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Method for producing surfactin by applying bacillus amyloliquefaciens

A technology of amyloliquefaciens and bacilli, which is applied in the field of industrial microorganisms, can solve the problems of high production cost and commercial production of biosurfactant, and achieve the effect of avoiding product feedback inhibition and strong product tolerance

Active Publication Date: 2017-05-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Expensive production costs are the main factor why biosurfactins are still not commercially available

Method used

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  • Method for producing surfactin by applying bacillus amyloliquefaciens
  • Method for producing surfactin by applying bacillus amyloliquefaciens
  • Method for producing surfactin by applying bacillus amyloliquefaciens

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 strain screening

[0036]Collect samples from Chinese liquor brewing high-temperature Daqu, take 5g samples and dissolve them in 10ml of 1% by mass sodium chloride solution, vortex and mix well, draw 1mL and inoculate them into the activation culture medium, at a temperature of 50-60°C, 200rpm Activate for 24-36 hours under the condition of rotating speed to make mature activated bacterial liquid.

[0037] Take the mature activated bacteria solution, add 1% sodium chloride solution by mass percentage to dilute. Choose a dilution gradient of 10 -2 -10 -6 The dilutions were sequentially spread on solid medium plates. Cultivate for 48-72 hours at a temperature of 30°C and a humidity of 40%-60%; a large number of single colonies are obtained. After the obtained single colony was inoculated into the nutrient medium for fermentation and culture, the content of biosurfactin in the fermentation liquid was detected, and finally a pure culture with high biosurfactin...

Embodiment 2

[0038] The assay method of embodiment 2 biosurfactins

[0039] Extraction of biosurfactin: centrifuge the fermentation broth at 8000rpm at 4°C for 10min. The fermentation supernatant was adjusted to pH 2.0 with 6mol / L hydrochloric acid, and placed in a refrigerator at 4°C for 2 hours. Then, centrifuge at 12000 rpm at 4°C for 30 min to obtain a precipitate. The crude product of biosurfactin was obtained after the precipitate was extracted with methanol.

[0040] Determination method of biosurfactin in fermentation broth: adopt ultra-high performance liquid chromatography to accurately quantify biosurfactin. A waters BEH C18 chromatographic column was used, the mobile phases were (A) 0.1% formic acid aqueous solution and (B) HPLC grade methanol, and the elution conditions were 30%-70% methanol for 5 min and 70%-100% methanol for 1 min. Samples were detected at 210 nm using a diode array detector. After the obtained chromatographic peaks were integrated, the interpolation met...

Embodiment 3

[0041] Example 3 The strains screened in Example 1 were identified.

[0042] The bacterial strain numbered MT45 obtained in Example 1 was subjected to strain identification.

[0043] The Bacillus amyloliquefaciens MT45 strain formed round and translucent colonies on the solid plate medium; the surface of the colonies was smooth and wrinkled, and no biofilm represented by wrinkled colonies was formed; the center of the colonies was slightly raised. Under the microscope, the cells were rod-shaped, spore-forming, and Gram staining was positive ( figure 1 ).

[0044] The strain 16r rRNA gene sequence was sequenced (SEQ ID NO.1), compared with the database, and phylogenetic tree analysis was carried out to determine that MT45 was Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ( figure 2 ).

[0045] Bacillus amyloliquefaciens MT45 was deposited on June 6, 2016 in the General Microbiology Center of China Committee for the Collection of Microorganisms, with the preservati...

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Abstract

The invention discloses a method for producing surfactin by applying bacillus amyloliquefaciens, and belongs to the technical field of industrial microbiology. The yield of the surfactin synthesized by applying the bacillus amyloliquefaciens with the collection number of CGMCC No.12593 and obtained by a screening method reaches up to 9.43g / L, and the method for producing the surfactin by applying the bacillus amyloliquefaciens has the characteristics of synchronization of surfactin synthesis and strain growth and no lag phase in a fermentation process. Strains and a fermentation method provided by the invention play an important role in industrial production of the surfactin and have broad application prospects.

Description

technical field [0001] The invention relates to a method for producing biosurfactin by using bacillus amyloliquefaciens, belonging to the technical field of industrial microorganisms. Background technique [0002] Biosurfactin, namely surfactin, is a long-chain fatty acid (C 12 -C 17 ) and a polypeptide containing 7 amino acids connected by a lactone bond, it has antibacterial, antiviral, antitumor, thrombolytic, cholesterol-lowering medicinal effects and superior surface active functions. The broad-spectrum antibacterial effect of biosurfactin is expected to make up for the widespread problem of antibiotic resistance today. The antiviral and anti-tumor functions of biosurfactin also provide some options for the design of new drugs, and biosurfactin is superior The surface active function (capable of reducing the surface tension of water molecules from 72mN / M to 27mN / M) makes it have a wide range of application prospects in cosmetics, medicine, emulsifiers, moisturizers an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/04A61K38/12A61P31/04A61P31/12A61P35/00A61P7/02A61P3/06C12R1/07
CPCA61K38/12C12P21/02C12R2001/07C12N1/205C07K7/06C12N1/20C09K8/582C09K8/584A61K35/742A61P31/04A61P31/10A61P31/12A61P35/00A61P3/06A61P7/02C07K7/64A61K35/74
Inventor 徐岩吴群郅岩
Owner JIANGNAN UNIV
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