Brewing yeast, microbial agents and their applications

Abstract

This invention relates to the field of microbiology, and more particularly to *Saccharomyces cerevisiae*, microbial inoculants, and their applications. This invention provides *Saccharomyces cerevisiae*, with accession number CCTCC NO: M 2026537. This invention provides a method for efficiently screening yeast strains with high γ-glutamylcysteine ​​synthase (γ-GCS) activity and high γ-GC production capacity by utilizing hexavalent chromium ions (Cr(VI)) as a stress factor.

Description

Technical Field

[0001] This invention relates to the field of microorganisms, and more particularly to brewer's yeast, microbial agents and their applications. Background Technology

[0002] γ-Glutamylcysteine ​​(γ-GC) is an important precursor for the synthesis of glutathione (GSH) and has wide applications in the pharmaceutical, food, and cosmetic fields. Currently, strategies to improve the production of γ-GC through microbial fermentation mainly include genetic engineering and mutagenesis breeding. While genetic engineering methods (such as knocking out the glutathione synthase gene) can directionally accumulate γ-GC, they are complex to operate and pose challenges to strain stability. Traditional mutagenesis breeding methods suffer from low screening throughput, high workload, and high susceptibility to chance.

[0003] Therefore, developing an efficient and targeted screening method to obtain yeast strains that produce high levels of γ-GC has significant industrial application value. Summary of the Invention

[0004] In view of this, the present invention provides brewing yeast, microbial inoculants, and their applications. The present invention addresses the problems of low efficiency and lack of specificity in existing methods for screening high-γ-GC-producing yeast strains, and provides a method that utilizes hexavalent chromium ions (Cr(VI)) as a stress factor to efficiently screen yeast strains with high γ-glutamylcysteine ​​synthase (γ-GCS) activity and high γ-GC production capacity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides Saccharomyces cerevisiae, with accession number CCTCC NO:M 2026537.

[0007] The present invention also provides microbial inoculants, comprising any one of the following and acceptable adjuvants:

[0008] (a) the above-mentioned brewing yeast (Saccharomyces cerevisiae); and / or

[0009] (b) Inactivated strains of the aforementioned Saccharomyces cerevisiae; and / or

[0010] (c) Metabolites, derivatives, fermentation broth, cultures, exosomes, lysates, or extracts of the aforementioned Saccharomyces cerevisiae.

[0011] The present invention also provides a method for preparing the above-mentioned microbial inoculant, wherein the starting strain is inoculated into a screening medium, subjected to multiple rounds of subculturing, and after expansion culture, the microbial inoculant is obtained;

[0012] The screening medium contains 10-30 mM of chromium ions.

[0013] In some embodiments of the present invention, the starting strain in the above preparation method is: Saccharomyces cerevisiae ATCC9763.

[0014] In some embodiments of the present invention, in the above preparation method, the number of rounds is 1 to 5; in the rounds, the concentration of chromium ions in the screening medium of the latter round is higher than that of the former round in adjacent rounds.

[0015] In some embodiments of the present invention, in the above preparation method, the number of rounds is 1 to 3; the concentration of chromium ions in the first round is 10 mM; the concentration of chromium ions in the second round is 20 mM; and the concentration of chromium ions in the third round is 30 mM.

[0016] The present invention also provides the application of the above-mentioned Saccharomyces cerevisiae, the above-mentioned microbial inoculum and / or the microbial inoculum obtained by the above preparation method in the preparation of γ-glutamylcysteine.

[0017] The present invention also provides a method for preparing γ-glutamylcysteine, wherein the above-mentioned Saccharomyces cerevisiae, the above-mentioned microbial agent and / or the seed liquid of the microbial agent obtained by the above preparation method are inoculated into a fermentation medium, and after fermentation, the supernatant is collected to obtain the γ-glutamylcysteine.

[0018] In some embodiments of the present invention, in the above preparation method, the fermentation medium comprises: 0.1-0.25M carbon source, 30-50g / L sodium glutamate, 15-35g / L L-cysteine, 2-4g / L dipotassium hydrogen phosphate and 1-3g / L magnesium chloride; the pH of the fermentation medium is 6.5-7.0.

[0019] In some embodiments of the present invention, the carbon source in the above preparation method includes glucose and / or sucrose.

[0020] In some embodiments of the present invention, in the above preparation method, the fermentation medium comprises: 0.167M glucose, 40 g / L sodium glutamate, 20 g / L L-cysteine, 3 g / L dipotassium hydrogen phosphate and 2 g / L magnesium chloride; the pH of the fermentation medium is 6.8.

[0021] In some embodiments of the present invention, in the above preparation method, the fermentation time is 12-36 hours and the temperature is 36-38°C.

[0022] In some embodiments of the present invention, the fermentation time in the above preparation method is 32 hours and the temperature is 37°C.

[0023] In some embodiments of the present invention, in the above preparation method, the inoculation amount is 1-3%, and the viable count is 8 × 10⁻⁶. 4 ~2.4×10 5 CFU / mL.

[0024] The beneficial effects of this invention include:

[0025] (1) High screening efficiency and strong purposefulness: By utilizing the specific inhibitory effect of Cr(VI) on γ-GCS activity, the selection pressure on the γ-GCS system of the strain is directly applied, making the screening direction clear and significantly improving the efficiency and success rate of obtaining high-yield strains.

[0026] (2) Significant improvement in strain performance: The yeast strains screened by the method of the present invention can express γ-GCS 3-6 times that of unscreened strains, and the γ-GC production can be greatly increased from about 5 g / L to more than 38 g / L.

[0027] (3) Simple operation and easy to promote: This method does not require complex gene manipulation equipment, is suitable for routine microbiology laboratories, and is easy to apply in industrial applications. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 The standard curve equation for γ-GCS enzyme activity assay is y = 0.6402x - 0.0034; where x is the molar mass of the standard (μmol / mL) and y is ΔA; defined as the amount of inorganic phosphorus produced per milliliter of liquid per hour by catalysis is one unit of enzyme activity; γ-GCS enzyme activity (μmol / h / mL) = [(ΔA + 0.0034) ÷ 0.6402 × V2] ÷ V1 ÷ T = 23.43 × (ΔA + 0.0034);

[0030] Figure 2Thin-layer chromatography was used to verify the results. The developing solvent was n-butanol:glacial acetic acid:anhydrous ethanol:water = 4:1:1:2. 0.4% ninhydrin was added for color development. A baseline was drawn 1 cm from the bottom of the GF254 silica gel plate, with sample spots spaced 1 cm apart. After the sample was air-dried, the developing solvent was placed at the bottom, not exceeding 1 / 2 of the baseline. The developing solvent was removed 1 cm from the top and dried with a hairdryer until complete color development. The left image shows the yield color development of strain GC01 F1, F3, F5 generations and the standard control (35 g / L), while the right image shows the yield color development of the original Saccharomyces cerevisiae ATCC9763 strain F1, F3, F5 generations and the standard control (5 g / L).

[0031] Figure 3 The chromatogram of the high performance liquid chromatography γ-GC standard with a concentration of 4.8452 g / L is shown. Chromatograph: Vanquish Core, column temperature: 40℃, detector wavelength: 220 nm, column: Acciaim 120 C18 (4.6*250 mm, 5 μm), mobile phase: A: 0.1% phosphoric acid aqueous solution; D: acetonitrile gradient elution conditions.

[0032] Figure 4 The chromatogram of the high performance liquid chromatography γ-GC standard with a content of 10.4680 g / L is shown. Chromatograph: Vanquish Core, column temperature: 40℃, detector wavelength: 220 nm, column: Acciaim 120 C18 (4.6*250 mm, 5 μm), mobile phase: A: 0.1% phosphoric acid aqueous solution; D: acetonitrile gradient elution conditions.

[0033] Figure 5 The chromatogram of the high performance liquid chromatography γ-GC standard with a content of 20.2973 g / L is shown. Chromatograph: Vanquish Core, column temperature: 40℃, detector wavelength: 220 nm, column: Acciaim 120 C18 (4.6*250 mm, 5 μm), mobile phase: A: 0.1% phosphoric acid aqueous solution; D: acetonitrile gradient elution conditions.

[0034] Figure 6 The chromatogram of the high performance liquid chromatography γ-GC standard with a content of 48.9761 g / L is shown. Chromatograph: Vanquish Core, column temperature: 40℃, detector wavelength: 220 nm, column: Acciaim 120 C18 (4.6*250 mm, 5 μm), mobile phase: A: 0.1% phosphoric acid aqueous solution; D: acetonitrile gradient elution conditions.

[0035] Figure 7The chromatogram of the high performance liquid chromatography γ-GC standard with a concentration of 100.4135 g / L is shown. Chromatograph: Vanquish Core, column temperature: 40℃, detector wavelength: 220 nm, column: Acciaim 120 C18 (4.6*250 mm, 5 μm), mobile phase: A: 0.1% phosphoric acid aqueous solution; D: acetonitrile gradient elution conditions.

[0036] Figure 8 The standard curve of γ-GC by high performance liquid chromatography is shown. γ-GC standard solutions at five different concentrations were analyzed by HPLC. With the standard concentration C (g / L) as the abscissa and the peak area A as the ordinate, a linear regression was performed using the least squares method to obtain the standard curve regression equation: A = 0.0217C + 0.0074, with a correlation coefficient R^2 = 0.9997. The results indicate that γ-GC exhibits good linearity within the five set concentration gradients and can be used for the quantitative analysis of γ-GC content in samples.

[0037] Figure 9 Chromatogram showing the yield of γ-GC synthesized by fermentation of strain GC01 using high performance liquid chromatography; Chromatograph: Vanquish Core, column temperature: 40℃, detector wavelength: 220nm, column: Acciaim 120 C18 (4.6*250mm, 5um), mobile phase: A: 0.1% phosphoric acid aqueous solution; D: acetonitrile gradient elution conditions.

[0038] Biological Preservation Instructions

[0039] Saccharomyces cerevisiae GC01, deposited on March 27, 2026, with accession number CCTCC No:M 2026537, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. Detailed Implementation

[0040] This invention discloses brewing yeast, microbial agents, and their applications.

[0041] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0042] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0043] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0044] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0045] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0046] The core of this invention lies in the discovery that hexavalent chromium ions (Cr(VI)) can bind to the sulfhydryl groups of γ-glutamylcysteine ​​synthase (γ-GCS), inhibiting its activity and thus consuming intracellular glutathione. Therefore, yeast strains that can survive in a high concentration of Cr(VI) may have higher expression or activity of γ-GCS, thereby possessing a stronger γ-GC synthesis capacity.

[0047] This invention provides a method for screening yeast strains that produce high levels of γ-glutamylcysteine ​​based on hexavalent chromium ion stress, comprising the following steps:

[0048] Stress screening: Using yeast strains as the starting strains, multiple rounds of subculture were carried out on solid plates or liquid culture media containing gradient concentrations of hexavalent chromium ions. In each round, the concentration of hexavalent chromium ions was gradually increased. The initial concentration of hexavalent chromium ions was 10 mM, and the final screening concentration was not less than 30 mM.

[0049] Strain isolation: After each round of screening, select colonies or bacterial solutions that grow vigorously under the highest stress concentration;

[0050] Fermentation validation: The strains selected in the final screening were inoculated into a fermentation medium without hexavalent chromium ions for fermentation, and the yield of γ-glutamylcysteine ​​was detected.

[0051] Preferably, the yeast strain is brewer's yeast (Saccharomyces cerevisiae).

[0052] Preferably, the gradient concentration of hexavalent chromium ions is 10 mM, 20 mM, 30 mM or higher.

[0053] Preferably, the fermentation medium contains a carbon source, a nitrogen source, monosodium glutamate, and L-cysteine; the carbon source is glucose or sucrose, and its concentration in the medium ranges from 0.1 M to 0.25 M.

[0054] The brewing yeast ATCC9763 of this invention was provided free of charge by Professor Yin Zhimin's laboratory at the School of Life Sciences, Nanjing Normal University.

[0055] In Examples 1 and 2 of this invention, all raw materials and reagents used can be purchased from the market.

[0056] The present invention will be further illustrated below with reference to the embodiments:

[0057] Example 1: Screening of brewer's yeast with high γ-GC production using hexavalent chromium ion plates

[0058] Stress screening: Saccharomyces cerevisiae (ATCC9763) was spread onto agar plates containing 10 mM Cr(VI) and incubated overnight at 30°C. The largest colonies on the plates were picked, resuspended in 100 μL of sterile water, and spread onto agar plates containing 20 mM Cr(VI) for further incubation overnight. This process was repeated to transfer the cells to agar plates containing 30 mM Cr(VI) for a third round of screening.

[0059] Expanded culture: The largest colony was picked from the third round of plates and inoculated sequentially into 1 mL liquid test tubes containing 30 mM Cr(VI) and 50 mL shake flasks for amplification culture.

[0060] Fermentation verification: The amplified strain was inoculated into 1 L of Cr(VI)-free fermentation medium and cultured at 30℃ for 30 hours; 40 g of sodium glutamate, 20 g of L-cysteine, 30 g of glucose, 3 g of dipotassium hydrogen phosphate, and 2 g of magnesium chloride were added per liter, pH 6.8, and the temperature was raised to 37℃ for 32 hours of fermentation.

[0061] Results: After fermentation, the supernatant was collected by centrifugation and detected by HPLC. The yields of γ-GC were 27.2 g / L, 25.6 g / L, 31.3 g / L, 28.9 g / L and 38.2 g / L, respectively. The strain with the highest yield was preserved.

[0062] Example 2: Cell stability experiment of a high-γ-glutamylcysteine-producing yeast strain

[0063] Experimental group: Preserved Saccharomyces cerevisiae ATCC9763 mutant strain (GC01 strain);

[0064] Control group: Original brewer's yeast ATCC9763;

[0065] Experimental objective: To verify the growth stability, enzyme activity stability, and γ-GC production stability of the screened strain under continuous stress-free passage conditions, and to evaluate the feasibility of industrial application.

[0066] Culture medium:

[0067] Subculture / activation medium (YM medium, Cr-free) 6+ ): Yeast extract 5 g / L, peptone 10 g / L, glucose 20 g / L, natural pH;

[0068] Fermentation medium: glucose 30 g / L, sodium glutamate 40 g / L, L-cysteine ​​30 g / L, dipotassium hydrogen phosphate 3 g / L, magnesium chloride 2 g / L, pH 6.8;

[0069] Instruments and equipment:

[0070] Clean bench, constant temperature incubator, high-speed centrifuge, ultraviolet spectrophotometer, electronic balance, pipette, petri dish, shake flask, etc.

[0071] Experimental methods:

[0072] 1. Continuous subculturing (Cr-free) 6+ (coercion)

[0073] 1) Primary activation: The freeze-dried strain was streaked onto YM plates and incubated at 30°C for 48 h; a single colony was picked and inoculated into YM liquid and incubated at 30°C and 220 rpm until OD200. 600 ≈0.8, denoted as F1 generation.

[0074] 2) Continuous subculturing: Each generation is inoculated with 1% fresh YM liquid and cultured under the same conditions until OD. 600 ≈0.8, continuously passaged to generation F5; control group passaged synchronously.

[0075] 2. Growth stability test

[0076] F1, F3, and F5 generations were used to determine OD. 600 Observe colony morphology (size, color, edge, smoothness) on plates; use F1 instead of OD. 600 Calculate the relative growth rate, setting it to 100%.

[0077] 3. γ-GCS enzyme activity stability assay

[0078] F1, F3, and F5 generation cells were centrifuged and cell walls were disrupted. The activity of γ-glutamylcysteine ​​synthase (γ-GCS) was measured using a γ-GCS activity assay kit. The relative enzyme activity was calculated with the F1 generation enzyme activity as 100%.

[0079] 4. Verification of γ-GC yield stability

[0080] F1, F3, and F5 generations were inoculated into 50 mL of fermentation medium at a 1% inoculum rate; cultured at 30℃ and 220 rpm for 30 h, and fermented for another 32 h; the supernatant was collected by centrifugation, and the γ-GC content was determined by thin-layer chromatography; the relative yield was calculated with F1 generation as 100%.

[0081] Experimental results:

[0082] 1. Growth stability

[0083] Table 1. Growth stability of strains

[0084]

[0085] Conclusion: After five consecutive passages, strain GC01 showed stable morphology and good growth stability with OD fluctuations of ≤4%.

[0086] 2. Stability of γ-GCS enzyme activity

[0087] The standard curve equation is: y = 0.6402x - 0.0034, where x is the molar mass of the standard (μmol / mL) and y is ΔA. Experimental results are as follows: Figure 1 As shown;

[0088] Calculation of γ-GCS activity in liquid:

[0089] Definition: One enzyme activity unit is defined as the amount of inorganic phosphorus produced per milliliter of liquid per hour by catalysis.

[0090] γ-GCS enzyme activity (μmol / h / mL) = [(△A + 0.0034) ÷ 0.6402 × V2] ÷ V1 ÷ T = 23.43 × (△A + 0.0034);

[0091] Table 2. Stability of γ-GCS enzyme activity (γ-glutamylcysteine ​​synthase (γ-GCS) activity assay kit)

[0092]

[0093] Conclusion: The F5 generation of GC01 bacteria showed a relative enzyme activity of 90.2%, indicating stable high enzyme activity.

[0094] 3. Stability of γ-GC production

[0095] Thin-layer chromatography, also known as "thin-layer chromatography analysis," is an analytical method that involves uniformly coating an adsorbent and a support onto a glass or plastic plate to form a thin layer, followed by chromatographic separation. After separating different types of compounds from a sample, the type of each component can be determined based on its R-value or fluorescence characteristics. The content of each component can be determined by using a thin-layer scanner based on the area of ​​the spots. It requires small sample volumes, offers rapid analysis, uses simple equipment, and does not alter the various components of the mixture during separation. Unidentified components can be transferred for further testing after separation, making it a rapid screening method for handling large quantities of different types of samples. In forensic identification, it is mainly used to test various high-molecular-weight organic compounds, such as organic components extracted from inks, dyes, paints, and soil in organic solvents, as well as oils, alkaloids, resins, synthetic fibers, organic explosives, and other organic substances.

[0096] Thin-layer chromatography verification:

[0097] The developing solvent was n-butanol:glacial acetic acid:anhydrous ethanol:water = 4:1:1:2, with 0.4% ninhydrin added for color development.

[0098] Draw a baseline 1 cm from the bottom of the GF254 silica gel plate, and place sample points every 1 cm. After the samples have air-dried, place the developing solvent at the bottom, not exceeding half of the baseline. Remove the developing solvent when it reaches 1 cm from the top, and use a hairdryer to heat until complete color development. The experimental results are as follows. Figure 2 As shown.

[0099] Table 3 Stability of γ-GC fermentation yield

[0100]

[0101] Conclusion: The F5 generation of strain GC01 yielded 36 g / L, with a relative yield ≥ 90%, and the yield was stable, meeting the requirements for industrialization.

[0102] In summary, the GC01 strain of this invention:

[0103] Growth stability: After five consecutive generations of passage without stress, the mutant strain showed no significant changes in growth rate or colony morphology, indicating good growth stability.

[0104] Enzyme activity stability: The relative enzyme activity of γ-GCS in the F5 generation of the mutant strain was 90.2%, and the high enzyme activity trait was stably inherited.

[0105] Yield stability: The F5 generation of the mutant strain yielded 36 g / L of γ-GC, with a relative yield ≥90%, demonstrating stable high-yield characteristics that meet industrial standards. The high-yielding γ-glutamylcysteine-producing Saccharomyces cerevisiae ATCC9763 mutant strain, screened under hexavalent chromium stress, exhibits excellent genetic stability and can be directly used for industrial fermentation production of γ-GC.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Saccharomyces cerevisiae, characterized by, Its accession number is: CCTCC NO: M2026537.

2. A microbial inoculant, characterized in that, Includes any of the following and acceptable adjuvants: (a) the brewing yeast (Saccharomyces cerevisiae) as described in claim 1; and / or (b) Inactivated strains of Saccharomyces cerevisiae as described in claim 1; and / or (c) Metabolites, derivatives, fermentation broths, cultures, exosomes, lysates, or extracts of Saccharomyces cerevisiae as described in claim 1.

3. The method for preparing the microbial inoculant as described in claim 2, characterized in that, The starting strain was inoculated into the screening medium, and after multiple rounds of subculturing and expansion culture, the microbial agent was obtained. The screening medium contains 10-30 mM of chromium ions.

4. The preparation method according to claim 3, characterized in that, The number of rounds is 1 to 5; in the multiple rounds, the concentration of chromium ions in the screening medium of the later round is higher than that of the previous round in any two adjacent rounds.

5. The application of the Saccharomyces cerevisiae as described in claim 1, the microbial agent as described in claim 2, and / or the microbial agent obtained by the preparation method as described in claim 3 or 4 in the preparation of γ-glutamylcysteine.

6. A method for preparing γ-glutamylcysteine, characterized in that, The seed culture of the Saccharomyces cerevisiae as described in claim 1, the microbial agent as described in claim 2, and / or the microbial agent obtained by the preparation method as described in claim 3 or 4 is inoculated into the fermentation medium. After fermentation, the supernatant is collected to obtain the γ-glutamylcysteine.

7. The preparation method according to claim 6, characterized in that, The fermentation medium comprises: 0.1-0.25M carbon source, 30-50g / L sodium glutamate, 15-35g / L L-cysteine, 2-4g / L dipotassium hydrogen phosphate and 1-3g / L magnesium chloride; the pH of the fermentation medium is 6.5-7.

0.

8. The preparation method according to claim 7, characterized in that, The carbon source includes glucose and / or sucrose.

9. The preparation method according to any one of claims 6 to 8, characterized in that, The fermentation time is 12-36 hours, and the temperature is 36-38℃.

10. The preparation method according to any one of claims 6 to 9, characterized in that, The inoculation amount is 1-3%, and the viable bacteria count is 8 × 10⁻⁶. 4 ~2.4×10 5 CFU / mL.

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