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Method for producing Bacillus subtilis inoculant by liquid-solid biphasic fermentation

A Bacillus subtilis, two-phase fermentation technology, applied in the direction of using spores, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problems that plague the industrial production of Bacillus subtilis inoculum, and it is difficult to obtain high-density live bacteria It can overcome the problems of spores and metabolites that cannot be fully utilized, reduce the probability of bacterial contamination, and improve the level of comprehensive utilization.

Inactive Publication Date: 2019-04-12
湖南慧谷农业生态研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, liquid submerged fermentation has high production costs, difficulty in obtaining high-density viable bacteria and spores, inability to fully utilize metabolites, complicated follow-up treatment (such as vacuum concentration of fermentation broth, adsorption of solid powder carrier, drying, etc.), and waste liquid discharge However, pure solid fermentation is not suitable for promotion and use due to the multi-stage solid expansion that increases the risk of bacterial contamination and the process is complex and the fermentation cycle is long.
How to increase the spore rate as much as possible while ensuring the number of effective viable bacteria while taking into account the production cost has become a bottleneck problem that plagues the actual industrial production of Bacillus subtilis inoculum

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A method for the production of Bacillus subtilis inoculum by liquid-solid two-phase fermentation of the present invention, specifically comprises the following steps:

[0037] (1) Shake flask seed liquid preparation: pick a ring of activated Bacillus subtilis lawn with an inoculation loop, inoculate it in 1.2LYPD seed medium for shaking table culture, shaking table culture conditions: temperature 36 ° C, shaking table speed 160r·min -1 , Culture time 12h, get shake flask seed solution.

[0038] (2) Submerged liquid fermentation culture in a fermenter: the shake flask seed solution obtained in step (1) is inserted into a 100L fermenter with an inoculation amount of 2% by volume. The composition of the liquid fermentation medium in the fermenter: 10g / L corn flour, 15g / L soybean flour, 3g / LNaCl, 1g / L KH 2 PO 4 , 1.5g / L K 2 HPO 4 , 3.5g / L MgSO 4 ·7H 2 O, 3g / L CaCO 3 , The initial pH is 7.0±0.2, the liquid volume is 60L, the tank pressure is 0.06Mpa, the temperature ...

Embodiment 2

[0045] A method for the production of Bacillus subtilis inoculum by liquid-solid two-phase fermentation of the present invention, specifically comprises the following steps:

[0046] (1) Shake flask seed liquid preparation: pick a ring of activated Bacillus subtilis lawn with an inoculation loop, inoculate it in 1.2LYPD seed medium for shaking table culture, shaking table culture conditions: temperature 36 ° C, shaking table speed 160r·min -1 , Culture time 12h, get shake flask seed solution.

[0047] (2) Submerged liquid fermentation culture in a fermenter: the shake flask seed solution obtained in step (1) is inserted into a 100L fermenter with an inoculation amount of 2% by volume. The composition of the liquid fermentation medium in the fermenter: 8g / L corn flour, 10g / L soybean flour, 1g / LNaCl, 0.5g / L KH 2 PO 4 , 0.5g / L K 2 HPO 4 , 1g / L MgSO 4 ·7H 2 O, 1g / L CaCO 3 , initial pH 6.7±0.2, filling coefficient 0.5, tank pressure 0.06Mpa, temperature 30°C, ventilation ra...

Embodiment 3

[0054]A method for the production of Bacillus subtilis inoculum by liquid-solid two-phase fermentation of the present invention, specifically comprises the following steps:

[0055] (1) Shake flask seed liquid preparation: pick a ring of activated Bacillus subtilis lawn with an inoculation loop, inoculate it in 1.2LYPD seed medium for shaking table culture, shaking table culture conditions: temperature 36 ° C, shaking table speed 160r·min -1 , Culture time 12h, get shake flask seed solution.

[0056] (2) Submerged liquid fermentation culture in a fermenter: the shake flask seed liquid obtained in step (1) is inserted into a 100L fermenter with an inoculation amount of 3% by volume. The composition of the liquid fermentation medium in the fermenter: 14g / L corn flour, 25g / L soybean flour, 4g / LNaCl, 1.5g / L KH 2 PO 4 , 2g / L K 2 HPO 4 , 5g / L MgSO 4 ·7H 2 O, 5g / L CaCO 3 , The initial pH is 7.3±0.2, the filling coefficient is 0.6, the tank pressure is 0.06Mpa, the temperature...

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Abstract

The invention discloses a method for producing a Bacillus subtilis inoculant by liquid-solid biphasic fermentation, comprising the steps of: inoculating an activated Bacillus subtilis strain in a seedculture medium, and performing shaker culture to obtain a seed liquid; inoculating the seed liquid into a liquid fermentation medium deep in a fermentor, and performing fermentation culture to obtaina liquid fermentation product; inoculating the liquid fermentation product into a porous shallow-tray solid fermentation medium, and performing shallow-tray solid fermentation to obtain a fermentation product of Bacillus subtilis; and drying the fermentation product of Bacillus subtilis under a ventilation condition, and then conducting crushing, so as to obtain the Bacillus subtilis inoculant. The invention utilizes an inexpensive carbon and nitrogen source as a medium and combines the medium with a liquid-solid biphasic fermentation process to produce the Bacillus subtilis inoculant, so that the effective viable bacterial count and spore yield are improved, and the raw material and post-treatment cost of the inoculant are reduced.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a method for producing Bacillus subtilis inoculum by liquid-solid two-phase fermentation. Background technique [0002] Bacillus is a type of spore-forming ( Endospore ) bacilli are widely distributed in soil, water, Air And animal intestines, etc., most spores are non-toxic, harmless to humans and animals, do not pollute the environment, can secrete antibacterial proteins, antibiotics, enzymes, polypeptides or lipids and other active substances, which can promote plant growth and prevent plant diseases. And it has the advantages of fast reproduction speed, simple nutrition, strong stress resistance, strong colonization and reproduction ability, etc., so it has become the most researched agricultural probiotic bacteria. Among them, Bacillus subtilis is a kind of Bacillus. [0003] Studies have shown that Bacillus subtilis can produce broad-spectrum antibacterial...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N3/00A01N63/02A01P21/00A01P1/00A01P3/00C12R1/125
CPCC12N1/20C12N3/00A01N63/10
Inventor 郑双凤谭石勇郭帅罗志威
Owner 湖南慧谷农业生态研究院有限公司
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