阅读说明：本技术 一种聚谷氨酸的生产工艺 (Production process of polyglutamic acid ) 是由 洪立芝 于 2020-05-26 设计创作，主要内容包括：本发明公开了一种聚谷氨酸的生产工艺,其包括：蜡样芽孢杆菌1205和解淀粉芽孢杆菌SFPG-123共发酵生产低分子聚谷氨酸；所述蜡样芽孢杆菌菌株的保藏号为：CCTCC NO:M2012382。所述解淀粉芽孢杆菌的保藏号为：CGMCC N0.3447。本公司研发团队在菌株筛选中发现,该株蜡样芽孢杆菌1205可以促进解淀粉芽孢杆菌SFPG-123生产聚谷氨酸的效率,并且显著降低分子量,可以一次性发酵得到低分子量,高生物活性的聚谷氨酸,而不需要进一步加工,节省成本和时间。(The invention discloses a production process of polyglutamic acid, which comprises the following steps: co-fermenting the bacillus cereus1205 and the bacillus amyloliquefaciens SFPG-123 to produce low-molecular polyglutamic acid; the preservation number of the bacillus cereus strain is as follows: CCTCC NO: M2012382. The preservation number of the bacillus amyloliquefaciens is as follows: CGMCC N0.3447. The research and development team of the company discovers that the strain of the bacillus cereus1205 can promote the efficiency of producing polyglutamic acid by the bacillus amyloliquefaciens SFPG-123, obviously reduce the molecular weight, can obtain the polyglutamic acid with low molecular weight and high biological activity by one-time fermentation without further processing, and saves the cost and time.)

1. A method for producing polyglutamic acid, comprising:

co-fermenting Bacillus cereus1205 (Bacillus cereus1205) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SFPG-123 to produce low-molecular polyglutamic acid;

the preservation number of the bacillus cereus strain is as follows: CCTCC NO of M2012382;

the preservation number of the bacillus amyloliquefaciens is as follows: CGMCC N0.3447.

2. The production method according to claim 1, characterized by the steps of:

(1) respectively activating:

respectively activating bacillus cereus1205 and bacillus amyloliquefaciens SFPG-123;

(2) seed culture:

transferring the activated strain into a seed culture medium, and carrying out shaking culture at 37 ℃ and 170rpm for 12 hours;

(2) and (3) shaking flask fermentation:

inoculating Bacillus cereus1205 into a fermentation tank according to Bacillus amyloliquefaciens SFPG-123, and performing ventilation stirring culture at 38 ℃ for 33-60 hours;

(3) and (4) separating the polyglutamic acid.

3. The production method according to claim 1, characterized in that the step (1) is:

respectively activating bacillus cereus1205 and bacillus amyloliquefaciens SFPG-123, wherein the used culture medium is standard MRS broth culture medium; the activation condition is shake culture at 37 ℃ for 12 h.

4. The production method according to claim 2 or 3, characterized in that the step (2) is:

transferring the activated strain into a seed culture medium, and carrying out shaking culture at 37 ℃ and 170rpm for 12 hours;

a seed culture medium comprising L-glutamic acid 20 g/L, glucose 60 g/L0.5.5 g, monopotassium phosphate 3 g/L, disodium hydrogen phosphate 2 g/L, magnesium sulfate heptahydrate 0.5 g/L, biotin 5 × 10-4 g/L and pH 7.0;

the bacterial load of the seed adjusting liquid is 107cfu/m L.

5. The production method according to any one of claims 2 to 4, characterized in that the step (3) is:

adjusting pH of the fermentation liquid to 6, centrifuging at 8000rpm and 4 deg.C for 20 min to obtain thallus, precipitating the supernatant with 4 times volume of anhydrous ethanol, and centrifuging at 6000rpm and 4 deg.C for 20 min to obtain precipitate. Dissolving with distilled water, dialyzing for 48 hours, and then freezing and drying in vacuum to obtain the crude product of the gamma-polyglutamic acid.

6. Polyglutamic acid obtained by the production method according to any one of claims 1 to 5.

Technical Field

The invention relates to a production process of polyglutamic acid, in particular to a production process for producing low-molecular polyglutamic acid by using microorganisms.

Background

Gamma-polyglutamic acid (gamma-PGA) is a polyamino acid compound formed by binding D-glutamic acid and L-glutamic acid through gamma-amide bonds, and is structurally and functionally different from proteins since it is different from general polypeptides in the way of linkage, although it is a polymer of amino acids.

The relative molecular mass (Mr) of γ -PGA prepared by microbial fermentation is generally about 100 × 10³～10 000×10³Each gamma-PGA molecule consists of about 500-5000 units of glutamic acid monomer, and gamma-PGAs with different Mr have different applications. Generally, as Mr increases, the viscosity of the solution increases, and the rheology is more difficult to control and to be made more difficult to be changedChemical reagent modification ultimately limits the production and application of gamma-PGA.

The gamma-PGA has a great amount of peptide bonds on the main chain, can be biologically degraded into small short peptide molecules and amino acid monomers in the nature or in the human body, does not generate toxic or side effect, has a great amount of free side chain carboxyl (-COOH) with higher activity on the molecular chain, is easy to combine with some medicines to generate stable compounds, has excellent biocompatibility and biodegradability, and is an ideal bioabsorbable medical high polymer material.

The gamma-PGA hydrogel is a colorless, tasteless, transparent and soft colloid, has a unique three-dimensional lattice structure, has super strong water absorption and moisture retention properties, and is very suitable for improving the moisture retention effect in cosmetics. However, the excessively strong water absorption and moisture retention properties of polyglutamic acid tend to compete for the moisture of the skin in a cold and dry environment, and the skin surface is dry and chapped. One method of adjusting the water absorption of γ -PGA is to obtain low molecular γ -PGA by moderate degradation.

The invention aims to provide a production process for producing low-molecular polyglutamic acid by using microorganisms.

Disclosure of Invention

The invention aims to provide a production process for producing low-molecular polyglutamic acid by using microorganisms.

Chinese patent 201010150439 discloses a Bacillus strain for high yield of gamma-polyglutamic acid, which is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SFPG-123, and the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11/16 th 2009 with the preservation number of CGMCC N0.3447, wherein the produced high molecular polymer is analyzed and identified as gamma-polyglutamic acid, the strain can convert glutamic acid, citric acid and the like to generate gamma-polyglutamic acid, and the yield of the gamma-polyglutamic acid in unit volume of fermentation broth can reach 15-20 g/L.

However, during production, the relative molecular mass (Mr) of the gamma-polyglutamic acid produced by the strain is higher and is about 2500-2600 × 10³The product has high viscosity and difficult post processing.

Research by research teams of the company discovers that the molecular weight of the product gamma-polyglutamic acid can be obviously reduced by adopting a method of co-fermentation of specific strains, and in addition, the yield can be further improved, and the fermentation time can be shortened.

Chinese patent 2012105218189 discloses a Bacillus cereus strain, which has been deposited in China center for type culture Collection (address: Wuhan university in Wuhan, China) at 26/9/2012 with the following deposition numbers: CCTCC NO, M2012382, with the name: bacillus cereus1205 (Bacillus cereus 1205). The known function of the strain is that phytophthora nicotianae has an inhibiting effect.

The research and development team of the company discovers in strain screening that the strain of Bacillus cereus1205 can remarkably promote the efficiency of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SFPG-123 in producing polyglutamic acid, remarkably reduce the molecular weight, can ferment at one time to obtain low-molecular-weight and high-bioactivity polyglutamic acid through co-fermentation, and can obtain the micromolecule polyglutamic acid with high bioactivity without further hydrolysis processing, thereby saving the cost and time.

The technical problem to be solved by the invention can be realized by the following technical scheme.

A method for producing polyglutamic acid, comprising:

the low molecular polyglutamic acid is produced by co-fermenting Bacillus cereus1205 (Bacillus cereus1205) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SFPG-123.

The preservation number of the bacillus cereus strain is as follows: CCTCC NO: M2012382.

The preservation number of the bacillus amyloliquefaciens is as follows: CGMCC N0.3447.

Preferably, the production method comprises the following steps:

(1) respectively activating:

respectively activating bacillus cereus1205 and bacillus amyloliquefaciens SFPG-123, wherein the used culture medium is standard MRS broth culture medium; the activation condition is shake culture at 37 ℃ for 12 h;

(2) seed culture:

transferring the activated strain into a seed culture medium, and carrying out shaking culture at 37 ℃ and 170rpm for 12 hours;

the seed culture medium comprises L-glutamic acid 20 g/L, glucose 60 g/L0.5.5 g, potassium dihydrogen phosphate 3 g/L, disodium hydrogen phosphate 2 g/L, magnesium sulfate heptahydrate 0.5 g/L, and biotin 5 × 10^-4g/L，pH7.0；

The bacterial load of the seed liquid is regulated as follows: 10⁷cfu/mL；

(2) And (3) shaking flask fermentation:

inoculating bacillus amyloliquefaciens SFPG-123 with the inoculation amount of 2 percent and bacillus cereus1205 with the inoculation amount of 1 percent into a fermentation tank, and performing ventilation stirring culture at 38 ℃ for 33-60 hours;

the formula of the fermentation medium is as follows:

30 g/L of sodium citrate, 20 g/L of sodium glutamate, 7 g/L of ammonium chloride, 10 g/L of soybean meal, 10 g/L of yeast powder, 3 g/L of potassium dihydrogen phosphate, 2 g/L of disodium hydrogen phosphate, 0.5 g/L of magnesium sulfate heptahydrate, 5 × 10 of biotin, 10 g/L of sodium glutamate^-4g/L，pH7.0；

(3) And (3) separating polyglutamic acid:

adjusting pH of the fermentation liquid to 6, centrifuging at 8000rpm and 4 deg.C for 20 min to obtain thallus, precipitating the supernatant with 4 times volume of anhydrous ethanol, and centrifuging at 6000rpm and 4 deg.C for 20 min to obtain precipitate. Dissolving with distilled water, dialyzing for 48 hours, and then freezing and drying in vacuum to obtain the crude product of the gamma-polyglutamic acid.

The invention relates to a method for measuring the molecular weight of gamma-polyglutamic acid, which comprises the following steps:

and measuring the molecular weight of the fermentation product gamma-polyglutamic acid by adopting gel permeation chromatography.

The yield of polyglutamic acid was calculated as crude polyglutamic acid (g) isolated/fermentation broth (L).

The invention has the advantages that:

the research and development team of the company discovers in strain screening that the strain of the Bacillus cereus1205 can promote the efficiency of producing polyglutamic acid by the Bacillus amyloliquefaciens SFPG-123 (the fermentation time is shortened from about 55 hours to about 33 hours), obviously reduce the molecular weight, and can obtain the polyglutamic acid with low molecular weight and high biological activity by one-time fermentation without further processing, thereby saving the cost and time.

Detailed Description

The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.

Specific examples of the present invention are described below.