Fermentation method for regulating and controlling molecular weight of gamma-polyglutamic acid by using metal ions

Abstract

The invention discloses a fermentation method for regulating and controlling the molecular weight of gamma-polyglutamic acid by using metal ions, and belongs to the technical field of microbial fermentation. According to the method, in the fermentation process of bacillus subtilis, metal ions are added, so that the bacillus subtilis is fermented to produce high-molecular-weight or low-molecular-weight gamma-polyglutamic acid, the yield of the gamma-polyglutamic acid is increased, and the metal ions are divalent manganese ions or ferric ions; the high-molecular-weight gamma-polyglutamic acid is gamma-polyglutamic acid with the relative average molecular weight of more than or equal to 3400kD, and the low-molecular-weight gamma-polyglutamic acid is gamma-polyglutamic acid with the relative average molecular weight of less than or equal to 220kD. According to the method, the metal ions are added into the fermentation culture medium, so that the simple synthesis of the high-molecular-weight or low-molecular-weight gamma-PGA is realized, and meanwhile, the yield of the gamma-PGA can be effectively improved.

Description

Technical Field

[0001] The invention relates to a fermentation method for regulating the molecular weight of gamma-polyglutamic acid by using metal ions, and belongs to the technical field of microbial fermentation. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is an extracellular polyamino acid produced by microorganisms such as Bacillus subtilis under specific conditions, which can provide protection and nutrition for microorganisms. γ-PGA is composed of glutamic acid monomers through dehydration condensation of hydrogen atoms on the α-amino group and hydroxyl groups on the γ-carboxyl group to form amide bonds, and its relative molecular mass is usually between 10kDa-8000kDa. Because γ-PGA is safe and non-toxic, biodegradable, highly absorbent, and non-immunogenic, γ-PGA and its derivatives are widely used in fertilizers, environmental protection, food, cosmetics, medicine, water treatment and other fields, and have great commercial value and application potential.

[0003] γ-PGA with different relative molecular weights has different properties, so the relative molecular weight of γ-PGA is closely related to its application field. In cosmetics, γ-PGA with different molecular weights has different effects. High molecular weight γ-PGA has film-forming properties and can effectively prevent water loss; while low molecular weight γ-PGA is conducive to transdermal absorption, can deeply moisturize and care for the skin. In the field of medicine, high molecular weight γ-PGA can be used in the clinical treatment of cancer, while low molecular weight γ-PGA can be used as a drug carrier. At present, most of the research on the production of γ-PGA with different molecular weights focuses on strain transformation, enzymatic and physical and chemical regulation. The process is complicated and lengthy, which is not conducive to the industrial production of γ-PGA. Therefore, a simple and convenient method for producing γ-PGA by fermentation is of great significance for the industrial production and application of γ-PGA. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions, thereby achieving a simple synthesis of high molecular weight or low molecular weight γ-PGA, and at the same time effectively increasing the yield of γ-PGA.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is: A fermentation method for regulating the molecular weight of gamma-polyglutamic acid by using metal ions comprises: adding metal ions during the fermentation process of bacillus subtilis to make the bacillus subtilis ferment to produce high molecular weight or low molecular weight gamma-polyglutamic acid and increase its yield.

[0006] Furthermore, the Bacillus subtilis is Bacillus subtilis FRD518, whose preservation number is CGMCC No.6772, and is preserved in the General Microbiology Center of China National Committee for the Preservation and Administration of Microbiological Cultures.

[0007] Furthermore, the metal ion is a divalent manganese ion or a trivalent iron ion.

[0008] Furthermore, the metal ions are added in the form of manganese sulfate monohydrate or ferric chloride hexahydrate.

[0009] Furthermore, the high molecular weight γ-polyglutamic acid is a γ-polyglutamic acid with a relative average molecular weight of ≥3400 kD, and the low molecular weight γ-polyglutamic acid is a γ-polyglutamic acid with a relative average molecular weight of ≤220 kD.

[0010] Furthermore, in the fermentation process of Bacillus subtilis, by adding divalent manganese ions to the fermentation medium, the yield of high molecular weight γ-polyglutamic acid produced by Bacillus subtilis is increased.

[0011] Furthermore, the concentration of the divalent manganese ions in the fermentation medium is 2.37-9.47 mmol / L.

