CN118325744B - Singular yeast and method for preparing gamma-aminobutyric acid by using singular yeast - Google Patents
Singular yeast and method for preparing gamma-aminobutyric acid by using singular yeast Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a singular yeast and a method for preparing gamma-aminobutyric acid by the singular yeast. Exotic yeast Saccharomyces paradoxus QYH, deposited under accession number GDMCC No:63592. the strain QYH5 is cultivated at 37 ℃ without special substrate and aerobic fermentation, and the fermentation yield is 67.95 mg/L. The gamma-aminobutyric acid prepared by the strain has the advantages of simple process, low cost of raw materials, environmental protection and potential industrial application prospect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a singular yeast and a method for preparing gamma-aminobutyric acid by using the same.
Background
Gamma-aminobutyric acid, the chemical name is 4-aminobutyric acid, GABA for short, CAS number is 56-12-2, molecular formula is C 4H9NO2, molecular weight is 103.1, is a non-protein amino acid. gamma-aminobutyric acid can stimulate the synthesis of hyaluronic acid in dermal fibroblasts, has moisturizing and repairing effects, can increase the production of intercellular glutathione, enhance the tolerance of the dermal fibroblasts to active oxygen, and protect the skin from being damaged by ultraviolet rays and the like (Biochimica et Biophysica Acta (BBA) -General Subjects, 2007, 291-296). Gamma-aminobutyric acid also can increase the concentration of chloride ions in human skin keratinocytes, inhibit depolarization injury of cell membranes, and maintain skin barrier homeostasis (Journal of Investigative Dermatology, 2002, 1041-1047). In addition, gamma-aminobutyric acid can improve sleep, enhance memory, and delay aging (Nutrients, 2021, 2544). Therefore, gamma-aminobutyric acid is widely applied to anti-wrinkle, tightening, repairing and anti-aging skin care products.
At present, most of the microbial synthesis of gamma-aminobutyric acid is performed by lactic acid bacteria, and few yeasts are used. Compared with saccharomycetes, lactobacillus has high requirement on nutrition, and amino acid, unsaturated fatty acid and vitamin are needed, so that the propagation of lactobacillus under the condition of low nutrition is limited, and the lactobacillus does not contain catalase, so that the lactobacillus is very sensitive to oxidation environment. The production process of gamma-aminobutyric acid by lactic acid bacteria has higher complexity and cost than that of saccharomycetes.
In order to solve the problems in the existing microbial synthesis of gamma-aminobutyric acid, some yeasts which are easy to culture and have simple culture medium formula are searched for preparing gamma-aminobutyric acid, so that the production cost can be reduced, the process is simplified, and the method has important practical significance.
Disclosure of Invention
A first object of the present invention is to provide a singular yeast Saccharomyces paradoxusQYH, which has a deposit number of: GDMCC No:63592.
The invention separates a strain Saccharomyces paradoxusQYH with high growth speed and gamma-aminobutyric acid secretion property from Hunan glutinous rice distiller's yeast. The bacterial colony features of the strain are shown in figure 1, and the bacterial colony is round, smooth in edge, milky white, raised in lawn, smooth in surface, wet and easy to pick up, has fragrance, and is cultured for 24 hours, and the diameter of the bacterial colony reaches 1-5 mm. The 18S rRNA sequence is shown as SEQ ID NO. 1. A microscopic photograph of the yeast Saccharomyces paradoxusQYH is shown in FIG. 2, and the yeast is oval, 10-30 μm in size, and can be used for growing the cell nucleus aerobically. The strain can produce gamma-aminobutyric acid under the condition of no special substrate and aerobic fermentation, and has the advantages of simple preparation process and low raw material cost.
The second purpose of the invention is to provide the application of the mirabilis Saccharomyces paradoxusQYH in the preparation of gamma-aminobutyric acid.
The third object of the present invention is to provide a method for preparing gamma-aminobutyric acid, which comprises fermenting with the above-mentioned yeast Saccharomyces paradoxusQYH.
