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Method for efficiently producing vitamin K2 by flavobacterium

A vitamin and Flavobacterium technology, applied in the field of high-efficiency production of vitamin K2 by using Flavobacterium, can solve the problems of reducing MK-4 production and product inhibition, and achieve the effects of high-efficiency biosynthesis, improved fermentation level, and convenient use

Inactive Publication Date: 2013-09-11
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, during the fermentation process of Flavobacterium, due to the accumulation of MK-4 in the cells of Flavobacterium, product inhibition is caused, which reduces the yield of MK-4, and new methods that can reduce product inhibition are needed.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] This embodiment includes the following steps:

[0024] (1) Using Flavobacterium 1.6859 as the starting strain, culture Flavobacterium in the following plate medium (g / L) for activation: glucose 15g / L, potato extract 180g / L, glycerol 5g / L, agar 20g / L, PH natural; temperature 37°C, culture time 3 days;

[0025] (2) Dilute the activated bacteria 100 times with sterile normal saline to achieve a bacterial concentration of 10 per ml 7 Use a micropipette to take 0.1 mL of the bacterial suspension and spread it evenly on a sterile plate, place it on an ultra-clean workbench to dry naturally, and put the plate into the target chamber of a low-energy ion implanter for N + Injection, the injection energy is 15kev, the dose is 2.0×10 15 N + / cm 2 ; At the same time, the sample that did not accept ion implantation under vacuum environment was used as a contrast; after ion implantation irradiation, the treated sample and the control sample were eluted with 1mL sterile saline on ...

Embodiment 2

[0029] This embodiment includes the following steps:

[0030](1) Using Flavobacterium 1.6859 as the starting strain, culture Flavobacterium in the following plate medium (g / L) for activation: glucose 15g / L, potato extract 180g / L, glycerol 5g / L, agar 20g / L, Natural PH; temperature 35°C, culture time 3 days;

[0031] (2) Dilute the activated bacteria 100 times with sterile normal saline until the concentration of the bacteria in the bacteria solution is 10 per milliliter 7 Use a micropipette to take 0.1 mL of the bacterial suspension and spread it evenly on a sterile plate, place it on an ultra-clean workbench to dry naturally, and put the plate into the target chamber of a low-energy ion implanter for Ar+ ion implantation. , the injection energy is 10kev, and the dose is 3.9×10 15 N + / cm 2 ; At the same time, the sample that did not accept ion implantation under vacuum environment was used as a contrast; after ion implantation irradiation, the treated sample and the contro...

Embodiment 3

[0035] This embodiment includes the following steps:

[0036] (1) Using Flavobacterium 1.6859 as the starting strain, culture Flavobacterium in the following plate medium (g / L) for activation: glucose 15g / L, potato extract 180g / L, glycerol 5g / L, agar 20g / L, Natural PH; temperature 30°C, culture time 3 days;

[0037] (2) Dilute the activated bacteria 100 times with sterile normal saline until the concentration of the bacteria in the bacteria solution is 10 per milliliter 7 Use a micropipette to take 0.1 mL of the bacterial suspension and spread it evenly on a sterile plate, place it on an ultra-clean workbench to dry naturally, and put the plate into the target chamber of a low-energy ion implanter for Ar + Ion implantation, the implantation energy is 60kev, the dose is 1.3×10 15 N + / cm 2 ; At the same time, the sample that did not accept ion implantation under vacuum environment was used as a contrast; after ion implantation irradiation, the treated sample and the control...

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Abstract

The invention discloses a method for efficiently producing vitamin K2 by flavobacterium. High-producing strains are bred by means of composite mutagenesis through a low-energy ion beam and a low-temperature plasma after original strains of the flavobacterium are activated on culture media, fermentation units of the high-producing strains are increased by 50-100% through preliminary screening and shake flask fermentation secondary screening of an analog flat plate with the vitamin K2, intracellular vitamin K2 is dissolved to an extracellular environment by adding surface active agents when the flavobacterium is fermented, and the yield of the vitamin K2 is improved by 50-200%. By the aid of the method, the fermentation level of the vitamin K2 can be greatly improved, high-efficiency biosynthesis of the vitamin K2 is realized, large-scale industrialized fermentation of the vitamin K2 is possible, and the method is convenient to use.

Description

technical field [0001] The invention relates to a method for mutagenic selection and improvement of fermentation level by using Flavobacterium, in particular to a method for efficiently producing vitamin K2 by using Flavobacterium. Background technique [0002] Vitamin K is a kind of fat-soluble vitamin, its structure is a series of polyene compounds containing 2-methyl-1,4-naphthoquinone, naturally occurring vitamin K can be divided into vitamin K1 and vitamin K2 according to the different forms of its side chains. Vitamin K1 is rich in green plants and animal livers. Vitamin K1 is a series of compounds, light yellow crystals, mainly synthesized by intestinal bacteria; vitamin K2 is defined according to the length of the isoprene side chain on C-3 , The isoprene side chain generally contains 4-13 isoprene units, represented by MK-n (n refers to the number of isoprene units on the side chain; the most important ones are MK-4 and MK- 7. The vitamin K2 biosynthesis pathway is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/66C12R1/20
Inventor 郑之明刘艳王鹏王丽赵根海贡国鸿刘会宋均营
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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