[0012] Furthermore, in the fermentation process of Bacillus subtilis, the yield of low molecular weight γ-polyglutamic acid produced by Bacillus subtilis is increased by adding trivalent iron ions to the fermentation medium.

[0013] Furthermore, the concentration of the trivalent iron ions in the fermentation medium is 0.296-0.739 mmol / L.

[0014] Furthermore, the fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: (1) Activation of strains: Inoculate Bacillus subtilis FRD518 strains on a solid culture medium plate and culture at 37°C for 24 hours to obtain activated Bacillus subtilis strains; (2) Preparation of seed solution: Take two rings of activated Bacillus subtilis strains, inoculate them into seed culture medium, and culture them under shaking to obtain Bacillus subtilis seed solution; (3) Shake flask fermentation: Add the metal ion solution to the fermentation medium to obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed liquid into the fermentation medium containing metal ions, ferment and culture, and obtain a fermentation liquid containing γ-polyglutamic acid.

[0015] Furthermore, in step (3), the components and contents of the fermentation medium are: glucose 20-120 g / L, peptone 5-25 g / L, inorganic nitrogen source 2-20 g / L, sodium glutamate 10-100 g / L, dipotassium hydrogen phosphate 0.2-5 g / L, citric acid 2-15 g / L and magnesium sulfate heptahydrate 0.1-2 g / L, and the rest is water, pH 6.0-8.0.

[0016] Furthermore, in step (3), the inoculation amount of the Bacillus subtilis seed liquid is 5%; the fermentation culture temperature is 37° C., the shaking culture is 72 h, the shaking speed is 250 rpm, and the amplitude is 26-50 mm.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention applies metal ions to the fermentation production of γ-polyglutamic acid, and produces γ-PGA by a one-step fermentation process, which can directly obtain relatively high molecular weight or low molecular weight γ-polyglutamic acid, so that the molecular weight of γ-polyglutamic acid produced by Bacillus subtilis is increased from about 2000 kD to more than 3400 kD or decreased to less than 220 kD, and the operation is simple and convenient.

[0018] (2) The present invention can increase the yield of γ-polyglutamic acid by 17-34% by simply adding metal ions under the condition of unchanged fermentation conditions, and has good commercial application value.

[0019] (3) The method of the present invention for producing high molecular weight or low molecular weight γ-polyglutamic acid by adding metal ions for fermentation has low cost, obvious effect and good application prospect. DETAILED DESCRIPTION

[0020] Example 1 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: (1) Activation of strains: Inoculate Bacillus subtilis FRD518 strains on a solid culture medium plate and culture at 37°C for 24 hours to obtain activated Bacillus subtilis strains; The solid culture medium comprises: 1-20 g / L glucose, 1-15 g / L yeast powder, 5-50 g / L sodium glutamate, 1-10 g / L dipotassium hydrogen phosphate, 0.1-2 g / L magnesium sulfate heptahydrate, 15-20 g / L agar powder, and the balance is water, with a pH value of 6.0-8.0; (2) Preparation of seed solution: Take two rings of activated Bacillus subtilis strains and inoculate them into a 250-mL Erlenmeyer flask containing 100 mL of seed culture medium. Incubate at 37°C and 250 rpm for 16 h to obtain Bacillus subtilis seed solution. The seed culture medium comprises: 1-20 g / L glucose, 1-15 g / L yeast powder, 5-50 g / L sodium glutamate, 1-10 g / L dipotassium hydrogen phosphate and 0.1-2 g / L magnesium sulfate heptahydrate, the balance being water, and a pH value of 6.0-8.0; (3) Shake flask fermentation: Add sterile monohydrate manganese sulfate solution to a 500 mL Erlenmeyer flask containing 100 mL fermentation medium, so that the final concentration of divalent manganese ions in the fermentation broth is 2.37 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculation rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in a shaking incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 26 mm, to obtain a fermentation broth containing γ-polyglutamic acid; The sterile manganese sulfate monohydrate solution is prepared by first preparing a 0.59 mol / L manganese sulfate monohydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: glucose 20-120 g / L, peptone 5-25 g / L, inorganic nitrogen source 2-20 g / L, sodium glutamate 10-100 g / L, dipotassium hydrogen phosphate 0.2-5 g / L, citric acid 2-15 g / L and magnesium sulfate heptahydrate 0.1-2 g / L, and the rest is water, with a pH of 6.0-8.0.

[0021] Example 2 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0022] (3) Shake flask fermentation: Add sterile monohydrate manganese sulfate solution to a 500 mL Erlenmeyer flask containing 100 mL fermentation medium, so that the final concentration of divalent manganese ions in the fermentation broth is 4.73 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculation rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in a shaking incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 26 mm, to obtain a fermentation broth containing γ-polyglutamic acid; The sterile manganese sulfate monohydrate solution is prepared by first preparing a 0.59 mol / L manganese sulfate monohydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: glucose 20-120 g / L, peptone 5-25 g / L, inorganic nitrogen source 2-20 g / L, sodium glutamate 10-100 g / L, dipotassium hydrogen phosphate 0.2-5 g / L, citric acid 2-15 g / L and magnesium sulfate heptahydrate 0.1-2 g / L, and the rest is water, with a pH of 6.0-8.0.

[0023] Example 3 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0024] (3) Shake flask fermentation: Add sterile monohydrate manganese sulfate solution to a 500 mL Erlenmeyer flask containing 100 mL fermentation medium, so that the final concentration of divalent manganese ions in the fermentation broth is 7.10 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculum rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in a shaking incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 26 mm, to obtain a fermentation broth containing γ-polyglutamic acid; The sterile manganese sulfate monohydrate solution is prepared by first preparing a 0.59 mol / L manganese sulfate monohydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: glucose 20-120 g / L, peptone 5-25 g / L, inorganic nitrogen source 2-20 g / L, sodium glutamate 10-100 g / L, dipotassium hydrogen phosphate 0.2-5 g / L, citric acid 2-15 g / L and magnesium sulfate heptahydrate 0.1-2 g / L, and the rest is water, with a pH of 6.0-8.0.

[0025] Example 4 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0026] (3) Shake flask fermentation: Add sterile monohydrate manganese sulfate solution to a 500 mL Erlenmeyer flask containing 100 mL fermentation medium, so that the final concentration of divalent manganese ions in the fermentation broth is 9.47 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculum rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in a shaking incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 26 mm, to obtain a fermentation broth containing γ-polyglutamic acid; The sterile manganese sulfate monohydrate solution is prepared by first preparing a 0.59 mol / L manganese sulfate monohydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: glucose 20-120 g / L, peptone 5-25 g / L, inorganic nitrogen source 2-20 g / L, sodium glutamate 10-100 g / L, dipotassium hydrogen phosphate 0.2-5 g / L, citric acid 2-15 g / L and magnesium sulfate heptahydrate 0.1-2 g / L, and the rest is water, with a pH of 6.0-8.0.

[0027] Example 5 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0028] (3) Shake flask fermentation: Add sterile ferric chloride hexahydrate solution to a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, so that the final concentration of trivalent iron ions in the fermentation liquid is 0.296 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed liquid into the fermentation medium at an inoculation rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in an oscillating incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 50 mm, to obtain a fermentation liquid containing γ-polyglutamic acid; The sterile ferric chloride hexahydrate solution is prepared by first preparing a 0.037 mol / L ferric chloride hexahydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: 20-120 g / L glucose, 5-25 g / L peptone, 2-20 g / L inorganic nitrogen source, 10-100 g / L sodium glutamate, 0.2-5 g / L dipotassium hydrogen phosphate, 2-15 g / L citric acid and 0.1-2 g / L magnesium sulfate heptahydrate, and the rest is water, with a pH of 6.0-8.0.

[0029] Example 6 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0030] (3) Shake flask fermentation: Add sterile ferric chloride hexahydrate solution to a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, so that the final concentration of trivalent iron ions in the fermentation liquid is 0.444 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculation rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in an oscillating incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 50 mm, to obtain a fermentation liquid containing γ-polyglutamic acid; The sterile ferric chloride hexahydrate solution is prepared by first preparing a 0.037 mol / L ferric chloride hexahydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: 20-120 g / L glucose, 5-25 g / L peptone, 2-20 g / L inorganic nitrogen source, 10-100 g / L sodium glutamate, 0.2-5 g / L dipotassium hydrogen phosphate, 2-15 g / L citric acid and 0.1-2 g / L magnesium sulfate heptahydrate, and the rest is water, with a pH of 6.0-8.0.

[0031] Example 7 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0032] (3) Shake flask fermentation: Add sterile ferric chloride hexahydrate solution to a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, so that the final concentration of trivalent iron ions in the fermentation liquid is 0.591 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed liquid into the fermentation medium at an inoculation rate of 5% (volume fraction), ferment at a temperature of 37°C, and culture in an oscillating incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 50 mm, to obtain a fermentation liquid containing γ-polyglutamic acid; The sterile ferric chloride hexahydrate solution is prepared by first preparing a 0.037 mol / L ferric chloride hexahydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: 20-120 g / L glucose, 5-25 g / L peptone, 2-20 g / L inorganic nitrogen source, 10-100 g / L sodium glutamate, 0.2-5 g / L dipotassium hydrogen phosphate, 2-15 g / L citric acid and 0.1-2 g / L magnesium sulfate heptahydrate, and the rest is water, with a pH of 6.0-8.0.

[0033] Example 8 A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0034] (3) Shake flask fermentation: Add sterile ferric chloride hexahydrate solution to a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, so that the final concentration of trivalent iron ions in the fermentation liquid is 0.739 mmol / L, and obtain a fermentation medium containing metal ions. Inoculate the Bacillus subtilis seed liquid into the fermentation medium at an inoculum amount of 5% (volume fraction), ferment at a temperature of 37°C, and culture in an oscillating incubator for 72 hours, with a shaker speed of 250 rpm and an amplitude of 50 mm, to obtain a fermentation liquid containing γ-polyglutamic acid; The sterile ferric chloride hexahydrate solution is prepared by first preparing a 0.037 mol / L ferric chloride hexahydrate solution, and then filtering and sterilizing it through a 0.22 μm sterile filter membrane; the components and contents of the fermentation medium are: 20-120 g / L glucose, 5-25 g / L peptone, 2-20 g / L inorganic nitrogen source, 10-100 g / L sodium glutamate, 0.2-5 g / L dipotassium hydrogen phosphate, 2-15 g / L citric acid and 0.1-2 g / L magnesium sulfate heptahydrate, and the rest is water, with a pH of 6.0-8.0.

[0035] Comparative Example 1 A fermentation method for producing γ-polyglutamic acid comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0036] (3) Shake flask fermentation: Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculum rate of 5% (volume fraction), culture at a fermentation temperature of 37°C in a shaking incubator for 72 h, with a shaker speed of 250 rpm and an amplitude of 26 mm to obtain a fermentation liquid containing γ-polyglutamic acid; The components and contents of the fermentation medium are: 20-120 g / L glucose, 5-25 g / L peptone, 2-20 g / L inorganic nitrogen source, 10-100 g / L sodium glutamate, 0.2-5 g / L dipotassium hydrogen phosphate, 2-15 g / L citric acid and 0.1-2 g / L magnesium sulfate heptahydrate, and the rest is water, with a pH of 6.0-8.0.

[0037] Comparative Example 2 A fermentation method for producing γ-polyglutamic acid comprises the following steps: Step (1) activation of the bacterial strain and step (2) preparation of the seed solution are the same as those in Example 1.

[0038] (3) Shake flask fermentation: Inoculate the Bacillus subtilis seed solution into the fermentation medium at an inoculum rate of 5% (volume fraction), culture at a fermentation temperature of 37°C in a shaking incubator for 72 h, with a shaker speed of 250 rpm and an amplitude of 50 mm to obtain a fermentation liquid containing γ-polyglutamic acid; The components and contents of the fermentation medium are: 20-120 g / L glucose, 5-25 g / L peptone, 2-20 g / L inorganic nitrogen source, 10-100 g / L sodium glutamate, 0.2-5 g / L dipotassium hydrogen phosphate, 2-15 g / L citric acid and 0.1-2 g / L magnesium sulfate heptahydrate, and the rest is water, with a pH of 6.0-8.0.

[0039] Experimental Example 1 The average molecular weight and content of the γ-polyglutamic acid prepared in Examples 1-8 and Comparative Examples 1-2 were determined by high performance liquid chromatography. The results are listed in Tables 1 and 2.

[0040] Table 1. Average molecular weight and content of γ-polyglutamic acid As can be seen from Table 1, in Comparative Example 1, Bacillus subtilis was fermented to obtain a relative average molecular weight of 2150 kD and a content of 46.87 g / L of γ-polyglutamic acid in the fermentation broth; while in Examples 1-4, fermentation was carried out under the same conditions, and the fermentation medium contained different concentrations of divalent manganese ions, and the relative average molecular weight of γ-polyglutamic acid in the obtained fermentation broth was higher than 3400 kD, the content of γ-polyglutamic acid was higher than 54 g / L, and the fermentation yield was increased by more than 17% compared with Comparative Example 1. It can be seen that the present invention can obtain high molecular weight γ-polyglutamic acid and increase the fermentation yield of γ-polyglutamic acid by adding divalent manganese ions to the fermentation medium.

[0041] Table 2. Average molecular weight and content of γ-polyglutamic acid As can be seen from Table 2, in Comparative Example 2, Bacillus subtilis was fermented to obtain a relative average molecular weight of 1998 kD and a content of 59.36 g / L of γ-polyglutamic acid in the fermentation broth; and in Examples 5-8, the fermentation medium contained different concentrations of trivalent iron ions, and the relative average molecular weight of γ-polyglutamic acid in the obtained fermentation broth was lower than 220 kD, and the content of γ-polyglutamic acid was higher than 76 g / L, and the fermentation yield was increased by more than 28% compared with Comparative Example 2. It can be seen that the present invention can obtain low molecular weight γ-polyglutamic acid and increase the fermentation yield of γ-polyglutamic acid by adding trivalent iron ions to the fermentation medium.

Claims

1. A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions, characterized in that: The method comprises the following steps: during the fermentation process of Bacillus subtilis, metal ions are added to make the Bacillus subtilis ferment and produce high molecular weight or low molecular weight gamma-polyglutamic acid, and improve the yield thereof.

2. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 1, characterized in that: The Bacillus subtilis is Bacillus subtilis FRD518, whose preservation number is CGMCC No.6772, and is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration.

3. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 1, characterized in that: The metal ion is a divalent manganese ion or a trivalent iron ion.

4. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 3, characterized in that: The metal ions are added in the form of manganese sulfate monohydrate or ferric chloride hexahydrate.

5. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 1, characterized in that: The high molecular weight γ-polyglutamic acid is a γ-polyglutamic acid with a relative average molecular weight of ≥3400 kD, and the low molecular weight γ-polyglutamic acid is a γ-polyglutamic acid with a relative average molecular weight of ≤220 kD.

6. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 1, characterized in that: In the fermentation process of Bacillus subtilis, the yield of high molecular weight gamma-polyglutamic acid produced by Bacillus subtilis is increased by adding divalent manganese ions to the fermentation medium.

7. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 6, characterized in that: The concentration of the divalent manganese ions in the fermentation medium is 2.37-9.47 mmol / L.

8. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 1, characterized in that: In the fermentation process of Bacillus subtilis, the yield of low molecular weight γ-polyglutamic acid produced by Bacillus subtilis is increased by adding trivalent iron ions to the fermentation medium.

9. The fermentation method for regulating the molecular weight of γ-polyglutamic acid by using metal ions according to claim 8, characterized in that: The concentration of the trivalent iron ions in the fermentation medium is 0.296-0.739 mmol / L.

10. A fermentation method for regulating the molecular weight of γ-polyglutamic acid using metal ions according to claim 1, characterized in that: The steps include: (1) Activation of strains: Inoculate Bacillus subtilis FRD518 strains on a solid culture medium plate and culture at 37°C for 24 hours to obtain activated Bacillus subtilis strains; (2) Preparation of seed solution: Take two rings of activated Bacillus subtilis strains, inoculate them into seed culture medium, and culture them under shaking to obtain Bacillus subtilis seed solution; (3) Shake flask fermentation: adding a metal ion solution to a fermentation medium to obtain a fermentation medium containing metal ions, inoculating Bacillus subtilis seed liquid into the fermentation medium containing metal ions, and fermenting to obtain a fermentation liquid containing γ-polyglutamic acid.

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