CN111996140B - Screening and application of buffalo rumen source bacillus amyloliquefaciens - Google Patents
Screening and application of buffalo rumen source bacillus amyloliquefaciens Download PDFInfo
- Publication number
- CN111996140B CN111996140B CN202010824185.3A CN202010824185A CN111996140B CN 111996140 B CN111996140 B CN 111996140B CN 202010824185 A CN202010824185 A CN 202010824185A CN 111996140 B CN111996140 B CN 111996140B
- Authority
- CN
- China
- Prior art keywords
- strain
- bacillus amyloliquefaciens
- silage
- cellulose
- rumen
- 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.)
- Expired - Fee Related
Links
- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 65
- 210000004767 rumen Anatomy 0.000 title abstract description 33
- 238000012216 screening Methods 0.000 title abstract description 8
- 108700023468 protein-bound SN-C polysaccharide Proteins 0.000 claims abstract description 66
- 239000004460 silage Substances 0.000 claims abstract description 39
- 229920002678 cellulose Polymers 0.000 claims abstract description 36
- 239000001913 cellulose Substances 0.000 claims abstract description 36
- 240000008042 Zea mays Species 0.000 claims abstract description 25
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 25
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 25
- 235000005822 corn Nutrition 0.000 claims abstract description 25
- 229920002488 Hemicellulose Polymers 0.000 claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 239000000835 fiber Substances 0.000 abstract description 18
- 238000000855 fermentation Methods 0.000 abstract description 15
- 230000004151 fermentation Effects 0.000 abstract description 15
- 230000015556 catabolic process Effects 0.000 abstract description 11
- 238000006731 degradation reaction Methods 0.000 abstract description 11
- 241000282849 Ruminantia Species 0.000 abstract description 10
- 239000000654 additive Substances 0.000 abstract description 10
- 238000002360 preparation method Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 6
- 230000029087 digestion Effects 0.000 abstract description 5
- 230000000996 additive effect Effects 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 238000011156 evaluation Methods 0.000 abstract description 3
- 239000003674 animal food additive Substances 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 30
- 238000012360 testing method Methods 0.000 description 29
- 239000000243 solution Substances 0.000 description 24
- 230000001018 virulence Effects 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 16
- 239000007788 liquid Substances 0.000 description 14
- 239000010902 straw Substances 0.000 description 14
- 238000007789 sealing Methods 0.000 description 13
- 108010059892 Cellulase Proteins 0.000 description 12
- 229940106157 cellulase Drugs 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 230000003321 amplification Effects 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 11
- 241000283690 Bos taurus Species 0.000 description 10
- 239000003599 detergent Substances 0.000 description 10
- 238000005303 weighing Methods 0.000 description 10
- 108020004465 16S ribosomal RNA Proteins 0.000 description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 229910052799 carbon Inorganic materials 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 238000001035 drying Methods 0.000 description 8
- 238000012257 pre-denaturation Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 230000007935 neutral effect Effects 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000000147 enterotoxin Substances 0.000 description 6
- 231100000655 enterotoxin Toxicity 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 241000193755 Bacillus cereus Species 0.000 description 5
- 101100394356 Bacillus cereus hblA gene Proteins 0.000 description 5
- 101710146739 Enterotoxin Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 5
- 238000004043 dyeing Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 239000002699 waste material Substances 0.000 description 5
- 240000007594 Oryza sativa Species 0.000 description 4
- 235000007164 Oryza sativa Nutrition 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 4
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 235000009566 rice Nutrition 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 206010016717 Fistula Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 239000003429 antifungal agent Substances 0.000 description 3
- 239000001768 carboxy methyl cellulose Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000003890 fistula Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000006041 probiotic Substances 0.000 description 3
- 235000018291 probiotics Nutrition 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 150000004666 short chain fatty acids Chemical class 0.000 description 3
- 235000021391 short chain fatty acids Nutrition 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 3
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 3
- OBMBUODDCOAJQP-UHFFFAOYSA-N 2-chloro-4-phenylquinoline Chemical compound C=12C=CC=CC2=NC(Cl)=CC=1C1=CC=CC=C1 OBMBUODDCOAJQP-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 229930186147 Cephalosporin Natural products 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 108010006464 Hemolysin Proteins Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 240000006024 Lactobacillus plantarum Species 0.000 description 2
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 2
- 240000003183 Manihot esculenta Species 0.000 description 2
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 229940126575 aminoglycoside Drugs 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 235000019728 animal nutrition Nutrition 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229940124587 cephalosporin Drugs 0.000 description 2
- 150000001780 cephalosporins Chemical class 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 2
- 230000001461 cytolytic effect Effects 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 239000002619 cytotoxin Substances 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 235000019621 digestibility Nutrition 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 229960000448 lactic acid Drugs 0.000 description 2
- 229940072205 lactobacillus plantarum Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000006996 mental state Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 235000019629 palatability Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000007660 quinolones Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011076 safety test Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ZNQVEEAIQZEUHB-UHFFFAOYSA-N 2-ethoxyethanol Chemical compound CCOCCO ZNQVEEAIQZEUHB-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 101001065065 Aspergillus awamori Feruloyl esterase A Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 241001251371 Betula chinensis Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000235646 Cyberlindnera jadinii Species 0.000 description 1
- 241000605056 Cytophaga Species 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241001486247 Etiella Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 208000012766 Growth delay Diseases 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- -1 aminoglycosides Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229920003123 carboxymethyl cellulose sodium Polymers 0.000 description 1
- 229940063834 carboxymethylcellulose sodium Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229960002227 clindamycin Drugs 0.000 description 1
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 108010041969 feruloyl esterase Proteins 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000004461 grass silage Substances 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 244000038280 herbivores Species 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004630 mental health Effects 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
- 229960000564 nitrofurantoin Drugs 0.000 description 1
- NXFQHRVNIOXGAQ-YCRREMRBSA-N nitrofurantoin Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)NC(=O)C1 NXFQHRVNIOXGAQ-YCRREMRBSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960005404 sulfamethoxazole Drugs 0.000 description 1
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229940072172 tetracycline antibiotic Drugs 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 108010047303 von Willebrand Factor Proteins 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 230000037303 wrinkles Effects 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
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of agricultural microorganism application, and relates to screening and application of buffalo rumen-derived bacillus amyloliquefaciens. The invention relates to the field of preparation of feed additive preparations and the field of rumen digestion development and utilization of ruminants. Relates to the strain separation and identification, safety evaluation and identification of silage fiber degradation capability of bacillus amyloliquefaciens which has the degradation effect on cellulose in the feed, and the development and application of bacillus amyloliquefaciens as a silage fermentation promoting additive. The strain is a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SN-C strain, and the preservation number is CCTCC NO: m2020137. The strain has the advantages of high growth speed, capability of remarkably improving the cellulose degradation rate in corn silage, capability of reducing the cellulose content by 3.10 percent and the hemicellulose content by 1.75 percent compared with a control group, strong stress resistance, safety and capability of preparing silage fermentation promotion additives.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganism application, and particularly relates to screening and application of buffalo rumen-derived bacillus amyloliquefaciens. The invention relates to the field of preparation of feed additive preparations and the field of ruminant rumen digestion biological additives, and provides separation identification, safety evaluation and identification of silage fiber degradation capability of a bacillus amyloliquefaciens strain capable of degrading cellulose in a feed, which can be used for promoting development and utilization of a feed silage fermentation additive.
Background
China is a big agricultural country, the annual output of agricultural byproducts is about 6.3 hundred million tons, the annual straw amount used as herbivore feed only accounts for 15-24 percent of the total output, and more than 70 percent of the straws are left unused or burnt, thereby causing huge waste and environmental pollution. The ruminant feed mainly comprises coarse feeds with high lignification such as corn, rice straw, wheat straw and the like, and even in beef cattle feeding, the proportion of the coarse feeds such as the corn straw, the rice straw and the like is not less than 50%. The straws are rich in cellulose, lack of protein, high in lignification degree and poor in palatability, are not beneficial to digestion and absorption of animals, and can digest and utilize only about half of cellulose in food even for ruminants with high digestion capability such as cattle, namely nearly half of cellulose in feed of the ruminants is not effectively utilized. Therefore, the utilization efficiency of the cellulose in the coarse feed for the ruminants has a 50% promotion space, and the novel strain with improved utilization of the cellulose can create great economic and social benefits.
In order to keep fresh and utilize the straws to the maximum extent, people adopt a silage fermentation method to treat the fresh straws. The silage is fermented to produce micromolecular saccharides, lactic acid and the like, so that the palatability of the straws can be improved, and the silage is also favorable for decomposing cellulose in the straws and improving the digestibility of the feed. However, the decomposition of cellulose is limited in the natural ensiling process, and ensiling additives have been developed in order to improve the ensiling effect. Silage additives are mainly classified into three major categories: bacterial preparations (e.g. lactobacillus plantarum, bacillus, etc.), chemical additives (e.g. formic acid, sorbic acid, benzoic acid, etc.) and enzyme preparations (cellulase, hemicellulase, amylase, etc.) (muck r.e. et al, 2018). Practice has shown that silage additive is effective, zahiroddin adds cellulase and amylase mixed preparation to barley silage, and the daily gain of cattle is significantly increased when cattle are fed with the silage (Zahiroddin H.i, 2004). Li et al Lactobacillus plantarum and cellulase producing feruloyl esterase were added as silage additives to corn silage and were found to reduce the fiber content of the feed (Li et al, 2019). Guohiming et al report that the simultaneous addition of ensiling bacteria, cellulolytic enzymes and carbon sources can better improve the quality of rice straw ensiling.
Ruminant rumen microorganisms can decompose part of cellulose in plant cell walls for self-reproduction, simultaneously generate a large amount of short-chain fatty acids for a host to utilize, and when the short-chain fatty acids reach the rear section of the host digestive tract, the short-chain fatty acids become a main protein nutrition source of the host. Although the rumen is a strictly anaerobic environment, and many bacteria are not easily isolated in an in vitro environment, some bacteria can still grow in vitro (christopher j. Et al, 2014).
Although the rumen of cattle can utilize roughage, cattle of different breeds vary in feed digestion efficiency. Research shows that buffalo has stronger coarseness resistance than other varieties of cattle. Changhakhoun found that buffalo rumen degraded lignocellulose more strongly and the number of lignocellulose decomposing bacteria in buffalo rumen was significantly higher than beef cattle (changhakhouna v. et al, 2012).
Disclosure of Invention
The invention aims to solve the problem of insufficient cellulose degradation in the fermentation of the existing silage, and is inspired by stronger degradation of lignocellulose by buffalo coarse feed and buffalo rumen, a bacterial strain with strong cellulose degradation capability is screened from the buffalo rumen, a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) capable of obviously improving the cellulose degradation rate in corn silage (compared with a control group, the cellulose content can be reduced by 3.10 percent and the hemicellulose content can be reduced by 1.75 percent) is obtained by screening, and the bacterial strain with high cellulase activity screened by the invention can be used for improving the cellulose degradation rate in the fermentation of silage. Through the optimization of a cellulose carbon source screening scheme, safety detection is carried out on the cellulose carbon source screening scheme, and detection of virulence genes, sensitivity tests of common antibacterial drugs and feeding tests of test animals are mainly carried out. Against common virulence genes of bacillus: detecting nonhemolytic enterotoxins NHE (nheA, nheB and nheC), hemolysin Hbl (hblA, hblC and hblD), cytotoxins CytK, enterotoxin T (BceT) and enterotoxin FM (EntFM), and finding that the strain of the bacillus amyloliquefaciens SN-C only contains nheA and nheC virulence genes; and the drug sensitivity test shows that the strain is very sensitive to common antibiotics such as penicillins, cephalosporins, quinolones, aminoglycosides, tetracyclines and the like, and the problem of spreading drug resistance genes does not exist. The mouse feeding test also proves that the strain is safe and nontoxic.
The applicant names the screened Bacillus amyloliquefaciens strain SN-C and Bacillus amyloliquefaciens SN-C, and the strain is delivered to China, wuhan university China Center for Type Culture Collection (CCTCC) for preservation in 5-21 months in 2020, with the preservation number of CCTCC NO: m2020137.
Drawings
FIG. 1: the invention screens the growth state of the rumen fluid culture on the plate with the only carbon source of the sodium carboxymethyl cellulose. Description of reference numerals: the rumen fluid culture has a moist and smooth surface and a needle tip size, and the bacterial colony is white.
FIG. 2: is the detection result of the screened SN-C strain of the bacillus amyloliquefaciens for producing the cellulase. Description of reference numerals: the screened strain of Bacillus amyloliquefaciens SN-C is marked with C in FIG. 2.
FIG. 3: the screened SN-C strain of the bacillus amyloliquefaciens is in a growth state on a domestication culture medium plate containing cellulase. Description of reference numerals: the bacillus amyloliquefaciens SN-C strain has a colony diameter of 3-4mm, white and opaque appearance, dry and flat surface and burr-shaped edge.
FIG. 4: the gram-positive reaction (x 1000) of the separated and screened strain of the bacillus amyloliquefaciens SN-C is realized.
FIG. 5 is a schematic view of: the invention relates to an electrophoretogram of a PCR amplification detection result of 16S rDNA gene of a bacillus amyloliquefaciens SN-C strain.
Lanes in FIG. 5 illustrate: m:2000bp Marker; lane 1, lane 2: bacillus amyloliquefaciens SN-C strain;
FIG. 6: the growth curve of the strain of the bacillus amyloliquefaciens SN-C is disclosed.
FIG. 7: electrophoresis diagram of positive result of virulence gene amplification of bacillus cereus.
Lanes in FIG. 7 illustrate: m:2000bp Marker; lane 1: nheA; lane 2: nheB; lane 3: nheC; lane 4: entFM; lane 5: a hblA; lane 6: a hblC; lane 7: a hblD; lane 8: cytK; lane 9: bceT; lane 10: and (5) negative control.
FIG. 8: electrophoresis diagram of the amplification result of the virulence gene of the strain SN-C of the bacillus amyloliquefaciens. Description of reference numerals:
FIG. 8A: electrophoresis pattern of the amplification result of nheA virulence gene of the SN-C strain of the bacillus amyloliquefaciens.
Lanes in FIG. 8A illustrate: m:2000bp Marker; lane 1: nheA positive control; lane 2: bacillus amyloliquefaciens SN-C strain; lane 3: negative control;
FIG. 8B: electrophoresis diagram of the amplification result of nheC virulence gene of the strain SN-C of the bacillus amyloliquefaciens.
Lanes in FIG. 8B illustrate: m:2000bp Marker; lane 1: nheC positive control; lane 2: bacillus amyloliquefaciens SN-C strain; lane 3: and (5) negative control.
FIG. 9: and (3) the result of drug sensitivity test of the strain SN-C of the bacillus amyloliquefaciens. Description of reference numerals:
fig. 9A and 9B: and (3) the result of drug sensitivity test of the strain SN-C of the bacillus amyloliquefaciens. FIG. 9A: upper left: compound sulfamethoxazole, lower left: nitrofurantoin; right lower: clindamycin.
FIG. 9B: upper left: kanamycin, bottom left: a tetracycline; right lower: gentamicin.
FIG. 10: and adding a bacillus amyloliquefaciens SN-C bacterial liquid and a corn micro-ensiling test result.
FIGS. 10A and 10B show corn micro-ensiling tests with the addition of Bacillus amyloliquefaciens SN-C bacterial liquid.
FIG. 11: and adding a bacillus amyloliquefaciens SN-C bacterial liquid and partial operation and materials of the corn micro-ensiling test.
FIG. 11A; and (3) determining the crushing of the silage corn crude fiber sample added with the bacterial liquid of the bacillus amyloliquefaciens SN-C by using a filter bag method.
FIG. 11B: the filter bag used for measuring the feed crude fiber is measured by a filter bag method.
FIG. 12: and (4) determining the Neutral Detergent Fiber (NDF) of the silage corn added with the bacterial liquid of the starch bacillus SN-C (digested with a neutral detergent).
FIG. 13 is a schematic view of: respectively adding SN-C, A, E and CO 4 Silage corn, neutral Detergent Fiber (NDF) assay of the strain, wherein: A. e, CO 4 For other strains isolated contemporaneously, the ordinate is the percentage of dry matter content of neutral detergent fiber NDF, and the abscissa is the strain type.
FIG. 14: respectively adding SN-C, A, E and CO 4 The result of the measurement of the strain silage corn acid washing fiber (ADF) is shown in the ordinate, the percentage of ADF in dry matter content is shown in the ordinate, and the strain type is shown in the abscissa.
FIG. 15: respectively adding SN-C, A, E and CO 4 The result of the cellulose determination of the strain silage corn is that the ordinate is the percentage of cellulose in dry matter content, and the abscissa is the strain type.
FIG. 16: respectively adding SN-C, A, E and CO 4 The result of measuring hemicellulose of corn ensiled by the strain has the ordinate of the content percentage of the hemicellulose in dry matter and the abscissa of the content percentage of the hemicellulose in dry matter.
Detailed Description
Description of the sequence listing:
Example 1: isolation and characterization of strains
1. Preparation of a culture medium:
TABLE 1 buffer A
Note: this buffer was used to prepare the enrichment medium.
TABLE 2 buffer B
Note: this buffer was used to prepare the enrichment medium.
TABLE 3 enrichment Medium
Note: the Resazurin is blue, the color of the sterilized culture medium can be changed into pink, and the solution is colorless when oxygen is not contained in the culture medium.
The antifungal agents were prepared according to the formulation shown in Table 4, and after completion of the preparation, they were sterilized by filtration through a 0.45 μm filter, and then dispensed in 1.0mL portions, and stored at 4 ℃.
TABLE 4 configuration of antifungal agents
Note: cycloheximide is highly toxic and can inhibit yeast, mold and protozoon; the solvent used for nystatin is DMSO, and a small amount of DMSO is dissolved and then mixed with the rest solution.
The prepared antifungal agent was added to 7.0mL of enrichment medium at a 1/100 ratio (10. Mu.L in 1 mL) (at which time the enrichment medium was not treated with oxygen to drive off, pink).
TABLE 5 plates using sodium carboxymethylcellulose (CMC-Na) as sole carbon source
Note: the plate is used for rumen bacteria sample culture, and rumen bacteria sample is diluted and then spread or streaked on the culture medium for separate culture.
TABLE 6 cellulase identification plate
Note: the plate was used for the identification of cellulase producing strains; the cellulase-producing strain is inoculated on the plate and cultured for 2-3d, and then stained by 0.1% Congo red staining solution, and a light yellow hydrolysis ring is generated after washing by 1.0mol/L NaCl solution.
TABLE 7 cellulase acclimatization Medium (volume 1.0L)
Note: used for domesticating, culturing and spotting to identify the strain producing hydrolysis loop.
0.1% congo red staining solution: weighing 0.1g of Congo red powder by using an analytical balance with one ten thousandth of precision, dissolving the powder in 100mL of distilled water, and sealing and storing at room temperature;
1.0mol/L NaCl solution: weighing 5.85g of NaCl by using a ten-thousandth precision analytical balance, dissolving in 100mL of distilled water, and sealing and storing at room temperature;
rumen sample collection:
preparing materials: 350mL (Mitsubishi MGC, japan) anaerobic gas generating bag, 10.0mL sterilized EP tube, sterilized gauze (four layers), 39 ℃ constant temperature incubator (vehicle mounted), medical sterilized alcohol and large and small size self-sealing bags.
The sampling points were 3 healthy buffalos fitted with rumen fistulas from northwest of Hubei Tan animal husbandry, inc., sanzhou, city.
The sampling method comprises the following steps: sampling for the first time before feeding in the morning, wearing protective gloves and protective clothing, after alcohol disinfection, opening a rumen fistula, soaking a fistula plug in hot water at about 80 ℃ for disinfection, then filtering part of rumen contents by using four layers of gauze, receiving filtrate in a 10mL sterilized EP tube, taking 1.0mL of filtrate and a small amount of rumen contents in 7.0mL of enrichment medium, sealing by using a sealing film after covering, placing on an EP tube frame, placing the tube frame and an anaerobic gas production bag together in a large self-sealing bag, sealing, placing in a 39 ℃ vehicle-mounted small thermostat, culturing, and bringing back to a test room.
Collection of the non-rumen fluid: taking rumen content, wrapping in four layers of gauze, extruding rumen liquid, storing in large size self-sealing bag, and storing on ice. Returning to the laboratory, filtering with four layers of gauze, centrifuging at 10000rpm/min for 10min, collecting supernatant as gastric juice without cytoma, and freezing at-20 deg.C. (the rumen fluid is used for preparing a culture medium and can be sterilized in the culture medium, so that sterilization is not needed when the rumen fluid is stored at the temperature of-20 ℃, and the external culture medium is mainly close to the real condition of the rumen when the cell-free rumen fluid is added into the culture medium)
2. Isolation of the Strain
Placing the collected sample in an anaerobic incubator at 39 ℃ for culturing for 1d, and taking a sample 10 -1 -10 -6 Diluting by multiple times (100 μ L culture medium is added into 900 μ L sterilized water for dilution by multiple times), and diluting with 10 μ L sterile water -4 、10 -5 、10 -6 Coating a single carbon source flat plate of sodium carboxymethylcellulose at three dilution degrees, placing the flat plate in an anaerobic box at 39 ℃ for culturing for 3d, and selecting a single colony, wherein the growth result of the strain is shown in figure 1;
picking a suspected single colony on a carboxymethylcellulose sodium sole carbon source plate by using a 10.0 mu L sterilization gun head, stabbing the suspected single colony on a cellulase spotting plate, culturing the single colony in an anaerobic box at 39 ℃ for 3 days, then, dyeing for 10-15min by using 0.1% Congo red dyeing solution (1.0-1.5 mL of the dyeing solution is dropwise added into each bacterial culture dish with the diameter of 9cm to ensure that the dyeing solution covers a thin layer of the plate and cannot immerse a lawn), washing for 2 times by using 1.0mol/L NaCl solution, and observing whether a light yellow hydrolysis ring is generated or not, wherein the dyeing result of the bacterial strain Congo red is shown in a figure 2 and a table 8;
TABLE 8 stain circle diameter
Selecting thallus Porphyrae producing yellowish hydrolysis ring with sterilized 1.0mL gun head, streaking and purifying for three times on cellulose acclimation plate, culturing in 39 deg.C anaerobic box for 3d, streaking and purifying growth state of SN-C on cellulose acclimation plate shown in figure 3 (single colony shape is 3-4mm diameter, white, opaque, wrinkle on surface, and burr on edge);
through identification of the strains, 4 strains producing faint yellow hydrolysis rings are obtained, and gram-positive bacteria are identified through gram staining, and the gram staining characteristics of the gram-positive bacteria are shown in figure 4. The applicant names A, SN-C, E and CO4; the strain C has the characteristics of blunt circles at two ends, short rod shape and bluish violet staining.
3. Identification of Strain species
On the basis of the identification, 16S rDNA gene sequence detection and species-specific gene detection are further carried out on the separated strain SN-C, and the species to which the strain belongs are verified and identified.
Extracting the isolated plant genome:
(1) 1mL of the culture solution was centrifuged at 10000rpm for 30 seconds, and the supernatant was discarded as much as possible to collect the cells.
(2) 200. Mu.L of buffer RB was added for resuspension, centrifuged at 10000rpm for 30s, and the supernatant was discarded.
(3) mu.L of lysozyme (20 mg/mL in 10mM Tris-HCl, pH 8.0) was added thereto, mixed by inversion, and incubated at 37 ℃ for 30-60min. Centrifuge at 12000rpm for 2min, discard the supernatant and resuspend in 180. Mu.L of buffer RB by pipetting.
(4) Adding 20 μ L proteinase K (20 mg/mL) solution, mixing well, adding 200 μ L binding solution CB, immediately vortex, mixing well, standing at 70 deg.C for 10min.
(5) After cooling, 100. Mu.L of isopropanol was added and mixed well immediately by vortexing, whereupon a flocculent precipitate may appear.
(6) Adding the mixture (including the precipitate if any) in the previous step into an adsorption column AC, centrifuging at 13000rpm for 30-60s (placing the adsorption column into a collection tube), and pouring off the waste liquid in the collection tube.
(7) 500. Mu.L of inhibitor removing solution IR was added, and centrifuged at 12000rpm for 30 seconds, and the waste solution was discarded.
(8) Add 700. Mu.L of the rinse WB (with addition of absolute ethanol), centrifuge at 12000rpm for 30s, and discard the waste.
(9) Add 500. Mu.L of the rinse WB (with addition of absolute ethanol), centrifuge at 12000rpm for 30s and discard the waste.
(10) And (4) putting the adsorption column AC back into an empty collection tube, centrifuging at 13000rpm for 2min, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction.
(11) Taking out the adsorption column AC, placing into a clean centrifuge tube, adding 50 μ L of elution buffer EB (the elution buffer is preheated in water bath at 67-70 deg.C in advance) into the middle part of the adsorption membrane, standing at room temperature for 3-5min, and centrifuging at 12000rpm for 1min. Adding the obtained solution into centrifugal adsorption column again, standing at room temperature for 2min, and centrifuging at 12000rpm for 1min.
(12) The DNA was stored at-20 ℃ until use.
(II) amplification of 16S rDNA Gene:
the 16S rDNA sequence of the isolate was amplified using 16S rDNA universal primers, which were synthesized by Wuhan Tianyihui Biotechnology Limited and have the following DNA sequences:
forward primer 27F:5 'AGAGAGTTTGATCCTGGCTCAG-3',
reverse primer 1492R:5 'GGTTACCTTGTTACGACTT-3';
TABLE 9 1696 rDNA sequence amplification PCR System
Amplification conditions: pre-denaturation at 95 ℃ for 5min, pre-denaturation at 95 ℃ for 50s, pre-denaturation at 55 ℃ for 35s, pre-denaturation at 72 ℃ for 1min,30 cycles, and elongation at 72 ℃ for 10min. The PCR product was electrophoretically detected on a 0.8% agarose gel (containing ethidium bromide) to amplify a fragment of about 1500 base pairs in size as expected (see FIG. 5)
(II) bacterial evolutionary tree analysis:
and sending the 16S rDNA obtained by amplification to Wuhan Kjeldahl biology Limited company for sequencing, performing Blast on a sequencing result at NCBI, selecting and downloading the sequences of the previous ten strains with high homology, and constructing a bacterial phylogenetic tree by using MEGA7.0 software to obtain a strain SN-C with the homology of 99 percent with the sequences of a plurality of strains of bacillus amyloliquefaciens. Generally, two strains can be regarded as microorganisms of the same species when the similarity of the 16S rDNA sequences reaches 97%, and the strains can be regarded as microorganisms of the same genus when the similarity reaches 95%, and the strains SN-C separated by the method is determined to be a bacillus amyloliquefaciens strain through the analysis of the phylogenetic tree constructed by the 16S rDNA sequencing result.
The applicant names the obtained strain SN-C as Bacillus amyloliquefaciens SN-C and Bacillus amyloliquefaciens SN-C, and delivers the strain SN-C to China, wuhan university China Center for Type Culture Collection (CCTCC) for preservation in 5 and 21 months in 2020, with the preservation number of CCTCC NO: m2020137.
4. Growth curve determination of SN-C strain of bacillus amyloliquefaciens
Inoculating the activated Bacillus amyloliquefaciens SN-C strain into LB according to the inoculation amount of 1% (v/v), performing shake cultivation at 37 ℃ at 200rpm/min, and sampling every 2h to determine OD600; the time is plotted on the abscissa and the OD600 of the bacteria is plotted on the ordinate to plot the growth curve.
The result shows that the growth delay period of the SN-C separated by the invention is about 1h, the SN-C enters a logarithmic growth period (0 h-4 h) quickly, the SN-C enters a stabilization period after 8h, and the number of bacteria can reach 10 11 -10 12 CFU/mL. The strain has strong reproductive capacity and is beneficial to industrial large-scale fermentation production, and the growth curve of the strain is shown in figure 6 and table 10.
TABLE 10 growth of the strain of Bacillus amyloliquefaciens SN-C at different times
5. Cryopreservation of strain of Bacillus amyloliquefaciens SN-C strain
Transferring SN-C (identifying single colony with cellulose degradation capability) bacterial liquid into a cellulose acclimation culture medium, culturing in an anaerobic box at 39 ℃ for 36h, and culturing according to V Bacterial liquid :V Glycerol And (4) =3, adding 50% glycerol to prepare 1.0mL, sealing in a 1.5mL EP tube, marking date and time and strains, and freezing at-80 ℃.
Example 2: safety evaluation of SN-C strain of bacillus amyloliquefaciens
1. Virulence gene amplification (identification) of virulence gene positive strains
Amplifying virulence gene positive bacillus cereus by using gene primers of nonhemolytic enterotoxin NHE (nheA, nheB, nheC), hemolysin Hbl (hblA, hblC, hblD), cytotoxin CytK, enterotoxin T (BceT) and enterotoxin FM (EntFM), detecting virulence genes contained in the bacillus cereus, and positively identifying the bacillus cereus to contain 9 virulence genes of nheA, nheB, nheC, entFM, hblA, hblC, hblD, cytK and BceT by using 1% agarose gel electrophoresis (120V, 90mA); the gene primer sequences are shown in Table 11, and the amplification results are shown in FIG. 7.
TABLE 11 virulence gene PCR primer sequences
(II) amplification of virulence genes of the SN-C strain of Bacillus amyloliquefaciens
A bacterial genome DNA rapid extraction kit (produced by Beijing Etiella biotechnology, inc.) is selected to extract genomes of an SN-C strain of the bacillus amyloliquefaciens and a bacillus cereus (a virulence gene positive control strain), and virulence gene types of the SN-C are amplified and identified by using nheA, nheB, nheC, hblA, hblB, hblC, hblD, cytK and BceT virulence gene primers.
TABLE 12 virulence gene PCR amplification System
Amplification conditions: pre-denaturation at 95 ℃ for 3min, pre-denaturation at 95 ℃ for 30s, pre-denaturation at 58 ℃ for 30s, pre-denaturation at 72 ℃ for 35s,30 cycles, and extension at 72 ℃ for 10min. The results were detected by 1% agarose gel electrophoresis (120V, 90mA) after amplification.
After the virulence genes were amplified by PCR, it was identified that Bacillus amyloliquefaciens SN-C contained only the virulence genes nheA and nheC, and the results are shown in FIG. 8A and FIG. 8B.
2. Drug susceptibility test of Bacillus amyloliquefaciens SN-C strain
Taking overnight cultured strain of Bacillus amyloliquefaciens SN-C (seed solution is inoculated into TSB according to 1% for culture), diluting with 15.0ml sterilized glass test tube, and using McLeod turbidimetric tube No. 3 (9.0 x 10) 8 one/mL) control, dilutedReleased to 9.0x10 6 piece/mL, apply 130. Mu.L with sterilized cotton swab on TSA plate; after the plate is dried, the drug sensitive test paper is clamped by a pair of sterilized tweezers and is placed on a culture plate (approximately three to four pieces of drug sensitive test paper can be placed on a plate with the diameter of 9 cm), and the plate is placed in an incubator at 37 ℃ for 12 hours to observe the result.
The susceptibility test shows that the strain of Bacillus amyloliquefaciens SN-C is sensitive to common penicillin, cephalosporins, quinolones, aminoglycosides and tetracycline antibiotics. The results of the susceptibility testing of the strain of Bacillus amyloliquefaciens SN-C are shown in FIG. 9 and Table 13.
TABLE 13 antibiotic susceptibility test results for the strain of Bacillus amyloliquefaciens SN-C
3. Safety test for mice
And (3) selecting a three-week-old female Kunming mouse to carry out a bacillus amyloliquefaciens SN-C strain virulence feeding test, wherein the test period is a three-day pre-feeding adaptation period and a two-week formal test period. Groups were divided by weight, and the test groups were 5 mice per group, 9 am each day during the test period: 00-10, 00 heavy suspension bacterial liquid 10 with normal saline for intragastric administration 9 Each of CFU, the blank control group was gavaged with physiological saline equal in volume to the test group, and the mice were weighed every 3 days, and the mental state and health condition of the mice were observed and recorded every day; at the end of gavage, mice were removed and subjected to autopsy, and internal organs were normal.
TABLE 14 mouse safety test
Through a mouse feeding test, compared with a blank control, the weight increase of a test group in the whole test period has no obvious difference, and meanwhile, the mouse has the phenomena of normal mental state, good fur and appearance, no diarrhea, death and the like, so that although the bacillus amyloliquefaciens SN-C strain has a few virulence genes, the bacillus amyloliquefaciens SN-C strain has no toxic action on the mouse, and the bacillus amyloliquefaciens SN-C strain can be inferred to be safe and available probiotics.
Example 2: application of the Bacillus amyloliquefaciens SN-C strain in corn silage
1. Procedure for corn silage test
After the bacterial liquid with the serial numbers of A, SN-C, E and CO4 is placed in a shaking table at 37 ℃ and 200rpm/min for 24 hours, the viable bacteria count is calculated (about 2.3x10 8 CFU/mL), packaging 500 g/bag of corn silage without rods by using a vacuum sealing machine, adding no bacteria solution into a blank control, and adding 10 mL/bag of test group bacteria solution (namely adding 10 percent) 9 CFU/bag), repeating three bacteria, placing at room temperature for storage and fermentation for 45d, and detecting the residual content of the coarse fiber of the silage. The package of the corn silage test is shown in figure 10
2. Crude fiber measuring method (Filter bag method)
Table 15NDF reagents: neutral detergent (3% sodium dodecyl sulfate)
Note: accurately weighing 18.61g of Na 2 EDTA and 6.81g sodium tetraborate into a 1.0L beaker, adding a small amount of distilled water, heating to dissolve, then adding 30.0g sodium dodecyl sulfate and 10.0mL ethylene glycol ethyl ether, accurately weighing 4.56g anhydrous Na 2 HPO 4 Adding a small amount of distilled water into the other beaker, slightly heating to dissolve, pouring into the first beaker, uniformly mixing, and then adding into a 1000.0mL volumetric flask to reach a constant volume of 1.0L.
ADF reagent: preparation of acidic detergent (2% cetyl trimethyl ammonium bromide)
20.0g of cetyltrimethylammonium bromide was weighed out and dissolved in 1000.0mL of 1.0mol/L sulfuric acid solution, and the solution was stirred and dissolved, and heated if necessary.
The bag opening treatment:
unsealing scissors, uniformly mixing, putting a feed sample of about 5.0g into a 100mL beaker, adding 45.0mL of water, uniformly stirring, soaking for 5min, and measuring the pH value of the solution by using a pH meter. The measured pH values are all between 3.78 and 4.2, which proves that the micro-silage effect is good; reserve 100g of sample and place in a refrigerator at-80 ℃.
Placing a certain mass of silage sample in a 500g beaker, placing the beaker in a 65 ℃ oven to dry until the total mass is constant, and subtracting the mass of the beaker to obtain the mass of dry matter. Taking all samples dried at 65 ℃, putting the samples into a crusher for crushing, sieving all the samples with a 40-mesh sieve after crushing, and collecting the samples in a self-sealing bag for the next measurement.
