CN112852664A - Saccharomyces cerevisiae and method for improving yield of gamma-aminobutyric acid produced by saccharomyces cerevisiae - Google Patents
Saccharomyces cerevisiae and method for improving yield of gamma-aminobutyric acid produced by saccharomyces cerevisiae Download PDFInfo
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 156
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 86
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 64
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 44
- 230000004151 fermentation Effects 0.000 claims abstract description 44
- 239000006228 supernatant Substances 0.000 claims abstract description 36
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 31
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 31
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 31
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 244000005700 microbiome Species 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 6
- 229940073490 sodium glutamate Drugs 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 8
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 230000003698 anagen phase Effects 0.000 claims description 5
- 108020004463 18S ribosomal RNA Proteins 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims 2
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 239000002609 medium Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000002906 microbiologic effect Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000007222 ypd medium Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 235000021107 fermented food Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000010855 food raising agent Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 210000005171 mammalian brain Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001508 neuroinhibitory effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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Abstract
The invention provides saccharomyces cerevisiae and a method for improving the yield of gamma-aminobutyric acid by saccharomyces cerevisiae, wherein the saccharomyces cerevisiae for producing GABA is obtained by culturing and screening as a fermentation microorganism, and the yield of GABA is improved by adding lactobacillus plantarum M912 cell-free fermentation supernatant from an external source. By adopting the method, the GABA concentration is 3.96-7.68 g/L; GABA-producing Saccharomyces cerevisiae SC-125 fermentation of the control group (no cell-free supernatant added to the fermentation medium) gave GABA concentrations of 1.24-1.60 g/L. Compared with the control group, the GABA yield is improved by 3.19 to 4.80 times.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to saccharomyces cerevisiae and a method for improving the yield of gamma-aminobutyric acid by the saccharomyces cerevisiae.
Background
Gamma-aminobutyric acid (GABA) is a nonprotein amino acid, is an important neuroinhibitory transmitter in mammalian brain tissues, and has important physiological active functions of tranquilizing nerves, improving sleep, regulating hormone secretion, improving brain functions and the like. Because of the effective physiological function of GABA, a microbial fermentation synthesis method is adopted, safe and functional microbial strains for producing GABA are screened from the nature, and a fermentation product rich in GABA is prepared by fermentation of the strains, so that the method is a safe and effective method with low cost.
The yeast is regarded as a food-grade safe microorganism and is an important leavening agent in fermented food, and a plurality of strains in the yeast have health care and treatment effects on human beings and animals and are closely related to the life activities of the human beings and the animals. Therefore, screening of GABA-highly producing yeast and its use as a fermentation strain for developing GABA-enriched fermented foods have been the hot spots of research.
The factors influencing the GABA synthesis of microorganisms are related to the characteristics of the strains on one hand and the physicochemical and biological conditions of the external environment on the other hand. These conditions directly or indirectly affect the ability of the microorganism to synthesize GABA. At present, the GABA yield of the fermentation microorganism is improved mainly by optimizing culture conditions and culture media, mutagenizing microorganisms by physical and chemical factors, constructing genetically engineered bacteria, co-culturing different microorganism strains and the like. At present, the method for improving the yield of GABA by adding the lactobacillus plantarum cell-free fermentation supernatant into a saccharomyces cerevisiae fermentation medium by taking functional saccharomyces cerevisiae capable of producing GABA as a fermentation microorganism is not reported.
Disclosure of Invention
Aiming at the defects of the prior art, the functional GABA-producing saccharomyces cerevisiae and lactobacillus plantarum which are obtained by culturing and screening from a naturally fermented fruit sample and have symbiotic effect are taken as fermenting microorganisms, and the yield of GABA is increased by adding lactobacillus plantarum cell-free fermentation supernatant from an external source.
The invention aims to provide a gamma-aminobutyric acid producing Saccharomyces cerevisiae SC-125 (which is preserved in China general microbiological culture collection and management Committee general microbiological center at 26.11.2020 and is named as CGMCC 21236 by classification in the northwest way 1 of the sunny region in Beijing, China academy of sciences microbiological research institute, Saccharomyces cerevisiae by classification) and a Lactobacillus plantarum M912 (which is preserved in China general microbiological culture collection and management Committee general microbiological center at 26.11.2020 and is named as CGMCC 21236 by classification in the northwest way 1 of the sunny region in Beijing, China academy of sciences microbiological research, is named as Lactobacillus plantarum Lancepacia by classification and CGMCC 21237) separated, cultured and identified from a naturally fermented fruit sample, and a Lactobacillus plantarum SC-125 fermentation supernatant is added into a Saccharomyces cerevisiae SC-912 fermentation medium, so as to improve the GABA yield and solve the problem of too low GABA yield in single saccharomyces cerevisiae SC-125 fermentation synthesis.
Specifically, GABA-producing saccharomyces cerevisiae SC-125 and lactic acid bacteria strain M912 are separated from the traditional fermented Sichuan pickle by an enrichment culture method, and are respectively identified as saccharomyces cerevisiae and lactobacillus plantarum by 18S rRNA and 16S rRNA after culture. According to the study on GABA producing capability of the strain, the GABA producing capability of the GABA producing saccharomyces cerevisiae SC-125 strain in a culture medium containing 5 g/LL-sodium glutamate is 1.12 g/L.
The specific technical scheme is as follows:
a method for improving the yield of gamma-aminobutyric acid by saccharomyces cerevisiae comprises the following steps:
(1) preparation of lactobacillus plantarum M912 cell-free fermentation supernatant: lactobacillus plantarum M912 is activated in MRS liquid medium and cultured for 10-12h to logarithmic growth phase at 30 ℃. 10000g, centrifuging at 4 ℃ for 10min, collecting supernatant, and filtering with a 0.22 mu M filter membrane to obtain lactobacillus plantarum M912 cell-free fermentation supernatant.
(2) Preparing fermentation seeds of GABA-producing saccharomyces cerevisiae SC-125: the GABA-producing saccharomyces cerevisiae obtained by culture and screening is identified by 18S rRNA, and the yeast strain is activated in YPD culture medium containing 5 g/LL-sodium glutamate and cultured for about 12h to logarithmic growth phase at 30 ℃.
(3) GABA is synthesized by fermenting Saccharomyces cerevisiae SC-125 which produces GABA: inoculating Saccharomyces cerevisiae SC-125 producing GABA into YPD medium containing 6g/L-12g/L L-sodium glutamate at an inoculation amount of 5% (v/v), adding 8-9% (v/v) Lactobacillus plantarum M912 cell-free fermentation supernatant into the YPD medium, and fermenting at 30 ℃ for 120-144 h.
By adopting the method, the GABA concentration is 3.96-7.68 g/L; GABA-producing Saccharomyces cerevisiae SC-125 fermentation of the control group (no cell-free supernatant added to the fermentation medium) gave GABA concentrations of 1.24-1.60 g/L. Compared with the control group, the GABA yield is improved by 3.19 to 4.80 times.
Drawings
FIG. 1 is a phylogenetic tree of strain SC-125 constructed based on yeast 18S rRNA gene sequence according to the present invention;
FIG. 2 is a phylogenetic tree of strain M912 constructed based on the gene sequence of lactobacillus 16S rRNA;
FIG. 3 is the effect of addition of Lactobacillus plantarum M912 cell-free supernatant on growth of GABA producing Saccharomyces cerevisiae SC-125 thallus of the examples;
in the figure:
■ GABA-producing Saccharomyces cerevisiae SC-125(6g/L L-sodium glutamate); ● GABA-producing Saccharomyces cerevisiae SC-125(6g/L L-sodium glutamate) by adding 8% cell-free supernatant;
FIG. 4 is the effect of addition of Lactobacillus plantarum M912 cell-free supernatant on GABA production by s.cerevisiae SC-125 of the examples;
in the figure:
■ GABA-producing Saccharomyces cerevisiae SC-125(6g/L L-sodium glutamate); ● GABA-producing Saccharomyces cerevisiae SC-125(6g/L L-sodium glutamate) by adding 8% cell-free supernatant;
Detailed Description
The specific technical scheme of the invention is described by combining the drawings and the embodiment.
Example 1:
(1) preparation of lactobacillus plantarum M912 cell-free fermentation supernatant: activating lactobacillus plantarum M912 in MRS liquid medium, culturing at 30 deg.C for 12h to logarithmic growth phase, with bacterial liquid concentration of 107CFU/mL. 10000g, centrifuging at 4 ℃ for 10min, collecting supernatant, and filtering with a 0.22 mu m filter membrane to obtain cell-free supernatant.
(2) And (3) culturing and preparing seeds of GABA-producing saccharomyces cerevisiae SC-125: activating gamma-aminobutyric acid-producing GABA saccharomyces cerevisiae SC-125 in YPD medium containing 5g/L L-sodium glutamate, culturing at 30 ℃ to logarithmic phase, wherein the concentration of bacterial liquid is 107CFU/mL。
(3) GABA is synthesized by fermenting Saccharomyces cerevisiae SC-125 which produces GABA: inoculating GABA-producing saccharomyces cerevisiae SC-125 into YPD liquid culture medium containing 6 g/LL-sodium glutamate according to the inoculation amount of 5%, and adding 8% (v/v) of lactobacillus plantarum M912 cell-free fermentation supernatant into the YPD liquid culture medium. Meanwhile, fermentation without adding cell-free fermentation supernatant in the fermentation medium is taken as a control. Fermenting at 30 ℃ for 144h respectively, and detecting and analyzing by HPLC to obtain the maximum GABA concentration of 3.96g/L and the GABA concentration of a control group of 1.24 g/L. Compared with a control group, the experimental group has 3.19 times higher GABA fermentation synthesis yield.
