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Bacillus subtilis and application thereof

A Bacillus subtilis, applied technology, applied in the field of microorganisms, can solve the problems of high cost, inability to meet large-scale industrial production, and low output, and achieve the effects of reducing sugar consumption, increasing production, and reducing production costs

Active Publication Date: 2011-12-21
XINFA PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But existing Bacillus subtilis strains vitamin B 2 The output is low and the cost is high, which cannot meet the needs of large-scale industrial production

Method used

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  • Bacillus subtilis and application thereof
  • Bacillus subtilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the mutagenesis of Bacillus subtilis of the present invention

[0030] Take Bacillus subtilis BS-1 with the preservation number CGMCC No.4019 and inoculate it on the slant for culture. After washing the slant of the strain with 10 mL of sterile normal saline in the ultra-clean bench, take 1 mL of the bacterial suspension to the slant containing 9 mL of sterile normal saline. In the test tube, dilute 10 times, mix well, then take out 1mL bacterial suspension into a test tube containing 9mL sterile normal saline, dilute 100 times, mix well, and then dilute to 10 -6 times.

[0031] Diluted to 10 -6 Spread the 100% bacterial suspension on a sterilized 9cm petri dish, and irradiate three petri dishes at a time with an 18W ultraviolet lamp at a distance of 28cm from the plate. The irradiation time is 30s. Draw 1mL of the treated bacterial suspension and smear it on a plate to separate the strains.

Embodiment 2

[0032] Embodiment 2: the primary screening of Bacillus subtilis of the present invention

[0033] 1. Primary screening culture medium

[0034] Medium A: 1% sucrose, 1% corn steep liquor and 0.5% peptone, the balance is distilled water;

[0035] Medium B: 1% glucose, 0.5% yeast powder and 0.5% bean cake powder, the balance is distilled water.

[0036] 2. Primary screening of strains

[0037] Inoculate the isolated strains after ultraviolet mutagenesis in medium A, and observe the growth of colonies after culturing at 37°C for 2 days. Pick a single colony that is yellow, opaque, with a rough surface, a colony diameter greater than 2mm, and the culture medium on the back of the colony is obviously yellow, and a total of 76 single colonies were picked. Inoculate the picked single colony into medium B, culture at 37°C for 2 days, select a single colony with a diameter of less than 1 mm, pick 8 colonies in total, and name them as target strains S1-S8.

Embodiment 3

[0038] Embodiment 3: the re-screening of Bacillus subtilis of the present invention

[0039] 1. Re-screening culture medium

[0040] Re-screening liquid medium: 5% glucose, 2% corn steep liquor, 0.05% ammonium sulfate, 0.5% yeast extract and 0.005% magnesium sulfate, and the balance is distilled water.

[0041] 2. Shake flask culture

[0042] The 8 target strains obtained from the primary screening were respectively inoculated in the liquid medium of the secondary screening, and cultured in shake flasks at 37°C and 128rpm for 40h.

[0043] 3. Vitamin B 2 Extraction and determination

[0044] Take out the culture medium in the shake flask, mix it and dilute it, accurately pipette a certain volume into a certain volume brown volumetric flask, add 0.01mol / L sodium hydroxide solution, and distilled water to volume. After shaking well, put it in a dark place for alkali dissolution. If there is vitamin B in the culture fluid taken by microscopic examination 2 For crystals, dis...

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Abstract

The invention relates to the field of microorganisms and discloses bacillus subtilis and application thereof. The strain is preserved in a China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.4018. The bacillus subtilis is a strain which is prepared by the following steps of: with bacillus subtilis BS-1 with the preservation number of CGMCC No.4019 as a starting strain, subjecting to primary screening, secondary screening and fermenting in a small tank and verification by using a method of ultraviolet mutation, wherein a primary screening culture medium includes a culture medium A containing saccharose, corn steep liquor, ammonium sulfate, yeast extract and a culture medium B containing glucose, yeast powder and bean cake powder; and a secondary screen culture medium mainly comprises glucose, corn steep liquor, ammonium sulfate, yeast extract and magnesium sulfate. Provided by the experiment, vitamin B2 produced by fermenting the bacillus subtilis with the preservation number of CGMCC No.4018 has high yield and low sugar consumption; and the invention can be applied to the industrial production of the vitamin B2 and is beneficial to improving the production efficiency and reducing the production cost.

Description

technical field [0001] The invention relates to the field of microbes, more specifically to a bacillus subtilis and its application. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is an aerobic bacterium of the genus Bacillus, which is named for its widespread distribution in soil and decaying organic matter, and is easy to reproduce in the withered grass juice. The single cell of Bacillus subtilis is 0.7~0.8×2~3μm, uniformly colored, without capsule, with perinatal flagella, able to move, and is a Gram-positive bacterium. The spores are 0.6~0.9×1.0~1.5μm, elliptical to columnar, located in the center of the thallus or slightly off, and the thallus does not expand after the formation of spores. The surface of the colony is rough and opaque, stained white or yellowish, and often forms wrinkles when growing in liquid medium. Bacillus subtilis can use protein, various sugars and starch, decompose tryptophan to form indole, and can also synthesize vitamin ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01C12P25/00C12R1/125
CPCC12R1/125C12P25/00C12N1/205C12R2001/125
Inventor 马成兵陈军王涛
Owner XINFA PHARMA
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