CN102329749A - Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis - Google Patents
Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis Download PDFInfo
- Publication number
- CN102329749A CN102329749A CN201110278064A CN201110278064A CN102329749A CN 102329749 A CN102329749 A CN 102329749A CN 201110278064 A CN201110278064 A CN 201110278064A CN 201110278064 A CN201110278064 A CN 201110278064A CN 102329749 A CN102329749 A CN 102329749A
- Authority
- CN
- China
- Prior art keywords
- subtilis
- bacterium powder
- bacillus subtilis
- strain
- enzyme
- 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.)
- Granted
Links
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 16
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 16
- 238000005516 engineering process Methods 0.000 title claims abstract description 16
- 238000009395 breeding Methods 0.000 title claims abstract description 15
- 230000001488 breeding effect Effects 0.000 title claims abstract description 15
- 230000035772 mutation Effects 0.000 title abstract 2
- 239000000843 powder Substances 0.000 claims abstract description 61
- 102000004190 Enzymes Human genes 0.000 claims abstract description 49
- 108090000790 Enzymes Proteins 0.000 claims abstract description 49
- 239000006041 probiotic Substances 0.000 claims abstract description 41
- 235000018291 probiotics Nutrition 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 23
- 230000008569 process Effects 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 63
- 230000001580 bacterial effect Effects 0.000 claims description 55
- 239000007788 liquid Substances 0.000 claims description 40
- 239000002609 medium Substances 0.000 claims description 19
- 241000194017 Streptococcus Species 0.000 claims description 15
- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 8
- 239000006071 cream Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 241000726221 Gemma Species 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 239000002131 composite material Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 229940061607 dibasic sodium phosphate Drugs 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 210000000582 semen Anatomy 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 2
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 claims description 2
- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical compound O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 230000001788 irregular Effects 0.000 claims description 2
- 150000002823 nitrates Chemical class 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 229960003487 xylose Drugs 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 25
- 241001465754 Metazoa Species 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 10
- 230000000529 probiotic effect Effects 0.000 abstract description 5
- 240000001046 Lactobacillus acidophilus Species 0.000 abstract description 3
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 abstract description 3
- 244000057717 Streptococcus lactis Species 0.000 abstract description 3
- 235000014897 Streptococcus lactis Nutrition 0.000 abstract description 3
- 229940039695 lactobacillus acidophilus Drugs 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 3
- 229920002261 Corn starch Polymers 0.000 abstract 1
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 239000008120 corn starch Substances 0.000 abstract 1
- 230000029087 digestion Effects 0.000 abstract 1
- 102000038379 digestive enzymes Human genes 0.000 abstract 1
- 108091007734 digestive enzymes Proteins 0.000 abstract 1
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 41
- 238000012216 screening Methods 0.000 description 16
- 239000000047 product Substances 0.000 description 15
- 108090000145 Bacillolysin Proteins 0.000 description 14
- 102000035092 Neutral proteases Human genes 0.000 description 14
- 108091005507 Neutral proteases Proteins 0.000 description 14
- 108010059892 Cellulase Proteins 0.000 description 11
- 229940106157 cellulase Drugs 0.000 description 11
- 239000000463 material Substances 0.000 description 9
- 238000004659 sterilization and disinfection Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 8
- 244000025254 Cannabis sativa Species 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- 241000287828 Gallus gallus Species 0.000 description 6
- 235000013330 chicken meat Nutrition 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 235000014590 basal diet Nutrition 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 244000144972 livestock Species 0.000 description 5
- 241000272814 Anser sp. Species 0.000 description 4
- 241000193755 Bacillus cereus Species 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 210000003608 fece Anatomy 0.000 description 4
- 230000003031 feeding effect Effects 0.000 description 4
- 210000003736 gastrointestinal content Anatomy 0.000 description 4
- -1 glycase Proteins 0.000 description 4
- 231100000219 mutagenic Toxicity 0.000 description 4
- 230000003505 mutagenic effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241001494479 Pecora Species 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000001079 digestive effect Effects 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 230000000050 nutritive effect Effects 0.000 description 3
- 239000004575 stone Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241001112741 Bacillaceae Species 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000005486 microgravity Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 235000002949 phytic acid Nutrition 0.000 description 2
- 239000000467 phytic acid Substances 0.000 description 2
- 229940068041 phytic acid Drugs 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 201000009032 substance abuse Diseases 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108010011619 6-Phytase Proteins 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000035101 Aspartic proteases Human genes 0.000 description 1
- 108091005502 Aspartic proteases Proteins 0.000 description 1
- 241000193833 Bacillales Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- OTGFEQJKSRFOED-UHFFFAOYSA-N [P].[V].[Mo] Chemical compound [P].[V].[Mo] OTGFEQJKSRFOED-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 101150039403 ams gene Proteins 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000003339 best practice Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002478 diastatic effect Effects 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 206010016256 fatigue Diseases 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
- 238000009472 formulation Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 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
- 210000001630 jejunum Anatomy 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229940085127 phytase Drugs 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 210000004767 rumen Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses Bacillus subtilis bred by a space mutation breeding technology. The strain is named Bacillus subtilis N9-1-35 and was collected in China Center for Type Culture Collection on August 31, 2011, and the collection number is CCTCC M2011301. The test shows that the Bacillus subtilis is an excellent probiotic strain. After entering intestinal tracts of animals, the Bacillus subtilis achieves the effects of a probiotic and can also generate a plurality of kinds of digestive enzymes in the growth and reproduction process of the animals, so that the digestion and absorption of the animals on feeds are improved, the conversion rate of the feeds is improved, and the cost of the feeds is reduced; and the Bacillus subtilis can be used for producing enzyme-producing probiotics, and is a strain which has excellent research and development value. The invention also discloses an enzyme-producing probiotic. The enzyme-producing probiotic consists of Bacillus subtilis powder, Streptococcus lactis powder, Lactobacillus acidophilus powder and corn starch.
Description
Technical field
The present invention relates to subtilis and the application thereof of a strain, belong to biological technical field through the seed selection of space breeding technology.
