CN114292763B - Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof - Google Patents
Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof Download PDFInfo
- Publication number
- CN114292763B CN114292763B CN202111565795.7A CN202111565795A CN114292763B CN 114292763 B CN114292763 B CN 114292763B CN 202111565795 A CN202111565795 A CN 202111565795A CN 114292763 B CN114292763 B CN 114292763B
- Authority
- CN
- China
- Prior art keywords
- glucan
- saccharomyces cerevisiae
- fermentation
- soluble beta
- gamma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 96
- 229920002498 Beta-glucan Polymers 0.000 title claims abstract description 75
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 title claims abstract description 70
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 70
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 56
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 56
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 53
- 230000004151 fermentation Effects 0.000 claims abstract description 53
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 230000000813 microbial effect Effects 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 17
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 5
- 229960002989 glutamic acid Drugs 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 4
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 4
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 2
- 230000036541 health Effects 0.000 claims description 2
- 231100000350 mutagenesis Toxicity 0.000 abstract description 21
- 238000002703 mutagenesis Methods 0.000 abstract description 21
- 238000012216 screening Methods 0.000 abstract description 16
- 239000006228 supernatant Substances 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 241000233866 Fungi Species 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 210000002421 cell wall Anatomy 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 229920001503 Glucan Polymers 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000007222 ypd medium Substances 0.000 description 4
- 108020004463 18S ribosomal RNA Proteins 0.000 description 3
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 3
- 241000235342 Saccharomycetes Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- CTYRPMDGLDAWRQ-UHFFFAOYSA-N phenyl hydrogen sulfate Chemical compound OS(=O)(=O)OC1=CC=CC=C1 CTYRPMDGLDAWRQ-UHFFFAOYSA-N 0.000 description 3
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 244000253724 Saccharomyces cerevisiae S288c Species 0.000 description 1
- 235000004905 Saccharomyces cerevisiae S288c Nutrition 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- -1 connected by beta-1 Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- UZIWRCBLXLKBID-UHFFFAOYSA-N dodecasodium sulfuric acid tetraborate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-].OS(O)(=O)=O UZIWRCBLXLKBID-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- YRTWNFXASSUQEW-UHFFFAOYSA-M sodium;methanol;acetate Chemical compound [Na+].OC.CC([O-])=O YRTWNFXASSUQEW-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microbial fermentation, in particular to a saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof. According to the invention, a saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan is obtained by screening in the natural world by combining ultraviolet mutagenesis, ARTP mutagenesis and ultraviolet-ARPT combined mutagenesis. The Saccharomyces cerevisiae of the present invention was deposited with the China center for type culture Collection, with a deposit address: the preservation number of the Chinese university of Wuhan in Wuhan is CCTCC NO: M20211406. The saccharomyces cerevisiae of the invention improves the GABA yield in the fermentation liquor to 7.4g/L, improves the soluble beta-glucan yield to 3.5g/L, and realizes the high-efficiency production of gamma-aminobutyric acid and soluble beta-glucan.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof.
Background
Gamma-aminobutyric acid (gamma-aminobutyric acid, GABA), also known as amino acid, is a natural amino acid widely existing in animals, plants and microorganisms, which is obtained by catalytic conversion of glutamic acid by glutamate decarboxylase (Glutamic acid decarboxylase, GAD). GABA is a natural anti-aging factor and has the effects of relieving muscles and reducing wrinkles. Currently, GABA is mainly obtained by three pathways, namely chemical synthesis, plant extraction and microbial fermentation. The cost of the raw material of the chemical synthesis GAB is high, the organic solvent is involved, the separation effect is poor, the safety threat is large, the application range is limited greatly, and the GAB can not be used in cosmetics, foods and medicines; the plant extraction yield is low and difficult to separate; in contrast, microbial fermentation to synthesize GABA is a safer and more economical method.
