CN112175889A

CN112175889A - A high-yielding vitamin K2 strain and its application

Info

Publication number: CN112175889A
Application number: CN202011249346.7A
Authority: CN
Inventors: 任路静; 彭程; 胡学超; 卢健尧
Original assignee: Nanjing Tech University
Current assignee: Jiangsu Yingyangxing Biotechnology Co ltd
Priority date: 2020-11-10
Filing date: 2020-11-10
Publication date: 2021-01-05
Anticipated expiration: 2040-11-10
Also published as: CN112175889B

Abstract

The invention discloses a high-yield vitamin K2 strain and its application. The strain is classified and named Bacillus subtilis ALE-Gly, and the preservation number is GDMCC No: 61234. The invention utilizes the medium containing the inhibitor to domesticate the original strain, subcultures it in the seed medium containing glyphosate, and then restores the culture in the common seed medium to obtain the domesticated strain. The domesticated strains were fermented, and the vitamin K2 production, biomass and yield were increased by 4 times, 2 times, and 2.4 times, respectively, compared with the original strains. At the same time, the spore-forming time of the domesticated strains was delayed by 24 hours, and the enzyme activity of EPSP synthase was improved. Compared with the original strain, the fermentation time is shortened, and the production cost is reduced.

Description

Strain capable of highly producing vitamin K2 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a strain for highly producing vitamin K2 and application thereof.

Background

Menaquinones (MK), also called vitamin K2, is a trace fat-soluble vitamin produced by probiotic flora in human intestinal tract, is a general name of a group of substances containing 2-methyl-1, 4-naphthoquinone ring, has phylloquinone bioactivity, is one of essential vitamins in human body, is an important fat-soluble blood coagulation vitamin, and mainly has the function of

Preventing and treating osteoporosis.

Preventing artery calcification and osteoarthritis.

And (3) resisting tumors.

Delaying senility

Repairing damaged cells and potentially treating the Parkinson's disease. The safety of the drug is determined by the authorities of the food and drug administration, the drug research institute and the European parliament and the European Union council. The natural menadione with higher activity can be obtained only by a microbial fermentation method, so the microbial fermentation method is concerned by scholars at home and abroad and has wide application prospect. The bacillus natto belongs to subspecies of bacillus subtilis, and has the advantages of safety, high growth speed, short fermentation period, easy culture, high menadione content and the like, so that the bacillus natto is the most main microorganism for preparing menadione by fermentation at present, and the product of the bacillus natto is mainly menadione-7 (MK-7).

Currently, most researchers mainly utilize bacillus natto to directly ferment and produce vitamin K2 or construct a metabolic pathway of vitamin K2 in bacillus subtilis to produce vitamin K. The prior art about the production of vitamin K2 by fermentation of Bacillus mainly comprises: mutation breeding of a VK2 production strain; 2. optimized with respect to medium composition. The traditional fermentation regulation and control process is difficult to stably control, and the fermentation regulation and control mainly changes the components of a culture medium or culture conditions, so that the performance of the strain is not fundamentally optimized. The traditional strain transformation mainly adopts a mutagenesis screening method, but the screening work is blind due to the lack of a screening way aiming at the strain with high VK2 content, so the strain screening workload is large, and the screening effect is not ideal. Therefore, the new strain performance improvement method has important significance for improving the fermentation content of VK 2.

Disclosure of Invention

Aiming at the technical problem of relative blindness in the strain breeding process in the prior art, the technology obtains a strain with high vitamin K2 yield by an oriented domestication method.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

strain capable of producing vitamin K2 with high yield, and its classification name isBacillus subtilis ALE-Gly with the deposit number GDMCC No: 61234.

the invention obtains a high-yield strain of vitamin K2 by an acclimatization method, adds a chemical reagent of glyphosate into an acclimatization culture medium, cultures bacillus natto in the culture medium containing the glyphosate for acclimatization, and screens out the ALE-Gly strain.

The invention also aims to provide application of the strain in fermentation production of VK2, wherein the strain is subjected to seed culture and fermentation culture to obtain a product.

Further, the seed culture mode is as follows: inoculating the strain to a seed culture medium for activation culture in an inoculation amount of 1% (V/V), and performing two-generation activation;

further, the conditions for activating each generation of seeds were 12 h, 37 ℃ and 200 rpm.

