Method for producing ultrahigh molecular weight gamma-polyglutamic acid through fermentation of bacillus subtilis
By using a compound molecular weight regulator and precise addition of concentrated nitrogen and carbon source solution during the fermentation process of γ-polyglutamic acid, combined with inexpensive raw materials such as beet molasses and corn steep liquor, the problems of single molecular weight control and high cost in the production of ultra-high molecular weight γ-polyglutamic acid have been solved. This has enabled the efficient and low-cost production of ultra-high molecular weight γ-polyglutamic acid, which is suitable for medical dressings and high-end cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-13
- Publication Date
- 2026-04-07
AI Technical Summary
Existing fermentation technologies for producing ultra-high molecular weight γ-polyglutamic acid suffer from limitations such as limited molecular weight control methods, high costs, and difficulty in achieving stable production. Traditional feeding strategies also lead to a mismatch between carbon and nitrogen source supply and cell synthesis, affecting product quality.
By employing a composite molecular weight regulator and a precise addition of concentrated nitrogen and carbon source solutions, combined with nutrient supply and molecular weight control during Bacillus subtilis fermentation, dynamic regulation is achieved by controlling the pH of the fermentation broth and the carbon source concentration. Inexpensive raw materials such as beet molasses and corn steep liquor are used as carbon sources to achieve phased carbon and nitrogen source replenishment.
It has enabled the targeted production of ultra-high molecular weight γ-polyglutamic acid, reducing production costs, increasing fermentation yield and product purity, ensuring molecular weight uniformity and production efficiency, and is suitable for medical dressings and high-end cosmetics.
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Figure CN121801982A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis, belonging to the field of bioengineering technology. Background Technology
[0002] γ-Polyglutamic acid (γ-PGA) is a polyamino acid biopolymer formed by linking glutamic acid monomers with amino groups via γ-carboxyl groups. Due to the large number of free carboxyl groups in its molecular chain, γ-PGA possesses excellent water solubility, water absorption and moisturizing properties, biodegradability, and biocompatibility, and is widely used in cosmetics, pharmaceuticals, agriculture, food, and environmental protection. Especially valuable is ultra-high molecular weight γ-PGA (molecular weight greater than 5 × 10⁻⁶). 6 Due to its higher viscosity and better film-forming properties, Dalton has greater application value in biomedical materials and high-end cosmetics.
[0003] However, current ultra-high molecular weight γ-PGA fermentation production technology still has many limitations. On the one hand, existing ultra-high molecular weight γ-PGA fermentation systems rely on single and limited methods for molecular weight regulation. These methods typically involve controlling fermentation pH, temperature, or adding a single substance to adjust molecular weight. There is a lack of complex molecular regulator systems that can synergistically regulate chain initiation, extension, and termination processes at the polymerization mechanism level. This results in a wide molecular weight distribution, a low proportion of ultra-high molecular weight components, and difficulty in consistently achieving ideal levels. On the other hand, there is a contradiction in the production routes for ultra-high molecular weight γ-PGA. It is difficult to achieve both high production costs and high performance. While using refined raw materials can guarantee the product's molecular weight, the raw material cost accounts for a very high proportion of the total cost, making it expensive and uneconomical. Using crude raw materials such as molasses to reduce costs can significantly lower costs, but the high impurity content and unstable composition result in low and fluctuating product molecular weights, making it difficult to stably produce ultra-high molecular weight products. In addition, in the current traditional fermentation production process, the feeding strategy is mostly based on time or cell density. However, the traditional timed or cell density-based feeding method fails to form a real-time and dynamic precise linkage with the key stages of product synthesis and the actual residual concentration of carbon source, nitrogen source and precursor in the fermentation broth. This results in the carbon and nitrogen source supply being out of sync with cell synthesis, which wastes raw materials and affects product quality.
[0004] Therefore, developing a method for the low-cost, efficient, and stable production of ultra-high molecular weight γ-PGA has significant industrial value and theoretical implications. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis, which can stably produce ultra-high molecular weight γ-PGA.