Beta-glucan (β -glucan) is a polysaccharide formed by the linkage of glucose monomers through β -glycosidic linkages, and is widely found in fungi, bacteria and plants. Fungi are one of the important sources of beta-glucans, which are predominantly composed of beta-1, 3 glycosidic linkages, with small amounts of beta-1, 6 glycosidic linkages also present. Part of the fungus beta-glucan is derived from fungus cell walls, wherein the glucan in the fungus cell walls is beta-1, 3-1, 6-glucan, and 65% -90% of the glucan is beta-1, 3-glucan.
Saccharomyces cerevisiae is a safe fungus widely used in cosmetics and is favored by people because of its abundant skin care factors. These skin care factors include yeast beta-glucan. Yeast beta-glucan is a cell wall structure polysaccharide mainly connected by beta-1, 3 glycosidic bonds and secondarily connected by beta-1, 6 glycosidic bonds, wherein about 85% of the yeast beta-glucan is water insoluble. The cell wall of Saccharomyces cerevisiae accounts for about 20% of the dry weight of the cell, has a thickness of about 100-200 nm, and is divided into three layers from outside to inside, and the components are mannose, protein and beta-glucan respectively. Beta-glucans in the cell wall of Saccharomyces cerevisiae can be divided into two classes: beta-1, 3 glucan as a main component forming a network structure; beta-1, 6 glucan is linked as a branch to other components.
The soluble yeast beta glucan has good moisturizing, antioxidation and cell repairing effects on damage caused by ultraviolet irradiation, and is favored in the cosmetic industry. At present, the preparation of the soluble yeast beta-glucan mainly takes yeast cell walls as raw materials and adopts acid extraction, alkali extraction, acid-alkali extraction and an enzyme method to prepare the soluble yeast beta-glucan. The methods have low production efficiency, low product purity, large loss of glucan activity and environmental pollution. Therefore, the use of yeast to autonomously secrete soluble yeast beta-glucan is critical to solving these problems.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof.
Aiming at the problems that the prior art for preparing gamma-aminobutyric acid and soluble beta-glucan by using the traditional method is low in yield, large in pollution, complex in steps and difficult to meet market demands, the invention provides the saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan. The invention obtains the saccharomyces cerevisiae which can produce the gamma-aminobutyric acid and the soluble beta-glucan with high yield by utilizing ultraviolet mutagenesis, ARTP mutagenesis and ultraviolet-ARTP combined mutagenesis.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: providing a strain of saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan, wherein the saccharomyces cerevisiae has been preserved in China center for type culture collection (China, with a preservation address) at 11-12 of 2021: the preservation number of the Chinese university of Wuhan in Wuhan is CCTCC NO: M20211406.
The invention also provides a microbial agent, which comprises the saccharomyces cerevisiae.
The invention also provides application of the saccharomyces cerevisiae in preparing gamma-aminobutyric acid or soluble beta-glucan.
The invention also provides application of the saccharomyces cerevisiae in preparing cosmetics containing gamma-aminobutyric acid or soluble beta-glucan.
The invention also provides application of the saccharomyces cerevisiae in preparing health care products containing gamma-aminobutyric acid or soluble beta-glucan.
The invention also provides a method for simultaneously producing gamma-aminobutyric acid and soluble beta-glucan, which is fermentation production by using the saccharomyces cerevisiae.
As a preferred embodiment of the method of the invention, the medium used for the fermentation comprises: 5-10 g/L yeast extract powder, 15-30 g/L peptone, 10-20 g/L malt extract powder, 10-30 g/L glucose, 10-30 g/L sucrose, 20-40 g/L sodium chloride, 4-10 g/L-glutamic acid and 0.2-0.8 g/L pyridoxal phosphate.
As a preferred embodiment of the method of the invention, the fermentation conditions are: the fermentation temperature is 25-32 ℃, the pH is controlled to be 4.5-6.5 in the fermentation process, the rotating speed of a fermentation tank is 200-800 rpm, the amount of dissolved oxygen in fermentation liquor is 20-80%, and the fermentation time is 48-72 h.
The invention has the beneficial effects that:
(1) The invention provides a saccharomyces cerevisiae capable of simultaneously producing gamma-aminobutyric acid and soluble beta-glucan at high yield. The invention adopts ultraviolet and ARTP mutagenesis method, combines with optimization of fermentation condition, and utilizes microorganism cells to realize high-efficiency production of gamma-aminobutyric acid and soluble beta-glucan.
(2) The saccharomyces cerevisiae strain of the invention improves the GABA yield in fermentation liquor to 7.4g/L and improves the soluble beta-glucan yield to 3.5g/L through optimizing a culture medium and optimizing fermentation conditions on a fermentation tank.
(3) According to the saccharomyces cerevisiae disclosed by the invention, after optimized condition culture is adopted, the oxidation resistance of fermentation broth is improved, namely, screened strains are inoculated into an optimized culture medium for fermentation, and after fermentation, the clearance of 30% concentration fermentation broth to DPPH free radicals is improved from 34.3% to 99.5% of the original strain.
Drawings
FIG. 1 is a graph showing the corresponding yield determinations of the primary screening of high-yielding GABA and soluble beta-glucan strains.
FIG. 2 is a graph of the corresponding yield assays for screening high-yielding GABA and soluble beta-glucan strains by UV mutagenesis.
FIG. 3 is a graph showing the corresponding yield assays for screening high-yielding GABA and soluble beta-glucan strains by ARTP mutagenesis.
FIG. 4 is a graph showing the corresponding yield assays for screening high-yielding GABA and soluble beta-glucan strains by ultraviolet-ARTP mutagenesis.
Detailed Description
The technical scheme of the invention is further described by specific examples. The examples are only for explaining the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials and reagents used, unless otherwise specified, are those commercially available.
1. GABA screening Medium (g/L): yeast extract 10.0, peptone 20.0, agar 20.0, glucose 20.0, bromocresol green 0.1, glyoxylate 2 and succinic acid 2.
2. High performance liquid phase method detects GABA: and preparing different concentrations of 0.5 g/L, 1g/L, 2g/L, 3 g/L, 4g/L and 5g/L by taking purchased GABA standard samples as standard samples, and preparing a standard curve. The GABA yield was calculated from the peak area by measuring the absorption peak at a wavelength of ultraviolet 338nm using a chromatographic column of C18 (5.0X1250 mm), mobile phase A (0.5% aqueous sodium acetate) and mobile phase B (0.5% aqueous sodium acetate-methanol).
3. Detection of soluble beta-glucan by phenol sulfate method: precisely sucking glucose standard solution 0.1, 0.2, 0.3, 0.4 and 0.5ml respectively, placing into 10ml test tubes with plugs, adding water to 0.5ml in each tube, adding sodium tetraborate-sulfuric acid solution 3ml into ice water bath respectively, mixing uniformly with vortex mixer, heating in boiling water bath for 5min, immediately cooling to room temperature after taking out, adding 0.1ml of 0.1% carbazole solution, shaking uniformly, boiling for 5min, cooling to room temperature, and measuring absorbance at 530 nm. And (3) carrying out linear regression by taking the concentration as an abscissa and the absorbance value as an ordinate to manufacture a standard curve. The test samples were examined in the same manner and the soluble β -glucan yield was calculated by substituting absorbance values into standard equations.
Example 1: screening of high-yield GABA Saccharomyces cerevisiae strains
20g of soil at a position about 5cm deep in a wet place in an orchard is taken, the soil is placed in a 500mL triangular flask, 200mL of sterile phosphate buffer solution (pH 5.0) is added, and then 20g/L sodium glutamate with final concentration is added, and after being stirred uniformly by a magnetic stirrer, the mixture is placed at 30 ℃ for 12h of incubation. Then taking appropriate amount of supernatant fluid and respectively diluting 10 -4 、10 -5 And 10 -6 After doubling 50. Mu.l of coated GABA screening plates were taken. After single colony grows out, picking round, moist, smooth and dark green single colony. And then streaking and purifying the selected single colony, screening to obtain 50 strains of GABA producing strains, and preserving for later use.
Example 2: screening of high-yielding GABA and soluble beta-glucan strains
The 50 GABA producing strains selected in example 1 were individually picked up as single colonies and inoculated into 250mL Erlenmeyer flasks containing 50mL YPD and cultured in a shaker at 220rpm at 30℃for 48 hours. After fermentation, taking fermentation liquor, centrifuging for 10min at 5000g, collecting supernatant, measuring gamma-aminobutyric acid by using a high performance liquid phase, and detecting the content of soluble beta-glucan by using a phenol sulfate method. From the results, it was found that 6 strains among the strains examined and analyzed were able to simultaneously synthesize GABA and soluble beta-glucan, the yields of which are shown in FIG. 1. Wherein the strain S6 has the highest GABA yield of 0.98g/L, and the yield of the soluble beta-glucan synthesized by the strain is 0.18g/L. GABA yield was 0.74g/L and soluble β -glucan yield was 0.38g/L in strain S5, which was the strain with the highest soluble β -glucan yield among 6 strains.
Example 3: identification of high-yielding GABA and soluble beta-glucan Saccharomyces cerevisiae strains
And identifying 6 strains which are obtained by screening and simultaneously produce GABA and soluble beta-glucan, using a Saccharomyces cerevisiae S288C genome as a template, amplifying an 18S rDNA sequence (shown as SEQ ID NO: 1) of the strain to be detected by using an 18S rDNA universal primer, and sequencing. The result shows that the similarity of the strain and the saccharomyces cerevisiae strain 18S rDNA sequence is up to 99.9% through NCBI website comparison, and the strain belongs to saccharomyces cerevisiae through phylogenetic tree clustering analysis.
Example 4: screening of Saccharomyces cerevisiae strains for high GABA and soluble beta-glucan production by UV mutagenesis
Taking and initially screening to obtain 6 strains of yeast strains capable of producing GABA and soluble yeast beta-glucan, respectively inoculating the strains into YPD culture medium, and culturing the strains in a shaking table at 30 ℃ and 220rpm for 8-12 hours to enter a logarithmic phase. Selecting saccharomycete cultured to logarithmic phase for mutagenesis treatment. In the darkroom, the ultraviolet lamp is started, and the equipment is preheated for 20min. 10-15 mL yeast suspensions with different dilutions are placed in a sterile empty culture dish. The dish cover is opened at a distance of 25cm under a 30W ultraviolet lamp by wearing an ultraviolet-proof sterile glove, and the bacterial suspension in the dish is gently shaken to be respectively irradiated for different times. And (3) respectively taking 1mL of treated bacterial liquid into sterile test tubes, placing the test tubes on ice, and cooling for 1-2 h, wherein the low-temperature condition can inhibit DNA repair in the bacterial bodies, and the mutation efficiency is improved. 200. Mu.L of the cooled and placed bacterial liquid was spread on YPD medium plates and cultured at 30℃for 3 days in the absence of light. Randomly growing normal single colony 500 strains, inoculating into a 96-well deep-hole plate filled with 2mL YPD culture medium, culturing at 30 ℃ for 72 hours, centrifuging, taking supernatant, and respectively detecting GABA and soluble beta-glucan yield by using a high performance liquid chromatography method and a phenol sulfate method. By detection, the yields of both products in most strains were not improved, even reduced. The 13 strains showed good results after mutation, and the two products showed different increases, the results of which are shown in FIG. 2. Wherein the maximum GABA yield was 2.6g/L (strain uvM) and the maximum soluble β -glucan yield was 0.45g/L (strain uvM).
Example 5: ARTP mutagenesis screening of Saccharomyces cerevisiae strains with high GABA and soluble beta-glucan yields
Taking and initially screening to obtain 6 strains of yeast strains capable of producing GABA and soluble yeast beta-glucan, respectively inoculating the strains into YPD culture medium, and culturing the strains in a shaking table at 30 ℃ and 220rpm for 8-12 hours to enter a logarithmic phase. Sucking 1mL of bacterial liquid cultured to logarithmic phase in an ultra-clean workbench, centrifuging for 10min at 6000g, discarding supernatant, washing 3 times with sterile physiological saline, sucking 10 mu L of the supernatant, placing the supernatant on a slide, placing the slide in a corresponding groove sequentially with sterile forceps, fixing a centrifuge tube filled with 1mL of YPD liquid culture medium below, and performing a mutagenesis test. Helium with a purity of 99.999% is selected as the working gas, the flow rate is 10sL/min, the power supply power is set to 120W, the operating temperature is 20 ℃, and the treatment time is 0, 20, 40, 60, 80, 100, 120, 140 seconds respectively. After mutagenesis, the centrifuge tube with the slide is placed on a vortex device to shake vigorously for 1min, the mutagenized bacteria are eluted, 200 mu L of the mutagenized bacteria are coated on a flat plate, and the culture is carried out in a 30 ℃ incubator for 72h in a dark place. 500 single colonies growing on the plate are randomly picked up, inoculated into a 96-well deep-hole plate filled with 2mL YPD culture medium, cultured for 72 hours at 30 ℃, and the supernatant is centrifugally taken to detect GABA and soluble beta-glucan yields, wherein 15 strains have good results after mutation, and the results are shown in figure 3. Wherein the maximum GABA yield was 2.9g/L (strain aM 13), and the maximum soluble β -glucan yield was 0.59g/L (strain aM 13).
Example 6: screening of Saccharomyces cerevisiae strains for high GABA and soluble beta-glucan production by ultraviolet-ARTP combined mutagenesis
In a darkroom, an ultraviolet mutagenesis instrument is started, and 10mL OD is taken after equipment is preheated 600 The yeast suspension of =0.2 was poured into a sterile dish with uv-proof sterile gloves, the dish lid was opened at a distance of 25cm under a uv lamp of 30W, and the dish was shaken smoothly clockwise to allow the uniform irradiation of uv light to the bacterial suspension in the dish. After ultraviolet mutagenesis treatment for 1 minute, 10 mu L of the irradiated strain suspension is taken and placed on an ARTP mutagenic instrument slide, the slide is sequentially placed into a corresponding groove by sterile forceps, and a centrifuge tube filled with 1mL of YPD liquid culture medium is fixed below, so that a mutagenesis test can be started, and the mutagenesis time is 100 seconds. After mutagenesis, the tube with slides was vigorously shaken on a vortex shaker for 1min, the mutagenized bacteria eluted, 200. Mu.L was applied to YPD solid plates and incubated in a 30℃incubator protected from light for 72h. After single colonies were grown, 500 single colonies were picked and inoculated into 96-well deep well plates containing 2mL of YPD medium, and fermented at 30℃and 220rpm for 72 hours. After fermentation, the fermentation broth was centrifuged at 5000g for 10min, and the supernatant was taken to determine the GABA and soluble beta-glucan contents. Among them, 10 strains were mutated to have good results, and the results are shown in FIG. 4. Wherein the GABA yield in the optimal strain uvaM10 is 4.9g/L, and the highest yield of the soluble beta-glucan is 1.4g/L.
The optimal strain uvaM10 of the invention was deposited at the China center for type culture Collection, address: the preservation number of the Chinese university of Wuhan in Wuhan is CCTCC NO: M20211406.
