Efficient breeding method of high-yield gamma-aminobutyric acid beer yeast strain

Abstract

The invention discloses an efficient breeding method of beer yeast with high yield of gamma-aminobutyric acid (GABA), and belongs to the technical field of strain breeding. The method comprises the following steps: carrying out ARTP mutagenesis treatment on beer yeast strains to construct a mutant strain library; rapidly screening from the mutant strain library by using a methyl red-methylene blue indicator method to obtain a growth dominant strain; and carrying out fermentation culture on the obtained primarily screened strain, accurately detecting gamma-aminobutyric acid by high performance liquid chromatography, carrying out secondary screening to obtain the beer yeast with high yield of gamma-aminobutyric acid, of which the yield is improved by 122.16% compared with that of the original strain, and determining flavor substances of the obtained secondarily screened strain through shake flask horizontal fermentation in a laboratory. The content of gamma-aminobutyric acid in the mutant strain for simulating beer fermentation is increased by 19.61%, and the main flavor is not obviously different from that of the original strain. The invention solves the problems of high difficulty and low efficiency of screening beer industrial yeast with high yield of gamma-aminobutyric acid at present.

Description

Technical Field

[0001] This invention belongs to the field of beer brewing technology, and in particular relates to an efficient breeding method for high-yield γ-aminobutyric acid (GABA) brewer's yeast strains. Background Technology

[0002] Beer, a globally popular alcoholic beverage, ranks first in consumption among all types of alcoholic drinks. This beverage, brewed primarily from malt and hops using brewer's yeast fermentation, not only contains a moderate amount of alcohol but also various trace amounts of bioactive components produced during fermentation. The malt and adjuncts in the raw materials are sources of carbohydrates, nitrogenous compounds, B vitamins, and dietary fiber, while hops contain polyphenols, flavonoids, and other nutrients. When beer is consumed in moderation, these active components may regulate bodily functions by activating specific enzymes. With the increasing popularity of healthy eating concepts, functional foods with specific physiological regulatory functions are gaining increasing favor among consumers.

[0003] National standards define functional factors as substances that can regulate human functions by activating enzyme activity or through other pathways. Gamma-aminobutyric acid (GABA) is a natural non-protein amino acid. As an important neurotransmitter, it exhibits a wide range of physiological regulatory effects in the human body, including regulating the central nervous system, relieving anxiety, and improving sleep. GABA affects neuronal excitability by activating GABA receptors, and has physiological functions such as regulating blood pressure and promoting growth hormone secretion; it is a natural functional factor. Studies have shown that mature or germinated barley grains contain GABA, which not only regulates blood pressure and relieves stress responses, but also plays a positive role in antidepressant effects, promoting water and electrolyte metabolism, and scavenging free radicals.

[0004] Gamma-aminobutyric acid (GABA) has various physiological functions, including antihypertensive and anti-stress effects on human health. It can be used as an antidepressant, diuretic, and antioxidant, and is widely applied in beverages, health products, and alcoholic beverages. Yeast produces GABA through glutamate decarboxylase, which catalyzes the decarboxylation of glutamate. Yeast also has the ability to produce GABA during wort fermentation. However, the natural content of the functional factor GABA in beer is usually low, making it difficult to meet the needs of industrial production. Ambient temperature and pressure plasma mutagenesis (ARTP) can obtain a large number of mutagenic strains. L-glutamate, under the action of glutamate decarboxylase (GAD), consumes a proton to produce GABA, which leads to an increase in the pH of the YPDS screening medium. The methyl red-methylene blue indicator turns red at pH less than 5.60 and blue-green at pH greater than 6.20. Based on the color change of the culture medium, mutagenic strains with different GABA synthesis capabilities can be quickly screened. Therefore, this invention uses industrial lager brewer's yeast as the starting strain and employs mutagenesis and high-throughput screening methods to enhance the synthesis capacity of the functional factor γ-aminobutyric acid (GABA) in industrial yeast, thereby increasing the GABA content in beer. This not only significantly enhances its health value but also better meets consumers' expectations for functional beverages, improves production efficiency, reduces production costs, and thus enhances the product's market competitiveness. This research direction not only aligns with market trends but also provides an important opportunity for innovation and upgrading in the beer industry. Summary of the Invention

[0005] This invention provides an efficient method for breeding high-yield γ-aminobutyric acid (GABA) brewer's yeast strains, which solves the current problems of high difficulty and low efficiency in screening high-yield GABA brewer's yeast.

[0006] To achieve the above objectives, the present invention provides a highly efficient method for breeding high-yield γ-aminobutyric acid (GABA) brewer's yeast strains, characterized by comprising the following steps: ARTP mutagenesis was performed on brewer's yeast strains to construct a mutant strain library; Strains with high γ-aminobutyric acid (GABA) synthesis capacity were rapidly screened from a mutant strain library using the methyl red-methylene blue indicator method. The obtained primary screening strains were fermented and cultured, and then re-screened by accurately detecting γ-aminobutyric acid using high performance liquid chromatography to obtain the re-screened strains, namely high-γ-aminobutyric acid-producing brewer's yeast. The high-yield γ-aminobutyric acid brewer's yeast Saccharomyces cerevisiae Lager-63 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M20251622.

[0007] Preferably, the ARTP mutagenesis treatment of the brewer's yeast strain specifically involves: Dilute the bacterial cells cultured to mid-log phase to 1×10⁻⁶. 6 ARTP mutagenesis was performed on yeast cells using a helium ion beam at a density of cells / mL.

[0008] Preferably, the conditions for the ARTP mutagenesis treatment are as follows: The helium flow rate was 10.0 L / min, the input power was 120 W, the processing distance was 2 mm, the processing temperature was 25℃, and the mutagenesis time was 110 s.

[0009] Preferably, the methyl red-methylene blue indicator method for initial screening of strains specifically includes: 1) Streak culture of brewer's yeast strain, pick single colonies for growth in YPD medium, centrifuge the cells, wash and resuspend to obtain cell suspension; 2) The cell suspension was subjected to ARTP mutagenesis to obtain the mutagenic bacterial solution; 3) Spread the mutant bacterial suspensions onto YPD plates and incubate at a constant temperature. Pick the mutant strains from the plates and incubate them in 96-well YPD liquid medium on a shaker until OD (dose elapsed). 600 It is 5; 4) Inoculate the above-mentioned mutant bacterial solution into YPDS screening medium containing methyl red-methylene blue indicator at an inoculation rate of 1%, and culture in a constant temperature shaking incubator. Stop the culture when the color of the original strain medium changes from red to blue-green. 5) Take an appropriate amount of the mutagenic bacterial solution and repeat steps 2)-4) above for at least 2-3 rounds. Store the final mutagenic strain in a glycerol tube at -80°C.

[0010] Preferably, in step 1), the colony growth temperature is 30°C and the culture time is 48 h; After centrifugation, the bacterial cells were washed and resuspended in sterile physiological saline containing 20% ​​glycerol, and the concentration was diluted to 1×10⁻⁶. 8 per mL.

[0011] Preferably, in step 2), the amount of cell suspension used is 10 μL; The ARTP mutagenesis treatment conditions were: helium flow rate 10 L / min, input power 120 W, treatment distance 4 mm, treatment temperature 25℃, and mutagenesis time 0-150 s.

[0012] Preferably, in step 4), the amount of the mutagenic bacterial solution taken is 100-120 μL; The YPDS screening medium is a YPD medium containing 40% 5 g / L L-glutamic acid, 1% methylene blue, and 2% methyl red. The constant temperature for incubation is 30℃, and the incubation time is 16-24 h.

[0013] As a preferred method, the γ-aminobutyric acid (GABA) content in the fermentation supernatant was detected by high performance liquid chromatography (HPLC), and the primary screening strains with a γ-aminobutyric acid content that increased by more than 1 times were selected as secondary screening strains.

[0014] Preferably, after obtaining the secondary screening strain, the method further includes a step of determining the flavor compounds of the obtained secondary screening strain by laboratory shake-flask horizontal fermentation.

[0015] As a preferred option, the obtained high-yield γ-aminobutyric acid (GABA) brewer's yeast has a γ-aminobutyric acid (GABA) yield that is more than 10% higher, and its main flavor is not significantly different from that of the initially screened brewer's yeast strain.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: This invention uses industrial lager brewer's yeast as the starting strain and optimizes the ARTP mutagenesis conditions and the high-throughput detection method for γ-aminobutyric acid (GABA). Through multiple rounds of ARTP mutagenesis and high-throughput screening, the synthesis capacity of the functional factor GABA in industrial yeast is enhanced, thereby increasing the GABA content in beer. Using a laboratory-scale shake-flask beer brewing system, a mutant strain with an increase in GABA of over 10% was finally obtained, and the main flavor of the fermentation broth from the mutant strain showed no significant difference from the starting strain. This invention represents a further optimization and improvement of the production strain and has the potential for application in beer industrial production. This not only significantly enhances its health value but also better meets consumer expectations for functional beverages, thereby improving the product's market competitiveness. Attached Figure Description

[0017] Figure 1 The graph shows the functional relationship between the lethality of the strain and the mutagenesis time when the mutagenesis conditions were established according to the embodiments of the present invention. Figure 2 This image shows the color changes of different concentrations of L-glutamic acid in YPDS culture medium. Figure 3 This is a colorimetric screening reaction diagram for the initial screening of high-yield γ-aminobutyric acid (GABA) strains. Figure 4 The graph shows the relationship between γ-aminobutyric acid (GABA) yield and fermentation time (a) and the γ-aminobutyric acid content of the re-screened strains (b). Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides an efficient method for breeding high-yield γ-aminobutyric acid (GABA) brewer's yeast strains, comprising the following steps: ARTP mutagenesis was performed on brewer's yeast strains to construct a mutant strain library; Strains with high γ-aminobutyric acid (GABA) synthesis capacity were rapidly screened from a mutant strain library using the methyl red-methylene blue indicator method. The obtained primary screening strains were fermented and cultured, and then re-screened by accurately detecting γ-aminobutyric acid using high performance liquid chromatography to obtain the re-screened strains, namely high-γ-aminobutyric acid-producing brewer's yeast. A high-yield γ-aminobutyric acid (GABA) brewer's yeast was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M20251622.

[0020] In the above scheme, the ARTP mutagenesis treatment of the brewer's yeast strain specifically involves: The bacterial strain was activated by streaking and inoculated into YPD medium until the strain reached the logarithmic growth phase. 1 mL of the bacterial suspension was centrifuged at 4°C for 10 min, and the supernatant was discarded. The bacterial pellet was washed and resuspended 2-3 times with an equal volume of 0.9% sterile physiological saline. 100 μL of 50% glycerol was added as a cryoprotectant, and the suspension was agitated and the OD was adjusted. 600 1. After sterilizing the ARTP-specific iron sheet (8mm), ignite both sides of the iron sheet over an alcohol lamp for 30 seconds. After the iron sheet cools to room temperature, take 10 μL of bacterial suspension and evenly spot it on the iron sheet. Then, use a helium plasma beam to induce mutagenesis in the yeast cells.

[0021] ARTP mutagenesis conditions: Helium flow rate QHe = 10.0 L / min, input power 120 W, treatment distance 4 mm, treatment temperature 25℃, and mutagenesis time 110 s. The metal slide was placed in 1 mL of sterile physiological saline and washed with shaking.

[0022] During the above mutagenesis treatment, under the premise of maintaining a helium flow rate QHe = 10.0 L / min, an input power of 120 W, a treatment distance of 4 mm, and a treatment temperature of 25℃, in order to obtain the optimal mutagenesis conditions, the treatment time was set to 0, 50, 70, 90, 110, 130, and 150 s, followed by shaking and elution of the mutagenic bacterial solution for 2 min. 10 μL of the bacterial solution was then diluted in 990 μL of 0.9% sterile physiological saline to a final concentration of 10 μL. -1 Repeat the steps to prepare 10 by continuous dilution -1 -10 -6Gradient concentration bacterial suspensions were prepared, with 100 μL of each suspension spread onto plates, three replicates per gradient. The plates were incubated at 30℃ for 2-3 days, and lethality curves were plotted to determine the optimal irradiation time. The lethality of the strain increased with increasing mutagenesis time. If the lethality was too low, effective improvement of the brewing yeast would be impossible; if the lethality was too high, significant changes in strain performance could occur, affecting the main flavor of the finished beer. To obtain mutant strains with a certain positive mutation rate, a lethality of over 95% was selected. Therefore, the optimal mutagenesis time was chosen as 110 s, resulting in a lethality of 95.64%. Figure 1 As shown.

[0023] In a preferred embodiment, the methyl red-methylene blue indicator method for initial screening of strains specifically comprises: 1) Streak culture of brewer's yeast strain, pick single colonies for growth in YPD medium, centrifuge the cells, wash and resuspend to obtain cell suspension; 2) The cell suspension was subjected to ARTP mutagenesis to obtain the mutagenic bacterial solution; 3) Spread the mutant bacterial solution onto YPD plates and incubate at a constant temperature. Pick the mutant strains from the plates and incubate them in a 96-well plate of YPD liquid medium on a shaker until the OD600 is 5. 4) Inoculate the above-mentioned mutant bacterial solution into YPDS screening medium containing methyl red-methylene blue indicator at an inoculation rate of 1%, and culture in a constant temperature shaking incubator. Stop the culture when the color of the original strain medium changes from red to blue-green. 5) Take an appropriate amount of the mutagenic bacterial solution and repeat steps 2)-4) above for at least 2-3 rounds. Store the final mutagenic strain in a glycerol tube at -80°C.

[0024] In a preferred embodiment, in step 1), the colony growth temperature is 30°C and the culture time is 48 h; After centrifugation, the bacterial cells were washed and resuspended in sterile physiological saline containing 20% ​​glycerol, and the concentration was diluted to 1×10⁻⁶. 8 per mL.

[0025] In a preferred embodiment, in step 2), the amount of cell suspension used is 10 μL; The ARTP mutagenesis treatment conditions were: helium flow rate 10 L / min, input power 120 W, treatment distance 4 mm, treatment temperature 25℃, and mutagenesis time 0-150 s.

[0026] In a preferred embodiment, in step 4), the amount of the mutagenic bacterial solution taken is 100-120 μL; L-glutamic acid YPDS culture medium solutions with different concentration gradients of 1.10 g / L, 1.30 g / L, 1.50 g / L, 1.70 g / L, 1.90 g / L, 2.10 g / L, 2.30 g / L, and 2.50 g / L were prepared, with three replicates for each concentration gradient. Methyl red-methylene blue indicator was added, and the optimal amount of L-glutamic acid was determined based on the color changes of the YPDS culture medium. The color changes of different concentrations of L-glutamic acid in YPDS culture medium are shown in the figure below. Figure 2 As shown.

[0027] The YPDS screening medium is a YPD medium containing 40% 5 g / L L-glutamic acid, 1% methylene blue, and 2% methyl red. The constant temperature for incubation is 30℃, and the incubation time is 16-24 h.

[0028] In a preferred embodiment, the high-performance liquid chromatography method for detecting γ-aminobutyric acid in the fermentation broth specifically includes: Sample pretreatment: Take 1 mL of sample solution, add 1 mL of 10% trichloroacetic acid (TCA) solution, mix well, and incubate at low temperature overnight to remove insoluble proteins and sugars and other macromolecules from the sample. Centrifuge at 10,000 rpm to collect the supernatant and filter through a 0.45 μm filter membrane.

[0029] Chromatographic conditions: Agilent 1200 HPLC system; Column: Agilent ODS 250 × 4.60 μm, 5 μm HPLC column; Detection wavelength: 338 nm; Column oven: 40℃; Injection volume: 10 μL. Mobile phase A: pH 7.20 aqueous phase (2 M sodium acetate aqueous solution, 0.02% triethylamine and 0.5% tetrahydrofuran); Mobile phase B: Organic phase (2 M pH 7.20 sodium acetate aqueous solution: methanol: acetonitrile = 1 : 2 : 2). Injection procedure: Using pre-column derivatization, 7 μL of 0.4 M pH 10.20 boric acid solution was drawn at 200 μL / min and mixed with 1 μL of sample solution. Then, 2 μL of o-phthalaldehyde (OPA) solution was added at 200 μL / min and mixed. Finally, 30 μL of water was drawn at 200 μL / min and 10 μL was injected. Gradient elution program: 0 min, 8.0% B, 0.70 mL / min; 31.50 min, 100.0% B, 0.70 mL / min; 32.0 min, 100.0% B, 0.90 mL / min; 35.0 min, 100.0% B, 0.90 mL / min; 35.50 min, 8.0% B, 0.70 mL / min; 40 min, 8.0% B, 0.70 mL / min.

[0030] In a preferred embodiment, after obtaining the secondary-screened strains, the method further includes a step of determining the flavor compounds of the obtained secondary-screened strains by horizontal fermentation in a laboratory shake flask. The specific method is as follows: 1) Propagate the strain at varying temperatures, then let it stand to collect the fermented slurry. Inoculate it into wort and seal.

[0031] 2) Let it stand for a period of time, and stop fermentation when the weight loss is less than 0.20 g.

[0032] 3) Let it stand and ferment for a period of time until the post-fermentation is complete.

[0033] 4) The content of main flavor substances such as n-propanol in the fermentation broth was detected by headspace gas chromatography, with 2-octanol as the internal standard.

[0034] In a preferred embodiment, in step 1), the inoculation amount is 1×10 6 CFU / mL; The sealing method is a fermentation plug seal followed by a water liquid seal.

[0035] In a preferred embodiment, in step 2), the static culture conditions are static culture at 12°C for 12-14 days; The pre-fermentation process is marked by a weight loss of less than 0.20 g in the fermentation broth.

[0036] In a preferred embodiment, in step 3), the static culture conditions are 4°C and static culture for 7 days.

[0037] In a preferred embodiment, in step 4), the main flavor substance includes ethyl acetate, isoamyl acetate, n-propanol, isobutanol, and isoamyl alcohol; The concentration of 2-octanol is 50 mg / L.

[0038] In a preferred embodiment, the γ-aminobutyric acid (GABA) yield of the obtained high-yield GABA brewer's yeast was increased by more than 10%, and the main flavor was not significantly different from that of the initially screened brewer's yeast strain.

[0039] Example 1: Establishment of Mutagenic Conditions The industrial brewer's yeast strain Lager-I7 was streaked to activate it and inoculated into YPD medium. The culture was then cultured until the logarithmic growth phase. One mL of the bacterial suspension was centrifuged at 4°C for 10 min, and the supernatant was discarded. The bacterial precipitate was washed and resuspended 2-3 times with an equal volume of 0.9% sterile physiological saline. 100 μL of 50% glycerol was added as a protectant, and the suspension was shaken thoroughly and the OD600 was adjusted to 1. An ARTP-specific iron plate (8 mm) was sterilized and then ignited on both sides over an alcohol lamp for 30 s. After the iron plate cooled to room temperature, 10 μL of the bacterial suspension was evenly spotted onto the iron plate, and the yeast cells were mutagenized using a helium plasma beam. Under the premise of maintaining a helium flow rate QHe = 10.0 L / min, input power 120 W, processing distance 4 mm, and processing temperature 25℃, in order to obtain the optimal mutagenesis conditions, the processing time was set to 0, 50, 70, 90, 110, 130, and 150 s, the mutagenic bacterial solution was eluted by shaking for 2 min, and 10 μL of the bacterial solution was diluted in 990 μL of 0.9% sterile physiological saline to a final concentration of 10 μL. -1 Repeat the steps to prepare 10 by continuous dilution -1 -10 -6 Gradient concentration bacterial suspensions were prepared, with 100 μL of each suspension spread onto plates, three replicates per gradient. The plates were incubated at 30℃ for 2-3 days, and lethality curves were plotted to determine the optimal irradiation time. The lethality of the strain increased with increasing mutagenesis time. If the lethality was too low, effective improvement of the brewing yeast would be impossible; if the lethality was too high, significant changes in strain performance could occur, affecting the main flavor of the finished beer. To obtain mutant strains with a certain positive mutation rate, a lethality of over 95% was selected; therefore, an optimal mutagenesis time of 110 s was chosen, resulting in a lethality of 95.64%.

[0040] Example 2: Establishment and effectiveness of a high-throughput initial screening method L-glutamate, under the action of glutamate decarboxylase (GAD), consumes one proton to generate γ-aminobutyric acid (GABA), which leads to an increase in the pH of the YPDS selection medium. The methyl red-methylene blue indicator appears red at pH less than 5.60 and blue-green at pH greater than 6.20. L-glutamate YPDS medium solutions with different concentration gradients (1.10 g / L, 1.30 g / L, 1.50 g / L, 1.70 g / L, 1.90 g / L, 2.10 g / L, 2.30 g / L, and 2.50 g / L) were prepared, with three replicates for each concentration gradient. The optimal amount of L-glutamate was determined by adding the methyl red-methylene blue indicator and observing the color change of the YPDS culture medium. Figure 2As shown, the red color was most prominent in YPD medium containing 2.5 g / L L-Glu, therefore 2.5 g / L L-Glu was chosen as the optimal addition amount for YPDS medium.

[0041] Selected ARTP-mutated strains with good growth were plated and stored, and then inoculated into 96-well plates containing 1 mL of YPD liquid medium and incubated at 30°C with shaking until OD. 600 The inoculum was 5%, and 1% (100 μL) was added to YPDS selection medium and cultured in a 30°C shaking incubator. Figure 3 As shown, the culture medium of the original strain was stopped when the color changed from red to blue-green. The mutant strain whose culture medium color changed to blue-green was subjected to the above steps for at least 2-3 rounds. The results showed that after three rounds of screening, a total of 11 mutant strains with high γ-aminobutyric acid production were obtained in the preliminary screening. The mutant strains were stored at -80℃ for subsequent re-screening.

[0042] Example 3: High Performance Liquid Chromatography (HPLC) for Secondary Screening of γ-Aminobutyric Acid Shake-flask fermentation experiments were conducted on the starting strain Lager-I7, and samples were taken every 12 hours to detect the γ-aminobutyric acid (GABA) content in the fermentation broth. The specific high-performance liquid chromatography (HPLC) method for detecting GABA in the fermentation broth was as follows: Sample pretreatment: Take 1 mL of sample solution, add 1 mL of 10% trichloroacetic acid (TCA) solution, mix well, and incubate overnight at low temperature to remove insoluble proteins and carbohydrates and other macromolecules from the sample. Centrifuge at 10,000 rpm and collect the supernatant. Filter through a 0.45 μm filter membrane. Chromatographic conditions: Agilent 1200 high-performance liquid chromatograph; Column: Agilent ODS 250 × 4.60 μm, 5 μm liquid chromatograph; Detection wavelength: 338 nm; Column oven: 40℃; Injection volume: 10 μL. Mobile phase A: pH 7.20 aqueous phase (2 M sodium acetate aqueous solution, 0.02% triethylamine and 0.5% tetrahydrofuran); Mobile phase B: Organic phase (2 M pH 7.20 sodium acetate aqueous solution: methanol: acetonitrile = 1 : 2 : 2). Injection procedure: Using pre-column derivatization, 7 μL of 0.4M pH 10.20 boric acid solution was drawn at 200 μL / min and mixed with 1 μL of sample solution. Then, 2 μL of o-phthalaldehyde (OPA) solution was added at 200 μL / min and mixed. Finally, 30 μL of water was drawn at 200 μL / min, and 10 μL was injected. Gradient elution program: 0 min, 8.0%B, 0.70 mL / min; 31.50 min, 100.0%B, 0.70 mL / min; 32.0 min, 100.0%B, 0.90 mL / min; 35.0 min, 100.0%B, 0.90 mL / min; 35.50 min, 8.0%B, 0.70 mL / min; 40 min, 8.0%B, 0.70 mL / min.