As shown in FIG. 3 and FIG. 4, FIG. 3 shows the effect of the addition of Lactobacillus plantarum M912 cell-free supernatant on the growth of GABA-producing Saccharomyces cerevisiae SC-125 cells, and the result of the number of cells at OD600 shows that the Lactobacillus plantarum M912 cell-free fermentation supernatant at a certain concentration has a significant effect on the growth of GABA-producing Saccharomyces cerevisiae SC-125. FIG. 4 shows the influence of the addition of Lactobacillus plantarum M912 cell-free supernatant on GABA production of Saccharomyces cerevisiae SC-125, and from the GABA results, it was found that the GABA yield of GABA production of Saccharomyces cerevisiae SC-125 can be significantly increased by using Lactobacillus plantarum M912 cell-free fermentation supernatant of a certain concentration.
Example 2:
(1) preparation of lactobacillus plantarum M912 cell-free fermentation supernatant: activating lactobacillus plantarum M912 in MRS liquid medium, culturing at 30 deg.C for 12h to logarithmic growth phase, with bacterial liquid concentration of 107CFU/mL. 10000g, centrifuging at 4 ℃ for 10min, collecting supernatant, and filtering with a 0.22 mu m filter membrane to obtain cell-free supernatant.
(2) And (3) culturing and preparing seeds of GABA-producing saccharomyces cerevisiae SC-125: activating gamma-aminobutyric acid-producing GABA saccharomyces cerevisiae SC-125 in YPD liquid culture medium containing 5g/L L-sodium glutamate, culturing at 30 ℃ to logarithmic phase, wherein the concentration of bacterial liquid is 107CFU/mL。
(3) GABA is synthesized by fermenting Saccharomyces cerevisiae SC-125 which produces GABA: inoculating GABA-producing saccharomyces cerevisiae SC-125 into an MRS liquid culture medium containing 12g/L L-sodium glutamate according to the inoculation amount of 5%, and adding 9% (v/v) of lactobacillus plantarum M912 cell-free fermentation supernatant into the YPD liquid culture medium. Meanwhile, fermentation without adding cell-free fermentation supernatant in the fermentation medium is taken as a control. Fermenting at 30 ℃ for 144h respectively, and analyzing by HPLC detection to obtain the maximum GABA concentration of 7.68g/L and the GABA concentration of a control group of 1.60 g/L. Compared with a control group, the GABA fermentation synthesis yield of the experimental group is improved by 4.80 times.
Claims (4)
1. The GABA-producing saccharomyces cerevisiae is characterized in that the strain name is SC-125, and the classification name is as follows: saccharomyces cerevisiae has been deposited in China general microbiological culture Collection center (CGMCC) at 26.11.2020, with the preservation number of CGMCC 21236.
2. A Lactobacillus plantarum having symbiotic activity with the GABA producing Saccharomyces cerevisiae SC-125 according to claim 1, having the strain name M912 and classified name: lactobacillus plantarum has been deposited in China general microbiological culture Collection center (CGMCC) at 26.11.2020, with the preservation number of CGMCC 21237.
3. A method for improving the gamma-aminobutyric acid production of Saccharomyces cerevisiae, which is characterized in that the GABA-producing Saccharomyces cerevisiae of claim 1 is obtained by culturing and screening as a fermentation microorganism, and the GABA production is improved by exogenously adding the Lactobacillus plantarum M912 cell-free fermentation supernatant of claim 2.
4. The method for improving the production of gamma-aminobutyric acid by saccharomyces cerevisiae according to claim 3, comprising the following steps:
(1) preparation of lactobacillus plantarum M912 cell-free fermentation supernatant: activating lactobacillus plantarum M912 in an MRS liquid culture medium, and culturing for 10-12h to logarithmic growth phase at 30 ℃; 10000g, centrifuging at 4 ℃ for 10min, collecting supernatant, and filtering with a 0.22 mu M filter membrane to obtain lactobacillus plantarum M912 cell-free fermentation supernatant;
(2) preparing a GABA-producing saccharomyces cerevisiae fermentation seed: carrying out 18S rRNA identification on GABA-producing saccharomyces cerevisiae obtained by culture and screening, activating a yeast strain in a YPD culture medium containing 5 g/LL-sodium glutamate, and culturing at 30 ℃ for 12h to logarithmic phase to obtain GABA-producing saccharomyces cerevisiae fermentation seeds;
(3) and (3) fermenting and synthesizing GABA by using GABA-producing saccharomyces cerevisiae: inoculating GABA-producing saccharomyces cerevisiae SC-125 into YPD culture medium containing 6g/L-12g/L L-sodium glutamate according to the inoculation amount of v/v 5%, adding 8-9% v/v of lactobacillus plantarum M912 cell-free fermentation supernatant into the YPD culture medium, and fermenting for 120-144h at 30 ℃.
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Cited By (3)
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| 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 |
| CN114317628A (en) * | 2021-12-28 | 2022-04-12 | 华熙生物科技股份有限公司 | Low-irritation gamma-aminobutyric acid product, preparation method thereof and application of reducing irritation |
| CN115812882A (en) * | 2022-11-19 | 2023-03-21 | 西华大学 | Preparation method of kiwi fruit fermented beverage rich in protocatechuic acid and GABA |
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| CN115812882B (en) * | 2022-11-19 | 2023-12-12 | 西华大学 | Preparation method of kiwi fruit fermented beverage rich in protocatechuic acid and GABA |
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