CN115386515B - 用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌及其应用 - Google Patents
用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌及其应用 Download PDFInfo
- Publication number
- CN115386515B CN115386515B CN202210941595.5A CN202210941595A CN115386515B CN 115386515 B CN115386515 B CN 115386515B CN 202210941595 A CN202210941595 A CN 202210941595A CN 115386515 B CN115386515 B CN 115386515B
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- calf
- intestinal
- yaks
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 43
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 40
- 244000309466 calf Species 0.000 title claims abstract description 26
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 23
- 230000001105 regulatory effect Effects 0.000 title abstract description 4
- 230000003647 oxidation Effects 0.000 title description 4
- 238000007254 oxidation reaction Methods 0.000 title description 4
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 10
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 239000006041 probiotic Substances 0.000 claims description 6
- 235000018291 probiotics Nutrition 0.000 claims description 6
- 230000010226 intestinal metabolism Effects 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 2
- 244000005700 microbiome Species 0.000 abstract description 13
- 238000002474 experimental method Methods 0.000 abstract description 10
- 230000004060 metabolic process Effects 0.000 abstract description 9
- 241000894006 Bacteria Species 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 abstract description 6
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 5
- 230000003859 lipid peroxidation Effects 0.000 abstract description 5
- 230000007760 free radical scavenging Effects 0.000 abstract description 4
- 238000012404 In vitro experiment Methods 0.000 abstract description 3
- 230000006872 improvement Effects 0.000 abstract description 3
- 230000009469 supplementation Effects 0.000 abstract description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 2
- 238000002835 absorbance Methods 0.000 description 13
- 210000001035 gastrointestinal tract Anatomy 0.000 description 10
- 230000002292 Radical scavenging effect Effects 0.000 description 9
- 235000013336 milk Nutrition 0.000 description 9
- 239000008267 milk Substances 0.000 description 9
- 210000004080 milk Anatomy 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 241000195576 Bacillus subtilis group Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 4
- 244000005709 gut microbiome Species 0.000 description 4
- 235000020778 linoleic acid Nutrition 0.000 description 4
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 208000018522 Gastrointestinal disease Diseases 0.000 description 3
- 241000282849 Ruminantia Species 0.000 description 3
- 230000009102 absorption Effects 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000008482 dysregulation Effects 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000010201 enrichment analysis Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000007366 host health Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000005502 peroxidation Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical group C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 241001202853 Blautia Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 208000027205 Congenital disease Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 108010026389 Gramicidin Proteins 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241001112693 Lachnospiraceae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241001135259 Rikenella Species 0.000 description 1
- 241000605947 Roseburia Species 0.000 description 1
- 108090000787 Subtilisin Proteins 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000003736 gastrointestinal content Anatomy 0.000 description 1
- 210000005095 gastrointestinal system Anatomy 0.000 description 1
- 229960004905 gramicidin Drugs 0.000 description 1
- ZWCXYZRRTRDGQE-SORVKSEFSA-N gramicidina Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](NC=O)C(C)C)CC(C)C)C(=O)NCCO)=CNC2=C1 ZWCXYZRRTRDGQE-SORVKSEFSA-N 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- -1 potassium ferricyanide Chemical compound 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 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
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- 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
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Birds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌（Bacillus subtilis）BS‑2022，保藏编号为CCTCC NO:M 2022073，该菌株分离于犊牦牛本身，首先分离的菌株经过体外实验包括DPPH自由基清除活性、羟基自由基清除活性、还原力、脂质过氧化抑制活性测定，证明该菌株具有很强的抗氧化能力，此外，牦牛本体实验证明补充枯草芽孢杆菌BS‑2022可以显著改善抗氧化能力并且其肠道微生物多样性和代谢也得到改善，肠道微生物的改善主要表现为期微生物多样性和丰度的显著提高，并且其有益细菌的丰度显著上升。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌(Bacillus subtilis)BS-2022及其应用。
背景技术
枯草芽孢杆菌(Bacillus subtilis)是目前应用最广泛的益生菌之一，广泛存在于动物胃肠道和自然界。与乳酸菌等益生菌相比，枯草芽孢杆菌具有易保存、抗逆性强的特点。研究表明，饲料中添加枯草芽孢杆菌可以改善多种动物包括肉鸡、鸭、羊的消化能力和生长性能。此外，枯草芽孢杆菌还被证明可以产生枯草菌素、多粘菌素和短杆菌肽等抗菌物质从而提高机体的抗病力。
牦牛是生活在青藏高原高海拔缺氧地区的珍惜牛种，具有耐低温和适低氧的特点。据统计，世界上大约90％以上分布在中国四川、青海、西藏、甘肃省。牦牛是当地牧民奶制品、肉类、皮革和交通工具的重要来源，与农业发展和人类文明息息相关。考虑到牦牛在青藏高原的重要性，任何危及牦牛健康和发展的因素都可能造成巨大的经济损失。然而，由于极端条件以及牧草和其他营养物质的短缺，牦牛的生产性能和健康受到严重威胁。
反刍动物肠道中含有数以万亿计的微生物，它们在新陈代谢、消化吸收、肠道稳态和宿主健康中发挥着关键作用。此外，肠道微生物群也已被证明在免疫系统成熟、通透性和肠道上皮分化中发挥作用。肠道内有益菌可通过分泌抗菌肽、调节肠道环境和竞争营养来限制外来病原体在肠道中的定植，被认为是抵御病原菌入侵的天然屏障。据统计，正常肠道含有超过1014种微生物，大约是宿主细胞总量的10倍。其中，肠道细菌约占总微生物群落的98％，其余则包含真菌(0.1％)、病毒和原生动物。稳定的肠道菌群是各种复杂的生理和代谢过程所必需的，而肠道菌群失调可能导致多种胃肠道疾病，包括腹泻、肠炎和肠易激综合征。尽管这些微生物在肠道内定植，但它们可能会导致全身效应。越来越多的证据表明，肠道微生物菌群失调对肠道功能的影响不仅限于胃肠系统，还会损害包括肝脏和大脑在内的其他器官的功能。因此改善动物肠道菌群对于宿主健康至关重要。先前的研究表明，反刍动物肠道微生物群在发育过程中动态变化并受年龄、营养和饮食的影响，并在成熟时达到稳定。由于肠道菌群不稳定和免疫系统不成熟，新生反刍动物更容易患腹泻等胃肠道疾病，但这种情况会随着年龄的增长而逐渐改善。重要的是，肠道菌群不成熟的新生牦牛在食物和营养短缺时容易出现生长迟缓和死亡。因此，改善和维持肠道微生物稳定性对新生牦牛的健康和生长具有重要意义。
发明内容
针对犊牦牛生长缓慢、易发生胃肠道疾病的问题，本发明从犊牦牛肠道分离出一株能够改善肠道菌群和代谢的枯草芽孢杆菌。
为了达到上述目的，本发明采取以下技术措施：
本发明从犊牦牛体内分离出一株具有很强抗氧化能力的枯草芽孢杆菌(Bacillussubtilis)BS-2022，保藏编号为CCTCC NO:M 2022073。
枯草芽孢杆菌BS-2022可用于促进犊牦牛肠道微生物多样性、改善犊牦牛肠道代谢以及提高犊牦牛抗氧化能力，因此可作为犊牦牛益生菌添加剂。
与现有技术相比，本发明具有以下优点：
本发明所述的枯草芽孢杆菌BS-2022分离于犊牦牛本身，首先分离的菌株经过体外实验包括DPPH自由基清除活性、羟基自由基清除活性、还原力、脂质过氧化抑制活性测定，证明分离菌株具有很强的抗氧化能力，此外，牦牛本体实验证明补充枯草芽孢杆菌BS-2022可以显著改善抗氧化能力并且其肠道微生物多样性和代谢也得到改善。肠道微生物的改善主要表现为微生物多样性和丰度的显著提高，并且有益细菌的丰度显著上升。此外，一些有益代谢物的丰度也显著上升。这表明该菌株可以用于牦牛的生产实践中，改善牦牛健康。
附图说明
图1为枯草芽孢杆菌BS-2022在LB琼脂培养基的菌落形态图。
图2为枯草芽孢杆菌BS-2022革兰氏染色结果。
图3为枯草芽孢杆菌BS-2022体外抗氧化结果。
图4为构建的系统发育进化树。
图5为枯草芽孢杆菌BS-2022对犊牦牛抗氧化指标的影响。CT、MT和BT分别代表21天的对照组、代乳粉组和枯草芽孢杆菌组；CF、MF和BF分别代表42天的对照组、代乳粉组和枯草芽孢杆菌组。
图6为枯草芽孢杆菌BS-2022对犊牦牛肠道微生物多样性的影响。CT、MT和BT分别代表21天的对照组、代乳粉组和枯草芽孢杆菌组；CF、MF和BF分别代表42天的对照组、代乳粉组和枯草芽孢杆菌组。Good's coverage代表高通量测序深度，Chao1和ACE指数代表肠道微生物的丰度，Shannon指数代表肠道微生物的多样性。
图7为差异肠道微生物群落分析。
图8为枯草芽孢杆菌BS-2022对犊牦牛肠道代谢的影响。A、B基于正负离子模式的CF组和BF组之间代谢途径的富集分析，C、D基于正负离子模式的MF组和BF组之间代谢途径的富集分析。
具体实施方式
实施例1枯草芽孢杆菌BS-2022的分离、筛选、鉴定和保藏
1、枯草芽孢杆菌的分离
申请人从青海地区采集大量的牦牛肠道和粪便样本，用于枯草芽孢杆菌的分离筛选。具体方法为将肠内容物和粪便样本置于灭菌的PBS溶液中，涡旋震荡均匀。随后，置于水浴锅中，85℃水浴加热15分钟，以消灭其他不耐热细菌。吸取100μL混合液，均匀涂布于LB琼脂平板中。将平板放于恒温培养箱中，24℃过夜培养。挑取疑似枯草芽孢杆菌的单个菌落(图1)，纯化培养，并进行革兰氏染色(图2)，该菌株的编号为BS-2022。
2、具有抗氧化性能的枯草芽孢杆菌的筛选
益生菌抗氧化能力可以通过一系列体外实验进行验证，如羟基自由基清除活性、DPPH自由基清除活性、还原力测定、脂质过氧化抑制活性。
羟基自由基清除活性：采用改进的芬顿反应方法进行羟基自由基清除实验。芬顿反应在以下混合物中进行：1.0mL 1,10-菲咯啉(0.75mM)，1.0mL 0.75mM FeSO4、2.0mL磷酸钠缓冲液(0.2M，pH 7.4)和1.0mL样品(细菌悬浮液，细胞浓度约为109CFU/mL)。通过加入1mL H2O2(0.01％，v/v)开始反应，在37℃下孵育1小时后，将带有细胞的混合物在4℃下离心(10,000×g)10分钟，并使用紫外可见分光光度计(UV-UV)在536nm下测量上清液的吸光度。羟自由基清除活性(％)的计算公式为(AS-A0)×100/(A-A0)，其中AS是样品的吸光度；A0是没有样品时对照的吸光度，A是没有H2O2的空白的吸光度，经过实验得出BS-2022的羟基自由基清除值大概为74.03％(图3A)。
DPPH自由基清除活性：将含有1.0mL样品(细菌悬浮液浓度约为109CFU/mL)和4.0mL 0.1mM DPPH溶液(溶于95％乙醇)的混合物在黑暗中放置30分钟。在10,000×g下离心10分钟后，在517nm下测量所得上清液的吸光度。所得的DPPH自由基清除活性(％)表示为[1-(AS-AB)/AC]×100，其中AS是样品的吸光度，AB是空白(乙醇和样品)的吸光度，AC是对照(去离子水和DPPH溶液)的吸光度，经过实验得出BS-2022的DPPH自由基清除活性大概为48.30％(图3B)。
还原力测定：取0.5mL样品(细菌悬浮液浓度约为109CFU/mL)与2.5mL磷酸钠缓冲液(0.2M,pH 6.6)和2.5mL 1％铁氰化钾混合，50℃孵育20min，加入10％(w/v)三氯乙酸2.5mL终止反应。以3000×g离心10min，上清(5ml)与去离子水5ml、0.1％氯化铁1ml混合。将混合物静置10分钟，在700nm处读取吸光度。以去离子水代替样品作为对照。在本实验中，观察到的吸光度的增加表明样品还原力的增加，经过实验得出BS-2022的DPPH还原力大概为364.54％(图3C)。
脂质过氧化抑制活性：抗氧化活性是通过硫代巴比妥酸(TBA)方法评估的，该方法基于监测细菌对亚油酸(被选为不饱和脂肪酸的来源)过氧化的抑制作用。亚油酸乳剂(20mL)由0.1mL亚油酸、0.2mL Tween 20和19.7mL去离子水组成。将等分试样(0.4mL)样品(细菌悬浮液浓度约为109CFU/mL)与0.5mL磷酸钠缓冲液(0.02M，pH 7.4)、1mL亚油酸乳剂和0.2mL 1％FeSO4混合。在37℃下孵育1小时后，将2mL混合物与0.2mL 4％三氯乙酸(TCA)、2mL 0.8％TBA和0.2mL 0.4％BHT混合。将该混合物在100℃下温育30分钟，并使其在冰上冷却。通过将悬浮液(2mL)与2mL丁醇涡旋混合1分钟，然后在1800×g离心10分钟。在相同条件下，将上层相(水相)再次离心10分钟，在532nm处读取上清液的吸光度。亚油酸过氧化的抑制百分比表示为(1-AS/AB)×100，其中AS为样品的吸光度，AB是去离子水而不是样品的吸光度。经过实验得出BS-2022的DPPH脂质过氧化抑制活性大概为33.28％(图3D)。以上结果都证明分离菌株BS-2022具有良好的抗氧化潜力。
3、枯草芽孢杆菌的分类鉴定
为了进一步对菌株进行鉴定，通过对BS-2022进行基因组DNA提取，利用16s rDNA的通用引物进行扩增，测序获得16s rDNA的基因序列。通过在NCBI网站进行BLAST分析，以及构建系统发育进化树(图4)，结果显示菌株BS-2022属于芽胞杆菌群中的枯草芽胞杆菌，该菌株已于2022年1月13号送至中国典型培养物保藏中心保藏，保藏编号为CCTCC:M2022073，地址:中国.武汉.武汉大学，分类命名:枯草芽胞杆菌(Bacillus subtillis)BS-2022。
实施例2枯草芽孢杆菌BS-2022对犊牦牛肠道菌群和代谢的影响
本实验在四川省阿坝藏族羌族自治州红原县(北纬31°51′-33°33′，东经101°51′-103°22′，平均海拔3507米)的一个牦牛养殖场进行，该养殖场拥有500多头牦牛。实验时间为2021年5月至6月。本实验筛选的牦牛都是畜牧场进行自我繁殖，并接受相同的免疫程序。此外，所有选择的牦牛在实验前都进行了身体检查，以确保没有畸形和其他先天性疾病。实验共使用年龄和初始体重相近的健康牦牛犊18头。适应性饲养三天后，将牦牛平均分为三组(n＝18)，即对照组、代乳粉组和枯草芽孢杆菌组。对照组牦牛自由采食，代乳粉组牦牛饲喂代乳粉，枯草芽孢杆菌组牦牛饲喂代乳粉和枯草芽孢杆菌，代乳粉饲喂量为每天500ml，枯草芽孢杆菌为每天1×109CFU。所选择的牦牛在同一草地上自由放牧，不补充任何精料，整个试验持续42天。此外，在第1、21和42天早晨喂食前记录各组每头牦牛的平均日增重和体重。在第21和42天早晨喂食前从颈静脉采集每头牦牛的血样，获得的血样随后使用离心机在3,500rpm下进行血清分离10分钟。同时，在第21天和第42天使用无菌拭子通过直肠收集粪便样本。将收集的血清和粪便样本置于干冰上并运送到实验室进行进一步分析。血清样本使用南京建成公司生产的试剂盒，测量其抗氧化指标包括T-AOC、SOD、MDA、GSH-PX、CAT，发现补充益生菌后犊牦牛的总抗氧化力、谷胱甘肽过氧化物酶、超氧化物歧化酶显著提高(图5)。将粪便用本进行16s RNA扩增子测序以及代谢组学分析，发现补充枯草芽孢杆菌BS-2022后犊牦牛微生物多样性指数显著升高(图6)，与消化吸收，短链脂肪酸生成的有益菌含量显著上升包Ruminococcaceae_UCG-004，Blautia，Roseburia，Ruminococcaceae_UCG-005，Parabacteroides，Ruminiclostridium_9，Lachnospiraceae_NK3A20_group，Lachnospiraceae_UCG-010，Bacteroides，Christensenellaceae_R-7_group，Bacillus，Romboutsia，Rikenellaceae_RC9_gut_group，Alistipes，Lachnospiraceae_NK4A136_group，Ruminococcaceae_UCG-010，Coprococcus_3，Ruminococcaceae_UCG-009，Butyricimonas，Ruminococcaceae_UCG-013 and Prevotellaceae_UCG-003(图7)。代谢组学分析发现，补充枯草芽孢杆菌显著改变了牦牛的肠道代谢，引起一系列通路的变化，包括次级胆汁酸生物合成、胆汁分泌、花生四烯酸代谢、5-羟色胺能突触、嘧啶代谢、脂多糖的生物合成、光合生物的碳固定、酪氨酸代谢、植物激素的生物合成和维生素的消化吸收(图8)。
Claims (5)
1.枯草芽孢杆菌（Bacillus subtilis）BS-2022，其特征在于，保藏编号为CCTCC NO:M2022073。
2.犊牦牛益生菌添加剂，其特征在于，含有枯草芽孢杆菌（Bacillus subtilis）BS-2022，保藏编号为CCTCC NO:M 2022073。
3.权利要求1所述的枯草芽孢杆菌BS-2022在制备促进犊牦牛肠道微生物多样性的产品中的应用。
4.权利要求1所述的枯草芽孢杆菌BS-2022在制备改善犊牦牛肠道代谢的产品中的应用。
5.权利要求1所述的枯草芽孢杆菌BS-2022在制备提高犊牦牛抗氧化能力的产品中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210941595.5A CN115386515B (zh) | 2022-08-03 | 2022-08-03 | 用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210941595.5A CN115386515B (zh) | 2022-08-03 | 2022-08-03 | 用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115386515A CN115386515A (zh) | 2022-11-25 |
| CN115386515B true CN115386515B (zh) | 2023-11-17 |
Family
ID=84119643
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210941595.5A Active CN115386515B (zh) | 2022-08-03 | 2022-08-03 | 用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115386515B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117187102A (zh) * | 2023-06-20 | 2023-12-08 | 广东南芯医疗科技有限公司 | 枯草芽孢杆菌bs02在制备抗氧化和抗衰老产品中的应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019002471A1 (en) * | 2017-06-30 | 2019-01-03 | Evonik Degussa Gmbh | DEBACILLUS SUBTILIS STRAIN HAVING PROBIOTIC ACTIVITY |
| CN109161509A (zh) * | 2018-10-10 | 2019-01-08 | 中国农业科学院兰州兽医研究所 | 一株能防治牛羊腹泻病的菌株 |
| CN110628669A (zh) * | 2019-09-04 | 2019-12-31 | 中国农业科学院特产研究所 | 枯草芽孢杆菌ly-5、菌剂及其应用和应用其的产品 |
| CN111187730A (zh) * | 2019-11-28 | 2020-05-22 | 中国农业科学院兰州兽医研究所 | 一株枯草芽孢杆菌及其应用 |
| CN112574924A (zh) * | 2020-12-31 | 2021-03-30 | 福建大北农水产科技有限公司 | 一种枯草芽孢杆菌菌株及其微生态制剂和应用 |
| CN115287224A (zh) * | 2022-06-24 | 2022-11-04 | 华中农业大学 | 一株改善本源动物肠道微生物发育的牦牛源罗伊氏乳杆菌及其应用 |
-
2022
- 2022-08-03 CN CN202210941595.5A patent/CN115386515B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019002471A1 (en) * | 2017-06-30 | 2019-01-03 | Evonik Degussa Gmbh | DEBACILLUS SUBTILIS STRAIN HAVING PROBIOTIC ACTIVITY |
| CN109161509A (zh) * | 2018-10-10 | 2019-01-08 | 中国农业科学院兰州兽医研究所 | 一株能防治牛羊腹泻病的菌株 |
| CN110628669A (zh) * | 2019-09-04 | 2019-12-31 | 中国农业科学院特产研究所 | 枯草芽孢杆菌ly-5、菌剂及其应用和应用其的产品 |
| CN111187730A (zh) * | 2019-11-28 | 2020-05-22 | 中国农业科学院兰州兽医研究所 | 一株枯草芽孢杆菌及其应用 |
| CN112574924A (zh) * | 2020-12-31 | 2021-03-30 | 福建大北农水产科技有限公司 | 一种枯草芽孢杆菌菌株及其微生态制剂和应用 |
| CN115287224A (zh) * | 2022-06-24 | 2022-11-04 | 华中农业大学 | 一株改善本源动物肠道微生物发育的牦牛源罗伊氏乳杆菌及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| Effect of Bacillus subtilis C-3102 supplementation in milk replacer on growth and rumen microbiota in preweaned calves;Satoshi Koike等;Anim Sci J.;第92卷(第1期);参见全文 * |
| 牦牛源枯草芽孢杆菌的益生作用研究;王琦等;江苏农业科学;第48卷(第11期);参见全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115386515A (zh) | 2022-11-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110982760B (zh) | 一株贝莱斯芽孢杆菌及其在防治猪腹泻中的应用 | |
| Ghasemi-Sadabadi et al. | The effects of fermented milk products (kefir and yogurt) and probiotic on performance, carcass characteristics, blood parameters, and gut microbial population in broiler chickens | |
| CN103005159A (zh) | 一种银杏叶生物饲料添加剂的制备方法 | |
| CN104673726A (zh) | 一种猪源嗜酸乳杆菌冻干制剂及其应用 | |
| Tang et al. | Effects of glucose oxidase on the growth performance, serum parameters and faecal microflora of piglets | |
| CN116103208B (zh) | 唾液乳杆菌在抗氧化上的应用 | |
| CN115386515B (zh) | 用于改善犊牦牛抗氧化能力和调节肠道菌群的枯草芽孢杆菌及其应用 | |
| CN113215019A (zh) | 一株粪肠球菌及其应用 | |
| Shah et al. | Effect of lactic acid bacteria‐treated King grass silage on the performance traits and serum metabolites in New Zealand white rabbits (Oryctolagus cuniculus) | |
| Wulandari et al. | Quality and probiotic lactic acid bacteria diversity of rabbit meat Bekasam-fermented meat | |
| CN113088468B (zh) | 干酪乳杆菌Ma.GLRGJ 1及其应用 | |
| CN112592870B (zh) | 一种凝结芽孢杆菌g4、其发酵液及制备方法和应用 | |
| CN115918909B (zh) | 一种猪源芽孢杆菌在制备抗氧化和提升免疫的制品中的应用 | |
| Pogány Simonová et al. | Enterocins as novel feed additives in rabbit Diet: Enterocin Ent M and Durancin Ent ED26E/7, their combination, and Effects on Microbiota, Caecal Fermentation, and enzymatic activity | |
| CN115925832A (zh) | 一种乳酸菌素及其在防控畜禽细菌感染的应用 | |
| CN115181711A (zh) | 人乳杆菌lhm11及菌剂和应用 | |
| CN115197885A (zh) | 格氏乳球菌lgm15及菌剂和应用 | |
| CN117343876B (zh) | 一株抑菌、抗炎的贝莱斯芽孢杆菌及其应用 | |
| RU2797587C1 (ru) | Пробиотическая кормовая добавка для коров и телят | |
| RU2794878C1 (ru) | Биологически активная кормовая добавка | |
| CN109971668B (zh) | 具有降低胆固醇功能的植物乳杆菌及其应用和微生态制剂 | |
| CN116218710B (zh) | 一种干酪乳杆菌及其发酵蔬菜尾菜制备高膳食纤维的方法 | |
| CN111961625B (zh) | 一株戊糖片球菌smm914及其筛选方法和应用 | |
| CN108795784B (zh) | 一种饲料添加剂及其制备方法、应用和制备饲料添加剂的培养基 | |
| RU2694256C2 (ru) | Кормовая добавка для профилактики бактерионосительства микроорганизмов рода Salmonella у сельскохозяйственной птицы и способ ее применения |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |