CN117106676A - Bacillus subtilis and application thereof in feed production - Google Patents
Bacillus subtilis and application thereof in feed production Download PDFInfo
- Publication number
- CN117106676A CN117106676A CN202311376288.8A CN202311376288A CN117106676A CN 117106676 A CN117106676 A CN 117106676A CN 202311376288 A CN202311376288 A CN 202311376288A CN 117106676 A CN117106676 A CN 117106676A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- strain
- powder
- fermentation
- soybean meal
- Prior art date
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- Granted
Links
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 108
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 108
- 238000004519 manufacturing process Methods 0.000 title abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 32
- 235000019764 Soybean Meal Nutrition 0.000 claims abstract description 18
- 239000004455 soybean meal Substances 0.000 claims abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims description 34
- 230000004151 fermentation Effects 0.000 claims description 34
- 230000001580 bacterial effect Effects 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 15
- 241001465754 Metazoa Species 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 9
- 238000001694 spray drying Methods 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 6
- 239000003674 animal food additive Substances 0.000 claims description 5
- 244000144972 livestock Species 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 229920002261 Corn starch Polymers 0.000 claims description 3
- 235000019733 Fish meal Nutrition 0.000 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 239000008120 corn starch Substances 0.000 claims description 3
- 239000013530 defoamer Substances 0.000 claims description 3
- 239000004467 fishmeal Substances 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 239000003223 protective agent Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000000643 oven drying Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 abstract description 16
- 230000015556 catabolic process Effects 0.000 abstract description 12
- 238000006731 degradation reaction Methods 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 108010059892 Cellulase Proteins 0.000 abstract description 8
- 229940106157 cellulase Drugs 0.000 abstract description 8
- 241000588724 Escherichia coli Species 0.000 abstract description 7
- 241000282887 Suidae Species 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 235000021053 average weight gain Nutrition 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 description 21
- 238000012360 testing method Methods 0.000 description 17
- 230000000844 anti-bacterial effect Effects 0.000 description 12
- 239000003833 bile salt Substances 0.000 description 12
- 238000000926 separation method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 9
- 210000003608 fece Anatomy 0.000 description 8
- 238000004321 preservation Methods 0.000 description 8
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 8
- 239000010871 livestock manure Substances 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 210000004051 gastric juice Anatomy 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 235000010288 sodium nitrite Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000004088 simulation Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 235000021050 feed intake Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000004211 gastric acid Anatomy 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 239000007818 Grignard reagent Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000404975 Synchiropus splendidus Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000001420 bacteriolytic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 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 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000004795 grignard reagents Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- 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
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- 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
-
- 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
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- 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
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- 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
-
- 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
-
- 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)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Birds (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Sustainable Development (AREA)
- Botany (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The novel bacillus subtilis BS-22 strain is separated from a pig farm, has higher cellulase production capacity, has stronger inhibition effect on staphylococcus aureus, escherichia coli K88 and K99 strains, is excellent in nitrite degradation, and has excellent heat resistance. After the BS-22 strain is used for fermenting soybean meal, proteins can be fully decomposed, and the proteins can be degraded into small peptide components with better absorption and utilization effects, and the small peptide content in the fermented soybean meal is more than or equal to 22.4%; the bacillus subtilis BS-22 strain powder is added on the basis of basic ration, so that daily average weight gain of the nursery pigs can be improved, and feed conversion ratio can be reduced.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a bacillus subtilis strainBacillus subtilis) And to the specific use thereof in feed production, for example as a starter, microecological preparation, etc.
Background
Bacillus subtilis @Bacillus subtilis) Also known as Bacillus subtilis, is a class of aerobic, endogenous stress-resistant spores of the rod-shaped gram-positive speciesThe sexual bacteria have no pathogenicity and can secrete enzymes such as amylase, protease, lipase, cellulase and the like. Bacillus subtilis widely exists in soil and plant body surfaces, can form spores, has good stress resistance and good stability in a spore state, can survive for about 20 minutes under high-temperature treatment at 120 ℃ generally, and can also survive in an acidic environment, so that the bacillus subtilis can keep higher activity in livestock and poultry breeding production and application, and belongs to a high-efficiency microecological preparation strain. In addition, researches show that bacillus subtilis produces a bacteriolytic substance and has antibacterial activity; the method can also grow nutrition cells in the intestinal tracts of livestock and poultry, and can be combined with intestinal mucosa sites for field planting to form biological barriers so as to effectively prevent the adsorption and growth of pathogenic microorganisms; by producing various digestive enzymes such as protease, lipase, amylase, etc., the conversion rate of the feed can be improved, and the growth of animals can be promoted. In recent years, researches show that the microecological preparation can rapidly degrade, adsorb and convert organic pollutants, nitrogen, phosphorus and the like in a water body, wherein bacillus subtilis can decompose animal excreta, residual feed, nitrate, phosphate and the like into micromolecular organic acid and amino acid, provide nutrition for algae in the water body, prevent eutrophication of the water body and maintain ecological balance of the water body; the concentration of harmful gases such as ammonia, indole and the like in the excrement can be effectively reduced, the odor of the excrement is lightened, the metabolic products of the excrement can inhibit the growth and reproduction of mosquitoes and flies, the culture environment is improved, and the recycling of the excrement is facilitated.
At present, the bacillus subtilis is widely applied in feed production, the application range of the bacillus subtilis in feed production and aquaculture is actively expanded by separating and obtaining new strains with better performance, the bacillus subtilis C-1 strain (CGMCC No: 4628) and the bacillus subtilis 1-C-7 strain (CGMCC No: 17348) are sequentially separated and obtained, and the invention patent CN20110116641. X and CN20201161705.6 are applied, wherein the bacillus subtilis has extremely strong antibacterial effect and cellulase production capability, can be used for livestock and poultry cultivation, and the bacillus subtilis is mainly concentrated in the aquatic product field and can be used for improving the health level of liver tissues of aquatic animals and enhancing the disease resistance of mandarin fish. There are also a number of other companies or university research units that have separated and identified bacillus subtilis and explored its use in feed production, and with the introduction and popularization of the "no-resistance" concept of feed, bacillus subtilis separation and use has been mentioned to an unprecedented new height. The bottleneck problem of large-scale application of the bacillus subtilis in the feed industry and the cultivation industry at present is still the separation and screening of excellent strains, so how to screen to obtain the strains which can effectively break through gastric acid and bile barriers, truly enter intestinal tracts to reproduce and exert the probiotics effect, and have strong enzyme production capability, high antibacterial activity and wide application range is the technical problem to be solved at present.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide the bacillus subtilis which has strong stress resistance, excellent probiotics and antibacterial effect and strong nitrite degradation capability, and explores the application of the bacillus subtilis in feed production.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a bacillus subtilis BS-22 strain, which is characterized in that the bacillus subtilis BS-22 strain is stored in the China general microbiological culture Collection center of the China general microbiological culture Collection center, named as BS-22 strain, and classified and named as follows: bacillus subtilis (Bacillus subtilis), accession no: CGMCC No:27370, the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The invention also claims a preparation method of the bacillus subtilis BS-22 strain bacterial powder, which is characterized by comprising the following steps:
step one, fermenting bacillus subtilis BS-22 strain:
(1) Preparing a fermentation medium: corn flour 0.5%, soybean cake powder 1%, sucrose 0.4%, fish meal 0.6%, KH 2 PO 4 0.1％，FeSO 4 .7H 2 O 0.025％，MgSO 4 .7H 2 O 0.05％，MnSO 4 0.024％，CaCO 3 0.1%, defoamer 0.05%, pH about 7.2;
（2）culturing in a fermentation tank: sterilizing at 121deg.C for 30min, cooling to 37deg.C, inoculating seed solution with bacterial age of 12 hr, inoculating 1% of seed solution, maintaining rotation speed of 220rpm during fermentation, ventilating 1:0.3vvm, and culturing at 0.05MPa for 24 hr as fermentation end point, and detecting that the viable count of bacillus subtilis BS-22 strain is not less than 1.0X10% 11 CFU/mL；
Step two, preparing bacterial powder by spray drying: adding 10% w/v corn starch as a protective agent into bacillus subtilis BS-22 strain fermentation broth, uniformly stirring, and then performing spray drying under the following spray drying conditions: the temperature of the air inlet is 160 ℃, the temperature of the air outlet is 85 ℃, and the feeding speed is 3L/h. Collecting to obtain bacillus subtilis dry powder preparation, and detecting that the viable count of bacillus subtilis BS-22 strain in the bacillus powder is more than or equal to 5.0X10 10 CFU/g。
The invention also discloses bacillus subtilis BS-22 strain bacterial powder, which is prepared by the method.
The invention also discloses application of the bacillus subtilis BS-22 strain in preparation of fermented soybean meal.
Further, the fermented soybean meal is prepared by the following method: uniformly mixing soybean meal, bran powder and water according to the mass ratio of 1:0.5:1, sterilizing at 121 ℃ for 15 minutes, cooling, inoculating bacillus subtilis BS-22 strain powder, and uniformly mixing to obtain a fermentation composition, wherein the addition amount of bacillus subtilis is 10 8 CFU/kg, fermenting at 32deg.C for 3 days, oven drying fermented soybean meal, and measuring the content of small peptide (not less than 22.4%).
The invention also claims the application of the bacillus subtilis BS-22 strain as a feed additive, which is characterized in that 0.1 per mill of bacillus subtilis BS-22 strain powder (the viable count is more than or equal to 5.0 multiplied by 10) is added into common daily ration 10 CFU/g) were used for feeding animals.
Further preferably, the animal is a livestock, poultry, ruminant, preferably a nursery pig.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
the novel bacillus subtilis BS-22 strain is separated from a pig farm, has relatively high cellulase production capacity, has a strong inhibition effect on staphylococcus aureus, escherichia coli K88 and K99 strains, is excellent in nitrite degradation, is safe and harmless as proved by a hemolysis test, has excellent heat resistance, can avoid the influence of high-temperature processing on the activity of the strain, and is more suitable for feed additives and feed processing processes. After the BS-22 strain is used for fermenting soybean meal, the soybean meal has relatively strong degradation capability, can fully decompose protein, degrade the protein into small peptide components with better absorption and utilization effects, and the small peptide content in the fermented soybean meal is more than or equal to 22.4%; the bacillus subtilis BS-22 strain bacterial powder is added on the basis of basic daily ration, so that the daily average weight gain of the nursery pigs can be improved and the feed conversion ratio can be reduced under the condition that the average daily feed intake is not changed greatly.
Description of the embodiments
Example 1: separation and screening of bacillus subtilis
1.1 Preliminary isolation of strains:
taking 30 parts of pig manure in pig farm pig manure in Heilongjiang area and Liaoning area, sampling 10 g parts of two-stage AO section sewage, biogas slurry returning soil and other environmental samples, respectively placing the samples in 100ml pig manure simulation culture medium (the pig manure simulation culture medium comprises 10 g/L of peptone, 10 g/L of yeast powder, 10 g/L of sodium acetate, 10 g/L of sodium citrate, 5 g/L of vegetable oil, 5 g/L of glycerol, 5 g/L of carboxymethyl cellulose, 2 g/L of ammonium chloride, 2 g/L of sodium nitrate, 2 g/L of dipotassium phosphate, 0.1 g/L of ferrous sulfate, 0.5 g/L of magnesium sulfate, 0.1 g/L of calcium chloride and 6.8-7.3 of pH), culturing for 5-6 days under the conditions of 25 ℃ and 200 rpm, then taking 10ml liquid culture medium, inoculating the 10 liquid culture medium into the new pig manure simulation culture medium, continuously enriching the liquid, carrying out subculture for further subculture in a large scale laboratory, and carrying out further subculture in Beijing laboratory for further subculture, and further identification by the laboratory, and further carrying out micro-biological culture in the North China.
Diluting enrichment culture solution by 10 times with sterile PBS, shaking, mixing to obtain bacterial suspension, placing into a water bath, heating at 90 ℃ for 15 min, collecting supernatant, centrifuging at 8000rpm for 10min, discarding supernatant, adding thallus precipitate into 10mL of artificial gastric juice (1% pepsin, 0.85% sodium chloride, adjusting pH to 2.0 with HCl solution, filtering, sterilizing, standing at 37deg.C for 2h, centrifuging at 6000rpm for 10min, discarding supernatant, adding thallus precipitate into 10mL of artificial bile salt (adding 0.3% pig bile salt into liquid broth culture medium, and sterilizing under high pressure, and standing at 37deg.C for 4 h). After shaking and mixing uniformly, the mixture is diluted by adopting sterile PBS in a gradient way and then is coated on an LB culture medium, and after culturing for 24 hours at 30 ℃, colonies are purified. 5 suspected bacillus subtilis strains are obtained through co-separation, and then the 5 strains obtained through preliminary separation are continuously separated and purified on an LB culture medium, and are respectively named as BS-05, BS-10, BS-17, BS-22 and BS-28.
1.2 Re-screening of strains
1.2.1 Hemolysis test of bacillus subtilis
The separated 5 strains of bacillus subtilis BS-05, BS-10, BS-17, BS-22 and BS-28 are respectively streaked and inoculated on fresh blood culture medium, and are cultured for 24 hours at 37 ℃, and after observation, no hemolytic ring appears on the 5 strains, which indicates that the separated 5 strains of bacillus subtilis have no hemolytic activity and are safe for animals.
1.2.2 Screening for cellulase-producing ability
The separated and purified strain is inoculated on LB-CMC culture medium of 0.5 percent carboxymethyl cellulose by an inoculating loop, cultured for 24 hours at 30 ℃, congo red is used for dyeing for 1 hour, 1M NaCl solution is used for washing for 1 hour after the dye is removed, the diameter of the transparent loop and the diameter of the colony are measured according to the existence of hydrolysis transparent loop around the colony, the ratio (H/C) of the transparent loop and the diameter of the colony is calculated, and meanwhile, bacillus subtilis C-1 strain (CGMCC No: 4628) and bacillus subtilis 1-C-7 strain (CGMCC No: 17348) obtained by early separation of the company are used as control strains. The specific detection results are shown in the following table 1:
TABLE 1 cellulase production ability test of Bacillus subtilis-transparent circle to colony diameter ratio (H/C)
Based on the test results, the separated BS-17 strain and BS-22 strain of the invention have higher cellulase production capacity, which is equivalent to that of the C-1 strain of the high-yield cellulase separated by the company, wherein the enzyme production capacity of the BS-22 strain is relatively highest.
1.2.3 Antibacterial ability measurement
And detecting the antibacterial activity of the separated bacillus subtilis by taking escherichia coli K88, escherichia coli K99 and staphylococcus aureus as indicator bacteria. Specifically, after the LB solid medium is melted, the solid medium is cooled to about 45 ℃, indicator bacteria culture solution (100 microliter of bacteria solution is added into each 100 milliliters of culture medium) for overnight culture is added, and the mixture is stirred and mixed uniformly, poured into a sterile flat plate, and poured into 15-20mL of each dish. 2 sterilized oxford cups are placed on each flat plate in advance, after agar is solidified, the oxford cups are taken out, and agar holes are formed on the flat plates. To the agar well, 150. Mu.L of the bacteria liquid to be tested was added, 3 replicates were set, the dish cover was covered, carefully transferred to a 37℃incubator for cultivation, the dish was placed in place, and the culture was allowed to stand. After 20 hours of incubation, the dish lid was opened and the diameter of the inhibition zone was measured with a caliper. Specific bacteriostatic effects are shown in table 2 below:
TABLE 2 determination of bacteriostatic Capacity of Bacillus subtilis
Based on the detection results of the antibacterial performance, the BS-17 strain and the BS-22 strain separated by the method have better antibacterial performance, wherein the antibacterial performance of the BS-22 strain is most prominent, the BS-17 strain and the BS-22 strain have strong antibacterial performance on staphylococcus aureus, escherichia coli K88 and escherichia coli K99 strain, and the antibacterial performance of the BS-17 strain and the BS-22 strain on escherichia coli is close to 2 times of that of the C-1 strain.
1.2.4 Nitrite degradation capability test of bacillus subtilis
Zhang Fengfeng (Beinong university journal, 2009, 24 (4): 218-221) and the like report that bacillus subtilis can rapidly reduce nitrate and nitrite in aquaculture water and has the effect of purifying water. The bacillus subtilis is an environmental sample separated from pig manure in a pig farm, a pig manure wastewater temporary storage tank, sewage in a two-stage AO working section, biogas slurry returning soil and the like, and the separation environment of the bacillus subtilis contains a large amount of nitrite, so that the bacillus subtilis has the capability of degrading nitrite by bacillus subtilis BS-17 and bacillus subtilis BS-22. The specific test method is as follows:
(1) Drawing a standard curve: the analytical pure sodium nitrite is weighed to prepare concentration gradient standard solutions, 200 mu L of each standard solution is respectively taken into a 96-well plate, and 20 mu L of Gris reagent A, B is sequentially added into each well. The absorbance at 550nm was measured with a microplate reader. And drawing a standard curve by taking the light absorption value as an abscissa and the sodium nitrite concentration as an ordinate.
(2) Preparing a nitrite degradation culture medium: 80mL of LB liquid medium, 2.5mg of sodium nitrite, adding distilled water to a volume of 1000mL, and subpackaging into 250mL triangular flasks with 100mL of each flask, wherein 5mL of paraffin oil is covered to isolate air. Autoclaving at 121℃for 20min.
(3) Measurement of nitrite degradation ability of the strain: the bacterial liquid cultured overnight is transferred into a nitrite degradation culture medium according to the inoculation amount of 1 percent, and 1ml of fermentation liquid is respectively taken out at the 0h, the 12h, the 24h and the 48h, and the fermentation liquid is centrifuged. 200. Mu.L of the supernatant was pipetted into a 96-well plate, 20. Mu.L each of Grignard reagent A, B was added, and the absorbance was measured at 550 nm. And calculating the concentration of sodium nitrite in the fermentation broth according to a standard curve. The specific results are shown in table 3 below:
TABLE 3 determination of the nitrite degrading Capacity of Bacillus subtilis
Based on the results shown in Table 3, the bacillus subtilis BS-22 strain separated by the method has the strongest nitrite degrading capability, can degrade 95.2% of nitrite in a culture medium within 12 hours, can degrade 98.4% of nitrite within 24 hours, has a degradation rate reaching 99.6% within 48 hours, and has relatively stronger nitrite degrading capability compared with the BS-17 strain and the existing C-1 strain and 1-C-7 strain.
Based on the above experiments, the BS-22 strain obtained by the separation has stronger cellulose degradation capability and antibacterial performance compared with other separated strains, is excellent in nitrite degradation, and is safe and harmless as proved by hemolysis experiments, so that the BS-22 strain is determined as the optimal strain obtained by the separation.
Example 2: identification and preservation (CGMCC No. 27370) of bacillus subtilis BS-22 strain
2.1 morphological and biochemical identification:
the bacillus subtilis BS-22 strain cells are observed to be in a rod shape, 0.9-1.1 mu m multiplied by 1.7-1.9 mu m, are uniformly colored, have no capsules, are formed by flagellum lateral growth and exercise, and are positive in gram staining. The spores are in the shape of elliptic cylinder, 0.6-0.7μm×0.8-1.2 μm. The colony is round, is not flashing and opaque, and after 48 hours of culture, the colony becomes thick and dry, the edge appears irregular, and the surface of the colony appears yellowish.
According to the Berger's bacteria identification handbook and the common bacteria identification handbook, the physiological and biochemical identification test of the bacillus subtilis BS-22 strain is carried out. Specific physiological and biochemical characteristics are shown in Table 4 below.
TABLE 4 physiological and biochemical characteristics of Bacillus subtilis BS-22 strain
Annotation: "+" indicates positive; "-" indicates negative.
2.2 16S rDNA sequencing
The bacterial 16s rDNA universal primer (the upstream primer is 5 '-AGAGTTTGATCCTGGCTCAG-3' -and the downstream primer is 5 '-GGTTACCTTGTTACGACTT-3') is utilized to amplify the bacterial 16s rDNA fragment, the sequence fragment is obtained after sequencing, the obtained sequence is analyzed and compared with the 16s rDNA sequence in Genbank, the result shows that the homology of the bacterium and bacillus subtilis C-1 strain is up to 99.5%, and the separated bacterium is further identified as bacillus subtilis by combining with the physiological and biochemical test result.
2.3 Preservation of Bacillus subtilis BS-22 strain
For the isolated strain, microorganisms were grown in China at 2023, 05 and 18 daysThe common microorganism center of the culture preservation management committee is used for preservation and is named as BS-22 strain, and the classification is named as: bacillus subtilis @Bacillus subtilis) The preservation number is: CGMCC No:27370, the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
Example 3: stress resistance detection of bacillus subtilis BS-22 strain
(1) Activating strains: the preserved bacillus subtilis BS-22 strain is streaked and inoculated on an LB culture medium inclined plane and cultured for 24 hours at 37 ℃.
(2) Seed liquid preparation: the single colony after activation was picked 2-loop inoculated into LB medium, shaking at 37℃and 150rpm for 24 hours.
(3) Preparation of fermentation liquor: inoculating the seed solution into LB culture medium according to 2% of inoculum size, and performing shaking culture at 37 ℃ and 150rpm for 24 hours to prepare bacillus subtilis BS-22 strain fermentation liquor.
3.1 temperature sensitivity test:
the fermentation broth is taken and subjected to heat preservation treatment for 10min at 25 ℃,80 ℃, 85 ℃,90 ℃, 95 ℃ and 100 ℃, the treatment group at 25 ℃ is taken as a control group, the bacterial liquid of each group is subjected to gradient dilution, uniformly coated on an LB solid culture medium plate, and after culturing for 48h at 37 ℃, colony counting is carried out, and the viable count and the survival rate of each treatment group are measured, and each group is repeated for 3 times. The specific results are shown in Table 5 below.
TABLE 5 determination of the heat resistance of Bacillus subtilis BS-22 strain
Based on the results of the table, the bacillus subtilis BS-22 strain obtained by separation has excellent heat resistance, and the survival rate is still up to 97.44 percent and is higher than 95.4 percent reported by the strain C-1 after being treated for 10 minutes at the temperature of 90 ℃; after being treated at 95 ℃, the survival rate is still up to 95.51 percent, and after being treated at 100 ℃ for 10 minutes, the number of residual viable bacteria is still up to 89.26 percent, and the test results show that the bacillus subtilis BS-22 strain obtained by separation has excellent heat resistance, can avoid the influence of high-temperature processing on the activity of the strain, and is more suitable for feed additives and feed processing processes.
3.2 Artificial gastric juice resistance and artificial bile salt test:
the preparation method of the artificial gastric juice comprises the following steps: 1% pepsin, 0.85% sodium chloride, and HCl to adjust pH to 2.0, and bacterial filter is used for filtration and sterilization.
The preparation method of the artificial bile salt comprises the following steps: adding 0.3% or 0.6% pig bile salt (analytically pure) into LB culture medium to obtain artificial bile salt culture medium with different concentrations, and autoclaving.
Taking 1mL of fermentation liquor, adding 9mL of artificial gastric juice with pH=2 as a test group, simultaneously taking 1mL of fermentation liquor, adding 9mL of physiological saline as a control group, standing and culturing for 5 hours at 37 ℃, carrying out gradient dilution on the distribution of the two groups of fermentation liquor, uniformly coating the fermentation liquor on an LB culture medium, and carrying out anaerobic culture for 48 hours at 37 ℃ and counting.
Survival (%) =viable count of artificial gastric juice treated group/viable count of control group×100%.
Taking 1mL of fermentation liquor, adding 9mL of 0.3% or 0.6% bile salt solution, taking 1mL of fermentation liquor, adding 9mL of physiological saline as a control group, standing and culturing for 4 hours at 37 ℃, carrying out gradient dilution on the distribution of the two groups of fermentation liquor, uniformly coating the two groups of fermentation liquor onto MRS culture medium, and carrying out anaerobic culture at 37 ℃ for 48 hours and counting.
Survival (%) = viable count of bile salt treated group/viable count of control group x 100%.
TABLE 6 results of artificial gastric and bile salt tolerance test
Based on the above experiments, the newly separated bacillus subtilis BS-22 strain has good tolerance to artificial gastric acid and bile salt, the survival rate of the newly separated bacillus subtilis BS-22 strain after artificial gastrointestinal fluid treatment is 94.14%, the viability of the newly separated bacillus subtilis BS-22 strain is not greatly influenced by 0.3% of bile salt treatment, the survival rate of the newly separated bacillus subtilis BS-22 strain after 0.6% of bile salt treatment is still more than 90%, and the newly separated bacillus subtilis BS-22 strain has better tolerance compared with the C-1 strain.
Example 4: preparation of bacillus subtilis BS-22 strain bacterial powder
4.1 Fermentation of bacillus subtilis BS-22 strain:
(1) Preparing a fermentation medium: corn flour 0.5%, soybean cake powder 1%, sucrose 0.4%, fish meal 0.6%, KH 2 PO 4 0.1％，FeSO 4 .7H 2 O 0.025％，MgSO 4 .7H 2 O 0.05％，MnSO 4 0.024％，CaCO 3 0.1%, defoamer 0.05%, pH about 7.2.
(2) Culturing in a fermentation tank: sterilizing at 121deg.C for 30min, cooling to 37deg.C, inoculating seed solution with bacterial age of 12 hr, inoculating 1% of seed solution, maintaining rotation speed of 220rpm during fermentation, ventilating 1:0.3vvm, and culturing at 0.05MPa for 24 hr as fermentation end point, and detecting that the viable count of bacillus subtilis BS-22 strain is not less than 1.0X10% 11 CFU/mL。
4.2 And (3) preparing bacterial powder by spray drying:
adding 10% w/v corn starch as a protective agent into bacillus subtilis BS-22 strain fermentation broth, uniformly stirring, and then performing spray drying under the following spray drying conditions: the temperature of the air inlet is 160 ℃, the temperature of the air outlet is 85 ℃, and the feeding speed is 3L/h. Collecting to obtain bacillus subtilis dry powder preparation, and detecting that the viable count of bacillus subtilis BS-22 strain in the bacillus powder is more than or equal to 5.0X10 10 CFU/g。
Example 5: application of bacillus subtilis BS-22 strain in preparation of fermented soybean meal
Uniformly mixing soybean meal, bran powder and water according to the mass ratio of 1:0.5:1, sterilizing at 121 ℃ for 15 minutes, cooling, inoculating bacillus subtilis powder, and uniformly mixing to obtain a fermentation composition, wherein the addition amount of bacillus subtilis is about 10 8 CFU/kg, fermentation at 32℃for 3 days. The blank group is a mixture of soybean meal and water without inoculating bacillus subtilis, the strain adopted by the control group is bacillus subtilis C-1 strain, and the test group is bacillus subtilis BS-22 strain.
And after the fermentation is finished, drying the fermented soybean meal, and measuring the content of the small peptide in the fermented soybean meal. The detection shows that the small peptide content in the blank group is 3.5%, the small peptide content in the control group is about 17.4%, and the small peptide content in the test group is more than or equal to 22.4%. Therefore, the BS-22 strain obtained by separation has relatively strong degradation capability, can fully decompose protein and degrade the protein into small peptide components with better absorption and utilization effects.
Example 6: application of bacillus subtilis BS-22 strain as feed additive
The test is carried out in a pig farm in Heilongjiang area of the company, 2 nursery pigs with equivalent initial weights and half male and female parts are randomly selected in a nursery house for test, the number of each nursery is 20, 1 nursery is set as a control group, and 1 nursery is set as a test group. Test group adds 0.1 per mill bacillus subtilis BS-22 strain powder (viable count is more than or equal to 5.0X10) into common ration 10 CFU/g). The control group is fed with common daily ration without adding strain. The test period is 31 days, the composition of common daily ration is the same, the feeding process is completed by the same breeder, and the weight of each pig before and after feeding is recorded. The specific results are shown in table 7 below:
TABLE 7 feeding results for nursery pigs
Based on the experiment, the bacillus subtilis BS-22 strain bacterial powder is added on the basis of basic daily ration, so that the daily average weight gain of the nursery pigs can be improved and the feed-meat ratio can be reduced without greatly changing the average daily feed intake. Therefore, the bacillus subtilis BS-22 strain bacterial powder can improve the digestion and absorption capacity of nursery pigs, enhance the digestion and conversion capacity of animals on feeds, promote the growth of the animals, remarkably improve the utilization rate of the feeds and improve the economic benefit of cultivation.
Claims (9)
1. A strain of bacillus subtilis (Bacillus subtilis) having a deposit number of: CGMCC No:27370.
2. the method for preparing the bacterial powder of the bacillus subtilis strain according to claim 1, comprising the following steps:
step one, fermenting bacillus subtilis strains:
(1) Preparing a fermentation medium: corn flour 0.5%, soybean cake powder 1%, sucrose 0.4%, fish meal 0.6%, KH 2 PO 4 0.1％，FeSO 4 .7H 2 O 0.025％，MgSO 4 .7H 2 O 0.05％，MnSO 4 0.024％，CaCO 3 0.1%, defoamer 0.05%, pH 7.2;
(2) Culturing in a fermentation tank: sterilizing at 121deg.C for 30min, cooling to 37deg.C, inoculating seed solution with bacterial age of 12 hr, inoculating 1% of seed solution, fermenting at 220rpm, introducing air flow of 1:0.3vvm, and culturing at 0.05MPa for 24 hr to obtain fermentation end point with bacterial count of 1.0X10 or more 11 CFU/mL；
Step two, preparing bacterial powder by spray drying: adding 10% w/v corn starch as a protective agent into bacillus subtilis strain fermentation broth, uniformly stirring, and then performing spray drying under the following spray drying conditions: the temperature of the air inlet is 160 ℃, the temperature of the air outlet is 85 ℃, the feeding speed is 3L/h, the bacillus subtilis dry powder preparation is obtained by collecting, and the number of viable bacteria of the bacillus subtilis in the bacterial powder is more than or equal to 5.0x10 after detection 10 CFU/g。
3. A bacillus subtilis strain powder, characterized in that the powder is prepared by the method of claim 2.
4. Use of the bacillus subtilis strain of claim 1 in the preparation of a feed or feed stock.
5. The use according to claim 4, wherein the feed material is fermented soybean meal.
6. The use according to claim 5, wherein the fermented soybean meal is prepared by the following method: uniformly mixing soybean meal, bran powder and water according to the mass ratio of 1:0.5:1, sterilizing at 121 ℃ for 15 minutes, cooling, inoculating the bacillus subtilis powder, and uniformly mixing to obtain a fermentation composition, wherein the bacillus subtilis is prepared by mixing the bacillus subtilis powder uniformlyThe addition amount of (2) is 10 8 CFU/kg, fermenting at 32deg.C for 3 days, oven drying fermented soybean meal, and measuring the content of small peptide (not less than 22.4%).
7. The use of the bacillus subtilis strain according to claim 1 as a feed additive, wherein the bacillus subtilis strain is added into common daily ration by 0.1 per mill for feeding animals, and the viable count of the bacillus subtilis strain is more than or equal to 5.0 multiplied by 10 10 CFU/g。
8. The use according to claim 7, wherein the animal is a livestock animal.
9. The use according to claim 8, wherein the animal is a nursery pig.
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