A method for synthesizing gamma-polyglutamic acid

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a method for synthesizing gamma-polyglutamic acid. A high-yield strain of gamma-polyglutamic acid, i.e., parabacillus licheniformis TR-1, is screened, and has been biologically preserved with a preservation number of CCTCC NO: M20231731. The strain is obtained from soil, has high activity and strong growth and metabolism, can be used in a fermenter for long-term fermentation, and can gradually reduce the amount of bacterial inoculum in the later stage. Fermentation substrates use low-cost high-carbon materials such as corn stalks, sweet potato stems and wheat stalks to produce fermented polyglutamic acid, and the yield is the highest when corn stalks are used as the fermentation substrate, which can reach more than 80 g / L, thereby seeking a new path for the comprehensive utilization of corn stalks. The culture medium for the fermentation production of gamma-polyglutamic acid is simple, easy to obtain, low in cost, short in fermentation period, and low in material requirement, and can be used for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for synthesizing γ-polyglutamic acid, using corn stalks as the fermentation substrate. Background Technology

[0002] γ-polyglutamic acid (γ-PGA) is a novel biopolymer composed of D-glutamic acid or L-glutamic acid linked by γ-amide bonds between α-amino and γ-carboxyl groups. Its relative molecular mass ranges from 10 kDa to 2000 kDa. γ-PGA possesses excellent biocompatibility, ion adsorption, and biodegradability, and is harmless to humans and the environment. It is commonly used in cosmetics, food processing, agriculture, medicine, and environmental protection.

[0003] The main methods for synthesizing γ-polyglutamic acid include extraction, chemical synthesis, enzymatic conversion, and microbial fermentation. Currently, microbial fermentation is the most widely used method. Patent application number CN202310371012 utilizes microbial fermentation to produce γ-polyglutamic acid. The fermentation medium used in this invention uses enzymatic hydrolysate as the main carbon source. The enzymatic hydrolysate is obtained by washing soybean meal and sago palm pith chips with acid, alkali, and water, followed by enzymatic hydrolysis with cellulase and Aspergillus niger acidic protease. However, the preparation of the enzymatic hydrolysate is cumbersome, requires numerous experimental materials, and is costly.

[0004] Patent application number CN202310187309 utilizes air-encapsulating agents for fermentation, including lecithin, cocoyl glucoside, soybean oil, and paraffin oil. The highest yield of γ-polyglutamic acid produced using this method is 35-42 g / L, but the air-encapsulation process is cumbersome and costly. Patent application number CN202210340897 discloses a strain of *Bacillus amyloliquefaciens* YZHY21.A02 that produces γ-polyglutamic acid through fermentation of soybean milk. It can also be produced through solid-state fermentation of soybeans, with a polyglutamic acid yield of 4.9 g / 100g soybeans. However, the purchase cost of soybean milk and soybeans is high, as is the fermentation cost. Patent application number CN202211354365 utilizes a strain of *Bacillus paralicheniformis* for microbial fermentation. Within a 24-hour fermentation cycle, the polyglutamic acid concentration can reach over 50 g / L, enabling glutamic acid-independent production of polyglutamic acid. The carbon and nitrogen sources used are laboratory reagents.

[0005] Currently, the fermentation substrate used for the synthesis of γ-polyglutamic acid is cumbersome to prepare and costly, making it unsuitable for large-scale production. Therefore, how to produce high-yield polyglutamic acid at a lower production cost is a key research focus. Corn stalks are agricultural solid waste. Returning corn stalks to the field results in slow decomposition, a long processing time, and poor utilization. Furthermore, the stalks contain numerous larvae and bacteria that are difficult to remove during the crushing process and can quickly grow after being buried in the soil, posing a threat of pests and diseases. Using corn stalks as a fermentation substrate can reduce production costs and solve the problem of corn stalk utilization. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for synthesizing γ-polyglutamic acid, using corn stalks as a fermentation substrate to produce γ-polyglutamic acid, while simultaneously providing Bacillus paralicheniformis (… Bacillus paralicheniform TR-1, has been bio-conserved with accession number CCTCC NO: M 20231731. This strain was obtained by screening from soil and can synthesize γ-polyglutamic acid using corn stalks, with a yield of up to 80 g / L or more.

[0007] The technical solution adopted is as follows: A method for synthesizing γ-polyglutamic acid involves screening for Bacillus paralichrysogenus TR-1, inoculating a seed culture containing Bacillus paralichrysogenus TR-1 into a fermentation medium, and then aerobically fermenting to obtain γ-polyglutamic acid.

[0008] Preferred is Bacillus paralicheniform TR-1, China Center for Type Culture Collection (CCTCC) accession number: M 20231731, deposited on September 18, 2023.

[0009] Preferably, the fermentation medium includes a carbon source, a nitrogen source, monosodium glutamate, dipotassium hydrogen phosphate, and distilled water, and the pH of the fermentation medium is 6.0~7.5; wherein the carbon source is any one or a mixture of corn stalks, sweet potato vines, and wheat stalks; and the nitrogen source is any one of soybean cake, soybean meal, and chicken manure. The C:N ratio used is 10~25:1.

[0010] Preferably, the aerobic fermentation method is shake flask fermentation or fermenter fermentation.

[0011] Preferably, when using shake flask fermentation, the fermentation conditions are: temperature 28~39℃, shaking speed 120~220rpm, and time 12~60h; When using a fermenter, the fermentation conditions are: temperature 28~39℃, fermentation speed 350~550rpm, fermentation time 24~60h, and aeration rate 0.8~1.2vvm.

[0012] Preferably, the volume ratio of seed culture to fermentation medium is 1~15:100.

[0013] Preferably, the inoculum amount is 1%~15%, the culture temperature is 30~40℃, the pH is 6.0~7.5, the stirring speed is 140~220rpm, and the culture time is 24~72h.

[0014] Preferably, the screening of Bacillus paralichrysogenum TR-1 includes the following steps: a. Sample preparation: Select soil covered with fallen leaves and branches, dig a depth of 10-15 cm, take a certain amount of soil, add it to sterile distilled water, shake to mix, then heat in a water bath for 10 min. After cooling, take the supernatant and perform serial dilution with sterile water at a dilution factor of 10. 1 10 2 10 3 10 4 10 5 10 6 10 7 The microbial community was obtained by incubating at 37℃ for 24 hours; the mass ratio of soil to sterile water was 1:9 (g / L). b. Initial screening: Take the diluted solution from step (1) at 10°C. 1 ~10 7 Take 100 μL of each sample and spread it on the primary screening plate medium. Invert the plate and incubate. Use an inoculation loop to pick up the strain with large colonies, high viscosity and easy stringing, and streak it to obtain a single colony pure culture. Preserve it in a new LB solid medium as the primary screening strain (the primary screening plate medium is also LB solid medium, so the single colony is cultured in a new LB solid medium). c. Screening and detection: The strains obtained from the initial screening were inoculated into seed culture medium and cultured at 37℃ and 180 rpm for 12 h. The seed liquid was then inoculated into the fermentation medium at a volume ratio of 2% and cultured at 37℃ and 180 rpm for 48 h. The fermentation broth was centrifuged at 4800 rpm for 25 min at 4℃. The supernatant was collected and the yield of γ-polyglutamic acid was detected by ultraviolet spectrophotometer. The strain with the highest yield of γ-polyglutamic acid was screened for expansion culture and preservation.

[0015] When using a UV spectrophotometer, the supernatant after centrifugation was taken, and then 4 times the volume of anhydrous ethanol was added. The mixture was placed in a 4°C refrigerator for 12 hours to precipitate. Then, it was taken out and centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was removed, and the precipitate was dissolved in ultrapure water. The absorbance was measured at 216 nm using a UV spectrophotometer, and the yield of γ-polyglutamic acid was calculated based on the standard curve.

[0016] The standard curve is plotted by measuring the absorbance at 216 nm using a UV spectrophotometer with different concentrations of γ-polyglutamic acid standard solutions, and plotting the absorbance as the ordinate and the γ-polyglutamic acid concentration as the abscissa.

[0017] Preferably, the initial screening plate medium is LB solid medium, with the following components: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 18 g / L, sterilized at 121℃ for 20 min.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention screened out a high-yield strain of γ-polyglutamic acid, namely Bacillus paralicheniformis (… Bacillus paralicheniform TR-1 has been biologically preserved with accession number CCTCC NO: M 20231731. This strain was obtained from soil screening. It has high activity and strong growth and metabolism capabilities. It can be used for long-term fermentation in a fermenter. The amount of strain added can be gradually reduced in the later stages. The fermentation substrate of this invention utilizes inexpensive high-carbon materials such as corn stalks, sweet potato vines, and wheat stalks to produce fermented polyglutamic acid. In particular, the yield is highest when corn stalks are used as the fermentation substrate, reaching up to 80 g / L or more, thus providing a new path for the comprehensive utilization of corn stalks. The culture medium for the fermentation production of γ-polyglutamic acid according to this invention has simple and readily available components, low cost, short fermentation cycle, and low requirements for materials, making it suitable for large-scale industrial production.