Further, the culture medium for seed culture comprises the following components: 30-60 g/L of glucose, 10-15 g/L of tryptone, 5-7 g/L of NaCl and 5-7 g/L of yeast powder.

Further, the components of a culture medium for fermentation culture are as follows: soybean peptone 60-100 g/L, glycerin 30-60 g/L, yeast powder 0.6-0.8 g/L, K₂HPO₄·3H₂O 6.55～8.55 g/L，MgSO₄·7H₂0.3-0.5 g/L of O and anhydrous CaCl₂ 0.0775～0.0975 g/L。

Further, adding a small molecule compound into a fermentation medium; the small molecular compound is selected from pyruvic acid, shikimic acid and sodium glutamate;

furthermore, the addition concentration of the small molecular compound is 2-4 g/L of pyruvic acid, 2-10 g/L of shikimic acid and 10-15 g/L of sodium glutamate. .

Furthermore, 4 g/L pyruvic acid, 4 g/L shikimic acid and 15 g/L sodium glutamate are added into the fermentation medium at the same time.

The invention has the beneficial effects that:

(1) the invention obtains a high-yield VK2 strain through a brand-new idea and method for domesticating and transforming strains, the domestication process does not relate to a genetic engineering method, and the domestication process is carried out for multiple generations by simply using an inhibitor of key enzyme in a biosynthetic metabolic pathway of vitamin K2. The method is environment-friendly, simple and convenient, and has no increase in manpower and material resources.

The technical principle of acclimatization may be that EPSP synthase is a key enzyme in metabolic pathway of synthesizing VK2 by bacillus, glyphosate is an inhibitor, if the strain grows in a seed culture medium containing glyphosate, the activity of EPSP synthase is improved in order to resist the poison of glyphosate, VK2 is an important electron transfer carrier in an oxygen breathing electron transfer chain, and enough VK2 must be synthesized to resist the severe environment in order to successfully carry out oxygen breathing. Only strains with high EPSP synthase activity can survive in the environment, and therefore, the method also plays a role in screening high-yield bacteria of VK 2.

(2) The strain obtained by domestication has vigorous growth in a normal culture medium, the carbon and nitrogen source consumption speed is accelerated, the fermentation period is short, the maximum yield is basically reached within 72 hours, the spore generation time is delayed for 24 hours, the biomass is 2 times higher than the original biomass, the VK2 yield is 4 times higher than the original yield, the EPSP synthase activity is enhanced by 2-3 times, the nitrogen source utilization rate is high, the domesticated strain has stable physiological and biochemical conditions and stable genetic performance, continuous passage is carried out for 5 times, the fermentation produced vitamin K2 is at a higher level, and the domesticated strain is an excellent strain with high vitamin K2 yield.

(3) The fermentation culture process is optimized, and pyruvic acid, shikimic acid and sodium glutamate are added into the fermentation culture medium, so that the yield of VK2 is improved.

Drawings

FIG. 1 is a graph showing the results of genetic stability of the acclimatized strain.

FIG. 2 shows MK-7 yields from fermentation of original A13 and domesticated ALE-40 (i.e., ALE-Gly) supplemented with different concentrations of small molecule compounds (shikimic acid, pyruvic acid, sodium glutamate).

FIG. 3 is a microscopic image of A13 and ALE-40 (i.e., ALE-Gly) fermentations.

The biological material of the invention is classified and namedBacillus subtilis ALE-Gly, which has been deposited in the Guangdong province culture Collection of microorganisms at 21.10.2020, with the deposit number GDMCC No: 61234, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

Detailed Description

The original strain related to the embodiment is obtained by laboratory self-screening, is disclosed in a patent CN104328064A previously applied in the laboratory, has a preservation number of CCTCC M2014405, and is named as Bacillus natto A13 in the embodiment.

Example 1

This example specifically illustrates the screening method of the strain of the present invention.

(1) Inoculating the original strain into seed culture medium at 1% (v/v) inoculation amount, culturing for 12 hr at 37 deg.C at 200 rpm to complete the first generation activation, and performing the second generation seed activation by the same method to obtain seed.