(II) Neutral Detergent Fiber (NDF) measurement
Drying the filter bag: marking a pencil, drying for 1h at 105 ℃, taking out, placing in a dryer, cooling for 30min, and weighing (one ten thousand balance);
weighing a sample: weighing 0.5g of sample in a filter bag (the sample is dried in a 65 ℃ oven to constant weight), weighing two bags of sample for repeated measurement, accurately recording the mass, and sealing by using a vacuum sealing machine (the sample can be stored at room temperature without immediate measurement);
fat removal: soaking the filter bag in acetone or petroleum ether for about 3h to remove fat, and replacing the filter bag with a liquid after 1h;
drying: taking out, naturally drying in the air (about 1h in a fume hood), and then putting into a 105 ℃ oven to dry to constant weight;
taking out after drying, placing in a beaker, adding 100mL of NDF (neutral detergent fiber) detergent into each filter bag, adding 1000 units of high-temperature alpha-amylase into each 100mL of solution, adding 3-5 drops of n-octanol, placing on an electric furnace to boil within 10min, and then slightly boiling for 1h (note: the filter bag is pressed by a flat dish and is completely immersed under the solution);
taking out, and washing with 70 deg.C distilled water for 5-6 times until pH is about 7.0;
after washing, placing the NDF in an acetone solution for soaking for 3-5min, naturally drying the NDF in the air, drying the NDF in an oven at 105 ℃ to constant weight, and accurately weighing and recording the mass after cooling to obtain the NDF mass.
(III) Acid Detergent Fiber (ADF) measurement
Putting the filter bags subjected to NDF measurement into beakers, adding 4 filter bags into each beaker, adding 100mL of ADF (acid detergent fiber) detergent into each filter bag, adding 3-5 drops of n-octanol, and placing the filter bags on an electric furnace to boil within 10min so as to slightly boil for 1h;
taking out, and washing with 70 deg.C distilled water for 3-4 times until pH is about 7.0;
and (3) after washing, placing the powder in an acetone solution for soaking for 3-5min, naturally airing, drying in an oven at 105 ℃ to constant weight, cooling, accurately weighing and recording the mass to obtain the ADF mass.
Method for calculating content of hemicellulose and cellulose
The cellulose content is within 10%, the allowable relative deviation is 0.4%, the cellulose content is more than 10%, and the allowable relative deviation is 4%.
The relative average deviation of A and B is calculated as | A- (A + B)/2 |/(A + B)/2;
calculating the content of hemicellulose:
hemicellulose (%) = NDF (%) -ADF (%)
Calculating the cellulose content:
cellulose = ADF (%) -weight of residue after 72% sulfuric acid treatment (%)
The cellulose and hemicellulose residual amounts were determined by corn silage and the results are shown in figures 13-16. After SN-C is added into corn silage, the difference of the residual amount of NDF and the blank group is obvious (P is less than 0.05), and the content of NDF is reduced by about 5.1% (see figure 13); the ADF residual was very different from the blank group (P < 0.01), reducing ADF content by about 3.5% (see fig. 14); the cellulose residual amount was very different from the blank group (P < 0.01), reducing the cellulose content by about 3.1% (see fig. 15); the amount of remaining hemi-cellulose was not significantly different from the blank group, and hemicellulose reduction was not significant, probably due to group-to-group differences, but a tendency toward hemicellulose reduction was still seen, with a reduction of about 1.75% (see fig. 16). Analysis of the results shows that SN-C can significantly reduce the NDF and ADF content of corn silage, and the NDF content is reduced by about 5.1%. And the total reduction of cellulose and hemicellulose was 4.85%, it can be considered that the strain of bacillus amyloliquefaciens SN-C of the present invention reduces NDF and ADF by reducing cellulose and hemicellulose in corn silage.
Reference to the literature
1. Von willebrand ruminants nutrition [ M ] scientific press, 2004, beijing;
2. optimization of technological parameters of composite ecological bacteria solid fermentation of cassava dregs and influence of mixed bacteria fermentation on nutrition and quality of the cassava dregs [ J ]. China livestock-raising journal 2016;52 (5) 55-59;
3. research on conditions and characteristics of fermentation of straw stalks by Zhang Yang, cytophaga sporogenes, bacillus subtilis and Candida utilis [ D ]. Hunan agriculture university, 2008;
4. eryanglan et al, influence of different combinations of ensiling bacteria, cellulolytic enzymes and carbon sources on the quality of ensiling fermentation of rice straws [ J ] animal Nutrition bulletin, 2019,31 (04): 489-497;
5. lejaya, lejaina, queen incense, etc. Bacillus amyloliquefaciens MN-8 degrades lignocellulose of corn stalks [ J ] application ecology report 2015,26 (5): 1404-1410;
6. royal red plum, etc. influence of different compatible enzyme preparations on growth performance and nutrient digestibility of meat sheep [ J ] chinese agricultural science, 2016, 49 (24): 4806-4813;
7. zhang Chong Yu, etc. a method for quickly measuring the contents of crude fiber, NDF, ADF and ADL in feed [ J ] Shandong zookeeper, 2015,000 (009): 20-22;
8. comparison of sample degreasing and non-degreasing treatments during determination of crude fiber content by Ankom filter bag method [ J ] heilongjiang stockbreeding veterinarian, 2017 (8): 275-277;
9.MuckR.E.,2,3E.M.G.Nadeau,T.A.McAllister,F.E.Contreras-Govea,M.C.Santos,and L.KungJr.Silagereview:Recent advances and future uses of silage additives J.DairySci.101:3980–4000；
10.LiF,DingZ,KeW,etal.Ferulic acidesterase-producing lacticacid bacteria and cellulase pretreatments of cornstalk silage at two different temperature:Ensiling characteristics,carbohydrates composition and enzy matics accharification[J].Bioresource Technology,2019,282:211-221；
11.CreeveyCJ,KellyWJ,HendersonG,etal.Determining the culturability of the rumen bacterial microbiome[J].Microbial Biotechnology,2014,7(5)；
12.V.Chanthakhouna M.Wanapata P.Kongmunb A.Cherdthonga Comparison of ruminal fermentation characteristics and microbial population in swamp buffalo and cattle Livestock ScienceVolume143,Issues2–3,February 2012,Pages172-176；
13.Zahiroddini,H.,etal.(2004)."Effect of aninoculant and hydrolytic enzymes on fermentation and nutritive value of whole cropbarley silage."Animal Feed Science and Technology 117(3-4):317-330；
14.DenisO,Krause,StuartE,Denman,RoderickI,Mackie,Mark,Morrison,AnnL,Rae,GraemeT,Attwoo d,ChristopherS,McSweeney.Opportunities to improve fiber degradation in the rumen:microbiology,ecology,andgenomics.[J].FEMS；
15.Muhammed A.Arowolo,Jianhua He.Use of probiotics and botanical extracts to improve ruminant production in the tropics:Areview[J].Animal Nutrition,2018,4(03):241-249；
16.GR,Ghorbani,DP,Morgavi,KA,Beauchemin,JAZ,Leedle.Effects of bacterial direct-fed microbials on ruminal fermentation,blood variables,and the microbial populations of feed lot cattle.[J].Journa lof animal science,2002,80(7):1977-85；
17.NithyaV,MuthukumarSP,HalamiPM.SafetyAssessment of Bacillusl icheniformis Me1 Isolated from Milk for Probiotic Application[J].International Journal of Toxicology,2012,31(3):228-237；
18.LengowskiMB,WitzigM,MHringJ,etal.Effects of corn silage and grass silage in ruminant rations on diurnal changes of microbial populations in the rumen of dairycows[J].Anaerobe,2016,42:6-16。
sequence listing
<110> university of agriculture in Huazhong
<120> screening and application of buffalo rumen source bacillus amyloliquefaciens
<141> 2020-08-17
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1449
<212> DNA
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<220>
<221> gene
<222> (1)..(1449)
<400> 1
catgcggcgt gctatacatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta 60
gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120
aaccggggct aataccggat ggttgtttga accgcatggt tcagacataa aaggtggctt 180
cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 240
caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360
gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga 420
acaagtgccg ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa 480
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540
taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 600
ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660
gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720
tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840
ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt 1020
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200
gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat 1260
ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct ttatggagcc agccgccgaa 1440
gtgacagat 1449
Claims (1)
1. The preservation number is CCTCC NO: m2020137 Bacillus amyloliquefaciens (B.amyloliquefaciens) (B.amyloliquefaciens)Bacillus amyloliquefaciens) Application of SN-C strain in reducing cellulose and hemicellulose in corn silage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010824185.3A CN111996140B (en) | 2020-08-17 | 2020-08-17 | Screening and application of buffalo rumen source bacillus amyloliquefaciens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010824185.3A CN111996140B (en) | 2020-08-17 | 2020-08-17 | Screening and application of buffalo rumen source bacillus amyloliquefaciens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111996140A CN111996140A (en) | 2020-11-27 |
| CN111996140B true CN111996140B (en) | 2023-04-07 |
Family
ID=73473889
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010824185.3A Expired - Fee Related CN111996140B (en) | 2020-08-17 | 2020-08-17 | Screening and application of buffalo rumen source bacillus amyloliquefaciens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111996140B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399156A (en) * | 2016-08-25 | 2017-02-15 | 中国热带农业科学院热带生物技术研究所 | Bacillus amyloliquefaciens subsp.plantarum and application thereof to scagassum biodegradation |
-
2020
- 2020-08-17 CN CN202010824185.3A patent/CN111996140B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399156A (en) * | 2016-08-25 | 2017-02-15 | 中国热带农业科学院热带生物技术研究所 | Bacillus amyloliquefaciens subsp.plantarum and application thereof to scagassum biodegradation |
Non-Patent Citations (2)
| Title |
|---|
| 西藏地区牦牛瘤胃中兼性厌氧纤维素降解菌的分离鉴定;李君风等;《草业学报》;20170620(第06期);第181页表3，第182页倒数第1段 * |
| 解淀粉芽孢杆菌高产纤维素酶菌株的筛选与鉴定;何深宏等;《福建农业学报》;20200715(第07期);第781-787页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111996140A (en) | 2020-11-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108587957B (en) | Screening and application of probiotic bacillus licheniformis capable of highly producing complex enzyme | |
| CN108208316B (en) | Siamese bacillus-containing feed | |
| CN111996139B (en) | Composite microecological preparation for improving production benefit of meat calves and application thereof | |
| CN103865846A (en) | Enterococcus faecium and preparation method thereof | |
| CN112662599B (en) | Poultry source Bacillus belgii CL-4 and application thereof | |
| CN111304119B (en) | Feeding bacillus subtilis for degrading fumonisins and application thereof | |
| CN110878265B (en) | Bacillus subtilis for degrading aflatoxin and application thereof | |
| CN113604404B (en) | Bacillus coagulans YSF17 and application thereof | |
| CN113215018A (en) | Lactobacillus plantarum subspecies and application thereof in mixed fermentation of soybean meal and artemisia vulgaris | |
| CN115418337B (en) | Lignin degrading bacterium and application thereof in rice straw micro-storage | |
| CN116179440B (en) | Bacillus gallinarum and application thereof | |
| CN113621533A (en) | Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof | |
| CN112314782A (en) | Compound microbial preparation for fermented feed and fermented puffed pellet feed | |
| CN107338203B (en) | Enterococcus faecalis for feed, microecological preparation for preventing and treating heat stress of livestock and poultry and preparation method thereof | |
| CN107603901B (en) | Bacillus subtilis for producing aflatoxin B1 degrading enzyme and application thereof | |
| CN113073064B (en) | Bacterial strains capable of efficiently degrading cellulose, silage bacterial agent and application of silage bacterial agent | |
| CN112625972A (en) | Bacillus amyloliquefaciens and fermentation product and application thereof | |
| CN111676153B (en) | Lactobacillus plantarum and application thereof in aquatic product fermented feed | |
| CN112877231B (en) | Lactic acid bacteria with antibacterial and antioxidant activities and application thereof | |
| CN111996140B (en) | Screening and application of buffalo rumen source bacillus amyloliquefaciens | |
| CN113151032A (en) | Bacillus subtilis with efficient gossypol degradation capability and application thereof | |
| CN114181867B (en) | Lactobacillus plantarum, and product and application thereof | |
| CN114480206B (en) | High-temperature-resistant enterococcus faecalis and preparation method and application thereof | |
| CN114854629A (en) | Lactobacillus plantarum C-37 and application thereof in fermenting corn germ meal | |
| CN112011484A (en) | Screening and application of probiotic bacillus |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20230407 |