[0019] Preservation instructions: Bacterial species name: Bacillus paralicheniformis Latin name: Bacillus paralicheniform Strain number: TR1 Preservation Institution: China Center for Type Culture Collection Abbreviation for depository institution: CGTCC Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postcode: 430072 Accession number: CCTCC NO: M 20231731 Date of preservation: September 18, 2023 Attached Figure Description

[0020] Figure 1 This is the standard curve for calculating the yield of γ-polyglutamic acid in this invention; Figure 2 This is a morphological diagram of Bacillus paralicheniformis TR-1 of the present invention. Detailed Implementation

[0021] The accompanying drawings are for illustrative purposes only; the invention will be described in detail below with reference to the embodiments; it should be understood that common knowledge or prior art in the embodiments may be omitted.

[0022] Example 1 A method for synthesizing γ-polyglutamic acid comprises the following steps: (1) Pretreatment of corn stalks: The corn stalks are dried at 60°C, and then crushed by a pulverizer and passed through a 40-mesh sieve to obtain corn stalk powder.

[0023] (2) Seed culture: A single colony of Bacillus paralichrysogenum TR-1 plate was picked and inoculated into liquid seed culture medium. The culture was carried out at 35℃ and 200 rpm for 18 h until the mid-logarithmic growth phase to obtain the seed culture. The components of the seed culture were: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and sterilized at 121℃ for 20 min.

[0024] The screening process for Bacillus paralichrysogenum TR-1 was as follows: Select soil with a thick layer of fallen leaves and branches, dig out 10-15cm of soil, take 10g of soil, add it to 90ml of sterile distilled water, shake for 30min to mix, then heat in a 100℃ water bath for 10min. After cooling, take the supernatant and perform serial dilutions with sterile water, with a dilution factor of 10. 1 10 2 10 3 10 4 10 5 10 6 10 7 The microbial community was cultured at 37℃ for 24 hours to obtain a preliminary microbial community, which was then screened.

[0025] First, perform an initial screening, selecting the dilutions from the above steps at 100%. 1 ~10 7100 μL of each sample was spread onto a primary screening plate and incubated upside down. The primary screening plate medium was LB solid medium. Because the γ-polyglutamic acid producing bacteria have high viscosity, the colony morphology in the medium was used as a screening marker during the initial screening of γ-polyglutamic acid producing strains. Large, viscous, and easily stringy colonies were picked up with an inoculation loop and streaked to obtain single colonies for pure culture preservation in LB solid medium as primary screening strains. Then, a secondary screening was performed. The strains obtained from the primary screening were inoculated into seed culture medium and cultured at 37°C and 180 rpm for 12 h. The seed culture was then inoculated into fermentation medium at a volume ratio of 2% and cultured at 37°C and 180 rpm for 48 h. The fermentation broth was centrifuged at 4800 rpm for 25 min at 4°C. The supernatant was used to detect the γ-polyglutamic acid yield, and high-yielding γ-polyglutamic acid bacteria were screened and inoculated into the seed culture.

[0026] Detection method and curve plotting: Add the supernatant to 4 times the volume of anhydrous ethanol, place it in a 4°C refrigerator for 12 hours to precipitate, then remove it, centrifuge at 8000 rpm for 15 minutes at 4°C, remove the supernatant, dissolve the precipitate in ultrapure water, detect the absorbance at 216 nm using a UV spectrophotometer, and calculate the yield of γ-polyglutamic acid based on the standard curve.

[0027] Preparation of the standard curve: Take 0.01g of γ-polyglutamic acid standard sample, add ultrapure water and dilute to 100ml in a volumetric flask to prepare a 0.1g / L γ-polyglutamic acid standard solution. Take 0, 5, 10, 15, 20, 25, and 30 ml of the standard solution respectively and dilute to 100ml. Measure the absorbance at 216nm using a UV spectrophotometer. Plotting the standard curve: Plot a line graph based on the absorbance corresponding to different concentrations of the standard sample to obtain the standard curve. The formula is y=0.0007x+0.017, R0. 2 =0.9976, where y is the absorbance and x is the concentration of γ-polyglutamic acid in the sample.

[0028] (3) Fermentation culture: Shake flask fermentation culture was used, and the flasks were sealed with breathable silicone stoppers, so aeration was not required. The seed culture obtained in step 2 was transferred to the fermentation medium (composed of corn straw, 200 g / L; chicken manure, 20 g / L; magnesium sulfate, 0.5 g / L; sodium glutamate, 20 g / L; potassium dihydrogen phosphate, 3 g / L and the remainder being distilled water, with an initial pH of 7.0 and sterilization at 121℃ for 30 min) at 35℃ and a stirring speed of 200 rpm for 48 h to obtain a fermentation broth containing γ-polyglutamic acid.

[0029] The yield of γ-polyglutamic acid was 80.65 g / L, which was obtained by detection at 216 nm using a UV spectrophotometer and then using a standard curve.

[0030] UV detection and calculation method: Take 1 ml of fermentation broth, centrifuge at 4800 rpm and 4℃ for 25 min to remove the cells, take the supernatant, then add 4 times the volume of pre-cooled anhydrous ethanol, place in a 4℃ refrigerator for 12 h to precipitate, then take it out, centrifuge at 8000 rpm for 15 min at 4℃, remove the supernatant, dissolve the precipitate in ultrapure water, and make up to 100 ml. Detect its absorbance at 216 nm using a UV spectrophotometer. Calculate the sample concentration using the formula y = 0.0007x + 0.017 (where y is absorbance and x is sample concentration). Multiply the result by 100 to get the final concentration.

[0031] Examples 2 and 3 γ-polyglutamic acid was produced according to the method of Example 1, except that the carbon source in step 3 of the fermentation process in Examples 2 and 3 was different from that in Example 1, as detailed in Table 1.

[0032] Table 1. Content of γ-polyglutamic acid after fermentation in step 3 of Examples 1-3 with different carbon sources. Examples 4 and 5 γ-polyglutamic acid was produced according to the method of Example 1, except that the nitrogen source in step 3 of the fermentation culture in Examples 4 and 5 was different from that in Example 2, as detailed in Table 2.

[0033] Table 2. Content of γ-polyglutamic acid after fermentation in step 3 of Examples 1, 4-5 with different nitrogen sources. Examples 6-9 γ-polyglutamic acid was produced according to the method of Example 1, except that the concentration of carbon source in step 3 of the fermentation culture in Examples 6-9 was different from that in Example 1, as detailed in Table 3.

[0034] Table 3. Corn straw concentration and γ-polyglutamic acid content after fermentation in steps 3 of Examples 1, 6-9. Examples 10-13 γ-polyglutamic acid was produced according to the method of Example 1, except that the concentration of chicken manure in step 3 of the fermentation culture in Examples 10-13 was different from that in Example 1, as detailed in Table 4.

[0035] Table 4. Chicken manure concentration and γ-polyglutamic acid content after fermentation in steps 3 of Examples 1, 10-13. Examples 14-17 γ-polyglutamic acid was produced according to the method of Example 1, except that the concentration of sodium glutamate in step 3 of the fermentation culture in Examples 14-17 was different from that in Example 1, as detailed in Table 5.

[0036] Table 5. Monosodium glutamate concentration and γ-polyglutamic acid content after fermentation in steps 3 of Examples 1, 14-17. Examples 18-21 γ-polyglutamic acid was produced according to the method of Example 1, except that the temperature in step 3 of the fermentation process in Examples 18-21 was different from that in Example 1, as detailed in Table 6, where the absorbance values ​​are omitted.

[0037] Table 6. Fermentation temperature and γ-polyglutamic acid content after fermentation in step 3 of Examples 1, 18-21 Examples 22-25 γ-polyglutamic acid was produced according to the method of Example 1, except that the inoculum amount in step 3 of the fermentation culture in Examples 22-25 was different from that in Example 1, as detailed in Table 7, where the absorbance values ​​are omitted.

[0038] Table 7. Inoculum size and γ-polyglutamic acid content after fermentation in step 3 of Examples 1, 22-25 Examples 26-29 γ-polyglutamic acid was produced according to the method of Example 1, except that the fermentation time in step 3 of Examples 26-29 was different from that in Example 1, as detailed in Table 8, where the absorbance values ​​are omitted.

[0039] Table 8. Fermentation time and γ-polyglutamic acid content after fermentation in step 3 of Examples 1, 26-29 Examples 30-33 γ-polyglutamic acid was produced according to the method of Example 1, except that the oscillation speed in step 3 of the fermentation culture in Examples 30-32 was different from that in Example 1, as detailed in Table 9, where the absorbance values ​​are omitted.

[0040] Table 9. Shaking speed and γ-polyglutamic acid content after fermentation in step 3 of Examples 1, 30-33 All experimental data in this invention examples use three parallel control groups, and the data used in the table are average values. According to the example data, the optimal conditions for Bacillus paralichrysum TR-1 to produce γ-polyglutamic acid using the material in Example 1 are: 35℃, inoculum size of 8% (v / v), corn stalk to chicken manure ratio of 10, and sodium glutamate addition of 20 g / L, with a maximum yield of 80.65 g / L.

[0041] Example 34 Fermentation in a fermenter: The seed culture from step 2 was inoculated into a 5L fermenter at an inoculation rate of 8% (v / v). The fermentation medium volume was 3L. The fermentation temperature was 35℃, the tank pressure was 0.02Mpa, the aeration ratio was 1:1.25, and the fermentation speed was 350 rpm. During fermentation, the pH value decreased. Whenever the pH value decreased, 6M NaOH was added to adjust it and maintain the pH value at around 7.2. The culture was carried out for 72 hours. The components and contents of the fermentation medium were: corn straw, 200g / L; chicken manure, 20g / L; magnesium sulfate, 0.5g / L; monosodium glutamate, 20g / L; and potassium dihydrogen phosphate, 3g / L.

[0042] In this example, the yield of γ-polyglutamic acid was 66.58 g / L.

[0043] The fermentation method described in Example 34 above was used to produce γ-polyglutamic acid, with the difference being the different rotation speeds during the fermentation process, as detailed in Table 10, where absorbance values ​​are omitted.

[0044] Table 10 Fermentation speed and γ-polyglutamic acid content after fermentation in Examples 34-38 As can be seen from the table above, the yield of γ-polyglutamic acid increases with the increase of rotation speed. This is because the dissolved oxygen content in the tank also increases with the increase of rotation speed, which is conducive to the effective conversion of fermentation culture.

[0045] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for synthesizing γ-polyglutamic acid, characterized in that, Bacillus paralichrysogenum TR-1 was screened, and seed culture containing Bacillus paralichrysogenum TR-1 was inoculated into fermentation medium for aerobic fermentation to obtain γ-polyglutamic acid; Bacillus paralicheniform TR-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231731 and deposit date of September 18, 2023. The fermentation medium includes a carbon source, a nitrogen source, monosodium glutamate, magnesium sulfate, dipotassium hydrogen phosphate, and distilled water. The pH of the fermentation medium is 6.0-7.

5. The carbon source is corn stalks, and the nitrogen source is chicken manure. The C:N ratio used is 10:1; Aerobic fermentation can be performed in shake flasks or fermenters. When using shake flask fermentation, the amount of corn stalks added is 175~200g / L, the amount of monosodium glutamate added is 20~30g / L; the inoculum amount is 8~11%, the fermentation temperature is 37℃, the fermentation time is 60h, and the shaking speed is 180~200 rpm. When using a fermenter, the fermentation temperature is 35~39℃, the fermentation speed is 450~550rpm, the fermentation time is 60h, and the aeration rate is 0.8~1.2vvm.

2. The method for synthesizing γ-polyglutamic acid according to claim 1, characterized in that, The volume ratio of seed culture to fermentation medium is 1~15:

100.

3. The method for synthesizing γ-polyglutamic acid according to claim 1, characterized in that, The screening of Bacillus paralichrysogenum TR-1 includes the following steps: a. Sample preparation: Select soil covered with fallen leaves and branches, dig a depth of 10-15 cm, take a certain amount of soil, add it to sterile distilled water, shake to mix, then heat in a water bath for 10 min. After cooling, take the supernatant and perform serial dilution with sterile water at a dilution factor of 10. 1 10 2 10 3 10 4 10 5 10 6 10 7 Incubate at 37℃ for 24 hours to obtain a preliminary bacterial community; b. Initial screening: Take 10% of the solution diluted in step a. 1 ~10 7 Take 100 μL of each sample and spread it on a primary screening plate. Invert the plate and incubate. Use an inoculation loop to pick up a strain with large colonies, high viscosity, and easy stringing and streak it to obtain a single colony pure culture and preserve it in LB solid medium as a primary screening strain. c. Detection and rescreening: The strains obtained from the initial screening were inoculated into seed culture medium and cultured at 37℃ and 180 rpm for 12 h. The seed culture was then inoculated into fermentation culture medium and cultured at 37℃ and 180 rpm for 48 h. The fermentation broth was centrifuged at 4800 rpm for 25 min at 4℃. The supernatant was collected and the yield of γ-polyglutamic acid was detected by ultraviolet spectrophotometer. A strain with high γ-polyglutamic acid production was screened for expansion culture and preservation.

4. The method for synthesizing γ-polyglutamic acid according to claim 3, characterized in that, The initial screening plate medium was LB solid medium, with the following components: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and agar 18 g / L, and sterilized at 121℃ for 20 min.

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