[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A method for producing ultra-high molecular weight γ-polyglutamic acid by Bacillus subtilis fermentation includes the following steps: (1) Inoculate the activated Bacillus subtilis seed culture into the fermentation medium and carry out fermentation culture; (2) When the pH of the fermentation broth drops below 7.0 after 9-12 hours of fermentation, add a compound molecular weight regulator to the fermentation broth and simultaneously add a concentrated nitrogen source solution. (3) When the fermentation culture has been carried out for 20-24 hours and the carbon source concentration in the fermentation broth is lower than 20 g / L, a concentrated carbon source solution is added, and the carbon source concentration in the fermentation broth is maintained at 20-25 g / L by controlling the addition rate; (4) Continue fermentation until the sodium glutamate in the fermentation medium is completely consumed, then stop fermentation and obtain a high-viscosity fermentation broth containing ultra-high molecular weight γ-PGA.
[0007] Furthermore, in step (1), the inoculation amount of the activated Bacillus subtilis seed liquid is 5-10%; the Bacillus subtilis is Bacillus subtilis FRD518, with the preservation number CGMCC No.6772.
[0008] Furthermore, in step (1), the fermentation medium contains the following components: 100-120 g / L beet molasses, 60-80 g / L sodium glutamate, 10-15 g / L ammonium sulfate, 2.5-7.5 g / L urea, 1-3 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L magnesium sulfate heptahydrate, 0.1-0.5 g / L calcium chloride, and an initial pH of 7.3-7.5.
[0009] Furthermore, in step (1), the method for preparing the activated Bacillus subtilis seed liquid is as follows: (a) Bacillus subtilis was inoculated onto LB solid slant medium and cultured in a constant temperature incubator at 37°C for 20 h to obtain activated strains; the components of the LB solid slant medium were: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar. (b) The activated bacterial culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a shaker at 37 °C and 250 r / min for 16 h until the OD600 reached 2.5-3.0; then, the seed culture was inoculated at a rate of 5% into a 1000 mL Erlenmeyer flask containing 400 mL of seed culture medium for expansion culture, and cultured in a shaker at 37 °C and 250 r / min for 6 h until the OD600 reached 3.0-3.5, to obtain activated Bacillus subtilis seed culture; the seed culture medium composition was: glucose 10 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium chloride 0.2 g / L; pH 7.3-7.5.
[0010] Furthermore, in step (2), the concentrated nitrogen source solution is a mixed aqueous solution of ammonium sulfate and urea, wherein the concentration of ammonium sulfate is 20-30 g / L and the concentration of urea is 10-15 g / L; the feed rate is 5-6 mL / (L·h). The feed-on concentrated nitrogen source maintains a low but continuous nitrogen supply during the product synthesis period, which can maintain the necessary metabolic activity of the cells, especially the turnover of proteins required for the synthesis of γ-PGA and the operation of the energy metabolism system, but does not provide excessive nitrogen source to promote excessive cell growth and compete with the substrate for product synthesis, while avoiding cell metabolic stagnation, aging or autolysis caused by insufficient nitrogen source.
[0011] Furthermore, in step (2), the composite molecular weight regulator is composed of glycerol and sodium citrate in a mass ratio of (2-4):1, and the total amount added is 1-3% of the fermentation broth volume. Sodium citrate and glycerol are mixed and added together to the fermentation broth. Glycerol can regulate the osmotic pressure of the fermentation broth, stabilize the cell membrane and synthase conformation, and provide a suitable microenvironment for long-chain polymerization. Sodium citrate, as a metal ion chelating agent, enters the cell and participates in the TCA cycle, regulating intracellular energy and reducing power levels. Simultaneously, it can inhibit the activity of γ-PGA degrading enzymes activated by metal ions in the fermentation broth, preventing long-chain breakage. The addition of the composite molecular weight regulator can directionally promote long-chain polymerization and inhibit degradation in the early stages of product synthesis.
[0012] Furthermore, in step (3), the concentrated carbon source solution is a mixed aqueous solution of beet molasses and corn steep liquor, wherein the mass ratio of beet molasses to corn steep liquor is (8-10):1. Beet molasses is rich in fermentable sugars such as sucrose and glucose, and is the core carbon source; corn steep liquor contains micronutrients such as amino acids, vitamins, and growth factors. The combination of the two can achieve synergistic supply, which can not only meet the carbon requirements for γ-PGA synthesis, but also supplement the key factors required by the bacterial synthesis enzyme system, maintain the high activity of Bacillus subtilis, and thus ensure the continuous progress of the γ-PGA polymerization reaction. At the same time, both beet molasses and corn steep liquor are industrial by-products with low cost.