(2) Inoculating the activated strain into 5 mu mol/L glyphosate acclimatization culture medium with the inoculation amount of 1%, wherein the culture time is 12 hours, the rotation speed is 200 rpm, and the temperature is 37 ℃, and then completing the first generation acclimatization. And then, streaking the first generation of domesticated strains on a solid domesticated culture medium with the same concentration, then selecting a single colony with larger growth to be inoculated into the next domesticated culture medium (the concentration of glyphosate is 5 mu mol/L) with the same concentration of glyphosate, taking the single colony as the second generation, and repeating the steps in the same way to carry out five generations of domestication.

Seed culture medium: 30-60 g/L of glucose, 10-15 g/L of tryptone, 5-7 g/L of NaCl and 5-7 g/L of yeast powder.

Domestication culture medium: 30-60 g/L of glucose, 10-15 g/L of tryptone, 5-7 g/L of NaCl and 5-7 g/L of yeast powder; 5, 10, 15, 20, 25 mu mol/L glyphosate.

(3) After five generations of domestication is completed, the strain streaked and purified on the domestication plate is inoculated into a domestication culture medium with the next concentration of 10 mu mol/L glyphosate, the culture time is 12 hours, the rotating speed is 200 rpm, and the temperature is 37 ℃, thus completing the first generation of domestication. And then streaking the first generation of domesticated bacterial strain on a 10 mu mol/L glyphosate solid domesticated culture medium, then selecting a single bacterial colony to be inoculated into the next domesticated culture medium with the same glyphosate concentration, and carrying out fifth generation domestication by analogy.

(4) Until the acclimatization of three concentrations of 15, 20 and 25 mu mol/L is completed in sequence. When the strain can contain 25 mu moI & L^-1When the glyphosate is normally grown in the domestication culture medium, domestication is continuously carried out for 40 generations, domesticated strains ALE-10, ALE-20, ALE-30 and ALE-40 obtained by domestication for 10 generations, 20 generations, 30 generations and 40 generations are inoculated into a normal culture medium without the glyphosate to recover the growth, and a subsequent fermentation experiment is carried out.

Example 2

This example illustrates the method of producing vitamin K2 by fermentation using Bacillus natto according to the present invention.

(1) Seed culture

The seed culture medium comprises the following components: 30-60 g/L of glucose, 10-15 g/L of tryptone, 5-7 g/L of NaCl and 5-7 g/L of yeast powder.

Inoculating the domesticated strain into a seed culture medium with the inoculation amount of 1%, culturing for 12 hours at the rotation speed of 200 r/min to complete first-generation activation, and performing second-generation seed activation by the same method to obtain seeds.

(2) Fermentation culture

Fermentation medium: soybean peptone 60-100 g/L, glycerin 30-60 g/L, yeast powder 0.6-0.8 g/L, K₂HPO₄·3H₂O 0.3-0.6 g/L，MgSO₄·7H₂0.3-0.5 g/L of O and anhydrous CaCl₂ 0.0775～0.0975 g/L。

Inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 1-5% (v/v), wherein the fermentation temperature is 37 ℃, the rotation speed is 200 rpm, and the fermentation is stopped when the fermentation is carried out for 6 days.

Example 3

This example illustrates the shake flask fermentation performance comparison of acclimatized and original strains.

Strains domesticated in a glyphosate culture medium in different generations are selected, fermentation is carried out under normal conditions, and the fermentation performances (VK 2 production capacity) of the different domesticated strains and the original strains are compared under the normal culture medium.

The fermentation process in this example is as follows:

(1) seed culture

The seed culture medium comprises the following components: 30 g/L of glucose, 10 g/L of tryptone, 5 g/L of NaCl and 5 g/L of yeast powder.

(2) Fermentation culture

Fermentation medium: soybean peptone 90 g/L, glycerin 60 g/L, yeast powder 0.6 g/L, K₂HPO₄·3H₂O 0.3 g/L，MgSO₄·7H₂O0.3 g/L, anhydrous CaCl₂ 0.08 g/L。

During the fermentation process, biomass, glycerol consumption, VK2 yield and the like are measured, and the yield after 72h fermentation tends to be stable and approaches to the maximum yield. The fermentation parameters of the different acclimatized and original strains are compared in table 1.

TABLE 1 comparison of fermentation parameters of original and acclimatized strains

The ALE-40 strain domesticated for 40 generations has the highest VK2 content and yield, and is preserved and named ALE-Gly.

Example 4

And (3) verifying the genetic stability of the domesticated strain Bacillus natto ALE-Gly.

The strain ALE-Gly is subjected to shake flask fermentation and continuous culture for 5 generations to detect the genetic stability of the strain ALE-Gly. The yield of VK2 was determined after fermentation using the original strain Bacillus natto A13 as a control. The results of the subculture experiments with the acclimatized strain are shown in FIG. 1. As can be seen from the figure, the strain ALE-Gly has no obvious influence on the fermentation level through passage, the VK2 production capacity is stable, and the same higher level is maintained, which indicates that the strain ALE-Gly has good genetic stability.

Example 5

Adding different precursor substances to strengthen VK2 for synthesis.

The precursor addition is carried out on the basis of shake flask fermentation in example 3, namely pyruvic acid, shikimic acid and sodium glutamate with different concentrations are added in the fermentation process of the original strain. The concentration of pyruvic acid is: 2, 4, 6, 8,10 g/L; the concentration of shikimic acid is: 2, 4, 6, 8,10 g/L; the concentration of sodium glutamate is as follows: 5, 10, 15, 20, 25 g/L, the fermentation temperature is 37 ℃, and the rotation speed is 200 rpm. The final fermentation results are shown in Table 2, and the highest yield of VK2 in fermentation medium supplemented with 4 g/L pyruvic acid, 4 g/L shikimic acid and 15 g/L sodium glutamate of original bacteria A13 is 50, 48 and 38 mg/L respectively.

TABLE 2 precursor supply enhancement of the original strains

Example 6

Influence of combined addition of precursor substances on VK2 production of different strains

Based on the optimal addition concentration of the precursor substances obtained in example 5, three precursor substances with corresponding concentrations, namely 4 g/L pyruvic acid, 4 g/L shikimic acid and 15 g/L sodium glutamate, are further added in the fermentation process of the original strain and the domesticated strain, and the fermentation results are shown in FIG. 2, wherein the maximum yield of VK2 of the original strain A13 and the domesticated strain ALE-Gly is 63 mg/L and 82 mg/L respectively.

Claims

1. A high-yield vitamin K2 bacterial strain is characterized in that, the classification name is Bacillus subtilis ALE-Gly, and the deposit number is GDMCC No: 61234.

2. the application of Bacillus natto described in claim 1 in producing vitamin K2.

3. The application according to claim 2, wherein the bacterial strain is subjected to seed culture and fermentation culture to obtain a product.

4 . The application according to claim 3 , wherein the seed cultivation method is as follows: inoculation of the bacterial strain with 1% (V/V) inoculation amount into the seed medium for activation and cultivation, and two generations of activation are carried out. 5 .

5. application according to claim 4, is characterized in that, the condition of each generation seed activation is 12 h, 37 ℃, 200rpm.

6. application according to any one of claim 3-4 is characterized in that, the medium composition of seed culture is: glucose 30～60 g/L, tryptone 10～15 g/L, NaCl 5～7 g /L, yeast powder 5～7 g/L.

7. The application according to any one of claims 3-4, wherein the medium components of the fermentation culture are: soybean peptone 60～100 g/L, glycerol 30～60 g/L, yeast powder 0.6～0.8 g/L, K ₂ HPO ₄ ·3H ₂ O 0.3-0.6 g/L, MgSO ₄ ·7H ₂ O 0.3-0.5 g/L, anhydrous CaCl ₂ 0.0775-0.0975 g/L.

8 . The application according to claim 3 , wherein a small molecule compound is added to the fermentation medium; the small molecule compound is selected from the group consisting of pyruvic acid, shikimic acid, and sodium glutamate. 9 .

9. application according to claim 8, is characterized in that, described small molecule compound addition concentration is pyruvic acid 2～4g/L, shikimic acid 2～10 g/L, sodium glutamate 10～15 g/L .

10. application according to any one of claim 7～9, is characterized in that, in fermentation medium, add 4 g/L pyruvate simultaneously, 4 g/L shikimic acid and 15 g/L sodium glutamate.