Method for producing gamma-aminobutyric acid through co-culture fermentation

Abstract

The invention provides a method for producing gamma-aminobutyric acid through co-culture fermentation, and belongs to the technical field of ferment.The method for producing gamma-aminobutyric acid through fermentation comprises the steps that corynebacterium glutamicum fermentation liquor is provided; adding the lactobacillus brevis liquid into the corynebacterium glutamicum fermentation liquid to obtain mixed fermentation liquid; fermenting the mixed fermentation liquor to obtain co-culture fermentation liquor; and treating the co-culture fermentation liquor to obtain the gamma-aminobutyric acid product. According to the method for producing gamma-aminobutyric acid through co-culture fermentation provided by the embodiment of the invention, gamma-aminobutyric acid is obtained through mutual cooperation of corynebacterium glutamicum and lactobacillus brevis, so that the biological safety is improved. Besides, the corynebacterium glutamicum can generate organic acid and carbon dioxide in the fermentation process, so that the pH value of the fermentation liquor is reduced, a suitable growth environment can be provided for the lactobacillus brevis, the fermentation process can be simplified, and the conversion efficiency can be improved.

Description

Technical Field

[0001] The present application relates to the field of fermentation technology, and in particular to a method for producing γ-aminobutyric acid through co-culture and fermentation. Background Art

[0002] γ-Aminobutyric acid (GABA) is a naturally occurring non-protein amino acid found in animals, plants, and microorganisms. It has numerous physiological functions, including relieving anxiety, regulating blood pressure, and improving insomnia. It holds broad application prospects in the pharmaceutical, food, and chemical industries. Glutamate decarboxylase (GAD) in microbial cells catalyzes the α-decarboxylation of glutamate, enabling the bioproduction of GABA.

[0003] Corynebacterium glutamicum is an important industrial strain and the primary producer of glutamate. However, it lacks the GAD system and cannot directly synthesize GABA. Genetic engineering is often used to integrate an exogenous GAD system for GABA fermentation. This modified strain has the advantage of eliminating the need for the added substrate glutamate or glutamate salts when fermenting glucose. However, GABA produced by genetically engineered strains carries potential risks and cannot be used as a "food-grade" product, limiting its application scenarios. Summary of the Invention

[0004] The embodiments of the present application provide a method for producing γ-aminobutyric acid by co-culture fermentation, which can improve the biosafety of the γ-aminobutyric acid product and expand the application scenarios of the γ-aminobutyric acid product.

[0005] The embodiments of the present application provide a method for producing γ-aminobutyric acid by co-culture fermentation, comprising: Providing Corynebacterium glutamicum fermentation broth; adding a Lactobacillus brevis bacterial liquid to a Corynebacterium glutamicum fermentation liquid to obtain a mixed fermentation liquid; fermenting the mixed fermentation broth to obtain a co-culture fermentation broth; The co-culture fermentation broth is treated to obtain a gamma-aminobutyric acid product.

[0006] Optionally, a Corynebacterium glutamicum fermentation broth is provided, comprising: inoculating Corynebacterium glutamicum into a first seed culture medium, and culturing the culture medium to obtain a Corynebacterium glutamicum seed solution; inoculating a Corynebacterium glutamicum seed liquid into a first fermentation medium to obtain a Corynebacterium glutamicum fermentation liquid; Wherein, the first fermentation medium includes at least one of glucose, molasses and hydrolyzed sugar.

[0007] Optionally, adding Lactobacillus brevis bacterial liquid to Corynebacterium glutamicum fermentation liquid comprises: After the Corynebacterium glutamicum fermentation liquid is fermented and cultured for 0-96 hours, Lactobacillus brevis bacterial liquid is added to the Corynebacterium glutamicum fermentation liquid.

[0008] Optionally, adding Lactobacillus brevis bacterial liquid to Corynebacterium glutamicum fermentation liquid comprises: After the Corynebacterium glutamicum fermentation liquid is fermented for 36 hours to 72 hours, Lactobacillus brevis bacterial liquid is added to the Corynebacterium glutamicum fermentation liquid.

[0009] Optionally, before adding the Lactobacillus brevis bacterial liquid to the Corynebacterium glutamicum fermentation liquid, the method further comprises: inoculating Lactobacillus brevis into the second seed culture medium, and culturing to obtain a Lactobacillus brevis seed liquid; inoculating the Lactobacillus brevis seed liquid into the second fermentation medium, and obtaining the Lactobacillus brevis fermentation liquid after fermentation; centrifuging the Lactobacillus brevis fermentation broth to obtain Lactobacillus brevis cells; The Lactobacillus brevis cells were resuspended to obtain a Lactobacillus brevis bacterial solution.

[0010] Optionally, resuspend the Lactobacillus brevis cells, including: The Lactobacillus brevis cells are resuspended in the first fermentation medium.

[0011] Alternatively, the OD of the Lactobacillus brevis culture 600 15-25.

[0012] Optionally, the mixed fermentation broth is subjected to fermentation, comprising: The pH of the mixed fermentation liquid is adjusted to 5-6, and the fermentation is carried out at a temperature of 30°C-40°C for 12h-96h.

[0013] Optionally, during the fermentation process of the mixed fermentation broth, glucose is supplemented in an amount of 5 g / L / d to 15 g / L / d to the mixed fermentation broth.

[0014] Optionally, the Corynebacterium glutamicum includes Corynebacterium glutamicum ATCC13032; And / or, the Lactobacillus brevis includes Lactobacillus brevis K203.

[0015] Beneficial effects of the embodiments of the present application: The method for producing gamma-aminobutyric acid by co-cultivation fermentation provided in the embodiment of the present application, comprising: providing Corynebacterium glutamicum fermentation broth; adding Lactobacillus brevis bacterial liquid to Corynebacterium glutamicum fermentation broth to obtain a mixed fermentation broth; fermenting the mixed fermentation broth to obtain a co-cultivation fermentation broth; processing the co-cultivation fermentation broth to obtain a gamma-aminobutyric acid product. The method for producing gamma-aminobutyric acid by fermentation provided in the embodiment of the present application, by making Corynebacterium glutamicum capable of producing glutamate, and Lactobacillus brevis capable of glutamate fermentation to produce gamma-aminobutyric acid, does not require genetic modification of Corynebacterium glutamicum and Lactobacillus brevis, but obtains gamma-aminobutyric acid by mutual cooperation between Corynebacterium glutamicum and Lactobacillus brevis, thereby improving biosafety. In addition, Corynebacterium glutamicum produces organic acids and carbon dioxide during the fermentation process, resulting in a decrease in the pH of the fermentation broth, which can provide a suitable growth environment for Lactobacillus brevis, can simplify the fermentation process, and improve conversion efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a diagram showing the mechanism of γ-aminobutyric acid production by co-culture of Corynebacterium glutamicum and Lactobacillus brevis; Figure 2 This is a comparison chart showing the effect of adding Lactobacillus brevis culture solution at different time points on the yield of γ-aminobutyric acid in Example 2 of the present application; Figure 3 This is a graph showing the changes in γ-aminobutyric acid production at different time points in the co-culture of Corynebacterium glutamicum and Lactobacillus brevis in Example 3 of the present application. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. In addition, it should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application.

[0019] Current methods for producing gamma-aminobutyric acid (GABA) include plant enrichment, chemical synthesis, and microbial fermentation. Plant enrichment is safe, but the GABA concentration is low, making it unsuitable for use as a pharmaceutical or food additive. Chemical synthesis, while technically mature, is less safe, produces chemical residues, and struggles to meet pharmaceutical and food industry standards. Microbial fermentation offers low costs, making it highly attractive and promising for industrial production.

[0020] The main microbial fermentation methods for GABA production include Escherichia coli, Corynebacterium glutamicum, and lactic acid bacteria. Escherichia coli can be genetically engineered to enhance GAD activity, thereby increasing GABA production. However, its own GAD activity is low and it may produce endotoxins, posing a safety risk. It is typically used to produce feed-grade GABA. Corynebacterium glutamicum can be genetically engineered to integrate an exogenous GAD system, allowing it to directly ferment glucose to produce GABA without the addition of glutamic acid or its salts. However, genetically engineered bacteria carry potential risks and are unsuitable for food-grade GABA production. Lactic acid bacteria are considered food-grade safe strains, and the GABA produced by Lactobacillus brevis and Lactobacillus hilversii can be used as a novel food ingredient.

[0021] The embodiments of the present application provide a method for producing γ-aminobutyric acid by co-culture fermentation, comprising: Providing Corynebacterium glutamicum fermentation broth; adding a Lactobacillus brevis bacterial liquid to a Corynebacterium glutamicum fermentation liquid to obtain a mixed fermentation liquid; fermenting the mixed fermentation broth to obtain a co-culture fermentation broth; The co-culture fermentation broth is treated to obtain a gamma-aminobutyric acid product.

[0022] The method for producing gamma-aminobutyric acid by co-cultivation fermentation provided in the embodiment of the present application, by making Corynebacterium glutamicum capable of producing glutamate, and Lactobacillus brevis capable of producing gamma-aminobutyric acid by glutamate fermentation, does not require genetic modification of Corynebacterium glutamicum and Lactobacillus brevis, but instead obtains gamma-aminobutyric acid by mutual cooperation between Corynebacterium glutamicum and Lactobacillus brevis, thereby improving biosafety. In addition, Corynebacterium glutamicum produces organic acids and carbon dioxide during the fermentation process, resulting in a decrease in the pH of the fermentation liquid, which can provide a suitable growth environment for Lactobacillus brevis, can simplify the fermentation process, and improve conversion efficiency.

[0023] Wherein, short lactobacillus (Lactobacillus brevis) can be used as new food raw material, and research finds that short lactobacillus strains from different sources can synthesize GABA, but the synthesis capacity difference is very large, and all need to add glutamic acid or glutamate as substrate, increase fermentation cost. And the embodiment of the application is by the mutual coordination of Corynebacterium glutamicum and short lactobacillus, utilizes Corynebacterium glutamicum fermentation to produce glutamic acid, and short lactobacillus can utilize the characteristics of glutamate fermentation to produce gamma-aminobutyric acid, obtains the system of co-cultivation, can shorten fermentation time, reduce production cost, and do not need to carry out genetic improvement to Corynebacterium glutamicum and short lactobacillus, improve biosafety, the gamma-aminobutyric acid product obtained can be applied to the fields such as food and health products, expands the application scene of gamma-aminobutyric acid product.

[0024] like Figure 1 As shown, Corynebacterium glutamicum can utilize substrates such as glucose and convert them into glutamate, while Lactobacillus brevis can utilize glutamate and convert it into γ-aminobutyric acid.

[0025] In some embodiments, a fermentation broth of Corynebacterium glutamicum is provided, comprising: inoculating Corynebacterium glutamicum into a first seed culture medium, and culturing the culture medium to obtain a Corynebacterium glutamicum seed solution; inoculating a Corynebacterium glutamicum seed liquid into a first fermentation medium to obtain a Corynebacterium glutamicum fermentation liquid; Wherein, the first fermentation medium includes at least one of glucose, molasses and hydrolyzed sugar.

[0026] The invention relates to a method in which Corynebacterium glutamicum is first inoculated into a first seed culture medium to obtain a Corynebacterium glutamicum seed liquid, and then the Corynebacterium glutamicum seed liquid is inoculated into a first fermentation culture medium for fermentation. The method can shorten the fermentation time, improve the fermentation efficiency, and expand the fermentation volume. The first fermentation culture medium includes at least one of glucose, molasses, and hydrolyzed sugars. Substances such as glucose, molasses, and hydrolyzed sugars can serve as carbon sources during the fermentation process, and Corynebacterium glutamicum can use at least one of glucose, molasses, and hydrolyzed sugars as a substrate to produce glutamate. This can improve the fermentation efficiency of glutamate production and provide sufficient glutamate as a substrate for Lactobacillus brevis to produce gamma-aminobutyric acid.

[0027] Illustratively, the formula of the first seed culture medium may be: 5 g / L glucose, 25 g / L yeast powder, 3 g / L urea, 2.5 g / L sodium chloride, 1 g / L potassium dihydrogen phosphate, and 0.5 g / L succinic acid.

[0028] The first fermentation medium can be CGXII medium, whose formula can be: 50g / L glucose, 20g / L ammonium sulfate, 5g / L urea, 1g / L potassium dihydrogen phosphate, 1g / L dipotassium hydrogen phosphate, 0.25g / L magnesium sulfate heptahydrate, 13.3mg / L calcium chloride dihydrate, 42g / L 3-morpholinepropanesulfonic acid, 0.2mg / L biotin, 1ml / L trace element solution, and the pH is adjusted to 7.0 with potassium hydroxide; wherein the trace element solution includes: 10g / L ferrous sulfate heptahydrate, 10g / L manganese sulfate monohydrate, 1g / L zinc sulfate heptahydrate, 313mg / L copper sulfate pentahydrate, and 20mg / L nickel chloride hexahydrate.

[0029] Corynebacterium glutamicum was inoculated into the first seed culture medium and cultured at a temperature of 30° C. and a rotation speed of 180 rpm for 24 hours to obtain a Corynebacterium glutamicum seed liquid. The Corynebacterium glutamicum seed liquid was then inoculated into the first fermentation medium at a 5% inoculum amount and fermented at a temperature of 30° C. and a rotation speed of 200 rpm to obtain a Corynebacterium glutamicum fermentation liquid.

[0030] In some embodiments, adding a Lactobacillus brevis bacterial solution to a Corynebacterium glutamicum fermentation solution comprises: After the Corynebacterium glutamicum fermentation liquid is fermented for 0 h to 96 h, the Lactobacillus brevis bacterial liquid is added to the Corynebacterium glutamicum fermentation liquid.

[0031] By adding Lactobacillus brevis bacterial liquid after fermentation and cultivation of Corynebacterium glutamicum fermentation liquid for 0-96 hours, Corynebacterium glutamicum can be fermented to produce glutamate, and Lactobacillus brevis uses glutamate as a substrate to produce gamma-aminobutyric acid, thereby improving the efficiency of fermentation to produce gamma-aminobutyric acid, reducing the fermentation time, and being able to use glucose as a substrate, reducing the cost of raw material investment.

[0032] It can be understood that adding the Lactobacillus brevis bacterial solution when the Corynebacterium glutamicum is fermented for 0 h means that the Lactobacillus brevis bacterial solution is added at the same time as the Corynebacterium glutamicum seed solution is inoculated into the first fermentation medium.

[0033] In some embodiments, adding a Lactobacillus brevis bacterial solution to a Corynebacterium glutamicum fermentation solution comprises: After the Corynebacterium glutamicum fermentation broth is fermented for 36 h to 72 h, Lactobacillus brevis bacterial liquid is added to the Corynebacterium glutamicum fermentation broth.

[0034] By adding Lactobacillus brevis bacterial liquid after the Corynebacterium glutamicum fermentation liquid has been fermented for 36 hours to 72 hours, that is, the Corynebacterium glutamicum fermentation liquid is first fermented for 36 hours to 72 hours and then the Lactobacillus brevis bacterial liquid is added, a certain concentration of glutamate can be accumulated in the Corynebacterium glutamicum fermentation liquid, and substances such as organic acids and carbon dioxide produced by the fermentation of Corynebacterium glutamicum can lower the pH, provide a more suitable environment for Lactobacillus brevis, improve conversion efficiency, shorten fermentation time, and increase the yield of gamma-aminobutyric acid.

[0035] Exemplarily, the Lactobacillus brevis bacterial solution can be added to the Corynebacterium glutamicum fermentation broth after fermentation and cultivation for 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h, 76h, 80h, 84h, 88h, 92h or 96h.

[0036] In some embodiments, before adding the Lactobacillus brevis bacterial liquid to the Corynebacterium glutamicum fermentation liquid, the method further comprises: inoculating Lactobacillus brevis into the second seed culture medium, and culturing to obtain a Lactobacillus brevis seed liquid; inoculating the Lactobacillus brevis seed liquid into the second fermentation medium, and obtaining the Lactobacillus brevis fermentation liquid after fermentation; centrifuging the Lactobacillus brevis fermentation broth to obtain Lactobacillus brevis cells; The Lactobacillus brevis cells were resuspended to obtain a Lactobacillus brevis bacterial solution.

[0037] That is, first be inoculated to the second seed culture medium by short lactobacillus and cultivate, obtain short lactobacillus seed liquid, then short lactobacillus seed liquid is inoculated to the second fermentation medium and carries out fermentation culture, to expand the culture amount of short lactobacillus.Then collect the short lactobacillus thalline in the short lactobacillus fermentation liquid, after resuspension, obtain short lactobacillus bacterium liquid.In the short lactobacillus bacterium liquid that the method for providing by the present embodiment obtains, can guarantee the content of short lactobacillus thalline, and guarantee short lactobacillus dispersibility in Corynebacterium glutamicum fermentation liquid, can guarantee the conversion efficiency of gamma-aminobutyric acid after adding Corynebacterium glutamicum.

[0038] Illustratively, the formula of the second seed culture medium may be: 5 g / L glucose, 5 g / L peptone, 20 g / L yeast powder, 0.05 g / L ammonium sulfate, and a pH of 4.5.

[0039] The formula of the second fermentation medium can be: 20.0 g / L glucose, 10.0 g / L yeast powder, 5.0 g / L peptone, 2.0 g / L sodium acetate, 0.02 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate tetrahydrate, 0.01 g / L sodium chloride, and pH 5.0.

[0040] Lactobacillus brevis is inoculated into the second seed culture medium and cultured at a temperature of 37°C and a rotation speed of 180 rpm for 24 hours to obtain a Lactobacillus brevis seed liquid. The Lactobacillus brevis seed liquid is then inoculated into the second fermentation culture medium at a 5% inoculum amount and cultured at a temperature of 36°C and a rotation speed of 180 rpm for 24 hours to obtain a Lactobacillus brevis fermentation liquid. The Lactobacillus brevis fermentation liquid is centrifuged, and the precipitate is retained and the supernatant is removed to obtain Lactobacillus brevis cells. The Lactobacillus brevis cells are resuspended to obtain a Lactobacillus brevis bacterial liquid.

[0041] In some embodiments, resuspending the Lactobacillus brevis cells comprises: The Lactobacillus brevis cells are resuspended in the first fermentation medium.

[0042] By resuspending the Lactobacillus brevis cells in the first fermentation medium, the activity of the Lactobacillus brevis cells can be ensured while reducing the introduction of impurities into the co-culture system, thereby improving the stability of the co-culture of Lactobacillus brevis and Corynebacterium glutamicum and increasing the conversion efficiency and yield of gamma-aminobutyric acid.

[0043] In some embodiments, the OD of the Lactobacillus brevis bacterial solution is 600 By making the OD of Lactobacillus brevis bacterial solution 600 The ratio of 15 to 25 can ensure the concentration of Lactobacillus brevis in the Lactobacillus brevis bacterial liquid and the conversion efficiency and yield of γ-aminobutyric acid.

[0044] For example, the OD of the Lactobacillus brevis bacterial solution is 600 It can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0045] In some embodiments, the mixed fermentation broth is fermented, comprising: The pH of the mixed fermentation liquid is adjusted to 5-6, and the fermentation is carried out at a temperature of 30°C-40°C for 12h-96h.

[0046] By controlling the pH of the mixed fermentation broth to 5-6, a suitable environment is created for Lactobacillus brevis, thereby increasing its conversion efficiency to GABA. A temperature of 30°C-40°C satisfies the fermentation growth conditions of Corynebacterium glutamicum and Lactobacillus brevis, thereby increasing GABA production. Fermentation for 12-96 hours ensures effective fermentation, fully utilizing the substrate in the mixed fermentation broth and increasing GABA production.

[0047] Illustratively, the pH of the mixed fermentation broth can be 5, 5.5 or 6, the fermentation culture temperature can be set to 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, and the fermentation time can be 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h, 76h, 80h, 84h, 88h, 92h or 96h.

[0048] In some embodiments, during the fermentation process of the mixed fermentation broth, glucose is supplemented in an amount of 5 g / L / d to 15 g / L / d to the mixed fermentation broth.

[0049] During the fermentation process, the mixed fermentation broth consumes glucose, converting it into glutamate, and then into γ-aminobutyric acid. By adding glucose to the mixed fermentation broth during the fermentation process, the consumed glucose can be replenished, thereby increasing the production of γ-aminobutyric acid.

[0050] Illustratively, during the fermentation process, glucose can be supplemented in the mixed fermentation broth at an addition amount of 5 g / L / d, 10 g / L / d, or 15 g / L / d.

[0051] It is understood that g / L / d represents how many grams of glucose are added per liter of mixed fermentation broth per day.

[0052] In some embodiments, Corynebacterium glutamicum includes Corynebacterium glutamicum ( Corynebacterium glutamicum ) ATCC13032. Corynebacterium glutamicum ATCC13032 has efficient glutamate fermentation capabilities and strong tolerance, adapting to diverse fermentation conditions. Using Corynebacterium glutamicum ATCC13032 in a co-culture system ensures sufficient glutamate production as a substrate while also ensuring co-culture stability.

[0053] In some embodiments, Lactobacillus brevis includes Lactobacillus brevis ( Lactobacillus brevis ) K203. Lactobacillus brevis ( Lactobacillus brevis ) K203 has efficient γ-aminobutyric acid production capacity and a high substrate conversion rate. In addition, Lactobacillus brevis K203 is a γ-aminobutyric acid-producing strain clearly specified in the new food raw material regulations. It has not undergone genetic engineering modification and is extremely safe.

[0054] The following examples are further described in conjunction with specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally based on the conditions recommended by the manufacturer.

[0055] In the following examples, Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032, and Lactobacillus brevis is Lactobacillus brevis K203.

[0056] Example 1 Seed solution preparation 1) Transfer the glycerol tube of Corynebacterium glutamicum into the first seed culture medium for activation culture at a temperature of 30°C and a shaker speed of 180 rpm for 24 hours to obtain a Corynebacterium glutamicum seed solution.

[0057] The Corynebacterium glutamicum seed liquid was inoculated into the first fermentation medium at a rate of 5% for fermentation culture at a temperature of 30° C. and a shaking speed of 200 rpm for 48 hours to obtain a Corynebacterium glutamicum 48-hour fermentation liquid.

[0058] The first seed culture medium formula is: glucose 5 g / L, yeast powder 25 g / L, urea 3 g / L, sodium chloride 2.5 g / L, potassium dihydrogen phosphate 1 g / L and succinic acid 0.5 g / L.

[0059] The formula of the second fermentation medium (CGXII) is as follows: glucose 50 g / L, ammonium sulfate 20 g / L, urea 5 g / L, potassium dihydrogen phosphate 1 g / L, potassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.25 g / L, calcium chloride dihydrate 13.3 mg / L, 3-morpholinepropanesulfonic acid 42 g / L, biotin 0.2 mg / L, trace element solution 1 ml / L, pH adjusted to 7.0 with potassium hydroxide; wherein, the trace element solution includes: ferrous sulfate heptahydrate 10 g / L, manganese sulfate monohydrate 10 g / L, zinc sulfate heptahydrate 1 g / L, copper sulfate pentahydrate 313 mg / L, and nickel chloride hexahydrate 20 mg / L.

[0060] 2) Transfer the glycerol tube of Lactobacillus brevis into the second seed culture medium for activation culture at 37°C and a shaker speed of 180 rpm for 24 hours to obtain the Lactobacillus brevis seed solution.

[0061] The Lactobacillus brevis seed liquid was inoculated into the second fermentation medium at a rate of 5% for fermentation culture at a temperature of 36° C. and a shaking speed of 180 rpm for 24 hours to obtain the Lactobacillus brevis fermentation liquid.

[0062] The second seed culture medium formula is: glucose 5 g / L, peptone 5 g / L, yeast powder 20 g / L, ammonium sulfate 0.05 g / L, pH 4.5.

[0063] The second fermentation medium formula is: glucose 20.0 g / L, yeast powder 10.0 g / L, peptone 5.0 g / L, sodium acetate 2.0 g / L, magnesium sulfate heptahydrate 0.02 g / L, ferrous sulfate heptahydrate 0.01 g / L, manganese sulfate tetrahydrate 0.01 g / L, sodium chloride 0.01 g / L, pH 5.0.

[0064] Example 2 Co-cultivation of Lactobacillus brevis and Corynebacterium glutamicum The Lactobacillus brevis fermentation liquid obtained in Example 1 was collected and the supernatant was discarded. The bacteria were resuspended in the first fermentation medium to obtain a Lactobacillus brevis bacterial liquid.

[0065] The Lactobacillus brevis bacterial solution was measured at OD 600 20% of the co-cultured strain was inoculated into a 24-hour fermentation broth of Corynebacterium glutamicum. The culture conditions were 37°C, a shaker speed of 180 rpm, and a continuous sugar supplementation of 10 g / L / d during the fermentation process. The culture was continued for 48 hours. Liquid chromatography detected a GABA peak in the sample, demonstrating that the co-culture system can produce GABA.

[0066] The seed liquid of Corynebacterium glutamicum was inoculated into the fermentation medium at a rate of 5% for fermentation culture. At 0h, 24h, 36h, 48h, 72h, and 96h, the OD 600 The culture conditions were 36°C, 180 rpm shaker speed, 10 g / L / d glucose supplementation during the fermentation process, and 48 h of culture. The GABA production was detected by liquid chromatography. The results were as follows: Figure 2 shown.

[0067] Figure 2 Indicates the GABA content when the fermentation liquid of Corynebacterium glutamicum is inoculated with Lactobacillus brevis at 0h, 24h, 36h, 48h, 72h, and 96h of culture and the culture is continued for 48h. Figure 2 It can be seen that at the 48th hour of cultivation of Corynebacterium glutamicum, the GABA production of Lactobacillus brevis was the highest, reaching 50g / L.

[0068] Example 3 The Corynebacterium glutamicum seed liquid was inoculated into the first fermentation medium at a 5% inoculation rate for fermentation to obtain the Corynebacterium glutamicum fermentation liquid. 600 The pH of the Lactobacillus brevis culture liquid was regulated to maintain at pH 5.5 for fermentation. The culture temperature was 36°C, the shaker speed was 180 rpm, and glucose was continuously supplemented at 10 g / L / d during the fermentation process. The co-culture time of Corynebacterium glutamicum and Lactobacillus brevis was 48 h. The glutamate and GABA production was detected by liquid chromatography. Figure 3The results of the fermentation of the co-culture system of Corynebacterium glutamicum and Lactobacillus brevis were shown. Glutamic acid accumulated in the early stage. After the addition of Lactobacillus brevis culture and the pH was adjusted to 5.5, the GABA production gradually increased. After 96 hours, the glutamate was completely consumed, and the maximum GABA production of 103g / L was obtained.

[0069] As can be seen from embodiment 1-embodiment 3, the present application embodiment, by co-culturing Corynebacterium glutamicum and brevis lactobacillus, can produce gamma-aminobutyric acid, and conversion efficiency is higher, and cost is lower, does not need to carry out genetic modification to Corynebacterium glutamicum and brevis lactobacillus, has guaranteed biosafety.In addition, relatively speaking, after Corynebacterium glutamicum fermentation 24h-72h, adds brevis lactobacillus bacterium liquid, can improve the output of gamma-aminobutyric acid, this is mainly due to Corynebacterium glutamicum fermentation liquid after fermentation for a period of time, can accumulate a certain amount of glutamic acid in fermented liquid, for brevis lactobacillus provides comparatively sufficient substrate.And Corynebacterium glutamicum fermentation liquid after fermentation for a period of time, can improve pH environment, more suitable for the growth of brevis lactobacillus and the conversion to glutamic acid, contribute to improving conversion efficiency.

[0070] The above is a detailed introduction to the embodiments of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of the present application. At the same time, for those skilled in the art, based on the ideas of the present application, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A method for producing γ-aminobutyric acid by co-culture and fermentation, characterized in that: include: Providing Corynebacterium glutamicum fermentation broth; adding the Lactobacillus brevis bacterial liquid to the Corynebacterium glutamicum fermentation liquid to obtain a mixed fermentation liquid; fermenting the mixed fermentation broth to obtain a co-culture fermentation broth; The co-culture fermentation broth is treated to obtain a gamma-aminobutyric acid product.

2. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 1, characterized in that: The method of providing Corynebacterium glutamicum fermentation broth comprises: inoculating Corynebacterium glutamicum into a first seed culture medium, and culturing the culture medium to obtain a Corynebacterium glutamicum seed solution; inoculating the Corynebacterium glutamicum seed liquid into a first fermentation medium to obtain a Corynebacterium glutamicum fermentation liquid; Wherein, the first fermentation medium includes at least one of glucose, molasses and hydrolyzed sugar.

3. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 1, characterized in that: The step of adding the Lactobacillus brevis bacterial liquid to the Corynebacterium glutamicum fermentation liquid comprises: After the Corynebacterium glutamicum fermentation liquid is fermented and cultured for 0 hours to 96 hours, the Lactobacillus brevis bacterial liquid is added to the Corynebacterium glutamicum fermentation liquid.

4. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 3, characterized in that: The step of adding the Lactobacillus brevis bacterial liquid to the Corynebacterium glutamicum fermentation liquid comprises: The Corynebacterium glutamicum fermentation liquid is fermented and cultured for 36 hours to 72 hours, and then the Lactobacillus brevis bacterial liquid is added to the Corynebacterium glutamicum fermentation liquid.

5. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 2, characterized in that: Before adding the Lactobacillus brevis bacterial liquid to the Corynebacterium glutamicum fermentation liquid, the method further comprises: inoculating Lactobacillus brevis into the second seed culture medium, and culturing to obtain a Lactobacillus brevis seed liquid; inoculating the Lactobacillus brevis seed liquid into a second fermentation medium, and obtaining a Lactobacillus brevis fermentation liquid after fermentation; centrifuging the Lactobacillus brevis fermentation broth to obtain Lactobacillus brevis cells; The Lactobacillus brevis cells are resuspended to obtain the Lactobacillus brevis bacterial liquid.

6. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 5, characterized in that: The step of resuspending the Lactobacillus brevis cells comprises: The Lactobacillus brevis cells are resuspended using the first fermentation medium.

7. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 5, characterized in that: The OD of the Lactobacillus brevis bacterial solution 600 15-25.

8. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 1, characterized in that: The method of fermenting the mixed fermentation liquid comprises: The pH of the mixed fermentation liquid is adjusted to 5-6, and the mixture is fermented at a temperature of 30° C.-40° C. for 12 h-96 h.

9. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to claim 8, characterized in that: During the fermentation process of the mixed fermentation broth, glucose is supplemented in the mixed fermentation broth at an addition amount of 5 g / L / d to 15 g / L / d.

10. The method for producing γ-aminobutyric acid by co-cultivation and fermentation according to any one of claims 1 to 9, characterized in that: The Corynebacterium glutamicum includes Corynebacterium glutamicum ATCC13032; And / or, the Lactobacillus brevis includes Lactobacillus brevis K203.

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