Strain capable of highly producing vitamin K2 (MK-7), screening method of strain and method for producing vitamin K2 (MK-7)

A high-yield strain, MK-7 technology, applied in the field of microbial fermentation, can solve the problems of not meeting the normal intake of the human body, low activity cis isomers, complex reaction steps, etc., achieve good passaging stability, and reduce pollution emissions , the effect of increasing production

Active Publication Date: 2021-12-07
NORTH CHINA PHARMA COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]In recent years, the preparation and industrialization of vitamin K2 (MK-7) has become a research hotspot. Currently, there are three ways to obtain vitamin K2 (MK-7): ( 1) Direct intake from fermented foods, such as natto, tempeh, cheese and other foods contain trace amounts of vitamin K2 (MK-7), but the content is very low and cannot meet the normal intake of the human body
(2) Chemical synthesis of vitamin K2 (MK-7). At present, vitamin K2 (MK-7) can be artificially chemically synthesized, but the chemical synthesis reaction steps are complicated, the yield is low, and the cis isomer with low activity will be produced. At the same time, a large number of by-products are produced, causing serious environmental pollution.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Screening of vitamin K2 (MK-7)

[0034] 1.1, preparation of the medium

[0035]LB medium preparation: 5 g of yeast extract, 10 g of tryptone, 5 g sodium chloride, 15 g of agar, is disposed to 1000 mL with distilled water, and then the pH is adjusted to 7.0 ± 0.2 with a mass fraction of 30% sodium hydroxide solution. In several test tubes, 20 min at 121 ° C is sterilized. After sterilization is completed, a slope medium is obtained, and a slope medium is obtained, and the sterilized Lb medium is poured into the plate to obtain an Lb medium.

[0036] Seed medium preparation: 10 g of glycerol, 5g yeast, 10g soy protein, distilled water to 1000 mL, then adjust pH to 7.0 ± 0.2 with a mass fraction of 30% sodium hydroxide solution, each 250ml triangle bottle 30 ml seed medium, 8-slice gauze seal, 20 min at 121 ° C for 20 min, and the seed medium was obtained.

[0037] Fermentation medium preparation: 180 g of glycerol, 15g yeast powder, 240 g soy protein, 7.5 g sodium chloride, 3...

Embodiment 2

[0045] Study on Genetic Stability of Vitamin K2 (MK-7)

[0046] (1) Paste atrium BK21018 prepared in Example 1 was inoculated in Example 1 in the case of the beveled medium prepared in Example 1, and the beveled strain was deeply accumulated under 37 ± 0.5 ° C, relative humidity 40% -60%. After mature, it is again cultured in a beveled medium and repeated four times;

[0047] (2) The five-cultured strains in step (1) each transferred to the seed medium prepared in Example 1, cultured at 37 ± 0.5 ° C, relative humidity 40% -60%, 220 rpm, 16 When the bacteria was ripe, each of the fermentation medium prepared by the preparation Example 1 was taken in 37 ± 0.5 ° C, the relative humidity was 40% -60%, 220 rpm, and the five groups contain vitamin K2 ( MK-7) fermentation liquid;

[0048] (3) The effect of vitamin K2 (MK-7) in the fermentation liquid in the fermentation liquid in HPLC, and the result is: 232 g / ml, 239 g / ml, 231 g / ml, 236 g / ml according to the sequence of culture....

Embodiment 3

[0051] Vitamin K2 (MK-7) high-strain fermentation process control

[0052] 3.1, Preparation of Medium

[0053] Seed tank medium preparation: 100g glycerol, 100g yeast powder, 100g soy protein, to 9L with tap water, then adjust pH to 7.0 ± 0.2 with a hydroxide solution of mass fraction of 30%, and carry out seed can For dismissal, sterilization of 30 min at 121 ° C, cooled by circulating water, and 10 L seed culture is obtained.

[0054] Fermentation tank medium preparation: 3000 g of glycerol, 250g yeast powder, 4000 g soy protein, 150 g sodium chloride, 500g sucrose, 1 g nicotinamide, 1 g aspartic acid, use tap water to 45L, then use mass fraction of 30 The% sodium hydroxide solution regulates pH to 7.0 ± 0.2, and the fermenter was eliminated, 30 min at 121 ° C, cooled by circulating water, and 50 L of fermentation medium was obtained.

[0055] Fermentation process control of high strains: BK21018 strain inoculated into the LB medium, and in 37 ± 0.5 ° C, relative humidity 40% -6...

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PUM

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Abstract

The invention discloses a strain capable of highly producing vitamin K2 (MK-7), a screening method of the strain and a method for producing the vitamin K2 (MK-7) by using the strain, and belongs to the field of microbial fermentation. The strain is preserved in China Center for Type Culture Collection on August 23, 2021, and the preservation number is CCTCC M20211066. Mutagenesis is performed by using nitrosoguanidine, and the strain is obtained through shake flask screening, and has the characteristics of high shake flask titer, good passage stability and the like. When the strain is used for fermentation, a fermentation unit can reach 240 mg/L or above in cooperation with an optimal fermentation culture medium and a control process, the fermentation unit is improved by 243% compared with that of an original strain, the yield of the vitamin K2 (MK-7) can be effectively increased, the cost of raw materials, water, electricity, steam and the like for fermentation is obviously reduced, pollution emission of the unit yield is reduced, and the strain has high economic benefits.

Description

Technical field [0001] The present invention is directed to a vitamin K2 (MK-7) high strain and its screening method and a method of producing vitamin K2 (MK-7), belonging to the field of microbial fermentation. Background technique [0002] Vitamin K2 is also referred to as methylene, is a fat-soluble vitamin, which is initially isolated from natto, and vitamin K2 is named MK-4 to MK-4 to MK-4 to MK- 13. The most important subtype is a naphthalene-7 (MK-7), that is, the vitamin K2 side chain contains 7 isoprene units, the MK-7 is the strongest, the most lastmost, the highest safety The lowest dose is the longest in the blood. [0003] Vitamin K2 (MK-7) has a certain role in preventing arterial calcification, promoting liver function recovery, reducing diabetes risks, preventing cancer and Parkinson, etc., which play an important role in preventing and treating osteoporosis. Vitamin K2 (MK-7) As a new generation of health food, its functions are being received more and more, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/01C12N1/02C12P7/66C12R1/125
CPCC12N1/20C12N15/01C12N1/02C12P7/66
Inventor 尹贵超段逸飞王成张军剪王淑琳李默晗高雨宁丁宝娟王云牛霄亮李景
Owner NORTH CHINA PHARMA COMPANY
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