Mutagenized high-yield natural vitamin K2 bacillus subtilis strain and application thereof

A Bacillus subtilis, strain technology, applied in the application, bacteria, microorganisms and other directions, can solve problems such as difficulty in meeting production needs, and achieve the effects of reducing fermentation costs, good strain stability, and reduced production performance

Active Publication Date: 2021-03-02
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production of vitamin K2 in wild strains is less than 3mg / L, which is difficult to meet the production needs

Method used

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  • Mutagenized high-yield natural vitamin K2 bacillus subtilis strain and application thereof
  • Mutagenized high-yield natural vitamin K2 bacillus subtilis strain and application thereof

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Experimental program
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preparation example Construction

[0027] (2) Preparation of starting strain bacterial suspension:

[0028] Pick a ring of colony from the preserved culture medium, inoculate it into a sterilized liquid culture medium and cultivate it to make it in the logarithmic growth phase, take a 50mL centrifuge tube, add 10mL of bacterial solution, and centrifuge at 5000r / min for 10min; discard the above To clear, add 10mL of sterilized normal saline to the precipitate, suspend and mix well, and repeat once; finally add 10mL of normal saline to the precipitate, suspend and mix well, the preparation of the bacterial suspension is completed, and store in the refrigerator at 4°C for later use.

[0029] (3) Ultraviolet mutagenesis and passage stability experiments of bacteria

[0030] Carry out ultraviolet irradiation: take 1ml of the above bacterial suspension in each of six 10ml centrifuge tubes, and place them under a 20W ultraviolet lamp at 10cm to irradiate for 0min, 5min, 10min, 15min, 20min, 25min (do 3 parallel experi...

Embodiment 1

[0039] This embodiment is a method for mutation breeding of Bacillus subtilis strain BB-MK-16 with high vitamin K2 yield, the steps are as follows:

[0040] (1) Cultivation of the starting strain:

[0041] Liquid medium: glucose 0.5%, soluble starch 0.5%, soybean protein 0.5%, peptone 1%, MgSO 4 0.5%, K 2 HPO 4 0.1%, NaCl0.5%, natural pH;

[0042] Solid medium: glucose 0.5%, soluble starch 0.5%, soybean protein 0.5%, peptone 1%, MgSO 4 0.5%, K 2 HPO 4 0.1%, agar 2%, NaCl 0.5%, natural pH;

[0043] The wild Bacillus subtilis in the laboratory was inoculated on the sterilized liquid medium and rejuvenated for 3 generations at 37°C to make it reach the normal growth cycle, and it was ready for use.

[0044] (2) Preparation of starting strain bacterial suspension:

[0045] Pick a ring of colony from the preserved culture medium, inoculate it into a sterilized liquid culture medium and cultivate it to make it in the logarithmic growth phase, take a 50mL centrifuge tube, a...

Embodiment 2

[0056] This embodiment is a fermentation method of Bacillus subtilis BB-MK-16 with high vitamin K2 production.

[0057] 2.1 Inoculate Bacillus subtilis BB-MK-16 on sterilized liquid medium for rejuvenation at 37°C for 3 generations to make it reach a normal growth cycle, and set aside for later use.

[0058] The composition of the liquid medium is: 0.5% glucose, 0.5% soluble starch, 0.5% soybean protein, 1% peptone, MgSO 4 0.5%, K 2 HPO 4 0.1%, NaCl 0.5%, natural pH; sterilized at 115°C for 30 minutes.

[0059] 2.2 Preparation of fermentation medium

[0060] Prepare fermentation medium for a 500mL Erlenmeyer flask: glucose 0.5%, soluble starch 0.5%, soybean protein 0.5%, peptone 1%, MgSO 4 0.5%, K 2 HPO 4 0.1%, agar 2%, NaCl 0.5%, natural pH; sterilized at 115°C for 30 minutes.

[0061] 2.3 Inoculation and culture

[0062] When the Erlenmeyer flask was cooled to 30°C, the Bacillus subtilis BB-MK-16 strain that was in the logarithmic growth phase through secondary c...

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Abstract

The invention discloses a mutagenized Bacillus subtilis strain and an application thereof. When cultured in a liquid medium at 37°C and 180r / min for 3 days, the content of vitamin K2 in the fermentation liquid of the strain of the present invention is above 62.08 mg / L, and the ability of the fermentation to produce vitamin K2 can be maintained for at least 10 generations Subculture. Compared with the currently known Bacillus subtilis, the ability of the bacterial strain of the present invention to produce vitamin K2 is increased by at least 24 times.

Description

technical field [0001] The invention relates to the modification of microorganisms, in particular to a stable and high-yield vitamin K2 bacillus subtilis strain and application thereof. Background technique [0002] Bacillus natto is a subspecies of Bacillus subtilis, which can secrete and synthesize a variety of organic acids, enzymes, physiologically active substances, etc., promote the proliferation of small intestinal mucosal cells, and acidify the intestinal tract to facilitate the absorption of iron, calcium and vitamin D. As an important probiotic for industrial production, Bacillus subtilis can synthesize enzymes such as a-amylase, protease, lipase, cellulase, etc., so it has been widely used in food, enzyme preparation, and feed industries. This bacterium is one of the 40 kinds of probiotics announced by the US FDA. It is safe to eat and has a clear genome. As early as 1995, Rowland et al. systematically clarified the genome of Bacillus subtilis. However, natural s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/66A23L33/15C12R1/125
CPCA23V2002/00A23L33/15C12N1/20C12P7/66C12N1/205C12R2001/125A23V2200/30A23V2250/7144
Inventor 李拖平周宇朦张岫姗李苏红
Owner SHENYANG AGRI UNIV
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