Preferably, the method comprises the following steps:
(1) Seed liquid culture: inoculating the strain Saccharomyces paradoxusQYH of the mirabilis into a seed culture medium for shake cultivation for 24-48 hours to obtain seed liquid;
(2) Aerobic fermentation: inoculating the seed liquid into a fermentation medium for culturing for 24-72 hours to obtain a fermentation liquid containing gamma-aminobutyric acid.
Preferably, the seed culture medium or the fermentation culture medium comprises the following components in percentage by weight: 4 g/L of potato extract powder and 20/g/L of glucose, wherein the solvent is water.
Preferably, the inoculation amount of the seed liquid is 1-5% by volume.
Preferably, the culture temperature of the culture is 30-37 ℃.
The fourth object of the present invention is to provide a microbial agent, which contains the above-mentioned yeast Saccharomyces paradoxusQYH or its fermentation broth.
The fifth object of the present invention is to provide the culture method of the singular yeast Saccharomyces paradoxusQYH, which comprises inoculating the singular yeast Saccharomyces paradoxusQYH to a culture medium of potato extract powder 4-10 g/L, glucose 20-30 g/L, agar 0-15 g/L, and water as a solvent.
The sixth object of the present invention is to provide a gamma-aminobutyric acid detection method, which comprises the following steps: taking the fermentation liquor of the mirabilis Saccharomyces paradoxusQYH, adding 3-4 times of absolute ethyl alcohol, standing for 2-3 hours, filtering by a filter membrane, removing the solvent by rotary evaporation under reduced pressure, adding water for dilution, and detecting by a liquid chromatography-mass spectrometer;
The chromatographic conditions are as follows: c18 column 2.1X100 mm,3.5 μm, mobile phase water: methanol volume ratio=90:10, flow rate 0.3 mL/min, column temperature 40 ℃;
The mass spectrum conditions are as follows: the ion source is ESI positive ion mode, the ionization voltage is 5500V, the cluster removal voltage is 50V, the qualitative ion pair is 104.1-87.1, the collision energy is 16V, 104.1-69.2, the collision energy is 23V, and the quantitative ion pair is 104.1-87.1.
The invention has the following beneficial effects:
the invention relates to a singular yeast
Saccharomyces paradoxus
QYH5 can be used for preparing gamma-aminobutyric acid by a fermentation method, and is a novel gamma-aminobutyric acid preparation method, and is environment-friendly. Yeast mirabilis
Saccharomyces paradoxus
QYH5 grows fast under the condition of no special substrate and oxygen, the required fermentation culture medium is simple and easy to obtain, the culture is simple, the industrial development is facilitated, and the method has important significance and value in the biotechnology field and the cosmetic field.
The exotic yeast Saccharomyces paradoxusQYH is cultivated at 37 ℃ without special substrate and aerobic fermentation, and the fermentation yield is 67.95 mg/L. The gamma-aminobutyric acid prepared by the strain has the advantages of simple process, low cost of raw materials, environmental protection and potential industrial application prospect.
Mirabilis Saccharomyces paradoxusQYH deposited at the cantonese microbiological strain collection center (GDMCC) at 2023, 6, 27, address: building 5 of Guangzhou City martyr of Guangdong province, no. 100 of the university of road 59, post code: 510070, accession number is: GDMCC No:63592.
Drawings
FIG. 1 is a photograph of colony morphology features of Saccharomyces mirabilis Saccharomyces paradoxusQYH.
FIG. 2 is a photomicrograph of Saccharomyces mirabilis Saccharomyces paradoxusQYH.
FIG. 3 shows the quantitative measurement of gamma-aminobutyric acid in fermentation broth of Saccharomyces mirabilis Saccharomyces paradoxusQYH.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1: screening of Gamma-aminobutyric acid producing strains
Grinding glutinous rice distiller's yeast collected in Hunan Yongzhou city, diluting with sterile water, coating the diluted suspension on nutrient agar culture medium (formula: potato extract 4 g/L, glucose 20 g/L, agar 15 g/L, solvent is water; preparation steps: dissolving each component in water, sterilizing at 121 deg.C 20 min, cooling to obtain) plate, culturing at 30 deg.C or 37 deg.C, selecting single colony after colony grows out, continuing separating and purifying until pure bacteria are obtained, determining the existence and yield of gamma-aminobutyric acid by liquid phase mass spectrometry, and screening to obtain the strain with highest gamma-aminobutyric acid yield, named QYH5 strain.