[0043] The results are as follows Figure 4 As shown in (a), the yield of γ-aminobutyric acid (GABA) initially increased and then decreased with fermentation time. The yeast strain rapidly synthesized GABA in YPD liquid medium, reaching its maximum content at 12 h. Subsequently, as fermentation progressed, the yield of GABA began to decline, possibly due to the depletion of glutamate substrate, the cell entering the death phase, and the reduced metabolic rate of the strain. The cell used GABA as a nitrogen source, and GABA was degraded under the catalysis of GABA-T, resulting in its consumption rate exceeding the synthesis rate. Therefore, 12 h was taken as the end time of fermentation.

[0044] Eleven initially screened strains and the starting strain were fermented in shake flasks for 12 h, with three replicates for each strain. The fermentation supernatant was collected and the γ-aminobutyric acid (GABA) content was determined by HPLC. The results are as follows: Figure 4As shown in (b), strain 63 showed a significantly higher γ-aminobutyric acid (GABA) production capacity than other mutant strains, reaching 37.47 (± 5.16) mg / L, which was 122.16% higher than the original strain (16.87 ± 2.66 mg / L). Strains 78 (34.51 ± 4.87 mg / L), 83 (26.88 ± 2.62 mg / L), and 57 (25.67 ± 0.40 mg / L) were the next highest, while the GABA content of other strains was all below 25 mg / L.

[0045] Example 4: Preparation of wort culture medium At 45℃, add water at a feed-to-water ratio of 1:4. Raise the temperature to 48℃ and hold for 30 min; raise the temperature to 65℃ and hold for 40 min; raise the temperature to 72℃ and hold for 10 min; after passing the iodine test, raise the temperature to 78℃ and hold for 10 min, completing saccharification. Filter while hot, boil for 1 h, adding hops in batches at a rate of 0.40 g / L during the wort boiling process. After cooling to room temperature, adjust the concentration to 12 °P to obtain clear wort.

[0046] Example 5 Laboratory shake-flask horizontal fermentation verification The bacterial strains were streaked to activate them. Single colonies of the starting strain Lager-I7 and the secondary screening strain 63 were picked from the plate and inoculated into 1 mL of wort medium for seed culture at 30°C and 220 rpm for 12 h. The 1 mL of bacterial culture was then transferred to 9 mL of the corresponding wort medium and incubated at 24°C and 220 rpm for 12 h. Finally, 10 mL of the bacterial culture was transferred to 90 mL of the corresponding wort medium and incubated at 12°C and 220 rpm for 12 h. The fermented sludge was then collected and inoculated into wort (1×10⁻⁶). 6 (CFU / mL). Secure with the fermentation plug and liquid seal. Incubate at 12℃ for 14 days, weighing daily until the weight loss is less than 0.20 g, at which point fermentation is stopped. Incubate at 4℃ for 7 days until fermentation is complete, at which point the post-fermentation phase ends.

[0047] Example 6: Determination of main flavor compounds in beer Take 4.5 mL of beer fermentation broth, add 0.5 mL of 500 mg / L 2-octanol, and determine the concentration using headspace gas chromatography. Headspace sampler equilibration temperature: 70℃, equilibration time: 30 min. Transfer line temperature: 130℃, injection time: 0.04 min, injection port temperature: 200℃, detector temperature: 250℃. Column initial temperature: 40℃, programmed to increase to 180℃ at 10℃ / min. Column flow rate: 1.2 mL / min, N2 flow rate: 30 mL / min, H2 flow rate: 47 mL / min, air flow rate: 400 mL / min.

[0048] Table 1. Main flavor indicators in the wine sample at the end of fermentation (mg / L)

[0049] Note: If the superscript in the same column contains the same letter, it means there is no significant difference between the two groups of data. P <0.05), for example, a and ab represent no significant difference between the two groups of data. The results showed that the γ-aminobutyric acid content (50.14 ± 1.89 mg / L) of mutant strain 63 in the simulated beer fermentation broth increased by 19.61%, and the main flavor was not significantly different from that of the starting strain.

Claims

1. A highly efficient breeding method for high-yield γ-aminobutyric acid (GABA) brewer's yeast strains, characterized in that, Includes the following steps: ARTP mutagenesis was performed on brewer's yeast strains to construct a mutant strain library; Strains with high γ-aminobutyric acid (GABA) synthesis capacity were rapidly screened from a mutant strain library using the methyl red-methylene blue indicator method. The obtained primary screening strains were fermented and cultured, and then re-screened by accurately detecting γ-aminobutyric acid using high performance liquid chromatography to obtain the re-screened strains, namely high-γ-aminobutyric acid-producing brewer's yeast. The high-yield γ-aminobutyric acid (GABA) brewer's yeast was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M20251622.

2. The efficient breeding method according to claim 1, characterized in that, The specific steps of performing ARTP mutagenesis on the brewer's yeast strain are as follows: Dilute the bacterial cells cultured to mid-log phase to 1×10⁻⁶. 6 ARTP mutagenesis was performed on yeast cells using a helium ion beam at a density of cells / mL.

3. The efficient breeding method according to claim 2, characterized in that, The conditions for the ARTP mutagenesis treatment are as follows: The helium flow rate was 10.0 L / min, the input power was 120 W, the processing distance was 2 mm, the processing temperature was 25 °C, and the mutagenesis time was 110 s.

4. The efficient breeding method according to claim 1, characterized in that, The specific strains screened using the methyl red-methylene blue indicator method are as follows: 1) Streak culture of brewer's yeast strain, pick single colonies for growth in YPD medium, centrifuge the cells, wash and resuspend to obtain cell suspension; 2) The cell suspension was subjected to ARTP mutagenesis to obtain the mutagenic bacterial solution; 3) Spread the mutant bacterial suspensions onto YPD plates and incubate at a constant temperature. Pick the mutant strains from the plates and incubate them in 96-well YPD liquid medium on a shaker until OD (dose-free ratio) is reached. 600 It is 5; 4) Inoculate the above-mentioned mutant bacterial solution into YPDS screening medium containing methyl red-methylene blue indicator at an inoculation rate of 1%, and culture in a constant temperature shaking incubator. Stop the culture when the color of the original strain medium changes from red to blue-green. 5) Take an appropriate amount of the mutagenic bacterial solution and repeat steps 2)-4) above for at least 2-3 rounds. Store the final mutagenic strain in a glycerol tube at -80°C.

5. The efficient breeding method according to claim 4, characterized in that, In step 1), the colony growth temperature is 30℃ and the culture time is 48h. After centrifugation, the bacterial cells were washed and resuspended in sterile physiological saline containing 20% ​​glycerol, and the concentration was diluted to 1×10⁻⁶. 8 per mL.

6. The efficient breeding method according to claim 4, characterized in that, In step 2), the amount of cell suspension used is 10 μL; The ARTP mutagenesis treatment conditions were: helium flow rate 10 L / min, input power 120 W, treatment distance 4 mm, treatment temperature 25 °C, and mutagenesis time 0-150 s.

7. The efficient breeding method according to claim 4, characterized in that, In step 4), the amount of the mutagenic bacterial solution taken is 100-120 μL; The YPDS screening medium is a YPD medium containing 40% 5g / L L-glutamic acid, 1% methylene blue, and 2% methyl red; The constant temperature for incubation is 30℃, and the incubation time is 16-24 hours.

8. The efficient breeding method according to claim 1, characterized in that, The content of γ-aminobutyric acid (GABA) in the fermentation supernatant was determined by high performance liquid chromatography (HPLC), and strains with a γ-aminobutyric acid content that increased by more than 1 times were selected as secondary screening strains.

9. The efficient breeding method according to claim 1, characterized in that, After obtaining the secondary screening strains, the method also includes a step of determining the flavor compounds of the obtained secondary screening strains by horizontal fermentation in laboratory shake flasks.

10. The efficient breeding method according to claim 9, characterized in that, The high-yield γ-aminobutyric acid (GABA) brewer's yeast obtained increased GABA production by more than 10%, and its main flavor was not significantly different from that of the initially screened brewer's yeast strain.

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