CN111944721A - Lactobacillus rhamnosus with immunoregulation effect and application thereof - Google Patents
Lactobacillus rhamnosus with immunoregulation effect and application thereof Download PDFInfo
- Publication number
- CN111944721A CN111944721A CN202010834655.4A CN202010834655A CN111944721A CN 111944721 A CN111944721 A CN 111944721A CN 202010834655 A CN202010834655 A CN 202010834655A CN 111944721 A CN111944721 A CN 111944721A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus rhamnosus
- lactobacillus
- rhamnosus
- strain
- immunoregulation
- 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.)
- Pending
Links
- 241000218588 Lactobacillus rhamnosus Species 0.000 title claims abstract description 101
- 230000000694 effects Effects 0.000 title claims abstract description 39
- 230000007365 immunoregulation Effects 0.000 title claims abstract description 25
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims abstract description 12
- 235000013618 yogurt Nutrition 0.000 claims abstract description 6
- 235000013311 vegetables Nutrition 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 3
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 claims description 2
- 230000002519 immonomodulatory effect Effects 0.000 claims 2
- 241000186660 Lactobacillus Species 0.000 abstract description 19
- 210000002540 macrophage Anatomy 0.000 abstract description 19
- 229940039696 lactobacillus Drugs 0.000 abstract description 17
- 210000004698 lymphocyte Anatomy 0.000 abstract description 16
- 210000000952 spleen Anatomy 0.000 abstract description 16
- 230000035755 proliferation Effects 0.000 abstract description 13
- 210000004027 cell Anatomy 0.000 abstract description 12
- 239000013641 positive control Substances 0.000 abstract description 10
- 230000001105 regulatory effect Effects 0.000 abstract description 8
- 230000009466 transformation Effects 0.000 abstract description 7
- 241000917009 Lactobacillus rhamnosus GG Species 0.000 abstract description 6
- 229940059406 lactobacillus rhamnosus gg Drugs 0.000 abstract description 6
- 230000000968 intestinal effect Effects 0.000 abstract description 5
- 239000003833 bile salt Substances 0.000 abstract description 4
- 230000002596 correlated effect Effects 0.000 abstract description 3
- 230000003187 abdominal effect Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 230000003393 splenic effect Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 14
- 108010062877 Bacteriocins Proteins 0.000 description 12
- 240000006024 Lactobacillus plantarum Species 0.000 description 12
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 12
- 229940072205 lactobacillus plantarum Drugs 0.000 description 12
- 229920001817 Agar Polymers 0.000 description 11
- 210000000683 abdominal cavity Anatomy 0.000 description 11
- 239000008272 agar Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000006870 function Effects 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 230000000242 pagocytic effect Effects 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 239000006041 probiotic Substances 0.000 description 7
- 235000018291 probiotics Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000009631 Broth culture Methods 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 230000003213 activating effect Effects 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 206010061598 Immunodeficiency Diseases 0.000 description 4
- 208000029462 Immunodeficiency disease Diseases 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 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 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 229960004397 cyclophosphamide Drugs 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 230000007813 immunodeficiency Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 210000000822 natural killer cell Anatomy 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 208000006313 Delayed Hypersensitivity Diseases 0.000 description 2
- 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 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-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
- 241001052560 Thallis Species 0.000 description 2
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000000091 immunopotentiator Effects 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229960001614 levamisole Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 235000021110 pickles Nutrition 0.000 description 2
- 230000000529 probiotic effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 230000005951 type IV hypersensitivity Effects 0.000 description 2
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- YLDCUKJMEKGGFI-QCSRICIXSA-N 4-acetamidobenzoic acid;9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one;1-(dimethylamino)propan-2-ol Chemical compound CC(O)CN(C)C.CC(O)CN(C)C.CC(O)CN(C)C.CC(=O)NC1=CC=C(C(O)=O)C=C1.CC(=O)NC1=CC=C(C(O)=O)C=C1.CC(=O)NC1=CC=C(C(O)=O)C=C1.O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC=NC2=O)=C2N=C1 YLDCUKJMEKGGFI-QCSRICIXSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000168254 Siro Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000002295 alkylating antineoplastic agent Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000010855 food raising agent Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000020604 functional milk Nutrition 0.000 description 1
- 210000003736 gastrointestinal content Anatomy 0.000 description 1
- 230000007236 host immunity Effects 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000008944 intestinal immunity Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002417 nutraceutical Substances 0.000 description 1
- 235000021436 nutraceutical agent Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
Images
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
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- 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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
-
- 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
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Lactobacillus rhamnosus (L.rhamnosus)1.0320 strain with immunoregulation effect and application thereof are applied to fermentation process of yoghourt or pickled vegetables or used for preparing freeze-dried bacterial powder. The lactobacillus rhamnosus provided by the invention has high intestinal wall adhesion rate (more than 11.76%), low pH resistance (pH is lower than 3) and bile salt resistance (the colony number is 10 after the lactobacillus rhamnosus is incubated in 0.3% high-concentration bile salt for 4 hours7CFU/mL). Transformation value of Lactobacillus rhamnosus 1.0320 (Lactobacillus: cells: 1) on splenic lymphocytes and abdominal cavityThe macrophage secretes NO with strong regulating ability, and the immunoregulation ability is obviously higher than that of a reference strain Lactobacillus rhamnosus GG (p is less than 0.05). The immunoregulation strength and concentration of lactobacillus rhamnosus 1.0320 are positively correlated, and when the ratio of lactobacillus rhamnosus 1.0320 to cells is 100: when 1 hour, the conversion value of the spleen lymphocyte is 7.5 times of that of the positive control group, and the proliferation index of the spleen lymphocyte reaches more than 1.
Description
Technical Field
The invention belongs to the technical field of lactobacillus, and particularly relates to lactobacillus rhamnosus with an immunoregulation effect and an application thereof.
Background
Cyclophosphamide (CTX) is one of the most commonly used alkylating antineoplastic drugs, and like most chemotherapy drugs, it also has an inhibitory effect on the immune system of a patient while destroying tumor cells, which is a problem in the application of the Cyclophosphamide in tumor chemotherapy. Immunosuppression can lead to increased incidence of hypersensitivity diseases, autoimmune diseases, infectious diseases, and tumor formation. The prior commonly used immunopotentiator in clinic comprises chemical synthesis medicines of levamisole, isoprinosine and the like, but the immunopotentiator has great harm to liver and kidney functions, and can cause adverse reactions such as nausea, vomiting, abdominal pain and the like after being taken by part of people. Therefore, it is crucial to find safe and stable natural immunomodulators.
The lactobacillus is one of the well-known food safety probiotics, and has the effects of resisting infection, regulating immunity, resisting oxidation, regulating intestinal microecology and the like. Many clinical and animal experiments have confirmed that lactic acid bacteria can regulate innate and acquired immune responses in a host, and oral administration of probiotics has a positive effect on the composition of intestinal flora and colonization resistance to pathogenic bacteria, and can produce appropriate immune regulation responses. Lactobacilli are important probiotics that are present in the gut in a fixed manner and can elicit specific and non-specific immune responses in the host by binding to pattern recognition receptors expressed by immune cells and many other tissues in the gut, including the intestinal epithelium. It was found that the expression of anti-inflammatory cytokines (e.g., IL-10) and immunoglobulin M (IgM) was induced by Lactobacillus. Meanwhile, lactobacillus can enhance macrophage phagocytic activity and stimulate secretion of lysosomal enzyme, and improve host immunity by up-regulating macrophage activity and promoting proliferation and differentiation of T cells. The metabolite bacteriocin of lactobacillus can kill pathogenic bacteria in intestinal tract to improve intestinal immunity. Lactobacilli and their metabolites have also been proposed as delivery vehicles for antigens in place of inactivated vaccines and in the production of nutraceuticals. At present, the number of probiotics which can be added into food according with the regulations of national ministry of health is quite limited, and the actual immune efficacy of different strains in the same genus is obviously different, and no clear rule can be followed.
Disclosure of Invention
The invention aims to solve the problems that probiotics which can be added into food at present are limited and the like, provides a probiotic functional lactobacillus rhamnosus with strong immunoregulation effect and application thereof, and defines the regulation effect and the optimal dosage of lactobacillus rhamnosus 1.0320 on immunodeficiency, provides theoretical support for developing safe and efficient immunoregulation biological agents, and has important significance for enriching national strain libraries.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the lactobacillus rhamnosus strain is a strain 1.0320 of lactobacillus rhamnosus (L.rhamnosus) with the immunoregulation function, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15557, the preservation address is No. 3 of No.1 Siro of Beijing Ing-oriented region, the institute of microbiology of China academy of sciences, and the preservation date is No. 08 of 2018, 04 months.
An application of the lactobacillus rhamnosus with the immunoregulation function is to apply the lactobacillus rhamnosus in the fermentation process of yoghourt or pickled vegetables.
An application of the lactobacillus rhamnosus with the immunoregulation function is that the lactobacillus rhamnosus is used for preparing freeze-dried bacterium powder.
Compared with the prior art, the invention has the beneficial effects that:
the lactobacillus rhamnosus of the invention has the capability of producing bacteriocin, high intestinal wall adhesion rate (more than 11.76 percent), low pH resistance (pH is lower than 3), and bile salt resistance (the colony count is 10 after the lactobacillus rhamnosus is incubated in 0.3 percent of high-concentration bile salt for 4 hours7CFU/mL)。
Compared with the immunoregulation effect of the rest 7 strains of lactobacillus, the lactobacillus rhamnosus provided by the invention has better regulation effects on spleen lymphocyte transformation, spleen lymphocyte proliferation, abdominal cavity macrophage phagocytosis and NO secretion of abdominal cavity macrophages, and the immunoregulation ability is obviously higher than that of a reference strain lactobacillus rhamnosus GG and has obvious difference. When the addition amount and the cell ratio are 100:1, the effect is optimal, the spleen lymphocyte transformation value is 7.5 times of that of a positive control group, the proliferation index of the spleen lymphocytes reaches more than 1, the phagocytic capacity of macrophages is not obviously different from that of the positive control group, and an unpredictable technical effect is obtained.
After the lactobacillus rhamnosus is cultured under optimized conditions (ultraviolet induction, pH, nitrogen source and NaCl addition), the diameter of an inhibition zone is increased from (26.24 +/-0.31) mm to (36.68 +/-0.51) mm, and is increased by 39.79%.
The lactobacillus rhamnosus provided by the invention is applied as a biological preparation with an immunoregulation function, and has the capabilities of recovering the body weight, the T lymphocyte proliferation capability, the abdominal cavity macrophage phagocytosis capability, the NK cell activity, the carbon particle clearance capability, the delayed hypersensitivity and the level of a cell inflammatory factor of an immunodeficient mouse, and the optimal dosage is as follows: (109CFU/mL)/0.02kg/d。
Drawings
FIG. 1 is a graph showing the effect of eight strains of Lactobacillus on the spleen lymphocyte transformation values of mice;
FIG. 2 is a graph showing the effect of eight strains of Lactobacillus on the mouse spleen lymphocyte Proliferation Index (PI);
FIG. 3 is a graph showing the effect of eight strains of Lactobacillus on the ability of macrophages in the abdominal cavity of a mouse to phagocytose neutral red;
FIG. 4 is a graph showing the effect of eight strains of Lactobacillus on the NO content secreted by macrophages in the abdominal cavity of a mouse;
FIG. 5 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on mouse body weight;
FIG. 6 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on mouse spleen T lymphocyte proliferation;
FIG. 7 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on the phagocytic capacity of mouse macrophages;
FIG. 8 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on NK cell activity in mice;
FIG. 9 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on carbon clearance index in mice;
FIG. 10 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on delayed type hypersensitivity in mice;
FIG. 11 is a graph showing the effect of Lactobacillus rhamnosus 1.0320 on the IL-12/IL-10 ratio in mouse serum;
FIG. 12 is a graph of the effect of Lactobacillus rhamnosus 1.0320 on IgG in mouse serum;
FIG. 13 is a colony morphology of Lactobacillus rhamnosus 1.0320;
FIG. 14 is a cell morphology of Lactobacillus rhamnosus 1.0320.
Detailed Description
The technical solutions of the present invention are further described below with reference to the drawings and the embodiments, but the present invention is not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
The probiotic lactobacillus rhamnosus (L.rhamnosus)1.0320 strain with high bacteriocin yield is a gram-positive bacterium separated from a traditional marmilk wine sample. Firstly, a lactobacillus rhamnosus 1.0320 with strongest regulating effects on spleen lymphocyte transformation, spleen lymphocyte proliferation, abdominal cavity macrophage phagocytosis and NO content secretion of abdominal cavity macrophages is obtained from traditional mare milk wine and intestinal tracts of healthy human bodies through wide screening, and the optimal dose of the lactobacillus rhamnosus on the effect of recovering immunodeficiency is determined.
The first embodiment is as follows: the embodiment describes a strain of lactobacillus rhamnosus with an immunoregulation effect, namely a lactobacillus rhamnosus (L.rhamnosus)1.0320 strain, wherein the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15557, the preservation address is No. 3 of Beijing university Hokko No.1 of sunward West Chen, and the preservation date is No. 08 of 2018 monthly 04.
The second embodiment is as follows: in the first embodiment, the lactobacillus rhamnosus with the immunoregulation function is separated from the traditional marmilk wine.
The third concrete implementation mode: in a specific embodiment, the lactobacillus rhamnosus with the immunoregulation function is shown in fig. 13 and 14, the bacterial colony of the lactobacillus rhamnosus is milk white, the edge is neat, the surface is smooth and convex, and the thallus is in a short rod shape.
The fourth concrete implementation mode: the application of the lactobacillus rhamnosus with the immunoregulation effect disclosed in any one of the first embodiment to the third embodiment is to apply the lactobacillus rhamnosus to the fermentation process of yoghourt or pickled vegetables.
The fifth concrete implementation mode: the application of lactobacillus rhamnosus with immunoregulation effect disclosed in any one of the first to third embodiments, wherein the lactobacillus rhamnosus is used for preparing freeze-dried bacterial powder.
Isolation and purification of Lactobacillus rhamnosus (L.rhamnosus) strain 1.0320: 1mL of a traditional marmilk wine sample and 1g of intestinal contents of a healthy human body are dissolved in 10mL of sterile PBS (0.01M, pH 7.4) respectively to prepare suspensions, the suspensions are inoculated into a sterilized MRS broth culture medium in an inoculation amount of 2%, the operations are repeated after the culture is carried out at a constant temperature of 37 ℃ for 18 hours, and the second generation is activated. Taking 100 mu L of fully activated second-generation bacterium liquid, carrying out three-zone streaking on a sterilized MRS-agar culture medium in a plate streaking mode, carrying out constant-temperature culture at 37 ℃ for 24h, and picking out a single colony. Inoculating the strain in a sterilized MRS broth culture medium for activation passage twice, and culturing at 37 ℃ for 12-16h at constant temperature to generate turbidity. Taking 100 mu L of bacterial liquid to perform three-zone lineation on a sterilized MRS-agar culture medium, culturing for 24h at the constant temperature of 37 ℃, selecting a single bacterial colony, observing the bacterial colony under a microscope, and observing the strain form and whether the bacterial colony is free from mixed bacteria pollution. After the strains are determined to be qualified strains, the strains are sent to Jilin Ku Mei Biotech limited in the form of bacterial liquid, and the species identification is carried out on the bacteria by using a 16S rDNA sequence sequencing method. 3 strains isolated from the marburg sample were identified and named Lactobacillus plantarum 69-2, Lactobacillus plantarum 1.0628, and Lactobacillus rhamnosus 1.0320; the 3 strains isolated from the intestinal tract of a healthy human body are named as lactobacillus plantarum 117-1, lactobacillus plantarum 23-1 and lactobacillus rhamnosus 118-1. Lactobacillus rhamnosus GG (ATCC53103), deposited in the American Type Culture Collection (ATCC) of the authorized depository, was purchased from the ATCC China office agency, Beijing Central plains, Inc. Lactobacillus plantarum P0002(CMCC-P0002) is purchased from China medical bacteria preservation and management center (CMCC), and researches prove that Lactobacillus rhamnosus GG and Lactobacillus plantarum P0002 have the function of regulating immunity and can be used as reference strains for screening Lactobacillus with the function of regulating immunity.
The spleen lymphocyte transformation, the spleen lymphocyte proliferation (equivalent cell culture solution as a negative control group; ConA with a final concentration of 5 mu g/mL as a positive control group), the phagocytic capacity of the abdominal cavity macrophages and the content of the abdominal cavity macrophages secreting NO are respectively carried out on lactobacillus plantarum P0002(CMCC-P0002), lactobacillus plantarum 69-2, lactobacillus plantarum 1.0628, lactobacillus plantarum 117-1, lactobacillus plantarum 23-1, lactobacillus rhamnosus GG (ATCC53103), lactobacillus rhamnosus 1.0320 and lactobacillus rhamnosus 118-1 (equivalent cell culture solution as a negative control group; LPS with a final concentration of 30 mu g/mL as a positive control group), so that the conversion value of the spleen lymphocytes and the content of the abdominal cavity macrophages secreting NO of lactobacillus rhamnosus 1.0320 (lactobacillus: cells 1:1) are remarkably higher than those of the other 7 strains of lactobacillus (P < 0.05) (FIG. 1, B), Fig. 4). Wherein, the immunoregulatory ability of lactobacillus rhamnosus 1.0320 is significantly higher than that of the reference strain lactobacillus rhamnosus GG. The immunoregulation intensity of lactobacillus rhamnosus 1.0320 is positively correlated with the concentration, the spleen lymphocyte transformation value of lactobacillus rhamnosus 1.0320 (lactobacillus: cells are 100: 1) is 7.5 times of that of a positive control group (figure 1), the spleen lymphocyte Proliferation Index (PI) reaches more than 1 (figure 2), and the phagocytic capacity of abdominal cavity macrophages is not significantly different from that of the positive control group (p is more than 0.05) (figure 3). The result shows that the immunoregulation capability of the lactobacillus rhamnosus 1.0320 is obviously stronger than that of the rest 7 strains of lactobacillus.
Example 1:
optimization of Lactobacillus rhamnosus 1.0320 bacteriocin yield based on orthogonal analysis
MRS basal medium preparation (initial pH 6.2): beef powder 5.0g, tryptone 10.0g, glucose 20.0g, yeast powder 4.0g, Tween 801.0mL, K2HPO42.0 g、CH3COONa 5.0g、C6H5O7(NH4)32.0g、MgSO4 0.2g、MnSO40.05 g, and 1000mL of distilled water.
Influence of ultraviolet mutagenesis on bacteriocin yield: a plurality of 10mL of activated three-generation lactobacillus rhamnosus 1.0320 thallus suspension is put in a sterilized plate in a dark room, and the plate is respectively irradiated by 25W ultraviolet lamps for 0s, 20s, 40s, 60s, 80s, 100s and 120s at a distance of 30 cm. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
Influence of initial pH of the medium on bacteriocin yield: inoculating activated second-generation Lactobacillus rhamnosus 1.0320 with 2% inoculum size in MRS culture medium with pH of 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, and 7.0, respectively, and culturing at 37 deg.C for 18 h. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
③ Effect of the ratio of two important nitrogen sources on the yield of bacteriocins: the second generation of activated lactobacillus rhamnosus 1.0320 was inoculated in 2% inoculum size in MRS medium with yeast powder and tryptone ratio (basal medium ratio of 4: 10) adjusted to 2.5:11.5, 3:11, 3.5:10.5, 4:10, 4.5:9.5, 5:9, 5.5:8.5, respectively, and cultured at 37 ℃ for 18 h. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
Influence of NaCl addition on bacteriocin yield: the second-generation activated lactobacillus rhamnosus 1.0320 is respectively inoculated in MRS culture media with NaCl addition of 0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% in an inoculation amount of 2%, and cultured for 18h at 37 ℃. Escherichia coli is used as an indicator bacterium, and an oxford cup double-layer agar diffusion method is adopted for carrying out a bacteriostasis test.
The order of magnitude of range R is given by the factors in Table 21＞R3＞R4＞R2The main and secondary relationship of influence on the yield of the lactobacillus rhamnosus 1.0320 bacteriocin is that ultraviolet induction is greater than nitrogen source ratio, NaCl is greater than pH. From the K-values it can be determined that the best combination of lactobacillus rhamnosus 1.0320 bacteriocin production is: ultraviolet induction for 80s, adjustment of nitrogen source ratio (yeast powder: tryptone ═ 3.5:10.5), addition of NaCl of 1%, and initial pH of 6.4. The diameter of the zone of inhibition is increased from (26.24 +/-0.31) mm to (36.68 +/-0.51) mm.
TABLE 1 influence of single factors on the yield of bacteriocin of Lactobacillus rhamnosus 1.0320
TABLE 2 orthogonal test design and results table for yield optimization of Lactobacillus rhamnosus 1.0320 bacteriocin
Example 2:
immunoregulation effect of lactobacillus rhamnosus 1.0320 on immunodeficient model mice
Activating: 1mL of Lactobacillus rhamnosus 1.0320 preserved at-80 ℃ is taken out, streaked on an MRS-agar medium, and cultured at a constant temperature of 37 ℃ for 24 hours, and a single colony is picked. Inoculating the strain in a sterilized MRS broth culture medium for activation passage twice, and culturing at 37 ℃ for 12-16h at constant temperature to generate turbidity. Taking 100 mu L of bacterial liquid to perform three-zone lineation on a sterilized MRS-agar culture medium, culturing for 24h at the constant temperature of 37 ℃, selecting a single bacterial colony, observing under a microscope, and obtaining the qualified product without the pollution of mixed bacteria.
Preparing a sample: determining viable count of Lactobacillus rhamnosus 1.0320 bacteria liquid by plate counting method, centrifuging (4000r/min, 10min), washing for 3 times, and resuspending with sterile PBS (0.01M, pH 7.4) to 5 × 107CFU/mL、5×108CFU/mL、5×10910mL of each CFU/mL bacterial suspension was prepared in sterile PBS (0.01M, pH 7.4) in an amount of 15 mL.
Thirdly, molding: SPF-grade BALB/c female mice (weight 18 + -1 g; 6 weeks old) were randomly assigned: a blank control group (N), a model control group (M), a positive control group (P), a lactobacillus rhamnosus 1.0320 low dose group (1.0320-L), a lactobacillus rhamnosus 1.0320 medium dose group (1.0320-M) and a lactobacillus rhamnosus 1.0320 high dose group (1.0320-H). Except for a blank control group (replaced by equal volume of sterile PBS), cyclophosphamide is injected at a dose of 80mg/kg every day, and the weight of the mice is observed to continuously decrease for three consecutive days, which indicates that the cyclophosphamide causes the damage of the immune system of the mice, and the establishment of an immunosuppression model is successful.
Fourthly, intragastric administration: equal volumes of each group were given: sterile PBS, levamisole (2.5mg/mL), Lactobacillus rhamnosus 1.0320 (5X 10)7CFU/mL), Lactobacillus rhamnosus 1.0320 (5X 10)8CFU/mL) and Lactobacillus rhamnosus 1.0320 (5X 10)9CFU/mL) of the strain, wherein the weight, the proliferation capacity of T lymphocytes, the phagocytic capacity of abdominal macrophages, the activity of NK cells, the carbon clearance capacity, delayed hypersensitivity and the level of a cell inflammatory factor of the mouse are measured after continuous intervention for 30 days, as shown in the figure, the lactobacillus rhamnosus 1.0320 is found to be capable of remarkably relieving the weight reduction caused by immunodeficiency (figure 5), the improvement of the activity of immune cells is positively correlated with the viable count of lactobacillus rhamnosus 1.0320, and the high dose ((10) is used for (10)9CFU/mL)/0.02kg) of lactobacillus rhamnosus 1.0320 can promote phagocytic ability of macrophages (fig. 7) and carbon particle clearance ability (fig. 9) well, and has no significant difference (p > 0.05) from a positive control group; the effects of promoting T lymphocyte proliferation (fig. 6) and enhancing NK cell activity (fig. 8) were 1.06-fold and 1.54-fold, respectively, that of the positive control group. The delayed type hypersensitivity results are shown in figure 10, and simultaneously, lactobacillus rhamnosus 1.0320 can regulate the level of the cell inflammatory factor, play a role in relieving immunodeficiency by increasing the ratio of the IL-10 (figure 11) of the anti-inflammatory cytokine to the IL-12 of the proinflammatory cytokine and the level of immunoglobulin (figure 12), and determine the optimal dosage as (10)9CFU/mL)/0.02kg/d。
Example 3:
application of lactobacillus rhamnosus 1.0320 as freeze-dried bacterium powder with immune strengthening function
Activating: 1mL of Lactobacillus rhamnosus 1.0320 preserved at-80 ℃ is taken out, streaked on an MRS-agar medium, and cultured at a constant temperature of 37 ℃ for 24 hours, and a single colony is picked. Inoculating to sterilized MRS broth culture medium, activating and passaging twice, culturing at 37 deg.C for 12-16h, observing under microscope, and transferring to 40L fermentation tank for amplification culture after detection reaches standard.
Determining the viable count: adding 0.5mL of activated and amplified fermentation broth into 4.5mL of sterilized physiological saline, sequentially performing 10-fold gradient dilution, and selecting 10-8、10-9、10-10Diluting, spreading 200 μ L of diluent on sterile MRS-agar culture medium, culturing at 37 deg.C for 24 hr, counting, and determining viable count of fermentation broth to be 109CFU/mL。
Freeze-drying: centrifuging the fermentation liquor (4000r/min, 10min), collecting thalli cells, resuspending the thalli cells by using an equal-volume sterile PBS solution, wherein a freeze-drying protective agent is 5% sucrose skim milk powder (the concentration of the skim milk powder is 20%, and the concentration of sucrose is 5% (w/v)), and the rhamnose lactobacillus 1.0320 suspension and the sucrose skim milk powder freeze-drying protective agent are mixed according to the proportion of 1: 1(v/v), placing in a low-temperature refrigerator, and pre-cooling to-40 ℃. And simultaneously, the temperature of a freezing chamber of the freeze dryer is reduced to be below-40 ℃. After the sample is put in, a vacuum pump of a freeze dryer is opened to start freeze drying, the freeze drying time is about 24-72h, and the freeze-dried powder of lactobacillus rhamnosus 1.0320 is stored at-80 ℃, and the survival rate is about 49.6%.
The freeze-dried powder of Lactobacillus rhamnosus 1.0320 has immunity enhancing effect, and can be directly used as health product for storage at 0-4 deg.C, with viable count of 2 × 108CFU/g. Or used as an immune enhancing factor to be added into functional milk powder and prebiotics solid beverage. Has effects in improving proliferation ability of immune immunocyte, regulating phagocytic ability of macrophage, reducing inflammatory reaction, and regulating intestinal microecology.
Example 4:
application of lactobacillus rhamnosus 1.0320 and commercial leavening agent in food compounding
Activating: 1mL of Lactobacillus rhamnosus 1.0320 preserved at-80 ℃ is taken out, streaked on an MRS-agar medium, and cultured at a constant temperature of 37 ℃ for 24 hours, and a single colony is picked. Inoculating to sterilized MRS broth culture medium, activating and passaging twice, culturing at 37 deg.C for 12-16h, and observing under microscope without contamination.
Centrifugation: the fermentation broth was centrifuged (4000r/min, 10min), the cells were collected and resuspended in an equal volume of sterile 5% glucose solution.
③ fermenting the yoghourt: pretreating and standardizing raw milk, cooling to a fermentation temperature, and then inoculating a starter with an inoculation amount of 2%, wherein the composite starter is lactobacillus rhamnosus: streptococcus thermophilus: lactobacillus bulgaricus ═ 2: 4: and 4, the temperature of the fermentation tank is 42-45 ℃, the fermentation time is 2.5-3h, and when the pH value reaches 4.7, the temperature is reduced to 0-7 ℃ for stirring, so that the immune fortified yoghourt is obtained.
Fourthly, fermentation of pickled vegetables: fully washing fresh raw materials, adding 4% of salt and 2.5% of sucrose (w/w), inoculating a compound fermentation agent (lactobacillus rhamnosus 1.0320: lactobacillus plantarum 1:1) in an inoculation amount of 3%, fermenting at a constant temperature of 30 ℃ for 5 days to complete pickle, and preserving the pickle fermented by using lactobacillus rhamnosus 1.0320 as the fermentation agent for a longer time.
Claims (5)
1. A lactobacillus rhamnosus strain with immunoregulation function is characterized in that: lactobacillus rhamnosus (L.rhamnosus)1.0320 strain, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15557, the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo area, and the preservation date is No. 08 of 04.2018 at the institute of microbiology of China academy of sciences.
2. The lactobacillus rhamnosus strain with immunoregulation effect according to claim 1, characterized in that: the lactobacillus rhamnosus is isolated from traditional marmilk wine.
3. The lactobacillus rhamnosus strain with immunoregulation effect according to claim 1, characterized in that: the bacterial colony of the lactobacillus rhamnosus is milk white, the edge is neat, the surface is smooth and convex, and the thallus is in a short rod shape.
4. Use of lactobacillus rhamnosus with immunomodulatory effects according to any of claims 1 to 3, characterized in that: the lactobacillus rhamnosus is applied to the fermentation process of yoghourt or pickled vegetables.