Background technology
21st century, people are increasingly high to food safety and ecotope requirement, and the feedstuff industry that is in the aquaculture forward position generally causes people's attention.Clearly propose " very urgent " in " the feed industrial development strategic research of 1996-2020 China " of the promulgation of whole nation fodder industry office, substitute these pollution substance best practice and utilize biotechnology exactly with chemosynthesis material abuses such as the alternative feeding antibiotics of Green Product.Probiotics is Modern High-Tech's product of biotechnologyization, is the functional living being fodder additives that probiotic bacterium, zymin, oligose, biological peptide are organically combined.This several fields is the research focus of current safety fodder additives, can realize still can't being substituted fully at present by single product the result of use of feeding antibiotic.In addition; Probiotics has safety, characteristics of high efficiency; In animal and fowl fodder, use; Not only solve the antibiotic remains of animal food, the safety-problems that the abuse situation is brought, and greatly reduced aquaculture communicable disease and pollution on the environment (culture to pollute and become the first source of pollution of China at present).
Characteristics such as that probiotics has is safe, pollution-free, noresidue, preventing disease can significantly improve the production performance of feed conversion rate and animal, are to substitute the first-selected green feed additive of chemosynthesis material such as feeding antibiotic.Probiotics is not only a product, and a complex art is the key that realizes Sustainable development, saving type livestock industry, resolving three rural isssues in the livestock industry field especially.A large amount of uses of probiotics can make Chinese livestock feed industry changed by traditional extensive style " quality, benefit, ecology and Sustainable development " type on Modern Significance, with the competition in world market power that improves China's animal product greatly.
The research and development of fodder enzyme preparation have received the great attention of feed industry and aquaculture; The zymin of having developed at present has cellulase, AMS, aspartic protease, neutral protease, phytic acid plum, zytase, LSD etc. multiple; But because shortcoming such as tolerances such as the high thermal resistance of this type of zymin, acid resistance are relatively poor, period of storage is short; The enzyme that has influenced product is lived; Thereby influenced the feeding effect of this series products, big limitations applying of fodder enzyme preparation, be badly in need of a kind of novel animal microecological formulation of exploitation.
Summary of the invention
To above-mentioned prior art, the invention provides the subtilis of a strain through the seed selection of space breeding technology, bacterial strain of the present invention is from materials such as animal intestinal contents, feces of livestock and poultry, soil; Through technology such as mutagenesis, transgenics; The probiotics bacterial strain of the high enzyme characteristic alive that filters out carries out the research of proof test, fermentation condition to bacterial strain, and carries out compound compatibility; Through animal experiment checking etc., the present invention has developed a kind of new probiotics-product enzyme probiotics.
The present invention realizes through following technical scheme:
One strain is through the subtilis of space breeding technology seed selection; This bacterial strain called after subtilis (Bacillus subtilis) N9-1-35; Be preserved in Chinese typical culture collection center on 08 31st, 2011, its deposit number is: CCTCC M2011301.
The characteristic of said subtilis N9-1-35 is following: the rounded or irregularity of bacterium colony, and the edge is irregular, and there is gauffer, coarse on the surface, and dirty white or little band are yellow, and protuberance is arranged; This bacterium cell size is 0.3 μ m～0.5 μ m * 1.4 μ m～3.0 μ m, is shaft-like, Gram-positive; Gemma is oval, and size is 0.6 μ m～0.9 μ m * 1.0 μ m～1.5 μ m, and sporangiocyst does not expand, and mobility is arranged; Can utilize glucose, wood sugar and pectinose etc., ability hydrolyzed starch, reduction nitrate salt; On the substratum that contains 7% sodium-chlor, can grow, can grow 50 ℃ of following cultivations, the V-P reaction is positive, and does not under anaerobic grow.
The righttest substratum of subtilis N9-1-35 of the present invention is: 0.5% glucose, 1.0% peptone, 0.5% Carnis Bovis seu Bubali cream, 0.5% sodium-chlor are transferred pH to 7.2, the 20min that sterilizes under 115 ℃ of conditions (said percentage ratio is mass percent).
Subtilis N9-1-35 of the present invention comes out through the space breeding technology screening from conventional subtilis, shows through experimental test, and it is very outstanding probiotics bacterial classification.After getting into animal intestinal; Except the function of bringing into play its probiotics, in its growth and breeding process, can also produce multiple digestive ferments such as glycase, neutral protease, cellulase, thereby improve animal digesting and assimilating feed; Improve food conversion ratio; Reduce feed cost, the production that can be used to produce the enzyme probiotics is that a strain has the bacterial strain that research and development are worth.
The method that said production prepares the enzyme probiotics is: subtilis Bacillus subtilis N9-1-35 is through fermentative processing; Obtain fermented liquid; Add W-Gum then and process subtilis bacterium powder through spraying drying; Composite again streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum promptly get and produce the enzyme probiotics.
The concrete steps of said preparation subtilis bacterium powder are following:
(i) bacterial classification: select subtilis Bacillus subtilis N9-1-35 for use;
(ii) slant culture: bacterial classification one ring is inoculated on the solid slant culture base, cultivates 18～24h down at 37 ℃;
(iii) first order seed is cultivated: get above-mentioned cultured inclined-plane, under aseptic condition, get a ring with transfering loop and be inoculated in 50mL～100mL seed liquid nutrient medium, under 37 ℃ of conditions, leave standstill and cultivate 12～16h, make primary seed solution;
(iv) enlarged culturing: with the inoculum size of 2% (v/v), primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 37 ℃ of conditions, leaves standstill and cultivate 8～14h, make secondary seed solution;
(v) fermentor cultivation: with the inoculum size of 2% (v/v), secondary seed solution is connected in the liquid fermentation medium, under 37 ℃ of conditions, aerobic culture 22～28h;
(vi) collect tunning: treat that (v) the gemma rate reaches 90% when above to step, collects fermented liquid;
(vii) in fermented liquid, add 4～8% (mass percent) W-Gum, spraying drying makes the bacterium powder immediately.
Said step is (iii), the seed liquid culture medium prescription in (iv) is: glucose 5g/L, and peptone 10g/L, Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L during use, regulates the 20min that sterilizes under ℃ condition of pH to 7.0～7.5,115; The solid slant culture base of said step in (ii) is to add 1.5～2.0% agar powder in the above-mentioned seed liquid nutrient medium; Said step (the liquid fermentation medium prescription v) is: Semen Maydis powder 60g/L, and dregs of beans 30g/L, Sodium phosphate, dibasic 6g/L, ammonium sulfate 3g/L, calcium chloride 2g/L, pH 7.0～7.5.
The product enzyme probiotics that obtains through method for preparing; Form by subtilis bacterium powder, streptococcus acidi lactici (Streptococcus lactis) bacterium powder, Lactobacterium acidophilum (Lactobacillus acidophilus) bacterium powder and W-Gum; Preferred prescription is: the mass ratio of subtilis bacterium powder, streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum is 6: 2: 1: 21, and the viable count of subtilis, streptococcus acidi lactici and Lactobacterium acidophilum is greater than or equal to 3 * 10 respectively
8Cfu/g, 1 * 10
8Cfu/g and 5 * 10
7Cfu/g.
Description of drawings
Bacterial strain called after subtilis provided by the invention (Bacillus subtilis) N9-1-35; Be preserved in Chinese typical culture collection center on 08 31st, 2011; Its deposit number is: CCTCC M 2011301; The preservation address is: Chinese Wuhan City, Hubei Province Wuhan University, postcode: 430072.
Fig. 1 is a subtilis N9-1-35 and subtilis N enzyme relatively synoptic diagram alive.
Fig. 2-1 is 10 generations of subtilis N9-1-35 inclined-plane and 1 generation enzyme relatively synoptic diagram alive, and wherein A is a neutral protease, and B is a glycase, and C is a cellulase, the U/mL of unit.
Fig. 2-2 is 10 generations of subtilis N9-1-35 inclined-plane and 1 generation viable count comparison synoptic diagram, and lgX is the logarithmic value of viable count.
Fig. 3 is the product enzyme curve of subtilis N9-1-35.
Fig. 4 is the technological process of production figure among the embodiment 4.
Embodiment
Below in conjunction with embodiment the present invention is further described.
The screening of embodiment one, bacterial classification
1, materials and methods
11 samples: ight soil, the bovine rumen content of animals such as soil, withered grass, animal intestinal content, ox, sheep, goose.
1.2 screening culture medium:
Produce the screening culture medium of amylase strain: W-Gum 1.0%, Carnis Bovis seu Bubali cream 0.3%, peptone 0.5%, agar powder 2.0%, pH7.2～7.4,121 ℃, sterilization 30min.
Produce the screening culture medium of neutral protease bacterial strain: soybean protein powder 1.0%, Carnis Bovis seu Bubali cream 0.5%, potassium hydrogenphosphate 0.05%, sal epsom 0.05%, calcium chloride 0.05%, tween-80 0.1%, agar powder 2.0%; PH7.2～7.4; 121 ℃, sterilization 30min.
The screening culture medium of cellulase-producing bacterial strain: CMC 99.5 1%, peptone 0.5%, potassium hydrogenphosphate 0.05%, sal epsom 0.02%, calcium chloride 0.01%, iron(ic)chloride 0.02%, agar powder 2.0%, pH7.2～7.4,121 ℃, sterilization 30min.
The screening culture medium of phytase generating bacterial strain: phytic acid ca 2.0%, peptone 0.5%, potassium hydrogenphosphate 0.1%, sal epsom 0.05%, Repone K 0.05%, calcium chloride 0.002%, ferrous sulfate 0.001%, manganous sulfate 0.001%, agar powder 2.0%; PH7.2～7.4; 121 ℃, sterilization 30min.
1.3 the sample primary dcreening operation: sample is put into beaker, adds the sterile saline of 10 times of amounts, handles 10min, does 10 then for 90 ℃
-1, 10
-2, 10
-3, 10
-4, 10
-5Dilution, each extent of dilution are coated with dull and stereotyped 3 of step, cultivate 24～72h, carry out the screening of various bacterium producing multi enzyme preparations for 37 ℃.Observe the size of transparent circle, calculate transparent circle and colony diameter ratio, the bacterium colony that picking transparent circle and colony diameter are bigger is done pure culture, is stored in the refrigerator subsequent use.
1.4 bacterial classification sieves again: 250mL triangular flask liquid amount 50mL, rotating speed 180rpm, 34 ℃ of temperature are cultivated 24h, survey enzyme and live.Shake bottle and sieve substratum again: ox peptone substratum.
Diastatic mensuration is according to the mensuration of bacterial vigor in " food zymetology principle and analytical procedure "; The mensuration of neutral protease adopts forint-phenol law; The mensuration of cellulase adopts Xylo-Mucine (CMC) saccharification force method, and the mensuration of Sumizyme PHY adopts vanadium molybdenum phosphorus method.
1.5 instrument: UV-2000 ultraviolet spectrophotometer; Thermostat water bath; Incubator; Opticmicroscope; Stopwatch.
2, result
2.1 primary dcreening operation result: see table 1, from 30 samples, be divided into and left 123 bacterial strains,, screened the comparatively ideal bacterial strain of 8 strains altogether through the screening of transparent circle with the diameter ratio.
Table 1 primary dcreening operation result
Annotate: 1 ,-: expression does not detect; 2, BL
0: be the bacillus cereus of from soil, screening; 3, BL
1: be the Bacillus licheniformis of from soil, screening; 4, Z
6: be the subtilis that from withered grass, filters out; 5, Z
8: be the bacillus pumilus that from withered grass, filters out; 6, Y: be the subtilis that from the sheep excrement, filters out; 7, N: be the subtilis that from cow dung, filters out; 8, M: be the subtilis that from stalk, filters out; 9, E: be the subtilis that from the goose excrement, filters out.
2.2 sieve the result again: see table 2, the N bacterial strain produces that the ability of 4 kinds of enzymes is best, and the E bacterial strain takes second place, and the two is all much higher than withered grass type strain, DM423,8501 enzymatic productivity.
Table 2 sieves the result again
Annotate: 1 ,-: expression does not detect; 2, BL
0: be the bacillus cereus of from soil, screening; 3, BL
1: be the Bacillus licheniformis of from soil, screening; 4, Z
6: be the subtilis that from withered grass, filters out; 5, Z
8: be the bacillus pumilus that from withered grass, filters out; 6, Y: be the subtilis that from the sheep excrement, filters out; 7, N: be the subtilis that from cow dung, filters out; 8, M: be the subtilis that from stalk, filters out; 9, E: be the subtilis that from the goose excrement, filters out.10, KB: be the withered grass type strain, available from DSMZ of Microbe Inst., Chinese Academy of Sciences; 11, DM423: be the bacillus cereus of the Kang Bai of Dalian Medical College professor seed selection; 4,8501: the bacillus cereus of awarding seed selection for what bright Puritanism of Sichuan Agricultural University.
The evaluation of embodiment two, bacterial classification and proof test
1, materials and methods
11 bacterial classifications: the mikrobe that separation obtains from samples such as cow dung and goose excrement filters out 2 strains altogether and produces the higher bacterial strains of enzymic activity ability such as neutral protease behind primary dcreening operation, multiple sieve, and numbering is respectively N and E.
1.2 the routine biochemistry of bacterium is identified: except that specifying, gross morphology, the test of Physiology and biochemistry proterties are all with reference to " uncle's Jie Shi Bacteria Identification handbook (the 9th edition) and " microbiology laboratory manual " is carried out.
1.3 proof test: with different concns (1 * 10
8Cfu/mL, 1 * 10
9Cfu/mL, 1 * 10
10Cfu/mL, 1 * 10
11Cfu/mL) the N bacterial strain and the fermented liquid of E bacterial strain carry out abdominal injection to small white mouse, 10 every group, observe death condition and the clinical symptom of seeing small white mouse 1 week continuously.
2, result
2.1 the evaluation of bacterial classification: through the biochemical reaction of bacterium, N bacterial strain and E bacterial strain meet the characteristic of subtilis fully, and preliminary judgement is a subtilis, and the result sees table 3.
Table 3 biochemical reaction result
Annotate :+: positive-: negative d: variable
2.2 proof test: the proof test through small white mouse can know, death condition does not all appear in blank control group and four concentration group N and E bacterial strain small white mouse, and except 1 * 10
11Cfu/mL group has utmost point discrete outside preceding 2 days lassitudes, and all the other all show well.The security of N bacterial strain and E bacterial strain that screening is described is good, is suitable for doing the alternative bacterial classification of follow-up test.
The seed selection of embodiment three, N bacterial strain mutagenesis bacterial classification
1, material and method
11 bacterial classifications: subtilis N bacterial strain
1.2 substratum
1.2.1 liquid seed culture medium
Peptone 10g, sodium-chlor 5g, Carnis Bovis seu Bubali cream 5g, water 1000mL, pH7.2.121 ℃, sterilization 30min.
1.2.2 liquid fermentation medium
Glucose 5g, peptone 10g, sodium-chlor 5g, Carnis Bovis seu Bubali cream 5g, water 1000mL, pH7.2.
Loading amount: the bottled 100mL of 500mL triangle, 121 ℃ of sterilization 30min.
1.3 enzyme activity determination method and instrument are with embodiment one.
1.4 mutafacient system:
The 18th st recoverable science and technology satellite of lift-launch satellites transmits in Chinese Jiuquan; Orbital motion through 18 days; Under particular surroundingss such as space microgravity, severe radiation, high energy particle, alternating magnetic field, make the sudden change of bacterial strain producer, at last strains separation screening to returning.
2 contents of the test and result
2.1 space breeding
The strain of starting is the subtilis N bacterial strain of the sharp next biological research institute of Shandong Bora seed selection.This bacterial strain carries the 18th st recoverable science and technology test satellite; Launch by No. 2 launch vehicle of the Long March in Chinese Jiuquan Satellite Launch Center 15: 20 on the 3rd November in 2003; Orbital motion through 18 days; Return capsule reclaims in the middle area, Sichuan 10: 1 on the 21st November in 2003 and lands, and return capsule is transported to Beijing space technology development and test centre morning on November 23rd, 2003.Fetched biological study institute of company December 6.Subtilis N bacterial strain morphs under the effect of microgravity, severe radiation, high energy particle, alternating magnetic field etc. at space.Then, the contriver has just begun the work of space breeding technology bacterial screening.
2.1.1 the primary dcreening operation of mutagenic strain
Undertaken by conventional primary dcreening operation program, the result sees table 4 ratio of colony diameter (transparent circle with).
Table 4 primary dcreening operation result
Can know by test-results that through space breeding, the variant of the N bacterial strain that filters out produces that enzyme performance is most to descend, and has only only a few to raise, obtain mutagenic fungi N1-1-4 and N9-1-35 that two strain transparent circles and the ratio of colony diameter are higher than starting strain.
2.1.2 the multiple sieve of mutagenic strain
Adopt liquid triangular flask warp to cultivate 24h for 37 ℃, measure neutral protease, glycase, cellulase, subtilis primary dcreening operation bacterial classification is carried out three tests (getting average), last, select enzyme best bacterial strain alive, the result sees table 5.
Table 5 sieves the result again
Can know that by test-results the neutral protease of mutagenic fungi N9-1-35, glycase, cellulase are higher than the strain N that sets out all far away, be set out 3.7 times, 2 times of strain N, more than 7 times, so selected N9-1-35 is as the alternative bacterial classification of follow-up test.
2.2 the evaluation of genus bacillus N9-1-35
Method is seen the bacterium routine biochemistry evaluation of embodiment 2 1.2.
Through identifying that N9-1-35 belongs to: bacterium circle (Bacteria), Firmicutes (Firmicutes), genus bacillus guiding principle (Bacilli); Genus bacillus order (Bacillales); Bacillaceae (Bacillaceae), bacillus (Bacillus), called after subtilis (Bacillus subtilis) N9-1-35; Be preserved in Chinese typical culture collection center on 08 31st, 2011, its deposit number is: CCTCC M 2011301.
2.3 the cultural characters of subtilis N9-1-35
No pod membrane, all flagellums can move.Gram-positive microorganism, gemma 0.6 μ m-0.9 μ m * 1.0 μ m-1.5 μ m, oval to column, position thalline central authorities or inclined to one side slightly, gemma forms the back thalline and does not expand.The bacterium colony surface irregularity is opaque, dirty white or little yellow, and when growing in the liquid medium within, the normal wrinkle mould that forms.Aerophil.
2.4 the enzyme of subtilis N9-1-35 system and viable count analysis
The bottled liquid fermentation medium 100mL of 500mL triangle, sterilization, cooling, inoculation, 37 ℃ of constant temperature culture 24h survey neutral protease, glycase, cellulase and viable count.Simultaneously, with the bacillus subtilis N that starts as controlled trial.The result sees table 6 and Fig. 1.
The enzyme of table 6 triangular flask liquid fermentation liquid is lived and viable count (three test MV)
The variant N9-1-35 that obtains through space breeding compares with original strain N, and triangular flask liquid lab scale neutral protease, glycase, cellulase and viable bacteria number average have raising significantly.
See that from above test-results subtilis N9-1-35 compares with the strain N that starts, the neutral protein enzymic activity has improved 23.7%, and cellulase activity has improved 47.2%, and the work of glycase enzyme has improved 82.5%, and viable count has improved 65.2%.
2.5 the genetic stability of subtilis N9-1-35
The bottled liquid fermentation medium 50mL of 250mL triangle; Sterilization, cooling; To encircle in the liquid triangular flask through go down to posterity 10 times subtilis N9-1-35 inoculation 1 of inclined-plane, 37 ℃ of constant temperature culture 24h survey neutral protease, glycase, cellulase and viable count.Simultaneously, with the subtilis N9-1-35 in 1 generation as controlled trial.The result sees table 7 and Fig. 2-1,2-2.
Table 7 subtilis N9-1-3510 generation and the alive and viable count of 1 generation enzyme
Comprehensive above test-results can be found out: subtilis N9-1-35 goes down to posterity 10 times through the inclined-plane, and it produces enzyme performance, the viable bacteria number average remains on higher level, this bacterial strain be a strain very the production of ideal product enzyme probiotics use bacterial strain.
2.6N9-1-35 the enzyme activity determination of the different cultivation stages of bacterium
250mL triangular flask liquid amount 50mL, rotating speed is 200rpm, 37 ℃ of temperature are lived respectively at the glycase and the neutral protease enzyme of 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 24h sampling and measuring fermented liquid.The result sees table 8 and Fig. 3.
The enzyme of table 8 different fermentations time is lived
Can know that by test-results N9-1-35 reaches peak at fermentation 18h left and right sides neutral protease and glycase.
2.7 feeding experiment
2.7.1N9-1-35 confirming of the best feeding concentration of bacterial strain:
2.7.1.1 material
Test strain: the subtilis N9-1-35 that process mutagenesis obtains;
Experimental animal: the young cock of the extra large blue brown egg of 1 age in days, livestock corporation buys by Eastern Mountain, Tai.Basal diet is seen table 9.
Table 9 basal diet prescription
2.7.1.2 method
N9-1-35 strain fermentation, centrifugal, drying are processed the bacterium powder.The bacterium powder that in feed, adds different concns carries out feeding experiment, and subtilis N9-1-35 bacterium powder final concentration is respectively 5 * 10 in the interpolation Preblend
4Cfu/g, 1 * 10
5Cfu/g, 2 * 10
5Cfu/g, 5 * 10
5Cfu/g, 1 * 10
6Cfu/g, 2 * 10
6Seven groups of cfu/g.Daily administration is normally carried out.
Raised in advance 3 days, and after weighing 350 young cocks were divided into seven groups at random, 50 every group.3 weeks of trial period, claim after the off-test that chicken is heavy, feed consumption rate, calculate feedstuff-meat ratio.
2.7.1.3 growth performance index result: see table 10
The best feeding concentration of table 10 bacterial strain N9-1-35
Can find out that from table 10 the 2nd, 3,4,5,6 group feedstuff-meat ratio has reduced by 4.26%, 9.69%, 8.14%, 11.6% than the 1st group (control group) respectively, wherein to have added N9-1-35 bacterium powder 5 * 10
5The cfu/g feed is best, and the excessive concentration feeding effect descends on the contrary.
2.7.2 produce of the influence of enzyme probiotics to growing pig enteron aisle enzymic activity
2.7.2.1 materials and methods
(1) produces the enzyme probiotics
Form by N9-1-35 bacterium powder, streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum, by the development and production of biological study institute of the sharp next biological Engineering stock Co., Ltd of Shandong Bora, referring to embodiment four.
(2) selection of experimental animal, grouping and feeding and management
20 body weight are about the white hybridized pig of length about 20Kg, are divided into two groups at random, and 10 every group, one group is test group, and another group is control group.Basal diet is identical, and (seeing table 11) adds 0.1% product enzyme probiotics in the test group feed, raise one week of phase in advance, three weeks of trial period.A pig free choice feeding, drinking-water carry out immunity by the pig farm normal procedure.From every group, get 3 pigs during off-test at random and butcher, asepticly from jejunum immediately take an amount of intestinal contents to carry out glycase and neutral protein enzymic activity to detect.Adopt AMS measuring method and folin-phenol method to measure respectively.
The preparation of crude enzyme liquid: intestinal contents is added an amount of corresponding buffered soln soak about 20min, centrifugal at (0～4 ℃) then, 5000rpm, 5min, supernatant is crude enzyme liquid.
Table 11 basal diet prescription
2.7.2.2 test-results and analysis: see table 12.
Table 12 produces the influence of enzyme probiotics to growing pig enteron aisle enzymic activity
Visible by table 12: the neutral protease of test group has improved 149.4% than control group, and glycase has improved 98.1%.Explain and produce the enzyme probiotics in the chitling road, except the function of performance probiotics, can also produce various digestive ferments, promote digesting and assimilating of nutritive substance, improve food conversion ratio, have remarkable economic efficiency.
Save feeding experiment 2.7.3 produce the enzyme probiotics:
2.7.3.1 materials and methods
(1) produces the enzyme probiotics
Form by N9-1-35 bacterium powder, streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum, by the development and production of biological study institute of the sharp next biological Engineering stock Co., Ltd of Shandong Bora, referring to embodiment four.
(2) selection of experimental animal, grouping and feeding and management
With the young cock of the extra large blue brown egg of 360 1 ages in days, be divided into 6 groups at random, promptly 1 control group and 5 test group are established 2 repetitions for every group, and each repeats 30 chickens.The same 2.7.1.1 of basal diet.Test design is seen table 13.Hen house is carried out disinfection and the immunity of chicken according to routine, adopt the mode of raising in cages, free choice feeding, drinking-water.28 days trial periods.Weigh, calculate feed consumption rate, chicken weightening finish and feedstuff-meat ratio during off-test.
Table 13 test design
2.7.3.2 test-results and analysis: see table 14.
Table 14 produces the feeding experiment of enzyme probiotics
The result of table 14 shows: the stone flour with 4.8% substitutes part 5% Preblend, and then adds 0.1% and 0.2% product enzyme probiotics (two groups, four groups), and the feeding effect of chicken is better than control group (a group).Medical stone powder with 9.2% substitutes part 5% Preblend, and then adds 0.1% and 0.2% product enzyme probiotics (three groups, five groups), to feeding effect and control group (a group) no significant difference of chicken.Substitute 5% Preblend group without stone flour, feedstuff-meat ratio is minimum, has reduced by 10.7% than control group.Explain after producing the enzyme probiotics gets into enteron aisle to promote digesting and assimilating of nutritive substance, obviously improve the transformation efficiency of feed, reduce feed cost, have remarkable economic efficiency.
3, conclusion
Can find out that from above test-results bacterial strain N9-1-35 is very outstanding probiotics bacterial classification, can produce various digestive ferments behind the entering enteron aisle, promote digesting and assimilating of nutritive substance, obviously improve the transformation efficiency of feed, reduce feed cost.
The enzyme probiotics is produced in embodiment four preparations
Method is: subtilis N9-1-35 obtains fermented liquid through fermentative processing, adds 6% (weight percentage) W-Gum (trade name: W-Gum then; Article number: 6920420601219; Manufacturer: process subtilis bacterium powder through spraying drying Shandong Jin Cheng limited-liability company); Composite again streptococcus acidi lactici (Streptococcus lactis) bacterium powder (trade name: streptococcus acidi lactici bacterium powder that goes up; Article number: 2006061303; Manufacturer: the Shandong Bora is sharp to come biological Engineering stock Co., Ltd), Lactobacterium acidophilum (Lactobacillus acidophilus) bacterium powder (trade name: Lactobacterium acidophilum bacterium powder; Article number: 2009061402; Production company: Bora profit in Shandong is come biological Engineering stock Co., Ltd) and W-Gum (trade name: W-Gum; Article number: 6920420601219; Manufacturer: be product enzyme probiotics Shandong Jin Cheng limited-liability company); Wherein, (W-Gum is meant the W-Gum that adds when composite here for subtilis bacterium powder, streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum; Not comprising the W-Gum that contains in the bacterium powder) mass ratio is 6: 2: 1: 21, the viable count of subtilis, streptococcus acidi lactici and Lactobacterium acidophilum is respectively 3 * 10
8Cfu/g, 1 * 10
8Cfu/g and 5 * 10
7Cfu/g.Produce in the enzyme probiotics at 500g, need subtilis bacterium powder 100g, streptococcus acidi lactici bacterium powder 33.33g, Lactobacterium acidophilum bacterium powder 16.67g and W-Gum 350g, it is composite that composite mode is that routine mixes.
The concrete steps of said preparation subtilis bacterium powder are (it is as shown in Figure 4 to produce the fermentation schema) as follows:
(i) bacterial classification: select subtilis Bacillus subtilis N9-1-35 for use;
(ii) slant culture: bacterial classification one ring is inoculated on the solid slant culture base, cultivates 20h down at 37 ℃;
(iii) first order seed is cultivated: get above-mentioned cultured inclined-plane, under aseptic condition, get a ring with transfering loop and be inoculated in 50mL～100mL seed liquid nutrient medium, under 37 ℃ of conditions, leave standstill and cultivate 14h, make primary seed solution;
(iv) enlarged culturing: with the inoculum size of 2% (v/v), primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 37 ℃ of conditions, leaves standstill and cultivate 12h, make secondary seed solution;
(v) fermentor cultivation: with the inoculum size of 2% (v/v), secondary seed solution is connected in the liquid fermentation medium, under 37 ℃ of conditions, aerobic culture 24h;
(vi) collect tunning: treat that (v) the gemma rate reaches 90% when above to step, collects fermented liquid;
(vii) in fermented liquid, add 6% W-Gum, spraying drying makes the bacterium powder immediately.
Said step is (iii), the seed liquid culture medium prescription in (iv) is: glucose 5g/L, and peptone 10g/L, Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L during use, regulates pH to 7.2, and 20min sterilizes under 115 ℃ of conditions; The solid slant culture base of said step in (ii) is to add 1.5% agar powder in the above-mentioned seed liquid nutrient medium; Said step (the liquid fermentation medium prescription v) is: Semen Maydis powder 60g/L, and dregs of beans 30g/L, Sodium phosphate, dibasic 6g/L, ammonium sulfate 3g/L, calcium chloride 2g/L, pH 7.2.
Claims (8)
1. a strain is through the subtilis of space breeding technology seed selection; It is characterized in that: this bacterial strain called after subtilis N9-1-35; Be preserved in Chinese typical culture collection center on 08 31st, 2011, its deposit number is: CCTCC M 2011301.
2. a strain according to claim 1 is through the subtilis of space breeding technology seed selection; It is characterized in that: the rounded or irregularity of the bacterium colony of said subtilis N9-1-35, the edge is irregular, and there is gauffer, coarse on the surface; Dirty white or little band are yellow, and protuberance is arranged; This bacterium cell size is 0.3 μ m～0.5 μ m * 1.4 μ m～3.0 μ m, is shaft-like, Gram-positive; Gemma is oval, and size is 0.6 μ m～0.9 μ m * 1.0 μ m～1.5 μ m, and sporangiocyst does not expand, and mobility is arranged; Can utilize glucose, wood sugar and pectinose etc., ability hydrolyzed starch, reduction nitrate salt; On the substratum that contains 7% sodium-chlor, can grow, can grow 50 ℃ of following cultivations, the V-P reaction is positive, and does not under anaerobic grow.
3. the described strain of claim 1 is being produced the application in the preparation product enzyme probiotics through the subtilis of space breeding technology seed selection.
4. application according to claim 3; It is characterized in that: preparing the method for producing the enzyme probiotics is: subtilis N9-1-35 is through fermentative processing; Obtain fermented liquid; Add W-Gum then and process subtilis bacterium powder through spraying drying, composite again streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum promptly get and produce the enzyme probiotics.
5. application according to claim 4 is characterized in that, the concrete steps of said preparation subtilis bacterium powder are following:
(i) bacterial classification: select subtilis Bacillus subtilis N9-1-35 for use;
(ii) slant culture: bacterial classification one ring is inoculated on the solid slant culture base, cultivates 18～24h down at 37 ℃;
(iii) first order seed is cultivated: get above-mentioned cultured inclined-plane, under aseptic condition, get a ring with transfering loop and be inoculated in 50mL～100mL seed liquid nutrient medium, under 37 ℃ of conditions, leave standstill and cultivate 12～16h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 2%, primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 37 ℃ of conditions, leave standstill and cultivate 8～14h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 2%, secondary seed solution is connected in the liquid fermentation medium, under 37 ℃ of conditions, aerobic culture 22～28h;
(vi) collect tunning: treat that (v) the gemma rate reaches 90% when above to step, collects fermented liquid;
(vii) in fermented liquid, add 4～8% W-Gums, spraying drying makes the bacterium powder immediately.
6. application according to claim 4 is characterized in that: said step (iii), the seed liquid culture medium prescription in (iv) is: glucose 5g/L, peptone 10g/L; Carnis Bovis seu Bubali cream 5g/L, sodium-chlor 5g/L is during use; Regulate the 20min that sterilizes under ℃ condition of pH to 7.0～7.5,115; The solid slant culture base of said step in (ii) is to add 1.5～2.0% agar powder in the above-mentioned seed liquid nutrient medium; Said step (the liquid fermentation medium prescription v) is: Semen Maydis powder 60g/L, and dregs of beans 30g/L, Sodium phosphate, dibasic 6g/L, ammonium sulfate 3g/L, calcium chloride 2g/L, pH 7.0～7.5.
7. one kind is produced the enzyme probiotics, it is characterized in that: be made up of subtilis bacterium powder, streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum.
8. product enzyme probiotics according to claim 7; It is characterized in that: the mass ratio of said subtilis bacterium powder, streptococcus acidi lactici bacterium powder, Lactobacterium acidophilum bacterium powder and W-Gum is 6: 2: 1: 21, and the viable count of subtilis, streptococcus acidi lactici and Lactobacterium acidophilum is greater than or equal to 3 * 10 respectively
8Cfu/g, 1 * 10
8Cfu/g and 5 * 10
7Cfu/g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102780644A CN102329749B (en) | 2011-09-19 | 2011-09-19 | Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102780644A CN102329749B (en) | 2011-09-19 | 2011-09-19 | Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102329749A true CN102329749A (en) | 2012-01-25 |
| CN102329749B CN102329749B (en) | 2013-01-16 |
Family
ID=45481715
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102780644A Active CN102329749B (en) | 2011-09-19 | 2011-09-19 | Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102329749B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104739919A (en) * | 2015-03-30 | 2015-07-01 | 山东宝来利来生物工程股份有限公司 | Fermented astragalus composite preparation as well as preparation method and application thereof |
| CN105475624A (en) * | 2015-11-26 | 2016-04-13 | 山东碧蓝生物科技有限公司 | Microbial fermentation feed as well as production method and application thereof |
| CN106190933A (en) * | 2016-09-28 | 2016-12-07 | 吉林省农业科学院 | The bacillus subtilis of the anti-pathogenic bacterium of wide spectrum and application thereof |
| CN106811429A (en) * | 2015-12-02 | 2017-06-09 | 北京大北农科技集团股份有限公司 | The application of one bacillus subtilis strain and its feed addictive and feed |
| CN109504632A (en) * | 2018-12-18 | 2019-03-22 | 河北农业大学 | Bacillus subtilis and application thereof |
| CN109536420A (en) * | 2018-12-29 | 2019-03-29 | 贵州大学 | One bacillus subtilis and its application |
| CN115806896A (en) * | 2022-07-27 | 2023-03-17 | 江苏三仪生物工程有限公司 | Bacillus subtilis capable of producing cellulase and xylanase as well as culture method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004044186A1 (en) * | 2002-11-11 | 2004-05-27 | Zakrytoe Aktsionernoe Obschestvo 'kul' | Probiotic additive and the production method thereof |
| CN101948763A (en) * | 2009-10-13 | 2011-01-19 | 中国农业科学院饲料研究所 | High-efficiency compound microbial inoculum for fermenting beds, and preparation method and application thereof |
-
2011
- 2011-09-19 CN CN2011102780644A patent/CN102329749B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004044186A1 (en) * | 2002-11-11 | 2004-05-27 | Zakrytoe Aktsionernoe Obschestvo 'kul' | Probiotic additive and the production method thereof |
| CN101948763A (en) * | 2009-10-13 | 2011-01-19 | 中国农业科学院饲料研究所 | High-efficiency compound microbial inoculum for fermenting beds, and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 宝来利来微生态制剂: "http://www.shuichan.cc/flea_view-154576.html", 《中国水产养殖网》, 26 May 2011 (2011-05-26), pages 1 * |
| 徐海燕等: "芽孢杆菌发酵代谢产物的研究", 《饲料广角 》, no. 9, 31 December 2006 (2006-12-31), pages 22 - 23 * |
| 陈继红等: "多杀菌素产生菌株航天育种效果研究", 《安徽农业科学》, vol. 36, no. 12, 31 December 2008 (2008-12-31) * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104739919B (en) * | 2015-03-30 | 2018-11-30 | 山东宝来利来生物工程股份有限公司 | A kind of fermentation Radix Astragali compound formulation and the preparation method and application thereof |
| CN104739919A (en) * | 2015-03-30 | 2015-07-01 | 山东宝来利来生物工程股份有限公司 | Fermented astragalus composite preparation as well as preparation method and application thereof |
| CN105475624A (en) * | 2015-11-26 | 2016-04-13 | 山东碧蓝生物科技有限公司 | Microbial fermentation feed as well as production method and application thereof |
| CN105475624B (en) * | 2015-11-26 | 2020-02-11 | 山东碧蓝生物科技有限公司 | Microbial fermentation feed and production method and application thereof |
| CN106811429B (en) * | 2015-12-02 | 2021-06-11 | 北京大北农科技集团股份有限公司 | Bacillus subtilis strain, application of feed additive thereof and feed |
| CN106811429A (en) * | 2015-12-02 | 2017-06-09 | 北京大北农科技集团股份有限公司 | The application of one bacillus subtilis strain and its feed addictive and feed |
| CN106190933B (en) * | 2016-09-28 | 2019-08-13 | 吉林省农业科学院 | The bacillus subtilis of the anti-pathogenic bacteria of wide spectrum and its application |
| CN106190933A (en) * | 2016-09-28 | 2016-12-07 | 吉林省农业科学院 | The bacillus subtilis of the anti-pathogenic bacterium of wide spectrum and application thereof |
| CN109504632A (en) * | 2018-12-18 | 2019-03-22 | 河北农业大学 | Bacillus subtilis and application thereof |
| CN109536420A (en) * | 2018-12-29 | 2019-03-29 | 贵州大学 | One bacillus subtilis and its application |
| CN109536420B (en) * | 2018-12-29 | 2022-09-09 | 贵州大学 | Bacillus subtilis and application thereof |
| CN115806896A (en) * | 2022-07-27 | 2023-03-17 | 江苏三仪生物工程有限公司 | Bacillus subtilis capable of producing cellulase and xylanase as well as culture method and application thereof |
| CN115806896B (en) * | 2022-07-27 | 2023-09-01 | 江苏三仪生物工程有限公司 | Bacillus subtilis capable of producing cellulase and xylanase as well as culture method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102329749B (en) | 2013-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103614327B (en) | A kind of subtilis and uses thereof | |
| CN101341927B (en) | Microbial feed additive liquid agent for pig and preparation technique | |
| CN103468613B (en) | Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum | |
| CN102329749B (en) | Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis | |
| CN106260540B (en) | Biological feed for creep feed and creep feed | |
| CN101638627B (en) | Bacillus subtilis and application thereof in biological feed additives | |
| CN101671638B (en) | New strain of bifidobacterium and fermentative preparation method and application thereof | |
| CN102517238B (en) | Acid-producing bacillus cereus and application thereof | |
| CN111838408A (en) | Functional amino acid enzyme and preparation method thereof | |
| CN101760431A (en) | Compound microbial culture starter and application thereof | |
| CN103981118A (en) | Bacillus subtilis feed additive and preparation method and application thereof | |
| CN102715339A (en) | Microorganism fodder production method based on pleurotus eryngii mushroom cultivating residues | |
| CN102226162A (en) | Preparation method and application of composite microbial feed additive | |
| CN104480052A (en) | Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding | |
| CN103039696A (en) | Algal biological feed and preparation method thereof | |
| CN103087964A (en) | Bacillus subtilis, microecological preparation thereof and application of bacillus subtilis in animal feed | |
| CN102599351A (en) | Bio-enzyme and microorganism containing compound feed additive and preparation process thereof | |
| CN103667128A (en) | Bacillus licheniformis and its use | |
| CN101974434A (en) | Multi-bacterium microbial compound preparations for aquaculture and preparation method thereof | |
| CN106566791A (en) | Porcine compound synbiotics micro-ecological preparation and preparation method thereof | |
| CN101392223A (en) | Breeding method of microbial feed additive strain | |
| CN107974421A (en) | A kind of lactobacillus acidophilus and its screening technique and application, a kind of microbial inoculum | |
| CN102212493A (en) | Barton fermentation bed complex microbial inoculant and preparation method thereof | |
| CN103184174A (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN102517227B (en) | Enterococcus faecalis and applications and feed additive and leavening agent thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bacillus subtilis bred by space mutation breeding technology and application of Bacillus subtilis Effective date of registration: 20180413 Granted publication date: 20130116 Pledgee: Bank of Tai'an Limited by Share Ltd. Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: 2018370000070 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20190729 Granted publication date: 20130116 Pledgee: Bank of Tai'an Limited by Share Ltd. Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: 2018370000070 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Bacillus subtilis strain bred by aerospace breeding technology and its application Effective date of registration: 20201209 Granted publication date: 20130116 Pledgee: Bank of Tai'an Limited by Share Ltd. Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2020980009026 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20211220 Granted publication date: 20130116 Pledgee: Bank of Tai'an Limited by Share Ltd. Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2020980009026 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Bacillus subtilis selected by aerospace breeding technology and its application Effective date of registration: 20220111 Granted publication date: 20130116 Pledgee: Taian Bank Co.,Ltd. Daizong sub branch Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2022980000292 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20221206 Granted publication date: 20130116 Pledgee: Taian Bank Co.,Ltd. Daizong sub branch Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2022980000292 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Bacillus subtilis strain selected by space breeding technology and its application Effective date of registration: 20221212 Granted publication date: 20130116 Pledgee: Taian Bank Co.,Ltd. Daizong sub branch Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2022980026502 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20231213 Granted publication date: 20130116 Pledgee: Taian Bank Co.,Ltd. Daizong sub branch Pledgor: SHANDONG BOLY-LELY BIOENGINEERING Co.,Ltd. Registration number: Y2022980026502 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |