CN113907208A - Feed additive for preventing piglet diarrhea and preparation method and application thereof - Google Patents
Feed additive for preventing piglet diarrhea and preparation method and application thereof Download PDFInfo
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- CN113907208A CN113907208A CN202111297786.4A CN202111297786A CN113907208A CN 113907208 A CN113907208 A CN 113907208A CN 202111297786 A CN202111297786 A CN 202111297786A CN 113907208 A CN113907208 A CN 113907208A
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- piglet diarrhea
- bacillus subtilis
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- 206010012735 Diarrhoea Diseases 0.000 title claims abstract description 51
- 239000003674 animal food additive Substances 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000003094 microcapsule Substances 0.000 claims abstract description 43
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 42
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 42
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 40
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims abstract description 34
- 241000894006 Bacteria Species 0.000 claims abstract description 25
- 150000004676 glycans Chemical class 0.000 claims abstract description 18
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 18
- 239000005017 polysaccharide Substances 0.000 claims abstract description 18
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims abstract description 17
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims abstract description 17
- 241001061264 Astragalus Species 0.000 claims abstract description 13
- 235000006533 astragalus Nutrition 0.000 claims abstract description 13
- 210000004233 talus Anatomy 0.000 claims abstract description 13
- 240000008042 Zea mays Species 0.000 claims abstract description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 10
- 235000005822 corn Nutrition 0.000 claims abstract description 10
- 235000013312 flour Nutrition 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 3
- 238000000855 fermentation Methods 0.000 claims description 53
- 230000004151 fermentation Effects 0.000 claims description 53
- 239000002244 precipitate Substances 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 239000000654 additive Substances 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 12
- 230000000996 additive effect Effects 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 239000002775 capsule Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 8
- 239000000661 sodium alginate Substances 0.000 claims description 8
- 235000010413 sodium alginate Nutrition 0.000 claims description 8
- 229940005550 sodium alginate Drugs 0.000 claims description 8
- 235000012424 soybean oil Nutrition 0.000 claims description 8
- 239000003549 soybean oil Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229920000136 polysorbate Polymers 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 4
- 101710170231 Antimicrobial peptide 2 Proteins 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 16
- 230000036039 immunity Effects 0.000 abstract description 7
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 241000700605 Viruses Species 0.000 abstract description 2
- 239000003242 anti bacterial agent Substances 0.000 abstract description 2
- 229940088710 antibiotic agent Drugs 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000004321 preservation Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000401 methanolic extract Substances 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000036737 immune function Effects 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000001540 sodium lactate Substances 0.000 description 2
- 229940005581 sodium lactate Drugs 0.000 description 2
- 235000011088 sodium lactate Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000563903 Bacillus velezensis Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000014590 basal diet Nutrition 0.000 description 1
- 238000009924 canning Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000001937 non-anti-biotic effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
Classifications
-
- 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
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
-
- 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
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
Abstract
The invention discloses a feed additive for preventing piglet diarrhea and a preparation method and application thereof, wherein the feed additive comprises the following raw materials in parts by weight: 8-12 parts of bacillus subtilis microcapsule, 8-12 parts of bacillus amyloliquefaciens microcapsule, 10-15 parts of astragalus polysaccharide, 5-8 parts of antimicrobial peptide No. 1, 5-8 parts of antimicrobial peptide No. 2 and 45-64 parts of corn flour. According to the invention, the bacillus subtilis, the bacillus amyloliquefaciens and the antibacterial peptide and the plant polysaccharide which are the products thereof are combined to cooperatively inhibit viruses and improve the immunity of piglet organisms, and live bacteria are wrapped in the microcapsule, so that the survival rate is improved, the piglet diarrhea can be effectively prevented and treated, the usage amount of antibiotics is reduced, meanwhile, the preparation method is simple and efficient, the bioavailability is high, and the effect of preventing and treating piglet diarrhea can be better realized.
Description
Technical Field
The invention relates to a feed additive for piglets, and in particular relates to a feed additive for preventing piglet diarrhea, and a preparation method and application thereof.
Background
Porcine Epidemic Diarrhea is an infectious disease mainly characterized by watery Diarrhea caused by Porcine Epidemic Diarrhea Virus (PEDV). The death rate after infection is high, and huge economic loss is caused to the pig industry. In the production practice, besides enhancing the feeding management of piglets and sows, the prevention of porcine epidemic diarrhea can adopt intramuscular injection of vaccines, and the injection of the vaccines can not generate protective force in time due to the incomplete development of the immune system of newborn piglets, so that the immune failure is caused. The treatment mainly comprises antibiotic medicines, but the long-term use of the antibiotic medicines can cause pathogenic microorganisms to generate drug resistance to reduce the curative effect of the medicines, and on the other hand, the antibiotic medicines can partially remain in bodies of piglets to cause the problem of food health.
Aiming at the problems of the existing method for preventing and treating the piglet epidemic diarrhea, a plurality of scholars are dedicated to the research of antibiotic-free additives for preventing and controlling piglet diarrhea. Can produce various antibacterial peptides and has good effects in resisting virus, bacteria, fungi and parasites. The plant polysaccharide has effects of enhancing specific and nonspecific immunity of organism.
The application of the plant polysaccharide, the bacillus subtilis, the bacillus amyloliquefaciens and the antibacterial peptide as a single feed additive in livestock and poultry can promote the growth and development of organisms, improve the immune function of the organisms, improve the morphological structure of animal intestinal tracts, promote the digestion and absorption of nutrient substances and maintain the balance of a micro-ecological system of the intestinal tracts. However, there are many problems in the application of single probiotics, for example, the single probiotics has high requirements for nutrient environmental conditions and is easily influenced by micro-ecology in the intestinal tract of animals, so that it is not easy to colonize in the intestinal tract of animals, thereby affecting the effect. Researches show that the polysaccharide and the bacillus subtilis have synergistic effect, jointly enhance the immune response of organisms, and improve the activity of immune cells to promote the increase of immune globulin of piglets, thereby improving the immunity of the piglets.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems of antibiotic residue, easy failure of vaccine immunity, poor prevention and treatment effect of the existing non-antibiotic additive and the like in the prior art, the invention provides the feed additive for treating piglet diarrhea.
The invention also provides a preparation method and application of the feed additive for preventing piglet diarrhea.
The technical scheme is as follows: in order to achieve the purpose, the invention provides a feed additive for preventing piglet diarrhea, which comprises the following raw materials in parts by weight: 8-12 parts of bacillus subtilis microcapsule, 8-12 parts of bacillus amyloliquefaciens microcapsule, 10-15 parts of astragalus polysaccharide, 5-8 parts of antimicrobial peptide No. 1, 5-8 parts of antimicrobial peptide No. 2 and 45-64 parts of corn flour.
Wherein the bacillus subtilis is bacillus subtilis JCL16 with a preservation number of CCTCC NO: and M2018336.
Wherein the bacillus amyloliquefaciens is the bacillus amyloliquefaciens CPLK1314 with the preservation number of CCTCC NO: M2017658.
Wherein the preparation of the bacillus subtilis microcapsule comprises the following steps: activating the bacillus subtilis preserved on the inclined plane, culturing the activated bacillus subtilis to obtain seed fermentation liquor, inoculating the seed fermentation liquor for fermentation, and collecting the fermentation liquor; centrifuging the fermentation liquor, removing supernatant, collecting and washing thallus precipitates, uniformly mixing the thallus precipitates with a sodium alginate solution, injecting the mixture into a calcium carbonate solution to obtain a bacterium-containing mixed solution, slowly adding the bacterium-containing mixed solution into soybean oil containing tween, stirring, slowly stirring, adding glacial acetic acid, standing, centrifugally deoiling, collecting microcapsules, washing to obtain wet capsules, and freeze-drying to obtain the freeze-dried bacillus subtilis microcapsules.
Wherein the preparation of the bevel bacillus amyloliquefaciens microcapsule comprises the following steps: activating strains of the bacillus amyloliquefaciens preserved on the inclined plane, culturing the activated bacillus amyloliquefaciens to obtain seed fermentation liquor, inoculating the seed fermentation liquor, fermenting and collecting the fermentation liquor; centrifuging the fermentation liquor, removing supernatant, collecting and washing thallus precipitates, uniformly mixing the thallus precipitates with a sodium alginate solution, injecting the mixture into a calcium carbonate solution to obtain a bacterium-containing mixed solution, slowly adding the bacterium-containing mixed solution into soybean oil containing tween, stirring, slowly stirring, adding glacial acetic acid, standing, centrifugally deoiling, collecting microcapsules, washing to obtain wet capsules, and freeze-drying to obtain the freeze-dried bacillus amyloliquefaciens microcapsule.
Preferably, the antimicrobial peptide No. 1 is obtained by separating fermentation liquor after fermentation culture of bacillus subtilis CCTCC NO: M2018336.
Preferably, 5-8 parts of the antimicrobial peptide 2 is obtained by separating fermentation liquor after fermentation culture of bacillus amyloliquefaciens CCTCC NO: M20117658.
The preparation method of the feed additive for preventing the piglet diarrhea comprises the following steps:
uniformly mixing the bacillus subtilis microcapsule, the bacillus amyloliquefaciens microcapsule, the astragalus polysaccharide, the antimicrobial peptide No. 1, the antimicrobial peptide No. 2 and the corn flour to obtain the feed additive for preventing piglet diarrhea.
The feed additive for preventing piglet diarrhea is applied to preparation of a feed or a medicament for preventing and treating piglet diarrhea.
Wherein 10g of additive is added into 500g of feed.
The invention provides a feed additive formula for preventing and treating piglet diarrhea, wherein bacillus subtilis and bacillus amyloliquefaciens are added into an organism to regulate a microecological flora structure in an intestinal tract of the organism and can activate the immune function of the organism, and antibacterial peptide has activities such as antibacterial activity, antiviral activity and the like, polysaccharide has an enhancement effect on the specific and nonspecific immune functions of the organism, and the three are singly used for treating piglet diarrhea, so that the three are mixed in proportion through experiments to obtain a better effect of preventing and treating piglet diarrhea.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the feed additive for preventing and treating piglet diarrhea has the advantages of high viable count, greenness, safety, no toxic or side effect and the like, can effectively prevent and treat piglet diarrhea, and reduces the use amount of antibiotics.
2. The bacillus subtilis and the bacillus amyloliquefaciens have the effects of improving the intestinal microbial environment, improving the immunity of organisms and the like, can be used as a vaccine adjuvant and a vaccine for combined use, and can promote the growth and development of piglets, enhance the immunity of the piglets, reduce the diarrhea rate and the like.
3. The astragalus polysaccharide, the bacillus subtilis and the antibacterial peptide have synergistic effect, jointly enhance the immune response of organisms, and improve the activity of immune cells to promote the increase of immune globulin of piglets, thereby improving the immunity of the piglets.
4. The bacillus subtilis and the bacillus amyloliquefaciens are wrapped in the microcapsule, so that the bacterium inactivation caused by gastric acid can be avoided, the bacterium can be released at a fixed point, and the bacterium survival rate is improved.
5. The preparation method is simple and efficient, has high bioavailability, and can realize better effect of preventing and treating piglet diarrhea
Detailed description of the invention
The present invention is further illustrated by the following examples.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
The Bacillus subtilis strain JCL16 of the invention is preserved in China center for type microorganism preservation, the preservation time is 06 months 01 years in 2018, the preservation number is M2018336, and the strain is publicly preserved in the applicant's prior patent CN 111411053A.
The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens subsp. plantarum) strain CPLK131 is preserved in a China center for type microorganism preservation with the preservation time of 2017, 11 and 06 days, the preservation number is CCTCC NO: M2017658, and the strain is already publicly preserved in the applicant's prior patent CN 109182197A.
Slant culture medium: 10g of beef extract peptone, 5g of yeast extract, 2g of sodium chloride, 0.1g of glucose, 5g of sodium lactate and 20g of agar, and dissolving in 1000mL of distilled water.
Seed culture medium and fermentation medium: 10g of beef extract peptone, 5g of yeast extract, 2g of sodium chloride, 0.1g of glucose and 5g of sodium lactate, and the beef extract peptone, the yeast extract and the sodium chloride are dissolved in 1000mL of distilled water.
Example 1
And (3) preparing the bacillus subtilis microcapsule.
The well preserved bevel bacillus subtilis strain CCTCC NO: m2018336 is inoculated in LB slant culture medium, cultured for 24h at 37 ℃ and activated. Inoculating the activated bacillus subtilis strain into a sterilized LB seed culture medium, and culturing for 24h at 37 ℃ at 200r/min in a constant-temperature shaking incubator to obtain seed fermentation liquor; 50L of fermentation tank is filled with 35L of LB culture medium, the seed fermentation liquor is inoculated according to the inoculation amount with the volume fraction of 5 percent, and fermentation is carried outThe control conditions are as follows: the temperature is 37 ℃, the number of the living bacteria to be detected reaches 1.0 multiplied by 109At cfu/mL, the fermentation broth is collected.
Centrifuging the fermentation liquor for 15min at 5000r/min, removing the supernatant, collecting the thallus precipitate, washing the thallus precipitate for a plurality of times by using 0.9% sterile normal saline, uniformly mixing the thallus precipitate with 2.0% (w/v) of sodium alginate solution in a ratio of 1:4(w/v), and mixing the mixed solution of the bacillus subtilis and the sodium alginate by using a sterile syringe according to a ratio of 1: 3(v: v) is injected into 2.0% (w/v) calcium carbonate solution to obtain bacteria-containing mixed solution, then the bacteria-containing mixed solution is slowly added into soybean oil containing tween 1.0% (v/v) according to the volume ratio of 1:2, the soybean oil is stirred for 15min at the temperature of 4 ℃ at the stirring speed of 400r/min, then glacial acetic acid is added under slow stirring to adjust the pH value to 5.5, the mixture is kept stand for 2h, then centrifugal deoiling is carried out at 8000r/min, microcapsules are collected, wet capsules are obtained after 3 times of washing by physiological saline, the wet capsules are pre-frozen for 12h at the temperature of minus 20 ℃, then the wet capsules are put into a vacuum freeze dryer at the temperature of minus 60 ℃ for freeze drying for 48h, and the freeze-dried bacillus subtilis microcapsules are obtained.
Example 2
And (3) preparing the bacillus amyloliquefaciens microcapsule.
The well preserved strain of the slant bacillus amyloliquefaciens with CCTCC NO of M20117658 is inoculated in a slant LB culture medium and cultured for 24 hours at 37 ℃ to activate the strain. Inoculating the activated bacillus amyloliquefaciens strain into a sterilized seed LB culture medium, and culturing for 24 hours at 37 ℃ at 200r/min in a constant-temperature shaking incubator to obtain seed fermentation liquor; 50L of fermentation tank LB culture medium 35L, inoculating the seed fermentation liquor according to the inoculation amount of 5% of volume fraction, the fermentation control conditions are as follows: the temperature is 37 ℃, the number of the living bacteria to be detected reaches 1.0 multiplied by 109At cfu/mL, the fermentation broth is collected.
Centrifuging the fermentation liquor for 15min at 5000r/min, removing supernatant, collecting thallus precipitate, washing the thallus precipitate for several times by using 0.9% sterile normal saline, uniformly mixing the thallus precipitate with 2.0% (w/v) of sodium alginate solution in a ratio of 1:4(w/v), and mixing the mixed solution of the bacillus amyloliquefaciens and the sodium alginate by using a sterile injector according to a ratio of 1: 3(v: v) is injected into 2.0% (w/v) calcium carbonate solution to obtain bacteria-containing mixed solution, then the bacteria-containing mixed solution is slowly added into soybean oil containing tween 1.0% (v/v) according to the volume ratio of 1:2, the soybean oil is stirred for 15min at the temperature of 4 ℃ at the stirring speed of 400r/min, then glacial acetic acid is added under slow stirring to adjust the pH value to 5.5, the mixture is kept stand for 2h, then centrifugal deoiling is carried out at 8000r/min, microcapsules are collected, wet capsules are obtained after 3 times of washing by physiological saline, the wet capsules are pre-frozen for 12h at the temperature of minus 20 ℃, then the wet capsules are put into a vacuum freeze dryer at the temperature of minus 60 ℃ for freeze drying for 48h, and the freeze-dried bacillus amyloliquefaciens microcapsules are obtained.
Example 3
Preparation method of antibacterial peptide No. 1
The well preserved bevel bacillus subtilis strain CCTCC NO: m2018336 is inoculated in a slant culture medium, cultured for 24h at 37 ℃ and activated. Inoculating the activated Bacillus subtilis strain into sterilized seed culture medium, and culturing at 37 deg.C for 24 hr at 200r/min in a constant temperature vibration incubator to obtain seed fermentation liquid with bacteria content of 107-108cfu/mL; fermenting and culturing in a 50L fermentation tank, fermenting and canning 35L of culture medium in a 50L fermentation tank, inoculating the seed fermentation liquid according to the inoculation amount of 5 percent, wherein the fermentation control conditions are as follows: the temperature is 37 ℃, and the time is 120h, thus obtaining the fermentation liquor.
The fermentation broth was centrifuged at 5000r/min for 15min and the supernatant was adjusted to pH2.0 with concentrated hydrochloric acid and precipitated overnight. Centrifuging at 5000r/min for 25min to obtain precipitate, extracting the precipitate with 10% methanol for 2 times (2 hr each time), and mixing the methanol extracts. Concentrating the methanol extract under reduced pressure, and freeze drying to obtain antibacterial peptide No. 1.
Example 4
Preparation method of antimicrobial peptide No. 2
The well preserved strain of the slant bacillus amyloliquefaciens with CCTCC NO of M20117658 is inoculated in a slant LB culture medium and cultured for 24 hours at 37 ℃ to activate the strain. Inoculating the activated Bacillus amyloliquefaciens strain into sterilized seed LB culture medium, and culturing at 37 deg.C for 24 hr at 200r/min in a constant temperature shaking incubator to obtain seed fermentation liquid with bacteria content of 107-108cfu/mL; fermenting and culturing in a 50L fermentation tank, filling 35L LB culture medium in a 50L fermentation tank, inoculating the seed fermentation liquid according to the inoculation amount of 5 percent, wherein the fermentation control conditions are as follows: the temperature is 37 ℃, and the time is 120h, thus obtaining the fermentation liquor.
The fermentation broth was centrifuged at 5000r/min for 15min and the supernatant was adjusted to pH2.0 with concentrated hydrochloric acid and precipitated overnight. Centrifuging at 5000r/min for 15min to obtain precipitate, extracting the precipitate with 1/10 volume of methanol for 2 times, each for 2 hr, and mixing the methanol extracts. Concentrating the methanol extract under reduced pressure, and freeze drying to obtain antibacterial peptide No. 2.
Example 5
8 parts of a bacillus subtilis microcapsule; 12 parts of bacillus amyloliquefaciens microcapsules; 13 parts of astragalus polysaccharide; 5 parts of antibacterial peptide No. 1; 8 parts of antibacterial peptide No. 2; 54 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 6
12 parts of bacillus subtilis microcapsules; 8 parts of bacillus amyloliquefaciens microcapsules; 10 parts of astragalus polysaccharide; 8 parts of antibacterial peptide No. 1; 6 parts of antibacterial peptide No. 2; 56 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 7
10 parts of a bacillus subtilis microcapsule; 10 parts of bacillus amyloliquefaciens microcapsules; 15 parts of astragalus polysaccharide; 6 parts of antibacterial peptide No. 1; 5 parts of antibacterial peptide No. 2; 54 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 8
8 parts of a bacillus subtilis microcapsule; 8 parts of bacillus amyloliquefaciens microcapsules; 10 parts of astragalus polysaccharide; 5 parts of antibacterial peptide No. 1; 5 parts of antibacterial peptide No. 2; 45 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Example 9
12 parts of bacillus subtilis microcapsules; 12 parts of bacillus amyloliquefaciens microcapsules; 15 parts of astragalus polysaccharide; 8 parts of antibacterial peptide No. 1; 8 parts of antibacterial peptide No. 2; 64 parts of corn flour. The components are mixed evenly to obtain the feed additive for preventing piglet diarrhea.
Test example 1
1. Test animals and test design
Selecting 140 healthy weaned piglets (28 +/-2) with day age, randomly dividing the selected piglets into 6 groups according to the principles of same breed and birth times, similar weight and half of the male parent and the female parent, and repeating for 2 times and 10 times for each group. The basal diets of each group were identical, and the trial started with 28 days old weaned piglets, for a period of 28 days.
The feed is fed by mixing the powder with water, and the feed is prepared by adding water and mixing with water. The piglets are fed regularly, and the feeding time is respectively 8, 12, 16 and 20 points per day. Piglets had free access to water during the trial period and piggery management was performed as per the trial piggery routine.
Continuously observing the defecation condition of piglets from 7:00 in the morning to 21:00 in the evening every day, carrying out sensory evaluation on the feces discharged by each piglet every time, counting the feces condition of the piglets recorded every day (according to 0-3 grade records, 0 grade-dry, 1 grade-soft, 2 grade-dilute and 3 grade-water samples, wherein 0 grade and 1 grade are normal, and 2 grade and 3 grade are diarrhea), calculating the diarrhea rate, and recording and evaluating the growth performance index ratios of feed intake, daily gain, feed meat and the like. The diarrhea rate (diarrhea rate ═ number of diarrhea heads/(number of piglets × number of test days) ] × 100%) and the final statistics were calculated as shown in table 1:
2. test feed
Group A: basal ration, commercially available, for routine feed (containing only conventional ingredients) for piglets.
Group B: conventional feed the additive of example 5 was added at 10g per 500g feed.
Group C: conventional feed the additive of example 6 was added at 10g per 500g feed.
Group D: conventional feed the additive of example 7 was added at 10g per 500g feed.
Group E: conventional feed additive (except for astragalus polysaccharide, the other components are the same as in example 5), and 10g of the additive is added into every 500g of feed.
And F group: conventional feed additives (except for Bacillus subtilis and Bacillus amyloliquefaciens, and the other components are the same as in example 5) are added into every 500g of feed by 10 g.
Group G: the conventional feed additive (except for antimicrobial peptide No. 1, the other components are the same as in example 5), and 10g of the additive is added to 500g of feed.
Group H: the conventional feed additive (except for antimicrobial peptide No. 2, the other components were the same as in example 5), and 10g of the additive was added per 500g of the feed.
Group I: the conventional feed additive (except for antimicrobial peptide No. 1 and antimicrobial peptide No. 2, the rest components are the same as in example 5), and 10g of the additive is added to 500g of feed.
Group J: conventional feed additive (Bacillus subtilis, Bacillus amyloliquefaciens culture fluid is centrifuged to obtain bacterial mud, the mud is not made into microcapsules, the mud is freeze-dried and added into the additive, and the rest components are the same as the components in the example 5), and 10g of the additive is added into 100g of feed.
TABLE 1 Effect of feed additives on piglet diarrhea
As can be seen from Table 1, the feed additive for preventing diarrhea of piglets has the obvious effect of reducing the diarrhea rate of the piglets. The diarrhea rate of the group E, the group F, the group G, the group H and the group I is far higher than that of the group B, C, D, which indicates that certain synergistic effect exists between astragalus polysaccharide and bacillus subtilis and bacillus amyloliquefaciens. The experimental results of group G show that the diarrhea rate of the two bacteria which are not prepared into the microcapsule is higher than that of the two bacteria which are prepared into the capsule, and the two bacteria are wrapped in the microcapsule, so that the inactivation of the viable bacteria can be reduced.
Claims (10)
1. The feed additive for preventing piglet diarrhea is characterized by comprising the following raw materials in parts by weight: 8-12 parts of bacillus subtilis microcapsule, 8-12 parts of bacillus amyloliquefaciens microcapsule, 10-15 parts of astragalus polysaccharide, 5-8 parts of antimicrobial peptide No. 1, 5-8 parts of antimicrobial peptide No. 2 and 45-64 parts of corn flour.
2. The feed additive for preventing piglet diarrhea according to claim 1, wherein the bacillus subtilis is bacillus subtilis JCL16, CCTCC NO: and M2018336.
3. The feed additive for preventing piglet diarrhea according to claim 1, wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens CPLK1314, CCTCC NO: M2017658.
4. The feed additive for preventing piglet diarrhea according to claim 1, wherein the bacillus subtilis microcapsule is prepared by: activating the bacillus subtilis preserved on the inclined plane, culturing the activated bacillus subtilis to obtain seed fermentation liquor, inoculating the seed fermentation liquor for fermentation, and collecting the fermentation liquor; centrifuging the fermentation liquor, removing supernatant, collecting and washing thallus precipitates, uniformly mixing the thallus precipitates with a sodium alginate solution, injecting the mixture into a calcium carbonate solution to obtain a bacterium-containing mixed solution, slowly adding the bacterium-containing mixed solution into soybean oil containing tween, stirring, slowly stirring, adding glacial acetic acid, standing, centrifugally deoiling, collecting microcapsules, washing to obtain wet capsules, and freeze-drying to obtain the freeze-dried bacillus subtilis microcapsules.
5. The feed additive for preventing piglet diarrhea according to claim 1, wherein the slant bacillus amyloliquefaciens microcapsule is prepared by the following steps: activating strains of the bacillus amyloliquefaciens preserved on the inclined plane, culturing the activated bacillus amyloliquefaciens to obtain seed fermentation liquor, inoculating the seed fermentation liquor, fermenting and collecting the fermentation liquor; centrifuging the fermentation liquor, removing supernatant, collecting and washing thallus precipitates, uniformly mixing the thallus precipitates with a sodium alginate solution, injecting the mixture into a calcium carbonate solution to obtain a bacterium-containing mixed solution, slowly adding the bacterium-containing mixed solution into soybean oil containing tween, stirring, slowly stirring, adding glacial acetic acid, standing, centrifugally deoiling, collecting microcapsules, washing to obtain wet capsules, and freeze-drying to obtain the freeze-dried bacillus amyloliquefaciens microcapsule.
6. The feed additive for preventing piglet diarrhea according to claim 1, wherein the antimicrobial peptide No. 1 is isolated from fermentation broth after fermentation culture of Bacillus subtilis JCL 16.
7. The feed additive for preventing piglet diarrhea according to claim 1, wherein the antimicrobial peptide 2 in the amount of 5-8 parts is isolated from fermentation broth after fermentation culture of bacillus amyloliquefaciens CPLK 1314.
8. The preparation method of the feed additive for preventing the piglet diarrhea according to claim 1, which comprises the following steps:
uniformly mixing the bacillus subtilis microcapsule, the bacillus amyloliquefaciens microcapsule, the astragalus polysaccharide, the antimicrobial peptide No. 1, the antimicrobial peptide No. 2 and the corn flour to obtain the feed additive for preventing piglet diarrhea.
9. The application of the feed additive for preventing piglet diarrhea according to claim 1 in preparing a feed or a medicament for preventing and treating piglet diarrhea.
10. Use according to claim 9, characterized in that preferably 10g of additive is added per 500g of feed.
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