Bacillus licheniformis fermentation medium for low-cost high-yield gamma-aminobutyric acid and application

Abstract

This invention belongs to the field of microbial fermentation engineering technology, specifically disclosing a low-cost, high-yield γ-aminobutyric acid (GABA) fermentation medium for Bacillus licheniformis and its application. The medium comprises: glucose 30-80 g / L, organic nitrogen source 5-40 g / L, ammonium sulfate 6-20 g / L, K₂HPO₄·3H₂O 1-12.5 g / L, KH₂PO₄ 1-2.5 g / L, VH 0.1-0.5 mg / L, VB₁ 0.1-0.5 mg / L, Trace Metal Mix A9 stock solution 1-20 mL / L, with the remainder being deionized water, and a pH of 7.0-7.5. This invention further provides the application of the medium, including segmented pH control and fermentation conversion by supplementing with glutamate or glutamate solution in the later stages of fermentation. Using the medium of this invention in a 20 L fermenter, the GABA yield can reach 70.0 g / L; further supplementation with glutamate or glutamate solution in the later stages of fermentation can further increase the final GABA yield. The culture medium of this invention is low in cost, the raw materials are readily available, and the fermentation yield is high. It can realize the coupled production of GABA by de novo fermentation and subsequent in-situ conversion, and has good prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation engineering technology, specifically relating to a low-cost, high-yield Bacillus licheniformis fermentation medium and its application. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid widely found in microorganisms, plants, and animals. GABA possesses various physiological functions, including regulating nerve conduction, lowering blood pressure, sedation, anti-anxiety effects, improving sleep, and regulating the body's acid-base balance. It has broad application prospects in the food, pharmaceutical, health product, agriculture, animal husbandry, and chemical industries. Furthermore, GABA serves as an important precursor for the synthesis of 2-pyrrolidone and the biodegradable material polyamide nylon 4, demonstrating significant industrial application value.

[0003] Currently, the main methods for preparing GABA include chemical synthesis, plant enrichment, enzymatic or whole-cell catalysis, and microbial fermentation. Chemical synthesis typically suffers from harsh reaction conditions, poor product safety, and complex post-processing. While plant enrichment offers better safety, it results in low product concentrations and limited production efficiency, making it unsuitable for large-scale industrial production. Enzymatic or whole-cell catalysis methods usually require the addition of large amounts of glutamate or monosodium glutamate as substrates and necessitate the separate preparation of catalytic enzymes or catalytic cells, further complicating production costs and processes.

[0004] Microbial fermentation can directly synthesize GABA using inexpensive carbon and nitrogen sources such as glucose. It offers advantages such as mild reaction conditions, environmental friendliness, controllable processes, and ease of scale-up, making it an important direction for the industrial production of GABA. Bacillus licheniformis, as an important industrial microorganism, possesses advantages such as high biosafety, strong stress resistance, good fermentation stability, and suitability for high-density cultivation, making it suitable as a substrate strain for GABA fermentation production.

[0005] Existing technology CN118995551A discloses a recombinant Bacillus licheniformis strain for the fermentation production of γ-aminobutyric acid (GABA), its construction method, and its applications. This technology achieves de novo fermentation synthesis of GABA through multi-site genetic engineering of Bacillus licheniformis. The GABA fermentation medium disclosed in this patent mainly includes components such as glucose, peptone, yeast extract, ammonium sulfate, phosphate, and Trace Metal Mix A9. This medium can support the synthesis of GABA by recombinant Bacillus licheniformis; however, the peptone and yeast extract used are expensive refined organic nitrogen sources, which would significantly increase the cost of the medium in large-scale industrial fermentation.

[0006] Furthermore, while peptone and yeast extract are rich in nutrients, their batch stability, price fluctuations, and industrialization costs all affect the economics of GABA production. In contrast, soybean meal and corn steep liquor are widely available and inexpensive industrial or agricultural processing byproducts, possessing the potential to serve as nitrogen sources for fermentation. However, soybean meal and corn steep liquor have complex compositions, with their available nitrogen, amino acid, vitamin, mineral, and growth factor compositions significantly different from those of peptone and yeast extract. Directly replacing refined nitrogen sources often leads to problems such as limited cell growth, insufficient expression of glutamate decarboxylase, changes in fermentation broth viscosity, or increased byproducts. Those skilled in the art would find it difficult to anticipate that simply replacing peptone and yeast extract with soybean meal and corn steep liquor would still yield higher GABA yields.

[0007] On the other hand, existing fermentation methods typically rely on bacterial strains to synthesize GABA de novo from glucose via metabolic pathways. Whole-cell catalysis, however, usually requires separate cultivation and collection of catalytic cells, followed by the addition of glutamate or glutamate as a substrate for conversion. If, in the later stages of fermentation, the high-density bacterial cells and highly active glutamate decarboxylase already present in the fermentation system could be utilized to directly supplement glutamate or glutamate for in-situ conversion, it would be possible to couple de novo fermentation synthesis with whole-cell catalytic conversion within the same fermentation system, thereby further increasing the final GABA yield and reducing overall production costs. However, current technologies do not disclose a coupled production process using a low-cost soybean meal-corn steep liquor medium combined with segmented pH control and subsequent glutamate supplementation.

[0008] Therefore, developing a culture medium and fermentation process that uses inexpensive and readily available raw materials, has a simple formulation, is compatible with Bacillus licheniformis GABA fermentation system, and can combine in-situ conversion of glutamate in the later stage of fermentation to further increase the final yield of GABA is of great significance for reducing the cost of industrial production of GABA, improving fermentation efficiency, and enhancing process competitiveness. Summary of the Invention

[0009] To address the problems of high culture medium cost, limited fermentation yield, and limited final product concentration in the de novo synthesis process of Bacillus licheniformis fermentation to produce GABA in existing technologies, this invention provides a low-cost, high-yield Bacillus licheniformis fermentation culture medium.

[0010] Another objective of this invention is to provide a low-cost, high-yield γ-aminobutyric acid (GABA) fermentation medium for Bacillus licheniformis, which utilizes the above-mentioned medium for segmented pH-controlled fermentation and supplements glutamic acid or glutamate in the later stage of fermentation to achieve in-situ conversion to produce GABA.

[0011] This invention enables recombinant Bacillus licheniformis to maintain good growth and GABA synthesis capacity in a low-cost culture medium by replacing peptone and yeast powder with soybean meal and corn steep liquor, and by adding specific proportions of ammonium sulfate, phosphate, vitamins and trace elements. Furthermore, by supplementing glutamate or glutamate in the later stage of fermentation, the fermentation system has both de novo synthesis and in situ conversion functions, thereby significantly increasing the final GABA yield.

[0012] To achieve the above objectives, the present invention adopts the following technical solution.

[0013] A low-cost, high-yield fermentation medium for Bacillus licheniformis containing: glucose 30-80 g / L, organic nitrogen source 5-40 g / L, (NH4)2SO4 6-20 g / L, K2HPO4·3H2O 1-12.5 g / L, KH2PO4 1-2.5 g / L, VH 0.1-0.5 mg / L, VB1 0.1-0.5 mg / L, Trace Metal Mix A9 stock solution 1-20 mL / L, with the remainder being deionized water, and a pH of 7.0-7.5. The Trace Metal Mix A9 stock solution consists of: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0 g / L, and NaMoO4·2H2O 0.2 g / L. The organic nitrogen source is soybean meal and / or corn steep liquor. The organic nitrogen source is 0.1 g / L CuCl·H2O, 4.0 g / L FeSO4·7H2O, 0.1 g / L AlCl3·6H2O, and 4.0 g / L CaCl2.

[0014] Preferably, the culture medium comprises the following components: The formula consists of: glucose 50-70 g / L, soybean meal 5-30 g / L, corn steep liquor 2-4 g / L, (NH4)2SO4 10-16 g / L, K2HPO4·3H2O 5-10 g / L, KH2PO4 1.5-2.0 g / L, vitamin H 0.4 mg / L, vitamin B1 0.2 mg / L, Trace Metal Mix A9 mother liquor 5-15 mL / L, with the remainder being deionized water at pH 7.0-7.5. The Trace Metal Mix A9 mother liquor composition is: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0 g / L, NaMoO4·2H2O 0.2 g / L, CuCl·H2O 0.1 g / L. g / L, FeSO4·7H2O 4.0 g / L, AlCl3·6H2O 0.1 g / L, CaCl2 4.0 g / L.

[0015] More preferably, the culture medium comprises the following components: The formula consists of: glucose 60 g / L, soybean meal 20 g / L, corn steep liquor 3 g / L, (NH4)2SO4 12.5 g / L, K2HPO4·3H2O 7.5 g / L, KH2PO4 1.8 g / L, vitamin H 0.4 mg / L, vitamin B1 0.2 mg / L, Trace Metal Mix A9 mother liquor 10 mL / L, and the remainder being deionized water, pH 7.0-7.5. The Trace Metal Mix A9 mother liquor composition is: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0 g / L, NaMoO4·2H2O 0.2 g / L, CuCl·H2O 0.1 g / L, FeSO4·7H2O 4.0 g / L. g / L, AlCl3·6H2O 0.1 g / L, CaCl2 4.0 g / L.

[0016] This invention also provides the application of the above-mentioned culture medium in the fermentation production of γ-aminobutyric acid by Bacillus licheniformis. The Bacillus licheniformis is preferably a recombinant Bacillus licheniformis carrying a glutamate decarboxylase expression element, wherein the recombinant Bacillus licheniformis is Bacillus licheniformis DW2 / pHY-GAD or a derivative strain expressing glutamate decarboxylase.

[0017] The application of the above-mentioned culture medium in the fermentation production of γ-aminobutyric acid by Bacillus licheniformis specifically includes the following steps: (1) Seed culture: Bacillus licheniformis was inoculated into seed culture medium and cultured to obtain seed liquid; (2) Fermentation culture: The seed liquid obtained in step (1) is inoculated into the above-mentioned culture medium for fermentation culture; the fermentation temperature is 37℃ and the fermentation time is 60-72 h; the pH is controlled in stages during the fermentation process: the early stage of fermentation is 0-48 h or 0-60 h, and the pH of the fermentation liquid is controlled at 7.0±0.2; the later stage of fermentation is 48-72 h or 60-72 h, and the pH of the fermentation liquid is controlled at 4.5-5.2; and glucose solution is added during the fermentation process to maintain the residual sugar concentration in the fermentation liquid at no less than 1.0 g / L; (3) After fermentation, a fermentation broth containing γ-aminobutyric acid is obtained.

[0018] In this invention, glutamic acid or glutamate solution is added to the fermentation system in the later stage of fermentation, so that glutamic acid is converted into γ-aminobutyric acid in situ in the fermentation system; the cumulative amount of glutamic acid or glutamate added is 100-300 g / L, calculated as L-glutamic acid.

[0019] In this invention, the mass concentration of the glutamic acid or glutamate solution is 10%-30%.

[0020] In this invention, the aeration rate during fermentation is 0.8-1.0 vvm, and the stirring speed is 300-600 r / min.

[0021] Compared with the prior art, the present invention has the following advantages: 1. Significantly reduced culture medium costs: This invention uses soybean meal and corn steep liquor to replace peptone and yeast powder as the main organic nitrogen source. Soybean meal and corn steep liquor are widely available and inexpensive, which can significantly reduce the cost of fermentation culture medium.

[0022] 2. Achieving high GABA production with low-cost raw materials: This invention does not simply replace peptone and yeast powder with soybean meal and corn steep liquor. Instead, it uses a specific ratio of soybean meal, corn steep liquor, ammonium sulfate, phosphate, vitamins and trace elements to enable Bacillus licheniformis DW2 / pHY-GAD to grow efficiently and synthesize GABA in a low-cost culture medium.

[0023] 3. Significantly higher yield compared to existing culture media: In a 20 L fermenter, using the culture medium of this invention to produce GABA, the addition of 20% glutamic acid in the later stage of fermentation can achieve complete conversion, with a final yield of 210.0 g / L; when using the control culture medium containing peptone and yeast extract, the GABA yield is 62.9 g / L. This invention increases the yield by 2.3 times compared to the control.

[0024] 4. Segmented pH control balances cell growth and enzyme catalytic activity: The pH is controlled at 7.0±0.2 in the early stage of fermentation, which is conducive to the growth of Bacillus licheniformis and the expression of glutamate decarboxylase; the pH is controlled at 4.5-5.2 in the later stage of fermentation, which is conducive to the catalysis of glutamate decarboxylase to produce GABA from glutamate.

[0025] 5. Achieving coupling of fermentation synthesis and in-situ transformation: In the later stage of fermentation, 10%-30% glutamic acid or glutamate solution is added to the fermentation system. The high-density cells and glutamic acid decarboxylase accumulated in the fermentation system are used for in-situ transformation. There is no need to prepare whole-cell catalysts separately, which can further improve the final yield of GABA.

[0026] 6. Promising prospects for industrial application: The process of this invention is simple, the raw materials are readily available, the cost is low, and the fermentation yield is high. It can significantly improve the economic efficiency of GABA fermentation production and is suitable for industrial-scale production. Attached Figure Description

[0027] Figure 1 A comparison chart showing the yield of GABA produced by fermentation in a 20 L fermenter using different culture media.

[0028] Figure 2 This diagram illustrates the GABA accumulation when the culture medium of this invention is used and 20% glutamic acid solution is added during the later stage of fermentation. Detailed Implementation

[0029] The present invention will be described in detail below through specific implementation examples, but these examples do not limit the scope of protection of the present invention. The Bacillus licheniformis G35 / pHY-GAD strain used in this implementation example is a conventionally genetically engineered strain that can be constructed using conventional molecular cloning methods; all reagents used are commercially available conventional reagents.

[0030] Example 1: Preparation of low-cost culture medium A low-cost culture medium suitable for the fermentation of Bacillus licheniformis DW2 / pHY-GAD to produce GABA was prepared, with the following composition: Glucose 60 g / L, soybean meal 20 g / L, corn steep liquor 3 g / L, ammonium sulfate 12.5 g / L, K2HPO4·3H2O 7.5 g / L, KH2PO4 1.8 g / L, vitamin H 0.4 mg / L, vitamin B1 0.2 mg / L, Trace Metal Mix A9 stock solution 10 mL / L, pH 7.2, the remainder being deionized water.

[0031] The Trace Metal Mix A9 mother liquor consists of: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0g / L, NaMoO4·2H2O 0.2 g / L, CuCl·H2O 0.1 g / L, FeSO4·7H2O 4.0 g / L, AlCl3·6H2O 0.1g / L, CaCl2 4.0 g / L.

[0032] After mixing all components of the culture medium except glucose, adjust the pH to 7.2 and sterilize at 121°C for 20-30 min. Prepare glucose separately, sterilize it, and aseptically add it to the culture medium after cooling.

[0033] Example 2: Production of GABA by shake-flask fermentation 1. Activation of microbial strains Take the working strain of Bacillus licheniformis G35 / pHY-GAD, thaw it at room temperature, and inoculate 100 μL of the bacterial suspension onto LB solid plates (with tetracycline antibiotic). Spread it evenly and incubate at 37℃ for 24 h to obtain activated single colonies.

[0034] 2. Seed culture After activation, single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL LB liquid medium (with tetracycline antibiotic), and cultured at 37 °C and 230 r / min for 12 h with shaking to obtain seed culture.

[0035] The LB liquid medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.0-7.2.

[0036] 3. Fermentation culture The seed culture was inoculated at a rate of 2%-10% into a 250 mL Erlenmeyer flask containing 45 mL of the low-cost culture medium described in Example 1, and cultured at 37°C and 230 r / min for 72 h with shaking.

[0037] 4. GABA detection The GABA content in the fermentation broth was determined by HPLC. Before detection, the sample was derivatized: 100 μL of the sample or its dilution was added to 200 μL of 0.5 mol / L Na₂CO₃-NaHCO₃ buffer and 100 μL of 80 g / L dansyl chloride solution. After mixing, the mixture was incubated at 80℃ in the dark for 40 min. The derivatized sample was then filtered through a 0.22 μm microporous membrane before HPLC analysis.

[0038] The HPLC conditions were as follows: Hypersil ODS2 C18 column, 250 mm × 4.6 mm; UV detection wavelength 254 nm; injection volume 20 μL; mobile phase A was methanol, and mobile phase B was tetrahydrofuran:methanol:0.05 mol / L sodium acetate buffer, pH 6.2, with a volume ratio of 5:75:420. The GABA content in the sample was calculated based on the peak area of ​​the GABA standard.

[0039] The results showed that when using the low-cost culture medium of the present invention for shake-flask fermentation, the GABA yield could reach 19.8 g / L.

[0040] Example 3: Effects of different nitrogen sources on GABA production To verify the effectiveness of soybean meal and corn steep liquor as low-cost organic nitrogen sources, shake-flask fermentation was conducted with different combinations of organic nitrogen sources. Except for the different nitrogen source composition of the culture medium, the other fermentation conditions were the same as in Example 2.

[0041] The results are shown in the table below. As shown in the table above, the increase in GABA yield is limited when only a portion of the inexpensive nitrogen source is used or when no optimized ratio is formed. However, the GABA yield is significantly increased when the combination of soybean meal, corn steep liquor, ammonium sulfate, phosphate, vitamins, and trace elements of this invention is used. This result indicates that the culture medium of this invention is not a simple replacement of the organic nitrogen source in existing culture media, but rather achieves a balance between low cost and high yield through the synergistic effect of specific components.

[0042] Example 4: Comparison of the culture medium of the present invention with a control culture medium containing peptone and yeast extract in a 20 L fermenter 1. Control culture medium The control culture medium is a GABA fermentation medium containing peptone and yeast extract, with the following composition: Glucose 60 g / L, peptone 6 g / L, yeast extract 6 g / L, ammonium sulfate 6 g / L, K2HPO4·3H2O 12.5 g / L, KH2PO4 2.5 g / L, Trace Metal Mix A9, pH 7.0, the remainder being water.

[0043] 2. The culture medium of the present invention The culture medium used in this invention is the low-cost culture medium described in Example 1.

[0044] 3. Fermentation conditions The qualified secondary seed culture was inoculated into a 20 L fermenter containing 10 L of fermentation medium at an inoculation rate of 10%-20%. The experimental results are as follows: Figure 1 As shown.

[0045] Fermentation conditions are: The temperature was 37℃, the aeration rate was 0.8-1.0 vvm, the stirring speed was 300-600 r / min, and the dissolved oxygen was controlled at 20%-40%. The pH was maintained at 7.0±0.2 for the first 60 h of fermentation, and then adjusted to 4.5-5.0 after 60 h. The total fermentation time was 72 h. A glucose solution was added during fermentation to maintain a residual sugar concentration in the fermentation broth of not less than 1.0 g / L.

[0046] 4. Experimental Results Experimental results show that in a 20 L fermenter, the GABA yield reached 70.0 g / L when using the low-cost culture medium of this invention; while the GABA yield was 60 g / L when using the control culture medium containing peptone and yeast extract. The culture medium of this invention increases GABA yield by 17%.

[0047] The results demonstrate that by replacing peptone and yeast powder with soybean meal and corn steep liquor, and combining them with a specific system of inorganic salts, vitamins and trace elements, the present invention not only reduces the cost of the culture medium but also significantly increases the fermentation yield, thus demonstrating outstanding industrial application value.

[0048] Example 5: Comparison of Culture Medium Costs Based on the prices of common industrial-grade raw materials on the market, a cost comparison was made between the control culture medium containing peptone and yeast extract and the low-cost culture medium of the present invention. As shown in the table above, compared with the control medium containing peptone and yeast extract, the basal medium cost of the low-cost medium of this invention is reduced from approximately RMB 1.06-1.36 / L to approximately RMB 0.36-0.46 / L, a cost reduction of approximately 60%-70%. Simultaneously, this invention increases GABA yield from 58-62 g / L to 70.0 g / L in a 20 L fermenter. Therefore, this invention not only reduces the cost of the medium but also significantly reduces the cost of the medium per unit of GABA product.

[0049] Example 6: In-situ conversion of GABA by adding 20% ​​glutamic acid during the later stage of fermentation. 1. Fermentation method Fermentation was carried out in a 20 L fermenter according to the method of the present invention group in Example 4. The pH was controlled at 7.0±0.2 in the early stage of fermentation, and the pH was adjusted to 4.5-5.2 after 60 h of fermentation.

[0050] After 60 hours of fermentation, 20% L-glutamic acid was added to the fermentation system. The addition was done in batches or continuously to maintain the glutamic acid concentration in the fermentation broth at 5-30 g / L, avoiding excessive glutamic acid concentration which could inhibit cell or enzyme activity. During the addition process, the pH was adjusted using ammonia or hydrochloric acid to maintain the pH of the fermentation broth at 4.5-5.2.

[0051] 2. Glutamic acid supplementation amount Different cumulative glutamate supplementation amounts were set, calculated as L-glutamate, at 100 g / L, 50 g / L, and 50 g / L respectively. After fermentation for 72 h, the residual amounts of GABA and glutamate in the fermentation broth were measured.

[0052] 3. Experimental Results The theoretical mass conversion coefficient of L-glutamic acid to GABA is approximately: GABA molecular weight / L-glutamic acid molecular weight = 103.12 / 147.13 ≈ 0.70.

[0053] Therefore, theoretically, adding 200 g / L L-glutamic acid can generate an additional approximately 140 g / L of GABA. In this example, based on the basal fermentation GABA yield of 70.0 g / L, the final GABA yield reached 200-210 g / L after adding 200 g / L L-glutamic acid, close to the theoretical conversion level, and the residual glutamic acid was less than 1.5 g / L, indicating that the added glutamic acid was basically converted into GABA. The experimental results are as follows: Figure 2 As shown.

[0054] Experimental results show that, based on low-cost culture medium and segmented pH control, this invention, by adding 20% ​​glutamic acid in the later stage of fermentation, can utilize the high-density cells accumulated in the fermentation system and glutamic acid decarboxylase to catalyze the in-situ conversion of glutamic acid into GABA, thereby achieving coupled production of de novo fermentation synthesis and glutamic acid conversion, and significantly increasing the final yield of GABA.

[0055] Example 7: The effect of different pH control methods on GABA production To investigate the effect of segmented pH control on GABA production, fermentation was carried out in a 20 L fermenter using the low-cost culture medium of the present invention described in Example 1, with different pH control methods set. The results showed that although a neutral pH throughout the process was beneficial to bacterial growth, the catalytic efficiency of glutamate decarboxylase was insufficient in the later stage; an acidic pH throughout the process was not conducive to the early growth and enzyme expression of Bacillus licheniformis. The present invention adopts a segmented pH control method of neutral in the early stage and acidic in the later stage, which can take into account both bacterial growth and GABA synthesis, and significantly improve the final GABA yield.

[0056] Example 8: The effect of glucose feeding on GABA production Fermentation was carried out using the low-cost culture medium described in Example 1. During the fermentation process, a glucose solution with a mass concentration of 20%-50% was added to maintain the residual sugar concentration in the fermentation broth at no less than 1.0 g / L, preferably at 5-8 g / L.

[0057] The results showed that if glucose was depleted during fermentation, the metabolic activity of the cells decreased and the rate of GABA synthesis decreased; by adding glucose to maintain an appropriate residual sugar level, the metabolic activity of the cells could be maintained, and the GABA yield and fermentation stability could be improved.

[0058] Example 9: Fermentation verification of the lower limit dosage of each component of the culture medium To verify the feasibility of using the lower limit amounts of each component in the culture medium of the present invention, the following lower limit culture medium was prepared: The composition consisted of 30 g / L glucose, 5 g / L organic nitrogen source (including 4 g / L soybean meal, 1 g / L corn steep liquor, 6 g / L ammonium sulfate, 1 g / L K2HPO4·3H2O, 1 g / L KH2PO4, 0.1 mg / L vitamin H, 0.1 mg / L vitamin B1, 1 mL / L Trace Metal Mix A9 stock solution, and the remainder was deionized water with a pH of 7.0.

[0059] Bacillus licheniformis G35 / pHY-GAD was cultured in a 20 L fermenter under the same fermentation conditions as in Example 4. GABA production was measured after 72 h of fermentation.

[0060] The results showed that when using the aforementioned lower limit culture medium, the GABA yield was 25.6 g / L. This indicates that even when all components of the culture medium in this invention are at their lower limit amounts, it can still support the fermentation and synthesis of GABA by Bacillus licheniformis.

[0061] Example 10: Fermentation verification of intermediate dosage of each component of the culture medium To verify the feasibility of intermediate dosages of each component in the culture medium of the present invention, the following intermediate dosage culture medium was prepared: The composition consisted of 55 g / L glucose, 22.5 g / L organic nitrogen source (including 20 g / L soybean meal, 2.5 g / L corn steep liquor, 13 g / L ammonium sulfate, 6.75 g / L K2HPO4·3H2O, 1.75 g / L KH2PO4, 0.3 mg / L vitamin H, 0.3 mg / L vitamin B1, 10.5 mL / L Trace MetalMix A9 mother liquor, and the remainder was deionized water with a pH of 7.2.

[0062] Bacillus licheniformis G35 / pHY-GAD was cultured in a 20 L fermenter under the same fermentation conditions as in Example 4. GABA production was measured after 72 h of fermentation.

[0063] The results showed that when using the above-mentioned intermediate dosage of culture medium, the GABA yield was 48.4 g / L. This indicates that the culture medium of the present invention can achieve a high level of GABA production at intermediate dosages of each component.

[0064] Example 11: Fermentation verification of the upper limit of each component of the culture medium To verify the feasibility of the upper limit dosage of each component in the culture medium of the present invention, the following upper limit culture medium was prepared: The concentration of the organic nitrogen source was 80 g / L, including 36 g / L soybean meal, 4 g / L corn steep liquor, 20 g / L ammonium sulfate, 12.5 g / L K2HPO4·3H2O, 2.5 g / L KH2PO4, 0.5 mg / L vitamin H, 0.5 mg / L vitamin B1, 20 mL / L Trace Metal Mix A9 mother liquor, and the remainder was deionized water. The pH was 7.5.

[0065] Bacillus licheniformis G35 / pHY-GAD was cultured in a 20 L fermenter under the same fermentation conditions as in Example 4. GABA production was measured after 72 h of fermentation.

[0066] The results showed that when using the above-mentioned high-limit culture medium, the GABA yield was 64.8 g / L. This indicates that even when all components of the culture medium of the present invention are at their upper limits, it can still support efficient GABA synthesis; however, due to the higher concentrations of organic nitrogen source and inorganic salt, the viscosity and osmotic pressure of the fermentation broth increased, resulting in a slightly lower yield than the preferred culture medium in Example 1.

[0067] Example 12: Validation of the lower limit, median value and upper limit of each single factor in the culture medium To further illustrate the rationality of the dosage range of each component in the culture medium of the present invention, based on the preferred culture medium of Example 1, only the dosage of one component was changed, while the dosage of the other components remained unchanged in Example 1, and a 20 L fermenter was used for verification. The results are shown in the table below. As shown in the table above, all components of the culture medium can support GABA fermentation production within the lower, intermediate, and upper limits defined by this invention; among them, the GABA yield is higher when the intermediate dosage or close to the preferred dosage of Example 1 is used. This result indicates that the range of culture medium components defined by this invention is reasonable, and that there is a synergistic effect between the components.

[0068] Example 13: Verification of the lower limit, median and upper limit of glutamate supplementation To verify the rationality of the range of glutamic acid or glutamate supplementation during the later stage of fermentation, the preferred culture medium described in Example 1 was used, and the fermentation conditions of Example 4 were followed for cultivation in a 20 L fermenter. After 60 h of fermentation, the pH was adjusted to 4.5-5.2, and different cumulative amounts of 20% L-glutamic acid solution were added.

[0069] The results are shown in the table below. The theoretical mass conversion coefficient of L-glutamic acid to GABA is approximately 0.70. Therefore, theoretically, adding 100 g / L, 200 g / L, and 300 g / L of L-glutamic acid can generate approximately 70 g / L, 140 g / L, and 210 g / L of GABA, respectively.

[0070] Experimental results showed that when 100-300 g / L of glutamic acid was added during the later stages of fermentation, it could be efficiently converted into GABA in situ within the fermentation system. Among them, the addition of 200 g / L of glutamic acid resulted in better conversion efficiency and fermentation stability; when 300 g / L of glutamic acid was added, although the final yield was further increased, the residual glutamic acid increased slightly due to the higher substrate concentration, and the conversion rate was slightly lower than that of the intermediate addition.

[0071] Example 14: Verification of the lower limit, median and upper limit of glutamate solution concentration To verify the rationality of the mass concentration range of glutamic acid or glutamate solution, fermentation was carried out using the culture medium described in Example 1. After 60 h of fermentation, L-glutamic acid solution with mass concentrations of 10%, 20%, and 30% was added, respectively, with a cumulative addition of 200 g / L for each L-glutamic acid solution.

[0072] The results are shown in the table below. The results showed that glutamic acid or glutamate solutions with a mass concentration ranging from 10% to 30% could be used for in-situ conversion to GABA production during the later stages of fermentation; among them, 20% glutamic acid solution showed better overall performance in terms of replenishment volume, conversion efficiency, and fermentation stability.

[0073] Example 15: Validation of Glutamate Supplementation To verify that glutamate is also applicable to the in-situ transformation process of this invention, the culture medium described in Example 1 was used for fermentation in a 20 L fermenter. After 60 h of fermentation, L-glutamate sodium solution was added to make the cumulative addition amount, calculated as L-glutamate, 100 g / L, 200 g / L, and 300 g / L.

[0074] The results are shown in the table below. The results show that glutamate can also be converted into GABA in situ in the fermentation system of this invention, which is suitable for the late-stage fermentation conversion process described in this invention.

[0075] Example 16: Verification of different organic nitrogen source compositions To demonstrate that the organic nitrogen source in this invention can be soybean meal and / or corn steep liquor, the basic conditions of Example 1 were used, but the composition of the organic nitrogen source was changed, and a 20 L fermentation tank was used for verification. The results showed that soybean meal or corn steep liquor alone can support GABA fermentation production when used as organic nitrogen sources; when used in combination, they can better provide available nitrogen sources, growth factors and micronutrients, thereby further increasing GABA yield.

[0076] This invention provides a low-cost culture medium for Bacillus licheniformis GABA fermentation using soybean meal and corn steep liquor as the main organic nitrogen sources. Compared with existing culture media using peptone and yeast extract, the cost of this medium is significantly reduced, while increasing GABA yield to 70.0 g / L in a 20 L fermenter, a 17% increase over the control. Furthermore, this invention achieves coupled production of de novo fermentation synthesis and in-situ catalytic conversion through segmented pH control and the addition of 20% glutamate or glutamate solution in the later stage of fermentation, further increasing the final GABA yield. This invention eliminates the need for separate preparation of whole-cell catalysts, offering advantages such as a short process, low cost, high yield, and ease of industrial scale-up. Therefore, this invention represents a significant substantive improvement over existing technologies.

Claims

1. A low-cost, high-yield fermentation medium for Bacillus licheniformis, comprising: glucose 30-80 g / L, organic nitrogen source 5-40 g / L, (NH4)2SO4 6-20 g / L, K2HPO4·3H2O 1-12.5 g / L, KH2PO4 1-2.5 g / L, VH 0.1-0.5 mg / L, VB1 0.1-0.5 mg / L, Trace Metal Mix A9 stock solution 1-20 mL / L, with the remainder being deionized water, pH 7.0-7.5; the Trace Metal Mix A9 stock solution comprises: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0 g / L, NaMoO4·2H2O 0.2 g / L. The organic nitrogen sources are: CuCl·H2O 0.1 g / L, FeSO4·7H2O 4.0 g / L, AlCl3·6H2O 0.1 g / L, and CaCl2 4.0 g / L; the organic nitrogen sources are soybean meal and / or corn steep liquor.

2. The culture medium according to claim 1, characterized in that, The culture medium contains the following components: glucose 50-70 g / L, soybean meal 5-30 g / L, corn steep liquor 2-4 g / L, (NH4)2SO4 10-16 g / L, K2HPO4·3H2O 5-10 g / L, KH2PO4 1.5-2.0 g / L, vitamin H 0.4 mg / L, vitamin B1 0.2 mg / L, Trace Metal Mix A9 stock solution 5-15 mL / L, with the remainder being deionized water, and a pH of 7.0-7.

5. The Trace Metal Mix A9 stock solution consists of: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0 g / L, NaMoO4·2H2O 0.2 g / L, CuCl·H2O 0.1 g / L. g / L, FeSO4·7H2O 4.0 g / L, AlCl3·6H2O 0.1 g / L, CaCl2 4.0 g / L.

3. The culture medium according to claim 1, characterized in that, The culture medium contains the following components: glucose 60 g / L, soybean meal 20 g / L, corn steep liquor 3 g / L, (NH4)2SO4 12.5 g / L, K2HPO4·3H2O 7.5 g / L, KH2PO4 1.8 g / L, VH 0.4 mg / L, VB1 0.2 mg / L, Trace Metal Mix A9 stock solution 10 mL / L, and the remainder is deionized water, pH 7.0-7.5; the Trace Metal Mix A9 stock solution consists of: CoCl2·6H2O 0.4 g / L, H3BO4 0.05 g / L, ZnSO4·7H2O 0.2 g / L, MnSO4·5H2O 1.0 g / L, NaMoO4·2H2O 0.2 g / L, CuCl·H2O 0.1 g / L, FeSO4·7H2O 4.0 g / L. g / L, AlCl3·6H2O 0.1 g / L, CaCl2 4.0 g / L.

4. The application of the culture medium according to any one of claims 1-3 in the fermentation production of γ-aminobutyric acid by Bacillus licheniformis.

5. The application according to claim 4, characterized in that, The Bacillus licheniformis is a recombinant Bacillus licheniformis carrying a glutamate decarboxylase expression element.

6. The application according to claim 5, characterized in that, The recombinant Bacillus licheniformis is Bacillus licheniformis DW2 / pHY-GAD or a derivative strain expressing glutamate decarboxylase.

7. The application according to claim 4, characterized in that, The application of Bacillus licheniformis fermentation to produce γ-aminobutyric acid (GABA) specifically includes the following steps: (1) Seed culture: Bacillus licheniformis was inoculated into seed culture medium and cultured to obtain seed liquid; (2) Fermentation culture: The seed liquid obtained in step (1) is inoculated into the culture medium described in any one of claims 1-3 for fermentation culture; the fermentation temperature is 37℃ and the fermentation time is 60-72 h; the pH is controlled in stages during fermentation: the early stage of fermentation is 0-48 h or 0-60 h, and the pH of the fermentation liquid is controlled at 7.0±0.2; the later stage of fermentation is 48-72 h or 60-72 h, and the pH of the fermentation liquid is controlled at 4.5-5.2; and glucose solution is added during fermentation to maintain the residual sugar concentration in the fermentation liquid at not less than 1.0 g / L. (3) After fermentation, a fermentation broth containing γ-aminobutyric acid is obtained.

8. The application according to claim 7, characterized in that, In the later stage of fermentation, glutamic acid or glutamate solution is added to the fermentation system to convert glutamic acid into γ-aminobutyric acid in situ in the fermentation system; the cumulative amount of glutamic acid or glutamate added is 100-300 g / L, calculated as L-glutamic acid.

9. The application according to claim 7, characterized in that, The mass concentration of the glutamic acid or glutamate solution is 10%-30%.

10. The application according to claim 7, characterized in that, During fermentation, the aeration rate is 0.8-1.0 vvm, and the stirring speed is 300-600 r / min.

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