Detection of gamma-aminobutyric acid: the pure bacteria are placed in a nutrient liquid culture medium (formula is that potato extract powder 4 g/L, glucose 20 g/L and solvent is water, the preparation steps are that each component is dissolved in water, sterilized 20 min at 121 ℃, cooled to obtain the fermentation liquor after culturing 48 h at 37 ℃ and 180 rpm). And (3) taking the fermentation liquid as a test sample, adding three times of volume of absolute ethyl alcohol into the test sample, standing for 2 hours, filtering with a 0.45 micrometer filter membrane, removing the solvent by rotary evaporation under reduced pressure, diluting with water for thousands times, and measuring the gamma-aminobutyric acid content by adopting a liquid phase mass spectrometry. Liquid phase conditions: the chromatographic column is a C18 column, the inner diameter and the length are 2.1X100 mm, and the particle size of the filling is 3.5 μm; column temperature is 40 ℃; the sample injection amount is 1 mu L; the mobile phase is water to methanol volume ratio = 90:10; the flow rate was 0.3 mL/min. Mass spectrometry conditions: the ion source was ESI positive ion mode with an ionization voltage of 5500V and a declustering voltage (DP) of 50V. The qualitative ion pair was 104.1 to 87.1 (collision energy (CE) was 16V), 104.1 to 69.2 (collision energy (CE) was 23V). The quantitative ion pair was 104.1.fwdarw.87.1.
Example 2: morphological observations of Strain QYH5
The strain QYH5 grows faster on a nutrient agar culture medium, can grow after being aerobically cultured for 24-48 hours at the temperature of 30 ℃ and 37 ℃, has the colony diameter of 1-5 mm, is round, smooth in edge, milky white, raised in lawn, smooth in surface, wet and easy to pick up, has fragrance, and has the colony morphological characteristics shown in figure 1. The strain is oval under a microscope, and has a size of 10-30 mu m, and the nuclei can be seen, see figure 2.
Example 3: molecular biological characteristics of Strain QYH5
Amplification, sequencing and sequence alignment of 18S rRNA of strain QYH 5:
Fresh thalli are collected, a total DNA template is extracted by a rapid extraction kit (a biological engineering (Shanghai) stock, B518227), a universal primer 18S-F/18S-R is used for carrying out 18S rRNA gene amplification, and after PCR products are detected and purified, the sequence determination is directly carried out, and the determination is carried out by Beijing Liuhua large gene technology Co.
The primer sequences were as follows:
18S-F：5'-CCAGTAGTCATATGCTTGTCT-3'；
18S-R：5'-ACCTTGTTACGACTTTTACTTCC-3'；
50. The μL PCR reaction system was as follows: 1 μl of DNA template; 1. Mu.L each of the upstream primer and the downstream primer; DREAM TAQ GREEN PCR MASTER Mix 25 μl; ddH 2 O22. Mu.L.
PCR conditions: 95℃for 5 min,94℃for 30 s,52℃for 2min, 72℃for 1 min; cycling was performed 30 times.
The full sequence results were measured as follows: the 18S rRNA sequence of the strain is shown as SEQ ID NO.1, and the obtained complete sequence is uploaded to NCBI website for sequence comparison, the similarity of the strain QYH5 and the saccharomyces mirabilis Saccharomyces paradoxus is found to be 99.59%, and the strain is identified as the saccharomyces mirabilis Saccharomyces paradoxus QYH by combining colony characteristics and physiological and biochemical characteristics. The strain has been deposited at the cantonese microbiological strain collection center (GDMCC) at 2023, 6, 27, address: building 5 of Guangzhou City martyr of Guangdong province, no. 100 of the university of road 59, post code: 510070, accession number is: GDMCC No:63592.
Example 4: preparation of gamma-aminobutyric acid by using saccharomycetes QYH5
A method for preparing gamma-aminobutyric acid by fermenting yeast Saccharomyces paradoxus QYH comprises the following steps:
(1) Seed liquid culture: inoculating a strain Saccharomyces paradoxus QYH of saccharomyces mirabilis into a seed culture medium, and culturing 48 h at 37 ℃ and 180: 180 rpm to obtain seed liquid; the formula of the seed culture medium is as follows: 4 g/L of potato extract powder and 20/g/L of glucose, wherein the solvent is water. The preparation method comprises the following steps: dissolving the above materials in water, stirring to dissolve, and sterilizing at 121deg.C 20 min.
(2) Aerobic fermentation: inoculating the seed solution into a fermentation culture medium in an inoculum size of 5% by volume, and culturing at 37 ℃ under 100 rpm ℃ for 48 h to obtain a fermentation broth containing gamma-aminobutyric acid. The formula of the fermentation medium is as follows: 4 g/L of potato extract powder and 20/g/L of glucose, wherein the solvent is water. The preparation method comprises the following steps: dissolving the above materials in water, stirring to dissolve, and sterilizing at 121deg.C 20 min.
(3) Detection of gamma-aminobutyric acid: taking the fermentation liquor of the mirabilis Saccharomyces paradoxus QYH as a test sample, adding three times of volume of absolute ethyl alcohol into the test sample, standing for 2 hours, filtering with a 0.45 micrometer filter membrane, removing the solvent by rotary evaporation under reduced pressure, diluting with water for thousands times, and measuring the gamma-aminobutyric acid content by adopting a liquid phase mass spectrometry method. Liquid phase conditions: the chromatographic column is a C18 column, the inner diameter and the length are 2.1X100 mm, and the particle size of the filling is 3.5 μm; column temperature is 40 ℃; the sample injection amount is 1 mu L; the mobile phase is water: methanol volume ratio = 90:10; the flow rate was 0.3 mL/min. Mass spectrometry conditions: the ion source was ESI positive ion mode with an ionization voltage of 5500V and a declustering voltage (DP) of 50V. The qualitative ion pair was 104.1 to 87.1 (collision energy (CE) was 16V), 104.1 to 69.2 (collision energy (CE) was 23V). The quantitative ion pair was 104.1.fwdarw.87.1.
As shown in FIG. 3, the yield of gamma-aminobutyric acid in fermentation broth of Saccharomyces mirabilis Saccharomyces paradoxus QYH was 67.95 mg/L.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
SEQ ID NO.1 (18S rRNA of Saccharomyces mirabilis Saccharomyces paradoxusQYH)
ATGGTTAAGAACTACGACGGTATCTGATCATCTTCGATCCCTAACTTTCGTTCTTGATTAATGAAAACGTCCTTGGCAAATGCTTTCGCAGTAGTTAGTCTTCAATAAATCAAAGAATTTCACCTCTGACAATTGAATACTGATGCCCCCGACCGTCCCTATTAATCATTACGATGGTCTAGAAACCAACAAAATAGAACCAAACGTCTATTCTATTATTCCATGCTAATATATTCGAGCAATACGCCTGCTTTGAACACTCTAATTTTTTCAAAGTAAAAGTCCTGGTTCGCCAAGAGCCACAAGGACTCAAGGTTAGCCAGAAGGAAAGGCCCCGTTGGAAATCCAGTACACGAAAAAATCGGACCGGCCAACCGGGCCCAAAGTTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGTATTTACATTGTACTCATTCCAATTACAAGACCCGAATGGGCCCTGTATCGTTATTTATTGTCACTACCTCCCTGAATTAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTTATTCCCCGTTACCCGTTGAAACCATGGTAGGCCACTATCCTACCATCGAAAGTTGATAGGGCAGAAATTTGAATGAACCATCGCCAGCACAAGGCCATGCGATTCGAAAAGTTATTATGAATCATCAAAGAGTCCGAAGACATTGATTTTTTATCTAATAAATACATCTCTTCCAAAGGGTCGAGATTTTAAGCATGTATTAGCTCTAGAATTACCACAGTTATACCATGTAGTAAAGGAACTATCAAATAAACGATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTATAAATTGCTTATACTAGACATGCATGGCTTAATCTTTGAGACAAGCATATGACTACTGGCAGGATCAACCAGATAACTATCGTAAAAGAAAAGAAGCAACAAGCGACAGGAAAAGAAACCGAAATCTCTTTTTCTCTCGCCTATTCCCTCTCTCTAGAAGATACTTATTGAGTTTGGAAACAGCAGAAAGTCCAGAAAAATTGCTTTCTCAGGTCTCTCTGCTGCCGGAAATGCTCTCTGTTCAAAAAGCTTTTACACTCTTGAACAGCGCACTCCGTCACCATACCATAGCACTCTTTGAGTTTCCTCTAATCAGGTTCCACCAAACAGATACCCCGGTGTTTCACGGAATGGTACGTTTGATATCACTGATTTGAGAGGAGGTTACACTTGGAGAATCACAGTCTTGCGACCGGCTATTCAACAAGGCATTCCCCCAAGTTTGAATTCTTTGAAATAGATTGCTATTAGCTAGTAATCCACCAAATCCTTCGCTGCTCACCAATGGAATCGCAAGATGCCCACGATGAGACTGTTCAGGTCAAACGTAAAGAAAAACACACTCTGAGAATAACTCCCAAATTGTATCTCTCAATACGCATTAACCCATGTCAATTAAACACGCTGTATAGAGACTAGGCAGATCTGACGATCACCTAGCGACTCTCTCCACCGTTTGACGAGGCCATTTACAAAAACATAACGAACGACAAGCCTACTCGAATTCGTTTCCAAACTCTTTTCGAACTTGT
SEQ ID NO.2
CCAGTAGTCATATGCTTGTCT
SEQ ID NO.3
ACCTTGTTACGACTTTTACTTCC
Claims (9)
1. The singular yeast Saccharomycesparadoxus QYH has a preservation number of: GDMCC No:63592.
2. The use of the yeast cell Saccharomycesparadoxus QYH of claim 1 in the preparation of gamma-aminobutyric acid.
3. A process for producing gamma-aminobutyric acid, which comprises fermenting a strain of Saccharomyces mirabilis Saccharomycesparadoxus QYH as claimed in claim 1.
4. A method according to claim 3, comprising the steps of:
(1) Seed liquid culture: inoculating the strain Saccharomycesparadoxus QYH of the mirabilis yeast of claim 1 into a seed culture medium for shake culture for 24-48 h to obtain seed liquid;
(2) Aerobic fermentation: inoculating the seed solution into a fermentation medium for culturing for 24-72 h to obtain a fermentation liquor containing gamma-aminobutyric acid.
5. The method according to claim 4, wherein the seed culture medium or the fermentation medium comprises the following components in percentage by weight: 4g/L of potato extract powder and 20g/L of glucose, wherein the solvent is water.
6. The method according to claim 4, wherein the seed liquid is inoculated in an amount of 1 to 5% by volume.
7. The method according to claim 4, wherein the culturing temperature of the culture is 30 to 37 ℃.
8. A microbial agent comprising the yeast cell Saccharomycesparadoxus QYH according to claim 1.
9. The method for culturing the mirabilis Saccharomycesparadoxus QYH as claimed in claim 1, which is characterized by comprising inoculating the mirabilis Saccharomycesparadoxus QYH5 as claimed in claim 1 into a culture medium of 4-10g/L potato extract powder, 20-30g/L glucose, 0-15g/L agar and water as a solvent.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008068635A2 (en) * | 2006-12-01 | 2008-06-12 | Sarco | Use of ure2 mutant yeasts for increasing the release of aromatic volatile thiols by yeast during fermentation |
| CN113969242A (en) * | 2021-10-28 | 2022-01-25 | 吉林省农业科学院 | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and application of saccharomyces cerevisiae in preparation of gamma-aminobutyric acid products |
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| CN114317628B (en) * | 2021-12-28 | 2024-03-08 | 华熙生物科技股份有限公司 | Gamma-aminobutyric acid product with low irritation, preparation method thereof and application of gamma-aminobutyric acid product with low irritation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008068635A2 (en) * | 2006-12-01 | 2008-06-12 | Sarco | Use of ure2 mutant yeasts for increasing the release of aromatic volatile thiols by yeast during fermentation |
| CN113969242A (en) * | 2021-10-28 | 2022-01-25 | 吉林省农业科学院 | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and application of saccharomyces cerevisiae in preparation of gamma-aminobutyric acid products |
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