A strain of Bacillus licheniformis and its application in the preparation of low-molecular-weight γ-polyglutamic acid

Abstract

The present invention relates to the field of microbial technology, and specifically relates to a strain of Bacillus licheniformis and its application in the preparation of low-molecular-weight γ-polyglutamic acid. The present invention provides Bacillus licheniformis PGA-BL19, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20221810. This strain of Bacillus licheniformis can efficiently produce γ-polyglutamic acid, and the γ-polyglutamic acid produced by it is low-molecular-weight γ-polyglutamic acid. In addition to being used in fields such as cosmetics and food, the low-molecular-weight γ-polyglutamic acid produced by this strain can also be used as a freeze-drying protectant for microorganisms, which can effectively protect bacteria and fungi during the freeze-drying process and reduce the damage to microorganisms during the freeze-drying process.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a strain of Bacillus licheniformis and its application in the preparation of low-molecular-weight γ-polyglutamic acid. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is a biological macromolecular carrier material. It was first discovered by Ivanovics et al. in the capsule of Bacillus anthracis. It is a secondary metabolite produced by microorganisms of the genus Bacillus (such as Bacillus subtilis, Bacillus licheniformis, etc.) during their life activities and can be prepared by microbial fermentation.

[0003] The molecular weight (Mw) of γ-PGA is mainly in the range of 10 - 10000 kDa. γ-PGA with different molecular weights has different functions and application fields. For example, low-molecular-weight γ-PGA (Mw < 400 kDa) can improve the taste of food, and when applied to the skin, it can keep the skin moist and smooth. It can be used in the fields of food, cosmetics, and also as a drug carrier to target drugs to cancer cells and release the encapsulated active ingredients to inhibit tumor growth; high-molecular-weight γ-PGA (Mw > 1000 kDa) has a higher viscosity and can be used as a flocculant to adsorb heavy metals in water and as a thickener for the preparation of biomaterials, etc. Currently, the application of low-molecular-weight γ-PGA in the fields of food, cosmetics, and pharmaceutical carriers is more extensive and in greater demand.

[0004] The characteristics of different γ-PGA-producing strains are different, and the γ-PGA yields and relative molecular weights of the strains also vary. γ-PGA-producing bacteria can be divided into two major categories: the genus Bacillus and other strains. Commonly used microorganisms for producing γ-PGA are microorganisms of the genus Bacillus. In addition, Escherichia coli expressing the γ-PGA synthesis gene heterologously is also used. Among the microorganisms of the genus Bacillus, Bacillus licheniformis and Bacillus subtilis are often used as γ-PGA-producing strains.

[0005] Currently, the preparation of low-molecular-weight γ-PGA mainly involves two approaches: intracellular regulation and in vitro regulation. Intracellular regulation is mainly achieved by changing fermentation conditions, such as adding sorbitol, inorganic salts for stress, and genetically modifying the production strains. However, current genetic modification methods mainly focus on the yield of γ-PGA, and there is less research and immaturity in terms of molecular weight. For example, although Pgds and GTT can reduce the molecular weight, they may affect the yield of γ-PGA. In vitro regulation mainly controls the molecular weight of γ-PGA by adding γ-PGA degrading enzymes during fermentation, or promotes the degradation of γ-PGA by ultrasonic method, acid-heat degradation method, etc., but there are drawbacks such as difficulty in controlling the hydrolysis molecular weight.

[0006] The Bacillus licheniformis strains for producing γ-PGA disclosed in the prior art mainly include: the relative molecular mass of γ-PGA fermented by Bacillus licheniformis ATCC9945A is between 500 kDa and 800 kDa, and the yield of γ-PGA is 5-17 g / L; another strain of Bacillus lichniformis is fermented for 5 days, the relative molecular mass of γ-PGA is 300 kDa, and the yield is 8.5 g / L; the molecular weight of γ-PGA synthesized by Bacillus licheniormis WX-02 is 100-1200 kDa. At present, there is still a lack of Bacillus licheniformis strains that can produce high-yield and low-molecular-weight γ-PGA. Summary of the Invention

[0007] The purpose of the present invention is to provide a strain of Bacillus licheniformis that can produce high-yield and low-molecular-weight γ-PGA.

[0008] The present invention uses Bacillus licheniformis that produces γ-polyglutamic acid screened from the soil as the starting strain, and performs ion beam implantation mutagenesis screening to obtain a strain of Bacillus licheniformis that produces high-yield γ-polyglutamic acid. This strain can efficiently convert the precursor glutamic acid into γ-polyglutamic acid, and the γ-PGA produced by this strain is a low-molecular-weight product with a molecular weight less than 265 kDa, which has excellent effects in the lyophilization protection of microorganisms and can be used as a lyophilization protectant for microorganisms.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In the first aspect, the present invention provides Bacillus licheniformis PGA-BL19, which was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: Wuhan, China, Wuhan University, postcode 430072) on November 17, 2022, with the deposit number CCTCC NO: M 20221810, and the taxonomic name is Bacillus licheniformis.

[0011] Bacillus licheniformis PGA-BL19 has the characteristic of producing high-yield γ-polyglutamic acid. The molecular weight of γ-polyglutamic acid fermented and produced by it is concentrated below 265 kDa, belonging to low-molecular-weight γ-polyglutamic acid, which has a wide range of uses in the fields of food, cosmetics, drug carriers, etc., and has excellent effects in the lyophilization protection of microorganisms.

[0012] In the second aspect, the present invention provides a fermentation product prepared by fermenting and culturing the above-mentioned Bacillus licheniformis PGA-BL19.

[0013] The fermented product contains Bacillus licheniformis PGA - BL19 or contains Bacillus licheniformis PGA - BL19 and its fermentation supernatant.

[0014] In a third aspect, the present invention provides a bacterial agent, which contains the above - mentioned Bacillus licheniformis PGA - BL19.

[0015] Preferably, Bacillus licheniformis PGA - BL19 in the above - mentioned bacterial agent exists in the form of viable bacteria.

[0016] Regarding the dosage form of the bacterial agent, the present invention has no special limitation, and it can be a dosage form common in the field of microbial agents, for example: solid bacterial agent or liquid bacterial agent. Among them, the solid bacterial agent can be freeze - dried powder.

[0017] In addition to containing Bacillus licheniformis PGA - BL19, the above - mentioned bacterial agent may also contain excipients or carriers allowed in the field of microbial agents.

[0018] In a fourth aspect, the present invention provides a preparation method of the above - mentioned bacterial agent, and the method includes the step of culturing Bacillus licheniformis PGA - BL19 to obtain a bacterial liquid.

[0019] Preferably, the culturing is carried out under the condition of 35 - 37°C.

[0020] The obtained bacterial liquid can be directly prepared into a liquid bacterial agent or prepared into a liquid bacterial agent by adding excipients or carriers allowed in the field of microbial agents. It can also collect the bacteria in the bacterial liquid and mix them with excipients or carriers allowed in the field of microbial agents such as freeze - drying protectant, and then be prepared into freeze - dried bacterial powder through vacuum freeze - drying.

[0021] In a fifth aspect, the present invention provides the application of the above - mentioned Bacillus licheniformis PGA - BL19 or the bacterial agent in the preparation of γ - polyglutamic acid.

[0022] Preferably, the molecular weight of the γ - polyglutamic acid ≤ 300 kDa.

[0023] More preferably, the molecular weight of the γ - polyglutamic acid ≤ 270 kDa.

[0024] More preferably, the molecular weight of the γ - polyglutamic acid ≤ 265 kDa.

[0025] In a sixth aspect, the present invention provides the use of the above-mentioned Bacillus licheniformis PGA-BL19 or the bacterial agent in the preparation of a microbial lyoprotectant.

[0026] In addition to γ-polyglutamic acid produced by the fermentation of Bacillus licheniformis PGA-BL19, the above-mentioned microbial lyoprotectant may further contain one or more other substances having a microbial lyoprotective function.

[0027] In a seventh aspect, the present invention provides a method for preparing γ-polyglutamic acid, the method comprising: culturing the above-mentioned Bacillus licheniformis PGA-BL19 to obtain a culture solution, and collecting γ-polyglutamic acid in the culture solution.

[0028] The temperature of the above-mentioned culture is preferably 35-37 °C. The pH is preferably 6.8-7.4. The rotation speed is preferably 100-280 rpm.

[0029] The above-mentioned culture is preferably carried out under salt stress conditions.

[0030] The culture medium used for the above-mentioned culture preferably comprises the following components: glucose or sucrose or glycerol 130-150 g / L, peptone or yeast powder or potassium nitrate 30-40 g / L, dipotassium hydrogen phosphate or potassium dihydrogen phosphate 1.5-2.5 g / L, sodium glutamate 50-80 g / L, citric acid monohydrate 3-5 g / L, magnesium sulfate or calcium chloride or manganese sulfate 0.4-0.6 g / L, sodium chloride 8-12 g / L, pH 6.5-7.2.

[0031] To collect γ-polyglutamic acid in the culture solution, after removing the bacterial cells from the culture solution, ethanol is added to the supernatant for precipitation, the precipitate is collected by centrifugation, and γ-PGA is obtained after dialysis for desalting. Among them, the addition amount of ethanol is preferably 1-5 times that of the supernatant.

[0032] In an eighth aspect, the present invention provides a γ-polyglutamic acid, the molecular weight of the γ-polyglutamic acid ≤ 270 kDa, wherein the mass percentage content of γ-polyglutamic acid with a molecular weight of 265 kDa-35 kDa is 86-97%, and the mass percentage content of γ-polyglutamic acid with a molecular weight greater than or equal to 18 kDa and less than 35 kDa is 1.5-2.5%, and the balance is γ-polyglutamic acid with a molecular weight ≤ 10 kDa.

[0033] Preferably, the molecular weight of the γ-polyglutamic acid ≤ 265 kDa, wherein the mass percentage of γ-polyglutamic acid with a molecular weight of 265 kDa - 35 kDa is 94 - 97%, the mass percentage of γ-polyglutamic acid with a molecular weight greater than or equal to 18 kDa and less than 35 kDa is 1.5 - 2.0%, and the balance is γ-polyglutamic acid with a molecular weight ≤ 10 kDa.

[0034] Preferably, the above-mentioned γ-polyglutamic acid is produced by fermentation of Bacillus licheniformis PGA-BL19.

[0035] More preferably, the above-mentioned γ-polyglutamic acid is prepared by the method for preparing γ-polyglutamic acid described above.

[0036] In a ninth aspect, the present invention provides a microbial lyoprotectant, which comprises the above-mentioned γ-polyglutamic acid.

[0037] In addition to the above-mentioned γ-polyglutamic acid, the above-mentioned microbial lyoprotectant may further comprise one or more other substances having a microbial lyoprotecting function.

[0038] The beneficial effects of the present invention are as follows: The Bacillus licheniformis provided by the present invention can efficiently produce γ-polyglutamic acid, and the γ-polyglutamic acid produced by it is a low-molecular-weight γ-polyglutamic acid.

[0039] The low-molecular-weight γ-polyglutamic acid produced by the Bacillus licheniformis provided by the present invention can be used not only in the fields of cosmetics, food, etc., but also as a microbial lyoprotectant. It has been experimentally verified that the low-molecular-weight γ-polyglutamic acid has a good protective effect on bacteria such as Lactobacillus plantarum during the freeze-drying process and can be applied to the preparation process of probiotic freeze-dried powder. It has an excellent protective effect on the freeze-drying of fungi such as Mortierella alpina and can be used in the process of strain preservation and microbial agent preparation. Detailed Embodiments

[0040] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0041] Example 1 Obtaining of Bacillus licheniformis with High Yield of Low-Molecular-Weight γ-PGA

[0042] The Bacillus licheniformis bacterial suspension was spread on a sterile petri dish and air-dried with sterile air in a laminar flow hood for later use. Plasma mutagenesis of the strain was carried out on the nitrogen ion implantation device in the Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences. N + The injection energy of the ion source was 15 keV, and the injection dose was 10×10 14 ions / cm 2 , and it was injected in a pulsed manner for 5 s, with an interval of 20 s, and a total injection time of 15 s. The vacuum degree in the target chamber was about 10 -3 Pa. The spread petri dish was placed on the target stage of the injector for ion beam injection. After mutagenesis, it was eluted with 2 mL of sterile water, serially diluted, and then spread on a plate containing the primary screening medium and cultured at 37 °C for 16 h. Colonies with viscous colonies were selected, activated through secondary seed activation, and inoculated into the rescreening fermentation medium for preservation at an inoculation amount of 1%. The strains obtained from the primary screening were inoculated into a 24-well plate containing the seed medium (liquid loading 3 mL / 10 mL), with 3 replicates for each strain, and cultured at 37 °C and 250 r / min for 12 h. The seed liquid was transferred to a 24-well plate containing the fermentation medium (liquid loading 3 mL / 10 mL) at a ratio of 4%, and cultured at 37 °C and 150 r / min for 48 h.

[0043] After fermentation, the yield and molecular weight of γ-PGA were determined to screen for strains with high yield and low molecular weight of γ-PGA.

[0044] The methods for determining the yield and molecular weight of γ-PGA are as follows:

[0045] Determination of γ-PGA yield: It was determined according to the method in QB / T 5189-2017;

[0046] Determination of γ-PGA molecular weight: The molecular weight of γ-PGA was determined using aqueous gel permeation chromatography (GPC). Three columns were connected in series, and the column models used were: Waters Ultrahydrogel TM 2000 (7.8 mm × 300 mm), Waters Ultrahydrogel TM 250 (7.8 mm × 300 mm), and Waters Utrahydrogel TM 120 (7.8 mm × 300 mm). Detection method: Deionized water was used as the mobile phase, the flow rate was 0.6 mL / min, the column temperature was 65 °C, and a differential refractive index detector (RI) was used for detection. Dextran with different molecular weights was used as the standard sample to establish the standard equation between molecular weight and retention time, and the molecular weight of the sample γ-PGA was calculated.

[0047] After continuous screening, a strain of Bacillus licheniformis with high yield of γ-PGA and relatively low molecular weight of the produced γ-PGA was obtained, and this strain was named PGA-BL19.

[0048] Bacillus licheniformis PGA-BL19 was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: Wuhan University, Wuhan, China, postal code 430072) on November 17, 2022, with the deposit number CCTCC NO: M20221810, and the taxonomic name is Bacillus licheniformis.

[0049] Example 2 Strain Fermentation Culture and Preparation of Low-Molecular-Weight γ-PGA (1)

[0050] Fermentation culture of Bacillus licheniformis PGA-BL19 was carried out to produce low-molecular-weight γ-PGA, and the specific method is as follows:

[0051] 1. Seed culture

[0052] Strain activation: Inoculate Bacillus licheniformis PGA-BL19 on a slant medium, and culture it at 37°C and 60% humidity for 16 - 18 h to prepare mature slant seeds.

[0053] The composition of the slant medium is: sucrose 60 g / L, peptone 60 g / L, sodium glutamate 80 g / L, sodium chloride 10 g / L, magnesium sulfate 1 g / L, calcium chloride 1 g / L, agar 15 g / L, pH 6.5, sterilized at 115°C for 20 min.

[0054] Preparation of seed medium and seed activation culture:

[0055] Preparation of primary seed liquid: Use 1 - 2 mL of sterilized 0.85% normal saline to elute the colonies on the slant, and inoculate them into a 250 mL Erlenmeyer flask containing 25 mL of liquid medium at an inoculation amount of 0.1% - 0.2%, and culture at 37°C and 200 rmp for 14 h until the logarithmic growth phase.

[0056] Preparation of secondary seed liquid: Inoculate the prepared primary seed liquid into a 250 mL Erlenmeyer flask containing 50 mL of liquid medium at an inoculation amount of 0.3%, and culture at 37°C and 200 rmp for 12 h until the logarithmic growth phase.

[0057] The composition of the primary and secondary seed media is: glucose 10 g / L, yeast powder 5 g / L, magnesium sulfate 0.25 g / L, dipotassium hydrogen phosphate 2 g / L, sodium glutamate 10 g / L, pH 7.5, sterilized at 115°C for 20 min.

[0058] 2. Fermentation culture

[0059] Prepare the fermentation medium: glucose 140 g / L, yeast powder 35 g / L, dipotassium hydrogen phosphate 2 g / L, sodium glutamate 60 g / L, citric acid monohydrate 4 g / L, calcium chloride 0.5 g / L, sodium chloride 10 g / L, pH 6.8.

[0060] The filling volume of the fermentation medium is 200 mL / 1 L baffled shake flask. Inoculate the secondary seed liquid in step 1 into the fermentation medium at an inoculation amount of 10%, and culture it in a constant temperature shaker at 37°C for 48 h. The rotation speed is 120 rmp from 0 to 12 h; 200 rmp from 12 to 24 h; 250 rmp from 24 to 48 h. After culturing for 48 h, the fermentation is completed. Detect that the yield of γ-polyglutamic acid in the fermentation broth reaches 45.8 g / L, among which the content ratio of γ-polyglutamic acid with molecular weight 265 kDa ≥ Mw ≥ 35 kDa is 96.5%, the content ratio of γ-polyglutamic acid between 35 kDa > Mw ≥ 18 kDa is 1.7%, and the rest is γ-polyglutamic acid with molecular weight ≤ 10 kDa.

[0061] 3. Extraction of γ-PGA from the fermentation broth

[0062] After centrifuging the fermentation broth to remove the thallus, add ethanol with a volume twice that of the fermentation broth to the supernatant, precipitate and then centrifuge to collect the precipitate, and obtain γ-PGA after dialysis for desalting.

[0063] Example 3 Fermentation culture of the strain and preparation of low molecular weight γ-PGA (2)

[0064] Ferment and culture Bacillus licheniformis PGA-BL19 to produce low molecular weight γ-PGA. The difference in the fermentation culture method from Example 2 is only that: use a 7 L fermenter with a filling volume of 1 / 2 for fermentation culture, culture it in a constant temperature shaker at 37°C for 48 h. The rotation speed is 150 rmp from 0 to 12 h; 220 rmp from 12 to 24 h; 250 rmp from 24 to 48 h. After culturing for 48 h, the fermentation is completed. Detect that the yield of γ-polyglutamic acid in the fermentation broth reaches 48.8 g / L, among which the content ratio of γ-polyglutamic acid with molecular weight 265 kDa ≥ Mw ≥ 35 kDa is 94.6%, the content ratio of γ-polyglutamic acid between 35 kDa > Mw ≥ 18 kDa is 1.9%, and the rest is γ-polyglutamic acid with molecular weight ≤ 10 kDa.

[0065] Example 4 Application of low molecular weight γ-PGA in freeze-drying protection of microorganisms

[0066] Use the γ-PGA prepared in Example 2 for freeze-drying protection of Lactobacillus plantarum and Mortierella alpina. The specific methods and results are as follows:

[0067] 1. Freeze-drying protection of Lactobacillus plantarum

[0068] The seed culture medium and activation culture medium of Lactobacillus plantarum adopt MRS medium. Lactobacillus plantarum grows on the MRS solid medium for 48 h and in the liquid medium for 16 h to the logarithmic phase. The cells are collected by centrifugation, washed twice with sterile water, resuspended with 2.5% γ-polyglutamic acid prepared in Example 2, frozen at -80 °C overnight, and freeze-dried into powder. It is sealed and placed at -20 °C for two days, diluted by gradient, subjected to plate counting, and the survival rate N% of the strain after freeze-drying is calculated as N% = N1 / N0 * 100; where N1 is the number of viable bacteria counted in the freeze-dried powder and N0 is the number of viable bacteria counted before freeze-drying.

[0069] 2. Lyophilization protection of Mortierella alpina

[0070] The seed culture medium and activation culture medium of Mortierella alpina adopt PDA medium. Mortierella alpina is cultured on the PDA slant medium for 4 days and in the liquid medium for 3 days to the mature stage. The cells are collected by centrifugation, washed twice with sterile water, resuspended with 2.5% γ-polyglutamic acid prepared in Example 2, frozen at -80 °C overnight, and freeze-dried into powder. It is sealed and placed at -20 °C for two days, diluted by gradient, subjected to plate counting, and the survival rate N% of the strain after freeze-drying is calculated as N% = lgN1 / lgN0 * 100%, where N1 is the number of viable bacteria counted in the freeze-dried powder and N0 is the number of viable bacteria counted before freeze-drying.

[0071] The experimental results are shown in Table 1.

[0072] Table 1 Lyophilization protection effects of γ-PGA with different molecular weights on Lactobacillus plantarum and Mortierella alpina

[0073]

[0074] Lyophilization is a drying method in which the cell suspension is frozen below the freezing point, thereby transformed into a solid crystalline state, and then the solid is sublimated and dehydrated under low temperature and low pressure (vacuum). Generally, the drying process will damage the cell membrane of microorganisms, resulting in a large number of cell deaths. Therefore, cryoprotectants need to be added to improve the uniformity of the cell suspension density and reduce the death of viable bacteria caused by mechanical effects and solute effects during freeze-drying. The cryoprotectant added before freeze-drying is a crucial decisive factor affecting the viability of the freeze-dried bacterial powder. It will affect both the activity and stability of cells during storage and the cell survival rate during rehydration.

[0075] In addition to being widely used in the fields of cosmetics, food, and pharmaceuticals, it has been reported in the literature that γ-polyglutamic acid can be used for the freeze-drying protection of probiotics. For example, CN112899204B discloses a probiotic freeze-drying outer shell composite protective agent containing γ-polyglutamic acid; the literature "Freeze-drying protection effect of the high molecular polymer γ-PGA on Bifidobacterium longum" discloses that the compounding of γ-PGA and whey powder can achieve a good freeze-drying protection effect. These studies all involve the compounding of γ-polyglutamic acid with other substances as a freeze-drying protective agent for microorganisms. After physicochemical experiments in the literature "Relationship between the Antifreeze Activities and the Chemical Structures of Oligo- and Poly(glutamic acid)s", it was found that the antifreeze property of 20KDa γ-PGA is the best. Indeed, for bacteria, the advantages of the γ-PGA prepared in the present invention and the ultra-low molecular weight γ-PGA in the freeze-drying protection effect on microorganisms are not obvious. However, compared with bacteria, fungi are more vulnerable to mechanical damage by ice crystals during the pre-freezing process of freeze-drying due to their large cell volume and lack of cell wall protection. Therefore, fungi are more likely to be inactivated during the freeze-drying process, with a higher cell death rate and higher requirements for the performance of the freeze-drying protective agent. The Bacillus licheniformis PGA-BL19 of the present invention can produce γ-polyglutamic acid products with a suitable molecular weight range and distribution, and this product can enhance the protective effect of γ-polyglutamic acid on the cells during freeze-drying.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Bacillus licheniformis PGA-BL19, characterized in that, It is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20221810.

2. A bacterial agent, characterized in that, It contains Bacillus licheniformis PGA-BL19 described in claim 1.

3. The preparation method of the microbial agent according to claim 2, characterized in that, The method includes the step of culturing Bacillus licheniformis PGA-BL19 to obtain a bacterial liquid.

4. Use of Bacillus licheniformis PGA-BL19 described in claim 1 or the bacterial agent described in claim 2 in the preparation of γ-polyglutamic acid.

5. The application according to claim 4, wherein The molecular weight of the γ-polyglutamic acid is ≤ 300 kDa.

6. Use of Bacillus licheniformis PGA-BL19 described in claim 1 or the bacterial agent described in claim 2 in the preparation of a microbial lyophilization protectant.

7. A method for preparing γ-polyglutamic acid, characterized in that, The method includes: culturing Bacillus licheniformis PGA-BL19 described in claim 1 to obtain a culture solution, and collecting γ-polyglutamic acid in the culture solution.

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