[0013] Furthermore, the beet molasses was purchased from Shandong Yimeng Hope Agriculture Co., Ltd.; and the corn steep liquor was purchased from Shanghai Yuantai Biotechnology Co., Ltd.
[0014] Furthermore, in step (4), the weight-average molecular weight (Mw) of the ultra-high molecular weight γ-PGA is greater than 5 million Daltons.
[0015] Furthermore, the conditions for the entire fermentation process are: temperature of 35-38℃, stirring speed of 500-800rpm, aeration rate of 1.0-1.5vvm, and fermentation time of 48-72h.
[0016] Furthermore, the ultra-high molecular weight γ-polyglutamic acid can be used to prepare medical dressings, high-end cosmetics, and drug sustained-release carrier materials. This γ-polyglutamic acid possesses ultra-high molecular weight and excellent film-forming, moisturizing, and thickening properties, making it suitable for preparing materials for medical, cosmetic, and other fields with stringent performance requirements.
[0017] This invention achieves the synthesis of ultra-high molecular weight γ-polyglutamic acid with a weight average molecular weight > 5 million Daltons by precisely controlling three core aspects of the fermentation process: nutrient supply, molecular weight directional regulation, and metabolic environment stabilization. It relies on the inherent metabolic pathway of Bacillus subtilis to promote the polymerization of long-chain γ-polyglutamic acid and inhibit its degradation. At the start of fermentation, the basal medium provides the initial carbon, nitrogen, and monosodium glutamate (MSG) sources to meet the rapid proliferation needs of the strain and build a sufficient population of enzyme-producing cells, laying the foundation for subsequent high synthesis efficiency. In the early stages of product synthesis, MSG serves as a direct precursor. With ATP as the energy source, the synthase catalyzes the formation of an amide bond between the γ-carboxyl group of one glutamate residue and the α-amino group of another glutamate residue, achieving linear long-chain polymerization. Simultaneously, a complex molecular weight regulator is added. At this point, γ-PGA synthesis has just begun, which can guide the directional connection of glutamate residues from the initial polymerization stage, reducing the generation of short-chain byproducts and promoting the formation of ultra-high molecular weight long chains. In the later stages of product synthesis, a complex carbon source is fed in, utilizing the carbohydrate metabolism of beet molasses and corn steep liquor to generate ATP and NADPH, continuously providing energy and metabolic intermediates for amide bond formation and long-chain elongation, preventing carbon source deficiency from causing the termination of γ-PGA synthesis or shortening of chain length.
[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention achieves the targeted production of ultra-high molecular weight γ-polyglutamic acid by precisely controlling the nutrient supply and molecular weight regulation during the fermentation process, adding a compound molecular weight regulator, and adding concentrated nitrogen source solution and carbon source solution, thereby increasing fermentation yield and production efficiency.
[0019] (2) By combining glycerol and sodium citrate and adding them at a specific fermentation stage, the present invention can synergistically promote synthesis and inhibit degradation, and can stably produce ultra-high molecular weight γ-PGA with a weight average molecular weight of more than 5 million Daltons, thus solving the bottleneck of low molecular weight in conventional methods.
[0020] (3) This invention uses inexpensive raw materials such as beet molasses and corn steep liquor as the main carbon source and feed source. Combined with precise feed control, it reduces the cost of raw materials by more than 30% while achieving a high level of γ-PGA fermentation yield of 55-65 g / L, significantly improving fermentation yield and reducing production costs, thus improving economic benefits.
[0021] (4) The present invention uses the pH of fermentation broth and carbon source concentration as key trigger signals to induce molecular weight and supplement carbon and nitrogen sources in stages, so that the process control is highly synchronized with the physiological metabolism of cells and the product synthesis rhythm, thereby improving process efficiency and molecular weight uniformity.
[0022] (5) The purity of the ultra-high molecular weight γ-PGA obtained by this invention can reach 96.4%. The product has good quality and high application value, laying the foundation for its application in high value-added fields such as biomedicine and high-end daily chemicals. Attached Figure Description
[0023] Figure 1 This is the kinetic curve of Bacillus subtilis fermentation for the production of ultra-high molecular weight γ-polyglutamic acid; Figure 2 It is the molecular weight GPC spectrum of ultra-high molecular weight γ-polyglutamic acid; Figure 3 This is a GPC spectrum of ultra-high molecular weight γ-polyglutamic acid content. Detailed Implementation
[0024] Example 1 A method for producing ultra-high molecular weight γ-polyglutamic acid by Bacillus subtilis fermentation includes the following steps: (1) Bacillus subtilis was inoculated onto LB solid slant medium and cultured in a constant temperature incubator at 37℃ for 20 h to obtain activated strains; The components of the LB solid slant culture medium are: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar; (2) The activated bacterial strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a shaker at 37℃ and 250 r / min for 16 h until the OD600 reached 2.5-3.0; then, the seed culture was inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of seed culture medium at a rate of 5% to expand the culture, and cultured in a shaker at 37℃ and 250 r / min for 6 h until the OD600 reached 3.0-3.5 to obtain activated Bacillus subtilis seed culture; The seed culture medium consists of: 10 g / L glucose, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 0.2 g / L calcium chloride; pH is 7.3-7.5. (3) The activated Bacillus subtilis seed liquid was inoculated into the fermentation medium at an inoculation amount of 5%, and fermentation was carried out at a temperature of 38℃, a stirring speed of 500 rpm, and an aeration rate of 1.5 vvm. The fermentation medium contains the following components: beet molasses 100-120 g / L, monosodium glutamate 60-80 g / L, ammonium sulfate 10-15 g / L, urea 2.5-7.5 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 0.5-1.5 g / L, calcium chloride 0.1-0.5 g / L, with an initial pH of 7.3-7.5; (4) When the pH of the fermentation broth drops below 7.0 after 9-12 hours of fermentation, add a compound molecular weight regulator to the fermentation broth. The compound molecular weight regulator is composed of glycerol and sodium citrate in a mass ratio of 2:1. The amount added is 3% of the volume of the fermentation broth. At the same time, start adding a concentrated nitrogen source solution. The concentrated nitrogen source solution is a mixed aqueous solution of ammonium sulfate and urea, with an ammonium sulfate concentration of 20 g / L and a urea concentration of 15 g / L. The flow rate is 5 mL / (L·h). (5) When the fermentation culture has been carried out for 20-24 hours and the carbon source concentration in the fermentation broth is less than 20 g / L, the concentrated carbon source solution is started to be added. The concentrated carbon source solution is a mixed aqueous solution of beet molasses and corn steep liquor, wherein the mass ratio of beet molasses to corn steep liquor is 10:1, and the carbon source concentration in the fermentation broth is maintained at 25 g / L by controlling the flow rate. (6) Continue fermentation until the sodium glutamate in the fermentation medium is completely consumed, then stop fermentation and obtain a high-viscosity fermentation broth containing ultra-high molecular weight γ-PGA.
