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Bacillus subtilis natto for high yield rate of vitamin K2 (MK-7) and application thereof

A technology of Bacillus natto and MK-7, applied in the field of microorganisms, can solve the problems of long fermentation period and high time cost, and achieve the effects of stable yield, saving time and cost, and good genetic stability.

Active Publication Date: 2018-08-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, at present, the fermentation cycle of bacterial strains to produce vitamin K2 is also relatively long, requiring more than 6 days, and the time cost is relatively large.

Method used

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  • Bacillus subtilis natto for high yield rate of vitamin K2 (MK-7) and application thereof
  • Bacillus subtilis natto for high yield rate of vitamin K2 (MK-7) and application thereof
  • Bacillus subtilis natto for high yield rate of vitamin K2 (MK-7) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Isolation and identification of initial bacterial strain Bacillus subtilis natto (Bacillus subtilis natto) ND-1 and preservation of bacterial strain

[0027] (1) Isolation and screening of Bacillus subtilis natto ND-1

[0028] The present invention buys natto from the market, weighs 1.0-2.0g soil sample in a 250mL triangular flask, suspends it with 10-50mL physiological saline, then carries out gradient dilution, and takes 0.1mL dilution and spreads it on a nutrient agar plate for cultivation . After culturing at 37°C for 8 hours, a single colony with better growth was selected for fermentation verification. Pick a single colony and inoculate it into a fresh liquid seed medium, cultivate it at 37°C and 90-120r / min for 8h, and then insert a 2% (w / w) inoculum into a fermentation medium with a liquid volume of 30mL / 250mL, After fermenting for 3-6 days at 37°C under static culture conditions, the fermented liquid is collected, extracted with an extractant and t...

Embodiment 2

[0034] Example 2: Utilizing ARTP to ND-1 mutagenesis and utilizing structural analogues to carry out preliminary screening of mutagenized strains

[0035] (1) Preparation of bacterial suspension

[0036] Pick a ring of Bacillus subtilis natto ND-1 strain on the nutrient agar medium, inoculate it in a 250mL Erlenmeyer flask containing 30mL seed medium, place it on a shaker at 120r at 37°C Cultivate at a rotational speed of 1 / min for 8 hours to the logarithmic phase, and centrifuge to suspend the bacteria with physiological saline. Properly dilute the bacteria with normal saline to an OD value between 0.8-1.2, put the metal slide on the outer flame of the alcohol lamp in the ultra-clean bench, burn it, put it on a sterilized glass plate after cooling, and take the bacteria suspension The solution was evenly applied to the slide, and the mutagenesis was carried out without air drying.

[0037] (2) ARTP mutagenesis

[0038] The operating compartment of the mutagenesis system is...

Embodiment 3

[0041] Example 3: Fermentative production of vitamin K2 (MK-7) by strains after preliminary screening by mutagenesis

[0042] (1) Preparation of cell liquid culture of mutagenized strains

[0043] Pick 31 mutagenic strains and the initial strain Bacillus subtilis natto (Bacillus subtilis natto) ND-1 on the medium containing structural analog resistance respectively and inoculate them in a 250mL Erlenmeyer flask containing 30mL seed medium. Cultivate on a shaker at 120 r / min for 8 hours to the logarithmic phase at a temperature of 100° C. to obtain a liquid cell culture of the mutagenized strain.

[0044] (2) The composition and proportioning of the fermentation medium are:

[0045] Glycerin 50g / L; yeast powder 50g / L; soybean peptone 189g / L; dipotassium hydrogen phosphate 0.6g / L;

[0046] (3) Shake flask fermentation:

[0047] Inoculate the liquid cell culture of the above bacterial strain in a 250mL Erlenmeyer flask with 30mL of sterilized fermentation medium at an inoculum...

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Abstract

The invention discloses bacillus subtilis natto for high yield rate of vitamin K2 (MK-7) and application thereof, and belongs to the field of microorganisms. The bacillus subtilis natto ND-1-A27 was collected into China Center for Type Culture Collection on March 14, 2018 in Wuhan University of China, and the collection number is CCTCC NO:M 2018131. The bacillus subtilis natto has the advantages that the yield rate of the vitamin K2 (MK-7) is greatly increased and is about 70mg / L, and the maximum yield rate can be reached by about three days; the fermenting cycle is greatly shortened, the timeis shortened, and the cost is reduced; the bacillus subtilis natto can be applied to industrialized fermenting production, and the genetic stability is good; after five generations are continuously transferred, the yield rate of the vitamin K2 (MK-7) is basically stable, and is maintained at the higher level; the bacillus subtilis natto can be used as a production bacterial strain for further study and development.

Description

technical field [0001] The invention relates to a high-yield vitamin K2 (MK-7) bacillus natto and application thereof, belonging to the field of microorganisms. Background technique [0002] Vitamin K2 (menaquinone, MK) is a fat-soluble vitamin, a derivative of a naphthoquinone group with biological activity of phylloquinone, and is one of the indispensable important vitamins in the human body. Vitamin K2 is a series of menaquinone compounds, light yellow crystals, there are 14 forms according to the length of the isoprene side chain at the C-3 position of its molecular structure, and MK-n refers to the isoprene on the side chain The number of units), among which MK-7 (natural vitamin K2) has the most significant biological activity. [0003] At present, vitamin K2 (MK-7) is mainly chemically synthesized, but the traditional chemical synthesis method has problems such as limited sources of chemical precursor raw materials, a large number of isomers produced by chemical reac...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/66C12R1/125
CPCC12N1/20C12P7/66C12N1/205C12R2001/125
Inventor 李会徐行刘瑞涵史劲松许正宏张晓梅龚劲松
Owner JIANGNAN UNIV
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