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Surfactin high-yield bacillus subtilis obtained through compound mutation and fermentation method thereof

A technology for bacillus subtilis and mutagenic strains, applied in the field of microorganisms, can solve the problems of high production cost, limited industrial production, low biosynthesis efficiency, etc., and achieves high mutagenesis power, good application and promotion prospects, and enhanced foaming performance. Effect

Pending Publication Date: 2021-08-20
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low biosynthetic efficiency and high production cost of surfactin limit its industrial production

Method used

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  • Surfactin high-yield bacillus subtilis obtained through compound mutation and fermentation method thereof
  • Surfactin high-yield bacillus subtilis obtained through compound mutation and fermentation method thereof
  • Surfactin high-yield bacillus subtilis obtained through compound mutation and fermentation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 Surfactin high-yield strain compound mutagenesis selection

[0045] S1. Strain preservation: the starting strain Bacillus subtilis subsp.Subtilis (Bacillus subtilis subsp.Subtilis, resource number: BNCC 194013) was cultured to the logarithmic phase with nutrient broth medium (NB), and the bacterial solution was mixed with glycerol at 1:1. Mix well to make the final concentration of glycerol 50%. After pre-cooling in a refrigerator at 4°C, transfer to -20°C and -80°C low-temperature refrigerators.

[0046] S2. Ultraviolet mutagenesis: Pick the Bacillus subtilis strains on the inclined surface and inoculate them in NB medium, cultivate to the logarithmic phase, dilute the bacterial solution to a suitable gradient, smear the blood plate, and carry out ultraviolet mutagenesis, with no irradiation as the control . Except for the control group, they were all wrapped in black paper, cultured at 37°C for 24-48 hours, and the ratio of hemolytic zone to colony dia...

Embodiment 2

[0053] Embodiment 2 detects the performance of Surfactin high-yielding mutagenic strain

[0054] 1. Detection of surfactin production: Take FHYB201030 bacteria liquid in the logarithmic phase, add it to the fermentation medium according to the inoculum amount of 5%, ferment at 40°C for 72 hours, use CPC-BTB colorimetric method to detect, and finally determine the concentration of surfactin in the fermentation liquid as 10.91g / L. Adopt HPLC to measure output, the result is as follows figure 2 As shown, the yield of surfactin was 11.2g / L, and the purity of extracted surfactin was 98.82%.

[0055] 2. Determination of genetic stability of mutagenized strains:

[0056] The high-yielding mutant strains screened by mutagenesis were subcultured on slant, and subcultured once every 24 hours for a total of 10 generations. The mutagenized strains were picked for each generation and fermented in shake flasks for 3 days. The passage of the strains was detected by the CPC-BTB colorimet...

Embodiment 3

[0074] Example 3 This example provides the fermentation method of the mutagenized strain of the present invention

[0075] The fermentation medium of the mutagenic strain of the present invention is: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, maltose 15g / L, sorbitol 10g / L, yeast extract 40g / L, casein 30g / L, Lactose 35g / L, lactic acid 5mmol / L, glutamic acid 1g / L, MnSO4 1.5mmol / L, medium pH 7.0.

[0076] The fermentation conditions of the mutagenic strain of the present invention are as follows: the temperature is 40° C., the rotating speed is 250 rpm, and the liquid filling volume is 50%.

[0077] Fermentation method: inoculate the seed solution obtained from the mutagenized strain into the above-mentioned fermentation medium at an inoculation amount of 5%, and cultivate for 3 days according to the above-mentioned fermentation conditions.

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Abstract

The invention provides a Surfactin high-yield mutant strain--bacillus subtilis, with the preservation number being GDMCC No: 61604. According to the invention, the high-yield mutant strain is obtained by a compound mutation method combining ultraviolet rays, diethyl sulfate and spaceflight, with high mutation success rate and remarkable effect. The strain can remarkably increase the yield of Surfactin, enhance the activity of Surfactin and effectively resist gram-negative pathogenic bacteria and gram-positive pathogenic bacteria, and is enhanced in foaming performance, improved in emulsifying capacity and enhanced in oil discharge performance. Therefore, the strain can be applied to the fields of food, cosmetics and daily necessities, and also can be applied to the fields of livestock and petroleum, with good application and popularization prospects.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacillus subtilis with high yield of surfactin obtained by compound mutagenesis and a fermentation method thereof. Background technique [0002] With the development of various industries, the world's demand for surfactants is increasing year by year. Studies have shown that during 2015-2022, the global surfactant sales growth rate is 5.4%, and the sales growth rate is 5.9%; by 2020, the global surfactant market will reach 42.7 billion US dollars, and the total market will reach 2280.2 tons. At present, most of the surfactants used are chemical surfactants, which are used in a very large amount, but they are prone to environmental pollution after use. Therefore, the development trend of surfactants in the future is towards the direction of green, pollution-free and high efficiency. Biosurfactants are the most economically favored biotechnology products, su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12P1/04A23K10/16A23K10/18A23L33/135A61K8/99A61K35/742A61P31/04A61P31/10A61P31/14A61Q19/00C12R1/125
CPCC12N1/20C12N13/00C12P1/04A23K10/16A23K10/18A23L33/135A61K8/99A61K35/742A61P31/04A61P31/10A61P31/14A61Q19/00
Inventor 胡文锋李光月李雪玲祁姣姣陈彤郭罕星胡学生朱剑锋
Owner SOUTH CHINA AGRI UNIV
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