[0025] Example 2 A method for producing ultra-high molecular weight γ-polyglutamic acid by Bacillus subtilis fermentation includes the following steps: (1) Bacillus subtilis was inoculated onto LB solid slant medium and cultured in a constant temperature incubator at 37℃ for 20 h to obtain activated strains; The components of the LB solid slant culture medium are: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar; (2) The activated bacterial strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a shaker at 37℃ and 250 r / min for 16 h until the OD600 reached 2.5-3.0; then, the seed culture was inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of seed culture medium at a rate of 5% to expand the culture, and cultured in a shaker at 37℃ and 250 r / min for 6 h until the OD600 reached 3.0-3.5 to obtain activated Bacillus subtilis seed culture; The seed culture medium consists of: 10 g / L glucose, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 0.2 g / L calcium chloride; pH is 7.3-7.5. (3) The activated Bacillus subtilis seed liquid was inoculated into the fermentation medium at an inoculation amount of 10%, and fermentation was carried out at a temperature of 35℃, a stirring speed of 800 rpm, and an aeration rate of 1.0 vvm. The fermentation medium contains the following components: beet molasses 100-120 g / L, monosodium glutamate 60-80 g / L, ammonium sulfate 10-15 g / L, urea 2.5-7.5 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 0.5-1.5 g / L, calcium chloride 0.1-0.5 g / L, with an initial pH of 7.3-7.5; (4) When the pH of the fermentation broth drops below 7.0 after 9-12 hours of fermentation, add a compound molecular weight regulator to the fermentation broth. The compound molecular weight regulator is composed of glycerol and sodium citrate in a mass ratio of 4:1. The amount added is 1% of the volume of the fermentation broth. At the same time, start adding a concentrated nitrogen source solution. The concentrated nitrogen source is a mixed aqueous solution of ammonium sulfate and urea, with an ammonium sulfate concentration of 30 g / L and a urea concentration of 10 g / L. The flow rate is 6 mL / (L·h). (5) When the fermentation culture has been carried out for 20-24 hours and the carbon source concentration in the fermentation broth is less than 20 g / L, the concentrated carbon source solution is added. The concentrated carbon source solution is a mixed aqueous solution of beet molasses and corn steep liquor, wherein the mass ratio of beet molasses to corn steep liquor is 8:1, and the carbon source concentration in the fermentation broth is maintained at 25 g / L by controlling the flow rate. (6) Continue fermentation until the sodium glutamate in the fermentation medium is completely consumed, and the fermentation ends, obtaining a high-viscosity fermentation broth containing ultra-high molecular weight γ-PGA.
[0026] Example 3 A method for producing ultra-high molecular weight γ-polyglutamic acid by Bacillus subtilis fermentation includes the following steps: (1) Bacillus subtilis was inoculated onto LB solid slant medium and cultured in a constant temperature incubator at 37℃ for 20 h to obtain activated strains; The components of the LB solid slant culture medium are: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar; (2) The activated bacterial strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured in a shaker at 37℃ and 250 r / min for 16 h until the OD600 reached 2.5-3.0; then, the seed liquid was inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of seed culture medium at a rate of 5% for expansion culture and cultured in a shaker at 37℃ and 250 r / min for 6 h until the OD600 reached 3.0-3.5, thus obtaining activated Bacillus subtilis seed liquid; The seed culture medium consists of: 10 g / L glucose, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 0.2 g / L calcium chloride; pH is 7.3-7.5. (3) The activated Bacillus subtilis seed liquid was inoculated into the fermentation medium at an inoculation amount of 7%, and fermentation was carried out at a temperature of 37℃, a stirring speed of 650 rpm, and an aeration rate of 1.3 vvm. The fermentation medium contains the following components: beet molasses 100-120 g / L, monosodium glutamate 60-80 g / L, ammonium sulfate 10-15 g / L, urea 2.5-7.5 g / L, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 0.5-1.5 g / L, calcium chloride 0.1-0.5 g / L, with an initial pH of 7.3-7.5; (4) When the pH of the fermentation broth drops below 7.0 after 9-12 hours of fermentation, add a compound molecular weight regulator to the fermentation broth. The compound molecular weight regulator is composed of glycerol and sodium citrate in a mass ratio of 3:1. The amount added is 2% of the volume of the fermentation broth. At the same time, start adding a concentrated nitrogen source solution. The concentrated nitrogen source is a mixed aqueous solution of ammonium sulfate and urea, with an ammonium sulfate concentration of 25 g / L and a urea concentration of 12 g / L. The flow rate is 5.5 mL / (L·h). (5) When the fermentation culture has been carried out for 20-24 hours and the carbon source concentration in the fermentation broth is less than 20 g / L, the concentrated carbon source solution is started to be added. The concentrated carbon source solution is a mixed aqueous solution of beet molasses and corn steep liquor, wherein the mass ratio of beet molasses to corn steep liquor is 9:1, and the carbon source concentration in the fermentation broth is maintained at 23 g / L by controlling the flow rate. (6) Continue fermentation until the sodium glutamate in the fermentation medium is completely consumed, and the fermentation ends, obtaining a high-viscosity fermentation broth containing ultra-high molecular weight γ-PGA.
[0027] Comparative Example 1 The steps are the same as in Example 3, except that in step (3) the fermentation medium is replaced with a conventional medium, and in step (4) no compound molecular weight regulator is added, no concentrated nitrogen source solution is added, and in step (5) no concentrated carbon source solution is added.
[0028] The conventional culture medium consists of: 40 g / L sucrose, 25 g / L sodium glutamate, 8 g / L yeast extract, 6 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, and 0.8 g / L magnesium sulfate heptahydrate.
[0029] Comparative Example 2 The steps are the same as in Example 3, except that no composite molecular weight regulator is added in step (4).
[0030] Comparative Example 3 The steps are the same as in Example 3, except that in step (4), a concentrated nitrogen source solution is not added.
[0031] Comparative Example 4 The steps are the same as in Example 3, except that in step (5), a concentrated carbon source solution is not added.
[0032] Comparative Example 5 The steps are the same as in Example 3, except that a concentrated nitrogen source solution was not added in step (4) and a concentrated carbon source solution was not added in step (5).
[0033] Comparative Example 6 The steps are the same as in Example 3, except that no composite molecular weight regulator is added, and no concentrated nitrogen source solution or concentrated carbon source solution is added.
[0034] Experimental Example 1 The fermentation broths obtained in Example 3 and Comparative Examples 1-6 were extracted and purified, and the yield, purity, and molecular weight of γ-PGA were determined. The results are listed in Table 1.
[0035] The extraction and purification method is as follows: Three volumes of anhydrous ethanol are added to the fermentation broth to precipitate γ-PGA. The precipitate is collected, washed twice with 70% ethanol, and then reconstituted with two volumes of purified water. The solution is incubated at 80℃ for 2 hours to denature and inactivate impurities. The solution is then decolorized with 1% activated carbon for 1 hour, followed by plate and frame filtration until the transmittance of the filtrate is ≥99%. The pH of the filtrate is then adjusted to 2.0 with concentrated hydrochloric acid, allowed to stand for 24 hours, and the precipitate is collected. This precipitate is then filtered through a plate and frame filter, collected, and reconstituted with purified water. The pH of the solution is adjusted to 5.0-6.0. Finally, pure γ-PGA is obtained by vacuum freeze-drying.
[0036] Table 1. Yield, purity, and molecular weight of γ-PGA As shown in Table 1, the yield, purity, and molecular weight of γ-PGA in Example 3 were significantly better than those in Comparative Example 1. This indicates that by optimizing the culture medium formulation, using high concentrations of beet molasses and monosodium glutamate, and introducing a precise staged fed-batch strategy, substrate limitations were greatly eliminated, significantly improving the cell productivity and product accumulation level. Comparative Example 2, lacking only the compound molecular weight regulator, had a higher yield than other comparative examples, but its molecular weight plummeted to 2.1 million Daltons, far lower than the 6.2 million Daltons of Example 3. This indicates that the compound molecular weight regulator can significantly improve the degree of polymerization of γ-PGA by regulating the polymerization activity of Bacillus subtilis γ-PGA synthase. Comparative Example 3, lacking only the nitrogen source fed-batch, had a yield reduced to 40.6 g / L and a molecular weight reduced to 4.25 million Daltons. This is because fermentation 9-12 hours is the logarithmic phase of γ-PGA synthesis. The fed-batch ammonium sulfate-urea mixed nitrogen source can maintain cell activity and stable expression of the synthase, avoiding metabolic stagnation and interruption of the polymerization reaction due to insufficient nitrogen source. Comparative Example 4, lacking only the carbon source feedstock, saw its yield drop to 30.5 g / L and molecular weight decrease to 3.82 million Daltons. This is because the 20-24 h feedstock of beet molasses-corn steep liquor carbon source maintained a stable carbon source concentration of 23 g / L in the fermentation broth, preventing premature termination of γ-PGA synthesis due to carbon source depletion and ensuring continuous product accumulation. Comparative Example 5, lacking both nitrogen and carbon source feedstocks, showed a significantly greater decrease in yield and molecular weight than Comparative Examples 3 and 4, demonstrating the synergistic effect of nitrogen and carbon source feedstocks in providing a continuous and stable substrate and energy supply for γ-PGA synthesis. Comparative Example 6, retaining only the fermentation medium from Example 3, exhibited extremely low γ-PGA yield and molecular weight. Therefore, this invention, through the addition of a composite molecular weight regulator and nitrogen / carbon source feedstock, can achieve high-yield and high-purity synthesis of ultra-high molecular weight γ-PGA.
[0037] Experimental Example 2 Following the steps in Example 3, the effect of the composite molecular weight regulator on the molecular weight of γ-PGA was investigated. The components and ratios of the composite molecular weight regulator were adjusted, and the obtained fermentation broth was extracted and purified. The molecular weight of γ-PGA was then determined. The results are listed in Table 2.
[0038] Table 2. Molecular weight of γ-PGA Table 2 shows that when only glycerol or sodium citrate is added as a molecular weight regulator, the molecular weight of γ-PGA increases slightly, but the increase is limited. However, when the mass ratio of glycerol to sodium citrate is (2-4):1, the molecular weight of γ-PGA increases significantly, far exceeding that of any single component, demonstrating a synergistic effect. As the mass ratio of glycerol to sodium citrate increases from 1:1 to 3:1, the molecular weight of γ-PGA increases. When the mass ratio of glycerol to sodium citrate exceeds 3:1, the molecular weight of γ-PGA begins to decrease. This indicates that when the composite molecular weight regulator is composed of glycerol and sodium citrate in a mass ratio of 3:1, it can maximize the synthesis of ultra-high molecular weight (>5 million Daltons) γ-PGA.
[0039] Experimental Example 3: Fermentation Kinetics Analysis Kinetic analysis was performed on the fermentation process of Bacillus subtilis to produce ultra-high molecular weight γ-polyglutamic acid. The results are shown in the attached figure. Figure 1 As shown.
[0040] from Figure 1 It is known that the fermentation period from 0 to 20 hours is the stage of cell proliferation and substrate consumption. During this stage, the OD600 rises rapidly, indicating that Bacillus subtilis grows and proliferates rapidly in the early stage of fermentation. The carbon source and monosodium glutamate (MSG) decrease rapidly because a large amount of substrate is used for cell growth and basal metabolism. γ-PGA accumulates slowly, indicating that cell proliferation is the main focus during this stage, and the amount of γ-PGA synthesized is relatively small. The pH initially decreases rapidly and then tends to stabilize, indicating that the cell metabolism produces acidic substances, leading to a decrease in pH. Subsequently, cell metabolism tends to stabilize, and pH changes slow down. Therefore, during this stage, a complex molecular weight regulator is added to the fermentation broth, and a concentrated nitrogen source solution is simultaneously added.
[0041] The fermentation period of 20-40 hours is the stage of massive γ-PGA synthesis. During this stage, γ-PGA levels rise rapidly, and the cells enter a stationary phase, shifting their metabolic focus to γ-PGA synthesis, resulting in a large accumulation of the product. The carbon source concentration continuously decreases, with some used for γ-PGA synthesis and the rest for maintaining cell metabolism. Monosodium glutamate (MSG) is rapidly depleted because it is a direct precursor to γ-PGA and is efficiently used for product synthesis. OD600 remains at a high level, indicating that the cells have entered a stationary phase and the concentration no longer increases significantly. Therefore, during this stage, a concentrated carbon source solution is fed in, and the carbon source concentration in the fermentation broth is maintained at 20-25 g / L by controlling the feeding rate.
[0042] The fermentation period of 40-60 hours is considered the late stage of fermentation. During this stage, γ-PGA growth slows down and tends to stabilize because the precursor monosodium glutamate (MSG) has been depleted, and product synthesis has ceased. The remaining carbon source is low, and the substrate is essentially consumed. OD600 decreases slightly, and some cells begin to die. pH fluctuates slightly before stabilizing: metabolic activity weakens, and pH changes become gradual. The highest γ-PGA yield was 64.3 g / L; the maximum cell density OD600 was 21.5; the γ-PGA yield was 0.92 g / g substrate; and the production intensity was 0.115 g / L / h.
[0043] Experimental Example 4: Analysis of Molecular Weight and Intrinsic Viscosity of Pure Product The fermentation broth obtained in Example 3 was extracted and purified to obtain pure γ-PGA. The extraction and purification methods were the same as in Experiment 1. The pure γ-PGA was then tested for molecular weight, purity, and intrinsic viscosity. The results are attached. Figure 2-3 As shown.
[0044] The testing method is as follows: Molecular weight: Determined by gel permeation chromatography (GPC), using sodium polypropylene sulfonate of different molecular weights as standards, and K2HPO4 aqueous solution with a concentration of 10 mmol / L and NaCl aqueous solution with a concentration of 38.6 mmol / L as mobile phases, with a flow rate of 0.5-1.0 mL / min and a test temperature of 35℃.
[0045] Intrinsic viscosity: Measured using an Ubbelohde viscometer. A certain amount of pure γ-PGA was weighed and dissolved in a 1.2 mol / L sodium chloride solution (t1 / t0 controlled between 1.3 and 1.5). The outflow time of polymer solutions at different concentrations (c) was measured, and the relative viscosity (ηᵣ) and specific viscosity (ηᵣ) were calculated. sp ), and with η sp Plotting / c or (lnηᵣ) / c against concentration c, and extrapolating to the intercept when the concentration is zero (c→0), gives the intrinsic viscosity [η]; where η r =t / t0;η sp =η r -1.
[0046] Tests showed that the weight-average molecular weight (Mw) of γ-PGA was 6.2 million Daltons, and its intrinsic viscosity was η=7.22. The intrinsic viscosity of commercially available γ-PGA samples with a molecular weight of 2 million Daltons was approximately 2.0. The γ-PGA obtained in this invention is significantly superior to commercially available γ-PGA, demonstrating that the γ-PGA of this invention possesses ultra-high molecular weight, high intrinsic viscosity, and strong rheological properties. Furthermore, from the attached... Figure 2-3 The GPC spectrum of γ-PGA shows that γ-PGA has high purity and good homogeneity.
Claims
1. A method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis, characterized in that: Includes the following steps: (1) Inoculate the activated Bacillus subtilis seed culture into the fermentation medium and carry out fermentation culture; (2) When the pH of the fermentation broth drops below 7.0 after 9-12 hours of fermentation, add a compound molecular weight regulator to the fermentation broth and simultaneously add a concentrated nitrogen source solution. (3) When the fermentation culture has been carried out for 20-24 hours and the carbon source concentration in the fermentation broth is lower than 20 g / L, a concentrated carbon source solution is added, and the carbon source concentration in the fermentation broth is maintained at 20-25 g / L by controlling the addition rate; (4) Continue fermentation until the sodium glutamate in the fermentation medium is completely consumed, then stop fermentation and obtain a high-viscosity fermentation broth containing ultra-high molecular weight γ-PGA.
2. The method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis according to claim 1, characterized in that: In step (1), the fermentation medium contains the following components: 100-120 g / L beet molasses, 60-80 g / L sodium glutamate, 10-15 g / L ammonium sulfate, 2.5-7.5 g / L urea, 1-3 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L magnesium sulfate heptahydrate, 0.1-0.5 g / L calcium chloride, and an initial pH of 7.3-7.
5.
3. The method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis according to claim 2, characterized in that: In step (2), the concentrated nitrogen source solution is a mixed aqueous solution of ammonium sulfate and urea, wherein the concentration of ammonium sulfate is 20-30 g / L and the concentration of urea is 10-15 g / L; the flow rate is 5-6 mL / (L·h).
4. The method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis according to claim 3, characterized in that: In step (2), the composite molecular weight regulator is composed of glycerol and sodium citrate in a mass ratio of (2-4):1, and the total amount added is 1-3% of the fermentation liquid volume.
5. The method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis according to claim 4, characterized in that: In step (3), the concentrated carbon source solution is a mixed aqueous solution of beet molasses and corn steep liquor, wherein the mass ratio of beet molasses to corn steep liquor is (8-10):
1.
6. The method for producing ultra-high molecular weight γ-polyglutamic acid by fermentation of Bacillus subtilis according to claim 5, characterized in that: In step (4), the weight-average molecular weight (Mw) of the ultra-high molecular weight γ-PGA is greater than 5 million Daltons.