CN116496969A - Method for improving lactic acid tolerance by exogenously adding arginine - Google Patents
Method for improving lactic acid tolerance by exogenously adding arginine Download PDFInfo
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- CN116496969A CN116496969A CN202310654272.2A CN202310654272A CN116496969A CN 116496969 A CN116496969 A CN 116496969A CN 202310654272 A CN202310654272 A CN 202310654272A CN 116496969 A CN116496969 A CN 116496969A
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- lactic acid
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- acid bacteria
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 239000004310 lactic acid Substances 0.000 title claims abstract description 41
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 41
- 239000004475 Arginine Substances 0.000 title claims abstract description 28
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 15
- 241000894006 Bacteria Species 0.000 claims abstract description 32
- 230000006518 acidic stress Effects 0.000 claims abstract description 21
- 230000003834 intracellular effect Effects 0.000 claims abstract description 21
- 241001537924 Tetracoccus <angiosperm> Species 0.000 claims abstract description 14
- 108010082340 Arginine deiminase Proteins 0.000 claims abstract description 8
- 239000002028 Biomass Substances 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims abstract description 7
- 230000037361 pathway Effects 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 claims description 3
- 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 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 229940087562 sodium acetate trihydrate Drugs 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 3
- 239000001393 triammonium citrate Substances 0.000 claims description 3
- 235000011046 triammonium citrate Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims 3
- 230000003213 activating effect Effects 0.000 claims 1
- 239000006872 mrs medium Substances 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 9
- 230000004151 fermentation Effects 0.000 abstract description 9
- 230000035882 stress Effects 0.000 abstract description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 abstract description 5
- 241000186660 Lactobacillus Species 0.000 abstract description 4
- 229940039696 lactobacillus Drugs 0.000 abstract description 4
- 238000013518 transcription Methods 0.000 abstract description 4
- 230000035897 transcription Effects 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- -1 ammonia ions Chemical class 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 235000013555 soy sauce Nutrition 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 241000500332 Tetragenococcus halophilus Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 235000016127 added sugars Nutrition 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Organic Chemistry (AREA)
- Biotechnology (AREA)
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- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
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- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for improving lactic acid bacteria acid tolerance by exogenously adding arginine, and relates to the technical field of microorganisms. The method for improving the acid tolerance of the lactic acid bacteria by using the tetracoccus halophilus as a research object specifically aims at improving the biomass of the lactic acid bacteria under acid stress and discovers that the exogenous arginine can better maintain the stability of the intracellular microenvironment of the lactic acid bacteria when further analyzing a response mechanism, and specifically shows higher intracellular pH value, higher ammonia ion concentration and enhancement of an arginine deiminase pathway on the transcription level. The invention provides theoretical basis and basis for improving fermentation performance of lactobacillus under stress state.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for improving lactic acid bacteria acid tolerance by exogenously adding arginine.
Background
The invention relates to tetracoccus halophilusTetragenococcus halophilusCGMCC 3792) is screened and separated in the laboratory, and is identified as tetracoccus in the family of lactic acid bacteria by 16s rDNA sequencing. Due to its excellent characteristics of shortening fermentation period and improving fermentation qualityIs widely applied to fermentation of high-salt traditional foods, such as soy sauce, fish paste, bean products and the like. The research shows that the addition of tetracoccus halophilus can obviously improve the content of flavor substances such as organic acid, aldehydes, esters and the like in the food in the production process of various foods, and has higher commercial value.
However, lactic acid bacteria are subjected to a number of stress problems during production, such as acid stress, salt stress, etc. However, acid stress is the most widespread problem for lactic acid bacteria, because lactic acid bacteria metabolize fermentable sugars to lactic acid by fermentation in the same row, thereby creating an acid stress environment. Therefore, the fermentation and production performances of the lactic acid bacteria in the acid stress environment are improved, and the method has high value significance.
Exogenous addition is a hot topic used in recent years to enhance the growth performance of microorganisms in a stress environment. Among them, it has been confirmed and reported by scholars that exogenously added sugar substances, protective compatible substances can effectively relieve the pressure of lactic acid bacteria in a stress environment, but a method for improving the acid stress tolerance of lactic acid bacteria by exogenously adding arginine has not been conducted. Therefore, the invention hopes to improve the growth performance of the tetracoccus halophilus in the acid stress environment by adding exogenous arginine, and improves the theoretical basis for improving the robustness of the lactic acid bacteria in industry. In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for improving the acid tolerance of lactic acid bacteria by adding arginine exogenously in order to improve the fermentation capacity and commercial value of lactic acid bacteria, which can improve the growth performance of tetracoccus halophilus in an acid stress environment.
The pH of the acid stress environment is 5.2, the lactic acid is regulated, the fermentation medium is MRS (Oxoid), and the concentration of the exogenous arginine is 200mM.
The MRS agar plate comprises: 10 g/L peptone, 4 g/L yeast extract, 5 g/L sodium acetate trihydrate, 60g/L sodium chloride, 8g/L beef extract, 20 g/L glucose, 1ml Tween 80, 2g/L dipotassium hydrogen phosphate, 2g/L tri-ammonium citrate, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganese sulfate tetrahydrate, 20 g/L agar, and pH 5.2.
The invention examines the growth performance of tetracoccus halophilus, expressed by biomass, with and without arginine under acid stress conditions.
The invention examines the change of intracellular pH of tetracoccus halophilus with and without arginine under acid stress conditions.
The invention examines the change of the intracellular ammonia ion concentration of tetracoccus halophilus with and without arginine under the condition of acid stress.
The invention examines the change of the intracellular arginine deiminase pathway of tetracoccus halophilus on the transcriptional level under the condition of acid stress with and without arginine.
The beneficial effects of the invention include as follows: the exogenous addition of 200mM arginine can effectively improve the growth performance of tetracoccus halophilus under acid stress, and simultaneously, exogenous arginine can be transported into lactic acid bacteria cells to activate the intracellular arginine deiminase pathway, so that more ammonia ions are grown, more hydrogen ions are consumed, and the increase of the intracellular pH of the lactic acid bacteria and the accumulation of the ammonia ions in the bags are caused. This suggests that the addition of arginine by external assistance is a reliable and effective strategy that can be used to boost the stress resistance of lactic acid bacteria in stress conditions.
Drawings
In order to more clearly illustrate the implementation of the examples of the present invention, the drawings used in the examples will be briefly described below. It is specifically noted that the following drawings illustrate only some examples of the invention and therefore are not to be considered limiting of its scope, for those of ordinary skill in the art may derive other relevant drawings from these drawings without undue burden.
FIG. 1 is biomass at pH5.2 for example 1 and for blank 1; FIG. 2 is the results of intracellular pH of example 1 and comparative example 1; FIG. 3 is the results of intracellular ammonia ion concentrations of example 1 and comparative example 1; FIG. 4 shows the results of transcription levels of the intracellular arginine deiminase pathway of example 1 and comparative example 1.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the present invention examples will be clearly and completely described as follows. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The features and capabilities of the present invention are described in further detail below in conjunction with examples.
Example 1: the method of the high-activity lactobacillus preparation provided in the example 1 is as follows: collecting tetracoccus halophilus preserved in glycerol storage solution at-80deg.CTetragenococcus halophilus) CGMCC No. 3792, thawing, inoculating in seed culture medium at 5% (v/v), standing at 30deg.C for 24 hr to mid-log phase, and culturing twice to obtain seed culture medium with stable growth.
Tetracoccus halophilus (Tetragenococcus halophilus) CGMCC3792, separated from soy sauce mash in laboratory, and subjected to morphological, physiological and biochemical property test and 16S rDNA sequencing identification, and preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC) at 29 th month 4 of 2010.
Seed solutions of the two amplifications were collected and inoculated into stress medium with or without 200mM arginine, pH5.2, lactate adjustment. The growth performance was then measured.
Wherein, the culture medium comprises the following components: 10 g/L peptone, 4 g/L yeast extract, 5 g/L sodium acetate trihydrate, 8g/L beef extract, 60g/L sodium chloride, 20 g/L glucose, 1ml Tween 80, 2g/L dipotassium hydrogen phosphate, 2g/L tri-ammonium citrate, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganese sulfate tetrahydrate, and pH 5.2; the concentration of exogenously added arginine groups was 200mM.
And (3) collecting thalli from the fermentation liquor treated by arginine at 8000 r/min and 4 ℃ for 5 min.
The biomass was measured every 6 hours until the lactic acid bacteria grew to stationary phase, as shown in fig. 1, and the biomass of lactic acid bacteria was significantly higher in the exogenously added arginine group than in the control group (blank), indicating that exogenously added arginine helps to improve the growth performance of lactic acid bacteria under acid stress.
The mid-log cells were collected and then the intracellular pH was measured, as shown in fig. 2, and the results were shown in the exogenously added arginine group, where the intracellular pH of the lactic acid bacteria was significantly higher than that of the control group, indicating that the exogenously added lactic acid bacteria helped to maintain the intracellular microenvironment stable under acid stress.
The mid-log cells were collected and then the intracellular ammonia ion concentration was measured, and the results are shown in fig. 3, in the exogenously added arginine group, the intracellular ammonia ion concentration of lactic acid bacteria was significantly higher than that of the control group, indicating that the exogenously added lactic acid bacteria helps to maintain the intracellular microenvironment stable under acid stress.
After collecting cells in mid-log phase, the expression of the intracellular arginine deiminase pathway at the transcription level is measured, and the result is shown in fig. 4, wherein the transcription level of the gene related to arginine deiminase is obviously up-regulated, which shows that the addition of arginine helps to activate the intracellular arginine deiminase pathway of lactobacillus so as to maintain the intracellular microenvironment stability of the lactobacillus under acid stress.
Blank examples: the blank example of the invention is that no arginine group is added under acid stress. The rest of the operations, the experimental group and the blank group are not different in operation.
Claims (6)
1. A method for improving the acid tolerance of lactic acid bacteria by exogenously adding arginine is characterized in that the lactic acid bacteria is tetracoccus halophilus CGMCC3792.
2. The method for exogenously adding arginine to improve lactic acid tolerance according to claim 1, characterized in that the acid stress environment is ph5.2, lactic acid is regulated, and the environment is in MRS (Oxoid) medium, the concentration of exogenously added arginine is 200mM.
3. The method for exogenously adding arginine to improve lactic acid tolerance according to claim 2, characterized in that the medium is an MRS medium comprising: 10 g/L peptone, 4 g/L yeast extract, 5 g/L sodium acetate trihydrate, 8g/L beef extract, 20 g/L glucose, 1ml Tween 80, 2g/L dipotassium hydrogen phosphate, 2g/L tri-ammonium citrate, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganese sulfate tetrahydrate, 60g/L sodium chloride, pH5.2, adjusted with lactic acid.
4. The method for improving lactic acid tolerance by exogenously adding arginine according to claim 3, wherein the treated lactic acid bacteria are collected by centrifugation for intracellular microenvironment assessment and biomass analysis.
5. The method for exogenously adding arginine to improve lactic acid bacteria acid tolerance according to claim 4, characterized in that the centrifugation conditions are as follows: rotational speed: 8000 r/min, temperature: 3-5 ℃, centrifugation time: 5 min.
6. The method for exogenously adding arginine to increase acid tolerance of lactic acid bacteria according to claims 1-5, wherein the exogenously adding arginine is effective in activating the arginine deiminase pathway within the cells of lactic acid bacteria, thereby regulating and maintaining the stability of the intracellular microenvironment and increasing the biomass of lactic acid bacteria under acid stress.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948316A (en) * | 2022-12-13 | 2023-04-11 | 四川大学 | Method for improving acid resistance of lactic acid bacteria |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948316A (en) * | 2022-12-13 | 2023-04-11 | 四川大学 | Method for improving acid resistance of lactic acid bacteria |
| CN115948316B (en) * | 2022-12-13 | 2024-03-22 | 四川大学 | Method for improving acid resistance of lactic acid bacteria |
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