5. Use of lactobacillus rhamnosus with immunomodulatory effects according to any of claims 1 to 3, characterized in that: the Lactobacillus rhamnosus is used for preparing freeze-dried powder.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010834655.4A CN111944721A (en) | 2020-08-17 | 2020-08-17 | Lactobacillus rhamnosus with immunoregulation effect and application thereof |
| PCT/CN2020/120519 WO2022036822A1 (en) | 2020-08-17 | 2020-10-13 | Lacticaseibacillus rhamnosus having immunoregulation function, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010834655.4A CN111944721A (en) | 2020-08-17 | 2020-08-17 | Lactobacillus rhamnosus with immunoregulation effect and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111944721A true CN111944721A (en) | 2020-11-17 |
Family
ID=73342913
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010834655.4A Pending CN111944721A (en) | 2020-08-17 | 2020-08-17 | Lactobacillus rhamnosus with immunoregulation effect and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111944721A (en) |
| WO (1) | WO2022036822A1 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940984A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Compound lactobacillus preparation for resisting helicobacter pylori, reducing blood sugar, conditioning intestines and stomach and increasing immunity and preparation method thereof |
| CN112940985A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Lactobacillus rhamnosus preparation for enhancing human immunity and preparation method thereof |
| CN113621572A (en) * | 2021-08-03 | 2021-11-09 | 东北农业大学 | Application of lactobacillus rhamnosus in adhering colon cells |
| CN113773984A (en) * | 2021-08-20 | 2021-12-10 | 江南大学 | Lactobacillus rhamnosus for improving polygonatum polysaccharide yield and regulating skin barrier and immunity |
| CN114293268A (en) * | 2022-01-10 | 2022-04-08 | 东北农业大学 | Encapsulated lactobacillus rhamnosus 1.0320 core-shell fiber and preparation method and application thereof |
| CN114774315A (en) * | 2022-04-13 | 2022-07-22 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparing immunity enhancing and/or eczema relieving product |
| CN115851535A (en) * | 2022-12-10 | 2023-03-28 | 万益生物科技(山东)有限公司 | Lactobacillus rhamnosus WFP52 with function of regulating immunity and application thereof |
| WO2024160209A1 (en) * | 2023-02-01 | 2024-08-08 | 北京三元食品股份有限公司 | Breast milk-derived lacticaseibacillus rhamnosus and use |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116333922A (en) * | 2023-02-10 | 2023-06-27 | 宁波希诺亚海洋生物科技有限公司 | Lactobacillus rhamnosus strain derived from deep sea squid and application thereof |
| CN116694503B (en) * | 2023-03-27 | 2024-01-05 | 上海华聿康生物科技有限公司 | Lactobacillus plantarum Lp-HZ55 with bowel relaxing and immunity improving functions |
| CN118530914B (en) * | 2024-07-22 | 2024-10-18 | 山东健源生物科技有限公司 | Lactobacillus rhamnosus for enhancing immunity and improving sleep and metaplasia thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421715A (en) * | 2013-08-03 | 2013-12-04 | 青岛蔚蓝生物集团有限公司 | Lactobacillus rhamnosus and application thereof |
| CN103937708A (en) * | 2014-03-24 | 2014-07-23 | 华东理工大学 | Lactobacillus rhamnosus and its application |
| CN109161496A (en) * | 2018-08-19 | 2019-01-08 | 东北农业大学 | Lactobacillus rhamnosus high density bacterium solution and its preparation method for embedding bacterium powder |
| CN109266584A (en) * | 2018-10-18 | 2019-01-25 | 扬州大学 | One plant of cilium type Lactobacillus rhamnosus and application thereof with mast cell activity adjustment effect |
| CN109266568A (en) * | 2018-08-19 | 2019-01-25 | 东北农业大学 | A kind of prebiotic function Lactobacillus rhamnosus and its application with high bacteriocinogeny |
| CN110122877A (en) * | 2018-02-09 | 2019-08-16 | 深圳市华大农业应用研究院 | Lactobacillus rhamnosus and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498549B (en) * | 2018-05-22 | 2020-06-26 | 浙江海正药业股份有限公司 | Composition for improving immune function and application thereof |
-
2020
- 2020-08-17 CN CN202010834655.4A patent/CN111944721A/en active Pending
- 2020-10-13 WO PCT/CN2020/120519 patent/WO2022036822A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421715A (en) * | 2013-08-03 | 2013-12-04 | 青岛蔚蓝生物集团有限公司 | Lactobacillus rhamnosus and application thereof |
| CN103937708A (en) * | 2014-03-24 | 2014-07-23 | 华东理工大学 | Lactobacillus rhamnosus and its application |
| CN110122877A (en) * | 2018-02-09 | 2019-08-16 | 深圳市华大农业应用研究院 | Lactobacillus rhamnosus and application thereof |
| CN109161496A (en) * | 2018-08-19 | 2019-01-08 | 东北农业大学 | Lactobacillus rhamnosus high density bacterium solution and its preparation method for embedding bacterium powder |
| CN109266568A (en) * | 2018-08-19 | 2019-01-25 | 东北农业大学 | A kind of prebiotic function Lactobacillus rhamnosus and its application with high bacteriocinogeny |
| CN109266584A (en) * | 2018-10-18 | 2019-01-25 | 扬州大学 | One plant of cilium type Lactobacillus rhamnosus and application thereof with mast cell activity adjustment effect |
Non-Patent Citations (2)
| Title |
|---|
| MUHAMMAD SHAHIDRIAZ RAJOKA等: "Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk", 《LWT》 * |
| 樊代明: "《整合医学 理论与实践 6》", 30 April 2019, 上海世界图书出版公司 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940984A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Compound lactobacillus preparation for resisting helicobacter pylori, reducing blood sugar, conditioning intestines and stomach and increasing immunity and preparation method thereof |
| CN112940985A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Lactobacillus rhamnosus preparation for enhancing human immunity and preparation method thereof |
| CN113621572A (en) * | 2021-08-03 | 2021-11-09 | 东北农业大学 | Application of lactobacillus rhamnosus in adhering colon cells |
| CN113773984A (en) * | 2021-08-20 | 2021-12-10 | 江南大学 | Lactobacillus rhamnosus for improving polygonatum polysaccharide yield and regulating skin barrier and immunity |
| CN113773984B (en) * | 2021-08-20 | 2023-08-25 | 江南大学 | Lactobacillus rhamnosus for improving rhizoma Polygonati polysaccharide yield and regulating skin barrier and immunity |
| CN114293268A (en) * | 2022-01-10 | 2022-04-08 | 东北农业大学 | Encapsulated lactobacillus rhamnosus 1.0320 core-shell fiber and preparation method and application thereof |
| CN114774315A (en) * | 2022-04-13 | 2022-07-22 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparing immunity enhancing and/or eczema relieving product |
| CN114774315B (en) * | 2022-04-13 | 2023-12-19 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparation of immunity enhancing product and/or eczema relieving product |
| CN115851535A (en) * | 2022-12-10 | 2023-03-28 | 万益生物科技(山东)有限公司 | Lactobacillus rhamnosus WFP52 with function of regulating immunity and application thereof |
| WO2024160209A1 (en) * | 2023-02-01 | 2024-08-08 | 北京三元食品股份有限公司 | Breast milk-derived lacticaseibacillus rhamnosus and use |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2022036822A1 (en) | 2022-02-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111944721A (en) | Lactobacillus rhamnosus with immunoregulation effect and application thereof | |
| CN112852679B (en) | Probiotic bacillus coagulans and application thereof | |
| CN112869169B (en) | Application of lactobacillus paracasei ET-22 in improving intestinal bacterial infection resistance and intestinal immunity | |
| CN110564638A (en) | Lactobacillus reuteri with probiotic characteristics and application thereof | |
| CN115851535B (en) | Lactobacillus rhamnosus WFP52 with immunity regulating effect and application thereof | |
| CN110157645B (en) | Lactobacillus salivarius Y4 and application thereof | |
| CN110484459B (en) | Lactobacillus plantarum and application thereof | |
| CN103540545A (en) | Pediococcus pentosaceus and application thereof | |
| CN114774326B (en) | Lactobacillus plantarum A21136 for inhibiting helicobacter pylori and application thereof | |
| CN116396890B (en) | Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer and application thereof | |
| CN109022313B (en) | Lactobacillus plantarum | |
| CN114480231A (en) | Lactobacillus reuteri for resisting helicobacter pylori infection and application thereof | |
| CN115851551B (en) | Pediococcus pentosaceus A21358 with helicobacter pylori resisting effect and application thereof | |
| CN112940985A (en) | Lactobacillus rhamnosus preparation for enhancing human immunity and preparation method thereof | |
| CN116004456A (en) | Lactobacillus reuteri A21325 for inhibiting helicobacter pylori infection and application thereof | |
| CN114774315B (en) | Application of lactobacillus rhamnosus strain LRa05 in preparation of immunity enhancing product and/or eczema relieving product | |
| CN115181682B (en) | Lactobacillus fermentum highly enriched with organic zinc | |
| CN113249244B (en) | Lactobacillus paracasei for antagonizing pharyngitis pathogenic bacteria beta hemolytic streptococcus | |
| CN118272251A (en) | Helicobacter pylori resistant lactobacillus reuteri and application thereof | |
| CN112029676B (en) | Probiotic composition beneficial to improving immunity and application thereof | |
| CN112940984A (en) | Compound lactobacillus preparation for resisting helicobacter pylori, reducing blood sugar, conditioning intestines and stomach and increasing immunity and preparation method thereof | |
| CN117327616A (en) | Lactiplantibacillus plantarum ZN1 and application thereof | |
| CN111743158A (en) | Probiotic tablet with function of enhancing immunity and preparation method thereof | |
| CN109362882B (en) | Lactobacillus fermentum CQPC07 and application thereof in preparing food or medicine for improving ulcerative colitis | |
| CN102399728B (en) | Lactobacillus and fermentation method thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201117 |
|
| RJ01 | Rejection of invention patent application after publication |