Example 7: genetic stability analysis of high-yield GABA and soluble beta-glucan saccharomyces cerevisiae
The optimal strain uvaM10 obtained by the mutation is inoculated in YPD liquid culture medium for culturing for 24 hours, and then the culture is sequentially carried out for 20 times according to the inoculum size of 2 percent, and each generation is cultured for 24 hours. The final passage strain was inoculated in YPD medium at an inoculum size of 10%, and cultured at 30℃for 72 hours to examine the production of GABA and soluble β -glucan. The results were consistent with those in example 6, and no yield decrease occurred. The mutant strain has stable genetic character and can stably produce GABA and soluble beta-glucan.
Example 8: high-yield GABA and soluble beta-glucan saccharomyces cerevisiae shake flask medium optimization
The optimal strain uvaM10 was inoculated at 10% inoculum size into the following medium: 15g/L of sucrose, 20g/L of glucose, 20g/L of malt extract powder, 20g/L of peptone, 10g/L of yeast extract powder, 10g/L of sodium chloride, 10g/L of L-glutamic acid and 0.2g/L of pyridoxal phosphate. After shaking culture for 72 hours, 5000g is centrifuged for 10min to obtain fermentation supernatant, and the GABA and soluble beta-glucan contents are determined. The GABA yield of the saccharomycetes in the fermentation supernatant is improved to 5.8g/L, and the yield of the soluble beta-glucan is improved to 2.1g/L.
Example 9: high-yield GABA and soluble beta-glucan saccharomyces cerevisiae are subjected to amplification culture in a 10L fermentation tank
The fermentation medium is (g/L): sucrose, glucose, malt extract powder 60, yeast extract powder 20, peptone 10, sodium chloride 10, L-glutamic acid 30, pyridoxal phosphate 2. The fermentation conditions of the 10L fermentation tank are as follows: the fermentation temperature is 30 ℃, the fermentation pH is 5.0, the fermentation initial rotating speed is 200rpm, and the fermentation time is 96 hours.
Single colonies of uvaM10 were picked up and inoculated into a triangular flask containing 200ml of YPD medium and cultured in a shaker at 30℃and 220rpm for 24 hours to obtain seed liquid. Inoculating the cultured seed liquid into a 10L fermentation tank according to 10% of inoculation amount, regulating pH by ammonia water in the fermentation process, and coupling dissolved oxygen with rotating speed to keep the dissolved oxygen at 20% -50% when the dissolved oxygen is reduced to below 20%. After 96h fermentation, the fermentation broth is centrifuged to determine the GABA and soluble beta-glucan content. The GABA yield of the saccharomycete in the fermentation supernatant is improved from 5.8g/L of shake flask fermentation to 7.4g/L, and the content of the soluble beta-glucan is improved from 2.1g/L of shake flask fermentation to 3.5g/L.
Example 10: analysis of antioxidant Activity of mutant Strain uvaM10 fermentation broth
DPPH 1mg was weighed and dissolved in 20mL of ethanol, and the mixture was sufficiently shaken to completely dissolve the mixture. And (3) taking a proper amount of DPPH solution, measuring the light absorption value A at 517nm, adjusting the light absorption value A=0.8-1.0 by using ethanol, and then keeping the solution away from light for later use. Taking the supernatant of the fermentation broth cultured in a shake flask, diluting with purified water to 30% concentration, taking 2mL of the supernatant, placing the supernatant in a centrifuge tube, adding 2mL of the prepared DPPH solution, rapidly and uniformly mixing, reacting for 30min under the dark condition, and measuring the light absorption value As of a corresponding sample at 517 nm; the blank was replaced with water and the absorbance was A0.DPPH radical clearance (%) = ((A0-As)/A0) 100. The free radical clearance of the fermentation broth supernatant of the uvaM10 mutant strain was calculated to reach 99.5%.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> boundary (Shanghai) Biotech Co., ltd
<120> Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof
<130> 2021.12.16
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 822
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
caagtttgct gggggccccc tacctgattt gaggtcaaac tttaagaaca ttgttcgcct 60
agacgctctc ttcttatcga taacgttcca atacgctcag tataaaaaaa gattagccgc 120
agttggtaaa acctaaaacg accgtacttg cattatacct caagcacgca gagaaacctc 180
tctttggaaa aaaaacatcc aatgaaaagg ccagcaattt caagttaact ccaaagagta 240
tcactcacta ccaaacagaa tgtttgagaa ggaaatgacg ctcaaacagg catgccccct 300
ggaataccaa ggggcgcaat gtgcgttcaa agattcgatg attcacggaa ttctgcaatt 360
cacattacgt atcgcatttc gctgcgttct tcatcgatgc gagaaccaag agatccgttg 420
ttgaaagttt ttaatatttt aaaatttcca gttacgaaaa ttcttgtttt tgacaaaaat 480
ttaatgaata gataaaattg tttgtgtttg ttacctctgg gccccgattg ctcgaatgcc 540
caaagaaaaa gttgcaaaga tatgaaaact ccacagtgtg ttgtattgaa acggttttaa 600
ttgtcctata acaaaagcac agaaatctct caccgtttgg aatagcaaga aagaaactta 660
caagcctagc aagaccgcgc acttaagcgc aggcccggct ggactctcca tctcttgtct 720
tcttgcccag taaaagctct catgctcttg ccaaaacaaa aaaatccatt ttcaaaatta 780
ttaaatttct ttaatgatcc ttccgcaggt tcaccctacg ga 822
Claims (8)
1. A strain of saccharomyces cerevisiae with high yield of gamma-aminobutyric acid and soluble beta-glucan, which is characterized in that the saccharomyces cerevisiae has been deposited in China center for type culture collection (tcc) at 11-12 of 2021, and the deposit address: the preservation number of the Chinese university of Wuhan in Wuhan is CCTCC NO: M20211406.
2. A microbial agent comprising the saccharomyces cerevisiae of claim 1.
3. Use of the saccharomyces cerevisiae according to claim 1 for the preparation of gamma-aminobutyric acid or soluble beta-glucan.
4. Use of the saccharomyces cerevisiae according to claim 1 for the preparation of cosmetics containing gamma-aminobutyric acid or soluble beta-glucan.
5. The use of the saccharomyces cerevisiae of claim 1 in the preparation of health products containing gamma-aminobutyric acid or soluble beta-glucan.
6. A method for simultaneously producing gamma-aminobutyric acid and soluble beta-glucan, which is characterized in that the method is fermentation production by using the saccharomyces cerevisiae according to claim 1.
7. The method of claim 6, wherein the medium used for the fermentation comprises: 5-10 g/L yeast extract powder, 15-30 g/L peptone, 10-20 g/L malt extract powder, 10-30 g/L glucose, 10-30 g/L sucrose, 20-40 g/L sodium chloride, 4-10 g/L-glutamic acid and 0.2-0.8 g/L pyridoxal phosphate.
8. The method of claim 6, wherein the fermentation conditions are: the fermentation temperature is 25-32 ℃, the pH is controlled to be 4.5-6.5 in the fermentation process, the rotating speed of a fermentation tank is 200-800 rpm, the amount of dissolved oxygen in fermentation liquor is 20-80%, and the fermentation time is 48-72 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111565795.7A CN114292763B (en) | 2021-12-20 | 2021-12-20 | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111565795.7A CN114292763B (en) | 2021-12-20 | 2021-12-20 | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114292763A CN114292763A (en) | 2022-04-08 |
| CN114292763B true CN114292763B (en) | 2023-12-01 |
Family
ID=80968582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111565795.7A Active CN114292763B (en) | 2021-12-20 | 2021-12-20 | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114292763B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116144516B (en) * | 2023-03-10 | 2023-08-08 | 江南大学 | Saccharomyces cerevisiae for producing succinic acid and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005015348A (en) * | 2003-06-24 | 2005-01-20 | Asahi Denka Kogyo Kk | Skin cosmetic |
| CN104263665A (en) * | 2014-09-23 | 2015-01-07 | 南京工业大学 | Lignin-tolerant saccharomyces cerevisiae and application thereof in bioethanol production |
| CN110373338A (en) * | 2019-08-15 | 2019-10-25 | 东莞东阳光药物研发有限公司 | Saccharomyces cerevisiae and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7751981B2 (en) * | 2001-10-26 | 2010-07-06 | The Regents Of The University Of California | Articles of manufacture and methods for modeling Saccharomyces cerevisiae metabolism |
-
2021
- 2021-12-20 CN CN202111565795.7A patent/CN114292763B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005015348A (en) * | 2003-06-24 | 2005-01-20 | Asahi Denka Kogyo Kk | Skin cosmetic |
| CN104263665A (en) * | 2014-09-23 | 2015-01-07 | 南京工业大学 | Lignin-tolerant saccharomyces cerevisiae and application thereof in bioethanol production |
| CN110373338A (en) * | 2019-08-15 | 2019-10-25 | 东莞东阳光药物研发有限公司 | Saccharomyces cerevisiae and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114292763A (en) | 2022-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103898011B (en) | A kind of method of methylotrophic bacteria and fermentative production pyrroloquinoline quinone thereof | |
| US20240228391A1 (en) | Exiguobacterium indicum and application thereof in synthesis of nano-selenium | |
| CN113980828B (en) | Bacillus chestnut brown bacillus mutant strain for high yield chlorogenic acid | |
| CN110218676A (en) | A kind of clostridium butyricum and its application | |
| CN111484954A (en) | Pseudomonas nigricans for producing alginate lyase | |
| CN114292763B (en) | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof | |
| CN111518710B (en) | Enterobacter strain and application thereof in preparation of microbial polysaccharide | |
| CN108441429B (en) | A kind of method of pyrenomycetes and its fermenting and producing scleroglucan | |
| CN110684691A (en) | Preparation process of microbial agent based on directional screening of microorganisms | |
| CN111909881B (en) | Bacillus pumilus capable of producing feruloyl esterase and application thereof | |
| CN103374537A (en) | Method for preparing enduracidin and strain produced thereby | |
| CN110616150B (en) | High-yield polysaccharide eupatorium adenophorum endophytic fungus and application thereof | |
| CN108823266B (en) | A method for preparing chitin by fermentation | |
| CN113430126B (en) | Aureobasidium pullulans and method for preparing melanin polysaccharide by using same | |
| CN112094762B (en) | Corynebacteria vinifera strain and application thereof | |
| CN112143681B (en) | Bacillus belgii capable of producing feruloyl esterase and application thereof | |
| CN109735473B (en) | Method for preparing water-soluble curcumin by fermentation | |
| CN109706100B (en) | Staphylococcus pasteuri mutant strain and application thereof in preparation of 5-aminolevulinic acid | |
| CN110541011A (en) | Fermentation method based method for preparing carmine sulforaphane | |
| CN102827791B (en) | Agrobacterium radiobacter mutant strain and method for preparing beta-1, 3 glucan as fish and shrimp disease-resistant factor feed additive through utilizing agrobacterium radiobacter mutant strain | |
| CN116590160B (en) | Phaffia rhodozyma mutant strain HCYJ-07 and application thereof | |
| CN117417869B (en) | Flavobacterium johnsonii W24H and application thereof in production of 2, 3-butanediol | |
| CN116376730B (en) | Phaffia rhodozyma and application thereof | |
| CN114276936B (en) | Eurotium cristatum strain and application thereof | |
| CN116286513B (en) | Lactobacillus johnsonii FR-1012 and method for industrially producing gamma-aminobutyric acid by same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |