG C, 12000rpm after extracting, receive Collect methanol extract；Extracting 3 times is repeated according to the above method, merges methanol extract, after the sterile filtering with microporous membrane of 0.22um, 4 DEG C save backup.
Surfactin content detection is carried out using HPLC；Chromatographic column ZORBAX SB-C18(5 μm, 4.6 × 250mm), column temperature 35 DEG C, sample volume 20 μ l, wavelength 205nm, flow velocity 0.8mL/min, mobile phase A pure water (contains 0.1%TFA), and Mobile phase B acetonitrile (contains 0.1%TFA)；Condition of gradient elution are as follows: 0 ~ 9 min, 40% mobile phase A and 60% Mobile phase B；9 ~ 20min, 7% mobile phase A and stream Dynamic phase B；It is shown by HPLC map, methanol extract sample HPLC appearance time (Fig. 2) and Surfactin standard items appearance time (Fig. 1) is essentially identical, and Surfactin standard items standard curve regression equation is Y=0.08132X-41.76, coefficient R2= 0.9937, it brings methanol extract sample HPLC peak area into regression equation calculation and obtains Surfactin concentration, and then converse Surfactin content in fermentation liquid.
Embodiment 3:Surfactin fermentation
Fermentation medium group becomes glucose 20g, glutamic acid 5g, yeast extract 4g, potassium dihydrogen phosphate 1g, potassium chloride 0.5g, seven water Magnesium sulfate 0.5g, copper sulphate 0.16mg, ferrous sulfate heptahydrate 0.15mg, manganese sulfate 5mg, 1L, pH7.0,115 DEG C of water sterilizings 30min。
Mono- ring of picking bacterial strain LM34S07, access are equipped in the 250mL triangular flask of 50mL beef-protein medium, in 33 DEG C, shaking table culture 18h under 175rpm, obtain seed liquor；Seed liquor is fermented by the inoculum concentration access of 7% (v/v) equipped with 150mL In the 500mL triangular flask of culture medium, shaking table culture 40h, obtains fermentation liquid under 33 DEG C, 175rpm；Through detecting, fermentation liquid Surfactin content is 9.78g/L.
Embodiment 4:Surfactin fermentation
Fermentation medium group becomes glucose 35g, glutamic acid 3g, yeast extract 1g, potassium dihydrogen phosphate 1.1g, potassium chloride 0.3g, seven Water magnesium sulfate 0.6g, copper sulphate 0.2mg, ferrous sulfate heptahydrate 0.2mg, manganese sulfate 3mg, 1L, pH7.0,115 DEG C of water sterilizings 30min。
Mono- ring of picking bacterial strain LM34S07, access are equipped in the 250mL triangular flask of 100mL beef-protein medium, in 35 DEG C, shaking table culture for 24 hours, obtains seed liquor under 200rpm；Seed liquor is fermented by the inoculum concentration access of 5% (v/v) equipped with 200mL In the 500mL triangular flask of culture medium, shaking table culture 48h, obtains fermentation liquid under 35 DEG C, 200rpm；Through detecting, fermentation liquid Surfactin content is 9.71g/L.
Embodiment 5:Surfactin fermentation
Fermentation medium group becomes glucose 15g, glutamic acid 8g, yeast extract 4g, potassium dihydrogen phosphate 0.7g, potassium chloride 0.6g, seven Water magnesium sulfate 0.3g, copper sulphate 0.1mg, ferrous sulfate heptahydrate 0.1mg, manganese sulfate 10mg, 1L, pH7.0,115 DEG C of water sterilizings 30min。
Mono- ring of picking bacterial strain LM34S07, access are equipped in the 250mL triangular flask of 30mL beef-protein medium, in 30 DEG C, shaking table culture 16h under 150rpm, obtain seed liquor；Seed liquor is fermented by the inoculum concentration access of 10% (v/v) equipped with 100mL In the 500mL triangular flask of culture medium, shaking table culture 28h, obtains fermentation liquid under 30 DEG C, 150rpm；Through detecting, fermentation liquid Surfactin content is 9.66g/L.
The present invention is not by any restriction of the explanation of the embodiment of foregoing invention and embodiment.It is also contained in the present invention Various modifications in the case where without departing from scope of the claimed in the range of those skilled in the art are easily envisaged that Mode.

Claims (5)

1. one plant of Surfactin superior strain, which is bacillus subtilisBacillus subtilisLM34S07, It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC 15620, and the bacterial strain is raw Production Surfactin rate is fast, yield is high.
2. application of the bacillus subtilis LM34S07 described in claim 1 in terms of producing Surfactin.
3. bacillus subtilis LM34S07 described in claim 1 is in microbiological feed, feed addictive, microbial manure, plant The application of object disease control etc..
4. bacillus subtilis LM34S07 described in claim 1 food, cosmetics, in terms of application.
5. bacillus subtilis LM34S07 described in claim 1 environmental protection, in terms of application.
CN201811026114.8A 2018-09-04 2018-09-04 Surfactin high-yield strain and application thereof Active CN109182170B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110904092A (en) * 2019-12-09 2020-03-24 济南大学 Lactobacillus brevis compound mutation breeding method for high yield of GABA
CN112980747A (en) * 2021-04-30 2021-06-18 南京理工大学 Bacillus subtilis for producing lipopeptide biosurfactant
CN113278543A (en) * 2021-05-06 2021-08-20 华南农业大学 Bacillus subtilis capable of producing surfactin in high yield and obtained through compound mutagenesis and fermentation method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103898038A (en) * 2014-03-28 2014-07-02 清华大学 Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof
CN104830713A (en) * 2015-04-09 2015-08-12 河南工业大学 Bacillus thuringiensis for combined production of antimicrobial lipopeptide and gamma-polyglutamic acid
CN105602863A (en) * 2015-12-14 2016-05-25 南京工业大学 Bacillus subtilis strain for high yield of lipopeptide antibiotic and poly-gamma-glutamic acid

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103898038A (en) * 2014-03-28 2014-07-02 清华大学 Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof
CN104830713A (en) * 2015-04-09 2015-08-12 河南工业大学 Bacillus thuringiensis for combined production of antimicrobial lipopeptide and gamma-polyglutamic acid
CN105602863A (en) * 2015-12-14 2016-05-25 南京工业大学 Bacillus subtilis strain for high yield of lipopeptide antibiotic and poly-gamma-glutamic acid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈亮等: "响应面法优化Bcillus subtilis抗菌脂肽发酵工艺条件", 《食品科技》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110904092A (en) * 2019-12-09 2020-03-24 济南大学 Lactobacillus brevis compound mutation breeding method for high yield of GABA
CN112980747A (en) * 2021-04-30 2021-06-18 南京理工大学 Bacillus subtilis for producing lipopeptide biosurfactant
CN113278543A (en) * 2021-05-06 2021-08-20 华南农业大学 Bacillus subtilis capable of producing surfactin in high yield and obtained through compound mutagenesis and fermentation method thereof

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