CN117568201A - Lactobacillus rhamnosus YFA-012 with high tolerance and application thereof - Google Patents
Lactobacillus rhamnosus YFA-012 with high tolerance and application thereof Download PDFInfo
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- CN117568201A CN117568201A CN202311219150.7A CN202311219150A CN117568201A CN 117568201 A CN117568201 A CN 117568201A CN 202311219150 A CN202311219150 A CN 202311219150A CN 117568201 A CN117568201 A CN 117568201A
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- 241000218588 Lactobacillus rhamnosus Species 0.000 title claims abstract description 68
- 235000013305 food Nutrition 0.000 claims abstract description 11
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 230000000813 microbial effect Effects 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 230000036541 health Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 206010019133 Hangover Diseases 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 40
- 239000003833 bile salt Substances 0.000 abstract description 12
- 238000002703 mutagenesis Methods 0.000 abstract description 7
- 231100000350 mutagenesis Toxicity 0.000 abstract description 7
- 210000003608 fece Anatomy 0.000 abstract description 4
- 235000013402 health food Nutrition 0.000 abstract description 4
- 239000002253 acid Substances 0.000 abstract description 3
- 210000002784 stomach Anatomy 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 33
- 230000001580 bacterial effect Effects 0.000 description 26
- 230000012010 growth Effects 0.000 description 20
- 239000001963 growth medium Substances 0.000 description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 238000012258 culturing Methods 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- 229940093761 bile salts Drugs 0.000 description 7
- 239000006041 probiotic Substances 0.000 description 7
- 235000018291 probiotics Nutrition 0.000 description 7
- 210000004051 gastric juice Anatomy 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 5
- 230000000529 probiotic effect Effects 0.000 description 5
- 241000186660 Lactobacillus Species 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000006872 mrs medium Substances 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 4
- 239000011241 protective layer Substances 0.000 description 4
- 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 description 3
- 230000001476 alcoholic effect Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- PTKRHFQQMJPPJN-UHFFFAOYSA-N dipotassium;oxido-(oxido(dioxo)chromio)oxy-dioxochromium;sulfuric acid Chemical compound [K+].[K+].OS(O)(=O)=O.[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O PTKRHFQQMJPPJN-UHFFFAOYSA-N 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000005491 wire drawing Methods 0.000 description 2
- 208000030090 Acute Disease Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
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- 241000192125 Firmicutes Species 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108091093078 Pyrimidine dimer Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- ASJWEHCPLGMOJE-LJMGSBPFSA-N ac1l3rvh Chemical class N1C(=O)NC(=O)[C@@]2(C)[C@@]3(C)C(=O)NC(=O)N[C@H]3[C@H]21 ASJWEHCPLGMOJE-LJMGSBPFSA-N 0.000 description 1
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- 230000002075 anti-alcohol Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
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- 229940099352 cholate Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 239000000571 coke Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
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- 238000011534 incubation Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
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- 230000000877 morphologic effect Effects 0.000 description 1
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- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000013635 pyrimidine dimer Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
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- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004460 silage Substances 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
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- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 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 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
- A61P25/32—Alcohol-abuse
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
- C12R2001/225—Lactobacillus
Abstract
The invention discloses lactobacillus rhamnosus YFA-012 with high tolerance and application thereof, and belongs to the technical field of biology. The lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012 has a preservation number of CCTCC M20231457, a preservation date of 2023, 8 months and 14 days, and a preservation unit of China center for type culture Collection. The strain is derived from feces of healthy human bodies, belongs to safe strains capable of being used for foods, has higher tolerance to human stomach environments after ultraviolet mutagenesis, has excellent acid resistance, bile salt resistance, ethanol resistance and other characteristics, has better ethanol consumption capability, belongs to national standard edible allowed strains, can be added into various health foods and health foods, and has wide application prospects.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to lactobacillus rhamnosus YFA-012 with high tolerance and application thereof.
Background
Lactobacillus rhamnosus (Lactobacillus rhamnosus) is one of the lactic acid bacteria and produces bacteriocins that inhibit many gram-positive and some gram-negative bacteria. Lactobacillus rhamnosus has high adhesion rate to intestinal tracts, strong field planting capability, high-efficiency cholesterol reduction and cell division promotion capability, has antagonism to pathogenic microorganisms in animal bodies, can competitively inhibit the pathogenic microorganisms, and plays important physiological health care functions of regulating intestinal tracts flora, removing toxins, preventing decayed teeth, improving organism immunity, resisting cancers and the like. The lactobacillus rhamnosus has good stress resistance and antibacterial property, can be used as a candidate strain of probiotics, and can be widely applied to the fields of silage, functional food, medical care and the like.
Along with the continuous improvement of the living standard and the living quality of people, people pay more attention to the nutrition and health of foods, and lactobacillus related foods are continuously and vigorously developed in the market. However, in the actual food production process and in the process of playing a probiotic role in human body, the lactobacillus can face the change of various environmental factors, such as adverse factors of nutrition stress, high temperature stress, acid-base stress, preservative stress, disinfectant stress and the like, and the conditions can influence the normal metabolic function of the lactobacillus, so that the growth period of the lactobacillus is prolonged, and the field planting difficulty is increased.
With the development of economy, drinking alcohol becomes a part of life of many people, but alcohol can cause various chronic diseases such as acute diseases and alcoholic liver injury; the high tolerance strain currently on the market only measures tolerance in a short time, and it is uncertain whether it can survive smoothly in the gastrointestinal tract for a long time and grow in a large amount to colonize the intestinal tract.
In view of the above, the present invention has been made, and aims to obtain a probiotic strain which is not only highly resistant to the gastrointestinal environment but also continues to grow under adverse stress conditions, while being resistant to alcohol and decomposing alcohol-consuming. The application of probiotics in the field of dispelling effects of alcohol is relatively lacking, so that the strain has practical significance for producing probiotic health-care foods and medicines in the aspect of dispelling effects of alcohol.
Disclosure of Invention
In order to solve the technical problems, the lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012 screened by the invention has high tolerance to gastrointestinal tract environment, can continuously grow in the environment, has higher alcohol consumption capability, and has application value in the aspects of anti-alcoholic probiotic medicines, probiotic health-care foods and the like.
The invention provides lactobacillus rhamnosus with high tolerance, which is named lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012 and has a preservation number of CCTCC M20231457.
The invention carries out ultraviolet mutagenesis on lactobacillus rhamnosus separated from healthy human excrement, and bacterial colonies and thalli of the bacterial strains are obtained after screening, and the characteristics are as follows: culturing on MRS culture medium for 24 hr, and the colony is white, semitransparent, raised, clear in edge, moist, glossy and 0.1-0.3 mm in diameter, and has wiredrawing, observation of cell morphology under optical microscope, gram staining in G+ and rod shape. The strain can generate an extracellular polysaccharide protective layer, can grow under the conditions of pH 3.0 and 72 hours, can tolerate 0.3% of bile salts and can keep 56.2% of thalli alive after 48 hours, particularly has excellent performance in high-concentration alcohol tolerance experiments, not only improves the alcohol tolerance degree compared with the existing strain (can survive and reproduce after being subjected to 18% alcohol stress treatment and 48 hours), but also has higher consumption capability on alcohol. Specifically:
the strain is subjected to stress treatment for 72 hours under culture conditions with different gradients of pH 3, 3.5, 4, 5, 6 and 7, and the final growth ratio of the strain at different pH is 4.0%, 16.5%, 59.7%, 91.4%, 99.7% and 100% in sequence.
The strain was subjected to stress treatment in MRS medium containing no bile salts and 0.3% bile salts, and the ratio of the cells grown after 48 hours of culture was 100% and 56.2%.
The strain is artificially simulated in gastric juice for treatment, the coated plates are sampled at 0, 20, 40 and 60min respectively, and the survival rates of the lactobacillus rhamnosus under the condition of artificial gastric juice treatment are 100%, 73.6%, 47.7% and 18.3% respectively.
The strain is subjected to alcohol stress treatment without alcohol and with 5%, 8%, 10%, 12%, 15% and 18% (volume fraction), and can survive and reproduce, and after 48 hours of culture, the bacterial cell ratio of the growth is 100%, 96.1%, 87.7%, 36.8%, 14.2%, 9.9% and 8.7% in sequence compared with the growth under normal conditions.
Dividing the strain into different bacterial gradients, adding into MRS culture medium containing 10% alcohol, wherein the bacterial gradients are 8×10 respectively 8 、2×10 9 、5×10 9 、8×10 9 、1×10 10 The ratio of alcohol consumption at different times of the highest concentration is 3.2%, 17.1%, 23.1%, 25.9% and 27.1%, respectively.
The invention provides a culture or processed product thereof comprising the lactobacillus rhamnosus.
The invention provides a pharmaceutical composition containing lactobacillus rhamnosus, a culture or a processed product thereof.
The invention provides a food composition containing lactobacillus rhamnosus, a culture or a processed product thereof.
The invention provides application of lactobacillus rhamnosus, a culture or a processed product thereof in preparation of anti-hangover food or medicine.
The invention provides application of lactobacillus rhamnosus, a culture or a processed product thereof in preparation of health care of gastrointestinal tract.
A microbial agent comprising the lactobacillus rhamnosus as described above.
Further, the microbial agent is a liquid microbial agent.
Further, the microbial agent is a solid microbial agent.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012 of the invention is derived from feces of healthy human body, and belongs to a safe and accepted strain which can be used for food. The lactobacillus rhamnosus disclosed by the invention can generate an extracellular polysaccharide protective layer after ultraviolet mutagenesis, has high tolerance to the outside, particularly can resist the outside environment, can continue to grow in a stress environment, and has high stability. Meanwhile, the strain has the characteristics of excellent acid resistance, cholate resistance, ethanol resistance and the like, has better ethanol consumption capability, belongs to edible strains allowed by national standards, can be added into various health foods and health foods, and has wide application prospects.
Preservation of biological materials
Lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012, wherein lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012 is preserved in China center for type culture Collection (CCTCC M20231457) at a preservation number of China center for type culture Collection (China general microbiological culture Collection), and a preservation address of university of Wuhan.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a colony morphology of Lactobacillus rhamnosus of example 2 of the present invention;
FIG. 2 is a scanning electron microscope image of Lactobacillus rhamnosus according to example 2 of the present invention;
FIG. 3 is a graph showing the growth of Lactobacillus rhamnosus of example 3 under different pH gradient stress conditions;
FIG. 4 is a graph showing the growth of Lactobacillus rhamnosus of example 3 according to the present invention under 0.3% bile salt stress conditions;
FIG. 5 shows the concentration of the bacterial cells grown after lactobacillus rhamnosus of example 3 of the present invention was cultured for 48 hours at different alcohol concentrations;
FIG. 6 shows the amounts of alcohol consumed by Lactobacillus rhamnosus of example 3 of the present invention at 10% alcohol concentration for different periods of time.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The following examples relate to the following media:
MRS liquid medium: 10g of peptone, 5g of beef extract, 4g of yeast extract, 2g of tween-801 mL of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 2g of tri-ammonium citrate, 20g of glucose, 0.05g of manganese sulfate, 5g of sodium acetate, 1L of distilled water and pH 6.2+/-0.2, wherein the glucose is easy to coke during sterilization, so that the glucose is sterilized at 115 ℃ for 20min, then added according to concentration under aseptic conditions, and other components of the culture medium are weighed and sterilized at 121 ℃ for 20min for later use.
MRS solid medium: adding 1.5% -2% of agar powder into MRS liquid culture medium.
The formulation of the potassium dichromate sulfuric acid process solution referred to in the examples below is as follows:
potassium dichromate solution: 5g of potassium dichromate is weighed and dissolved in 50mL of water, 10mL of concentrated sulfuric acid is added, and the volume is fixed to 100mL after cooling.
Ethanol standard solution: 2g of analytically pure absolute ethanol is weighed into a 100mL volumetric flask and the volume is fixed to 100mL.
Example 1 isolation and identification of lactic acid bacteria
The following experiments were all run under sterile conditions.
1. Enrichment of strains
The strain is derived from feces of healthy human bodies, 25g of feces sample is taken, 225mL of sterile physiological saline is added for uniform mixing, the obtained bacterial liquid is sequentially subjected to 10-time gradient dilution, 50 mu L of each gradient dilution is coated on an MRS solid flat plate, and the bacterial liquid is placed in a constant temperature incubator at 37 ℃ for inverted sealing culture for 48 hours, and colony growth conditions are observed.
2. Isolation of strains
And (3) picking the bacterial colony obtained in the step (1), carrying out microscopic examination by a gram staining method, observing the morphological characteristics of the bacterial colony, and selecting different bacterial colony morphologies and gram positive bacteria as target bacterial strains.
3. Purification of strains
And (3) picking the bacterial colony obtained in the step (2), scribing on an MRS flat plate, repeatedly purifying until other mixed bacteria are absent, recording the states of bacterial colonies of different strains, and selecting to gently pick up the bacterial colony which is in a wiredrawing shape and is sticky by using a toothpick.
4. Identification of strains
After the purified strain in the step 3 is inoculated into a small bottle for culturing for 24 hours, genome is extracted by using a genome extraction kit to carry out 16SrDNA sequencing, and sequence homology is compared and analyzed through a BLAST retrieval system of NCBI, and the comparison result with the highest similarity is shown as lactobacillus rhamnosus, and the homology is 98.14%.
The 16SrDNA sequence of Lactobacillus rhamnosus YFA-012 (Lactobacillus rhamnosus) of the present invention is shown below (SEQ ID NO. 1):
ACTTAAGTTGTCACCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGGCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGGCACGTATTTAGCCGTGCTTTCTGGTTGGATACCGTCACGCCGACCACAGTTACTCTGCCGACCATTCTTCTCCAACAGCAGAGTTTTACGAACCCGAAAAGCCTTCTTCACTTGCAGACGAGGTTGCTCCATCAGAACTTGCGTTCATATGGTGAGAATTTCCCTGACTTGGCTTGGCCCTCCCGATAAGG
EXAMPLE 2 acquisition of highly tolerant Lactobacillus rhamnosus Strain
1. Acquisition of bacterial suspension
The lactobacillus rhamnosus strain obtained in step 4 of example 1 was picked up as a single colony from a plate, inoculated with an MRS vial, and subjected to stationary culture in an incubator at 37℃for 24 hours for activation, and inoculated with an MRS flask at 37℃for 24 hours in an inoculum size of 2%. 1mL of the fermentation broth was centrifuged at 8000rpm for 5min to collect the cells, which were washed three times with 0.9% physiological saline, resuspended in 1mL of 0.9% physiological saline, and transferred to a sterile petri dish.
2. Ultraviolet mutagenesis
Ultraviolet mutagenesis is performed at 254nm (this is the peak of absorption of nucleic acid), and after DNA absorbs ultraviolet light, DNA molecules form pyrimidine dimers and cause distortion of double-stranded structures, which hinder normal pairing between bases, and cause cell mutation or death. At the same time, dimer formation may interfere with double strand break-off, thereby affecting DNA replication and transcription. In many cases, such changes result in the death of cells in a short period of time due to the inability of the cells to reproduce and metabolize normally, but a small number of surviving cells contain mutations in genes and have a certain viability, and useful variants are selectively selected for expansion culture to give the desired strain.
The dish cover of the bacterial suspension obtained in the step 1 is opened under the aseptic condition, and ultraviolet (30W, 30 cm) is respectively irradiated for 5, 10, 15 and 20 seconds. Centrifuging the bacterial solution subjected to ultraviolet mutagenesis at 8000rpm for 5min to remove physiological saline, adding 800 mu L of MRS liquid culture medium, and placing in a 37 ℃ incubator for resuscitation for 4-6 h, wherein the process is operated under red light.
3. Obtaining highly tolerant strains
Diluting the resuscitated bacterial liquid by a certain multiple according to different mutagenesis time, respectively coating 50 μl of the resuscitated bacterial liquid on a substrate containing 5% alcohol by volume, pH 5.0, 0.3% bile salt, and 1% CaCO 3 After 48h incubation at 37℃the colonies were observed and selected for smoother and more transparent colonies on different selection media, with a transparent ring around them, and the more stringy and viscous colonies were gently picked up using a toothpick (see FIG. 1). The strain was cryopreserved in 25% glycerol tubes to a-80 ℃ refrigerator.
The strain is sent to a scanning electron microscope for observation, and the outside of the strain is found to have more extracellular secretion, and is detected to be an extracellular polysaccharide protective layer (shown in figure 2), the colony is semitransparent and filiform, and the polysaccharide protective layer is the reason that the strain has high tolerance. The strain was sent to storage.
EXAMPLE 3 high tolerance assay of Lactobacillus rhamnosus
1. Determination of acid resistance
Lactobacillus rhamnosus preserved in example 2 is taken from frozen glycerol tubes, streaked and activated on an MRS culture medium, placed in a 37 ℃ incubator for culturing for 24 hours, single colonies of the cultured strain are picked up and inoculated into the MRS liquid culture medium, the strain is cultured at 37 ℃ until the end of logarithmic growth, and 2% of the inoculum size is added into the liquid MRS culture medium with the pH of 3, 3.5, 4, 5, 6 and 7 for culturing in the 37 ℃ incubator. OD determination after 72h of stress 600 The growth conditions were compared (FIG. 3). The final growth ratio at different pH was 4.0%, 16.5%, 59.7%, 91.4%, 99.7%, 100% in this order compared to growth under normal conditions.
TABLE 1 growth of Lactobacillus rhamnosus under different pH conditions for 72h
2. Determination of bile salt resistance
The human stomach environment contains about 0.3% bile salts, so that the strain is cultivated in MRS medium containing 0.3% bile salts, lactobacillus rhamnosus strain cultivated to the end of logarithmic growth is inoculated into MRS liquid medium containing 0.3% bile salts with an inoculum size of 2%, and after cultivation for 3 hours, 5 hours, 8 hours, 12 hours, 24 hours and 48 hours, the ratio of grown bacteria after 48 hours is 100% and 56.2% compared with that of the strain grown in MRS medium without bile salts (FIG. 4).
TABLE 2 growth of different time control and 0.3% bile salt addition experimental groups
3. Tolerance determination to simulated gastric fluid
Preparing 0.9% physiological saline, adjusting pH to 3 with hydrochloric acid, sterilizing at 121deg.C for 20min, and adding 3g/L pepsin under aseptic condition to obtain artificial simulated gastric juice.
Taking lactobacillus rhamnosus well stored in the embodiment 2 from a frozen glycerol pipe, streaking and activating the lactobacillus rhamnosus on an MRS culture medium, placing the lactobacillus rhamnosus on a 37 ℃ incubator for culturing for 24 hours, picking single bacterial colonies of the cultured strain, inoculating the single bacterial colonies into the MRS liquid culture medium, culturing for 24 hours at 37 ℃, taking 1mL of lactobacillus rhamnosus culture solution, centrifuging at 8000rpm for 2min to collect thalli, adding the thalli into 5mL of simulated gastric juice, shaking uniformly, placing the lactobacillus rhamnosus into the 37 ℃ incubator for culturing, sampling at 0min, 20min, 40min and 60min respectively, coating the sample on the MRS solid culture medium after proper dilution, culturing for 24 hours, counting the viable count of the flat bacterial colonies, and calculating the survival rate of the viable count. The colony growth results are shown in Table 3, and the survival rates of the lactobacillus rhamnosus are respectively 100%, 73.6%, 47.7% and 18.3% in 0min, 20min, 40min and 60min under the artificial gastric juice treatment condition by taking the colony number growing at 0min as 100%. The result shows that lactobacillus rhamnosus has better tolerance to artificial simulated gastric juice.
TABLE 3 Lactobacillus rhamnosus growth conditions in artificially simulated gastrointestinal fluids for different time periods
4. Determination of alcohol stress tolerance
Strains at the end of log growth were selected, and alcoholic stress was performed in an incubator at 37 ℃ for 48h with 2% inoculum size added to MRS liquid medium with different levels (5%, 8%, 10%, 12%, 15%, 18%) of alcohol, while the non-alcoholic MRS medium culture group was used as a negative control. Determination of OD after stress 600 The growth conditions were compared.
The pure bacteria obtained in example 2 were subjected to alcohol treatments of different concentrations, and the tolerance of lactobacillus rhamnosus tended to decrease as the alcohol concentration increased, and the bacteria were still viable and propagated by alcohol stress treatments of no alcohol, 5%, 8%, 10%, 12%, 15%, 18% (volume fraction), and after 48 hours of cultivation, the bacterial cell ratios of growth were 100%, 96.1%, 87.7%, 36.8%, 14.2%, 9.9%, 8.7% in this order. Belongs to a strain having a very high alcohol stress resistance as compared with the tolerance of 10% in most documents (as shown in FIG. 5).
TABLE 4 growth of Lactobacillus rhamnosus under different alcohol concentrations for 48h
5. Determination of alcohol consumption Capacity
Lactobacillus rhamnosus well preserved in example 2 is taken from a frozen glycerol pipe, streaked and activated on an MRS culture medium, placed in a 37 ℃ incubator for culturing for 24 hours, the cultured strain is selected to inoculate single colony into the MRS liquid culture medium, the culture is carried out for 24 hours at 37 ℃, the OD value is measured, the colony CFU number is obtained according to the conversion of the OD value, the culture solutions of lactobacillus rhamnosus with different gradients are taken, centrifuged for 15 minutes at 8000rpm under the aseptic condition, washed three times with sterile 0.9% normal saline, bacterial sludge is added into the MRS liquid culture medium containing 10% alcohol by volume fraction, sampling is carried out at the moments of 0 hour, 0.5 hour, 1 hour, 2 hours and 3 hours respectively, and the alcohol content is measured by adopting a potassium dichromate sulfuric acid method (as shown in table 5, the smaller value represents more consumed alcohol), and the highest bacterial concentration is respectively 3.2%, 17.1%, 23.1%, 25.9% and 27.1% (as shown in figure 6).
TABLE 5 determination of absorbance values of reaction solution by Potassium dichromate sulfuric acid method after Lactobacillus rhamnosus was cultured at 10% alcohol concentration for various times
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. Lactobacillus rhamnosus with high tolerance, characterized in that: the lactobacillus rhamnosus (Lactobacillus rhamnosus) YFA-012 with a preservation number of CCTCC M20231457.
2. A culture or processed product thereof comprising the lactobacillus rhamnosus of claim 1.
3. A pharmaceutical composition characterized by: comprising the lactobacillus rhamnosus of claim 1 or the culture of claim 2 or a processed product thereof.
4. A food composition characterized by: comprising the lactobacillus rhamnosus of claim 1 or the culture of claim 2 or a processed product thereof.
5. Use of lactobacillus rhamnosus according to claim 1, a culture according to claim 2 or a processed product thereof for the preparation of an anti-hangover food.
6. Use of lactobacillus rhamnosus as claimed in claim 1, a culture as claimed in claim 2 or a processed product thereof for the manufacture of a medicament for alleviating hangover.
7. Use of lactobacillus rhamnosus according to claim 1, a culture according to claim 2 or a processed product thereof for the preparation of a product for maintaining the health of the gastrointestinal tract.
8. A microbial agent is characterized in that: comprising the lactobacillus rhamnosus of claim 1.
9. The microbial agent of claim 8, wherein: the microbial agent is a liquid microbial agent.
10. The microbial agent of claim 8, wherein: the microbial agent is a solid microbial agent.
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