CN107312732A - A kind of probiotic feed additive - Google Patents
A kind of probiotic feed additive Download PDFInfo
- Publication number
- CN107312732A CN107312732A CN201710595231.5A CN201710595231A CN107312732A CN 107312732 A CN107312732 A CN 107312732A CN 201710595231 A CN201710595231 A CN 201710595231A CN 107312732 A CN107312732 A CN 107312732A
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- China
- Prior art keywords
- bacterium
- lactobacillus plantarum
- seed
- addicted
- drink
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- 239000006041 probiotic Substances 0.000 title claims abstract description 46
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 46
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 29
- 239000003674 animal food additive Substances 0.000 title claims abstract description 28
- 241000894006 Bacteria Species 0.000 claims abstract description 115
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 59
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 59
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 59
- 238000000855 fermentation Methods 0.000 claims abstract description 54
- 230000004151 fermentation Effects 0.000 claims abstract description 54
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims abstract description 46
- 241000194040 Lactococcus garvieae Species 0.000 claims abstract description 40
- 241000222122 Candida albicans Species 0.000 claims abstract description 30
- 206010007134 Candida infections Diseases 0.000 claims abstract description 30
- 201000003984 candidiasis Diseases 0.000 claims abstract description 30
- 235000020246 buffalo milk Nutrition 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 19
- 235000013365 dairy product Nutrition 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000012153 distilled water Substances 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 13
- 239000008272 agar Substances 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 241000222175 Diutina rugosa Species 0.000 claims description 10
- 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 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 10
- 229940099596 manganese sulfate Drugs 0.000 claims description 10
- 239000011702 manganese sulphate Substances 0.000 claims description 10
- 235000007079 manganese sulphate Nutrition 0.000 claims description 10
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 239000001632 sodium acetate Substances 0.000 claims description 10
- 235000017281 sodium acetate Nutrition 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 241000509461 [Candida] ethanolica Species 0.000 claims description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 9
- 229920000053 polysorbate 80 Polymers 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 239000000654 additive Substances 0.000 claims description 6
- 230000000996 additive effect Effects 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 235000013372 meat Nutrition 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 241000186660 Lactobacillus Species 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 229940039696 lactobacillus Drugs 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 3
- 210000000481 breast Anatomy 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 2
- 244000131522 Citrus pyriformis Species 0.000 claims 2
- 241000196324 Embryophyta Species 0.000 claims 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 2
- 229910052739 hydrogen Inorganic materials 0.000 claims 2
- 239000001257 hydrogen Substances 0.000 claims 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- 239000011575 calcium Substances 0.000 claims 1
- 229910052791 calcium Inorganic materials 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 235000020247 cow milk Nutrition 0.000 claims 1
- 239000013505 freshwater Substances 0.000 claims 1
- 238000005057 refrigeration Methods 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 8
- 241001465754 Metazoa Species 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 4
- 230000036039 immunity Effects 0.000 abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 229940088710 antibiotic agent Drugs 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 241000287828 Gallus gallus Species 0.000 abstract 1
- 241001494479 Pecora Species 0.000 abstract 1
- 238000009374 poultry farming Methods 0.000 abstract 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 14
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 8
- 229910000019 calcium carbonate Inorganic materials 0.000 description 7
- 235000014590 basal diet Nutrition 0.000 description 6
- 235000013336 milk Nutrition 0.000 description 6
- 239000008267 milk Substances 0.000 description 6
- 210000004080 milk Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 239000008186 active pharmaceutical agent Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000010876 biochemical test Methods 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013406 prebiotics Nutrition 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- ZGBSOTLWHZQNLH-UHFFFAOYSA-N [Mg].S(O)(O)(=O)=O Chemical compound [Mg].S(O)(O)(=O)=O ZGBSOTLWHZQNLH-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000010855 food raising agent Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Husbandry (AREA)
- Birds (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to feed addictive and its processing technique field, more particularly to a kind of probiotic feed additive, the application be by the Lactobacillus plantarum being independently isolated, fold candida bacterium, be addicted to drink candidiasis and Lactococcus garvieae and buffalo's milk be that fermentation substrate mixed fermentation is made, the application preparation technology is simple, can be widely used in the herding poultry farming such as dairy buffalo, pig, chicken, ox, sheep.Animal intestinal tract microorganism species can be significantly improved, immunity is improved, the use of antibiotics can be reduced, medicament residue is eliminated, applied to production, breeding cycle can be shortened, feed is saved, so as to improve cultivation income.
Description
【Technical field】
The present invention relates to feed addictive and its processing technique field, more particularly to a kind of probiotic feed additive.
【Background technology】
Probiotics is defined as a kind of microorganism with bioactivity by FAO (Food and Agriculture Organization of the United Nation) and the World Health Organization, benefit
The species of raw bacterium is various, is generally segmented into lactobacillus, Bifidobacterium and gram-positive cocci three major types.Numerous studies
Confirm that probiotics has to change prevention and improve diarrhoea, promote enteron aisle digestive system health, strengthen immunity, reduction serum courage to consolidate
All multiactions such as alcohol, prevention genital system infection.
In recent years, the drawbacks of largely using antibiotic to be brought as feed addictive is more and more obvious, and the medicine caused
Remain and very big influence is brought to human and animal.Prohibited and use by many countries successively with antibiotic, it is prebiotic
Bacterium will preferably be developed as the substitute of antibiotic.
Additive for microbe feedstuff is a kind of microorganism for replacing or balancing one or more fungus strains in the animal ecosystem
Product.It after it enters in livestock and poultry body as feed, can rapidly breed, promote to form the dominant microflora of lactic acid bacteria in animal and bird intestines,
The microecological balance of its enteron aisle is maintained, the growth of livestock and poultry can be effectively facilitated, the consumption of feed is reduced.At present, there are many passes the country
The report on animal feed is applied in probiotics：For example, what the A of Chinese patent CN 106906145 were announced《One kind is used to send out
The probiotics and production method of ferment ensilage》This method is fermented using probiotics to ensilage, reaches that reduction is dynamic
The purpose of thing disease of digestive system, still, the problems such as fermented feed still suffers from complex manufacturing technology, is inconvenient to store, carries,
And physical characteristics of this method also not for the buffalo that gives milk are studied, therefore, produce a kind of technique and simply, conveniently take
Band, the feed addictive of storage, to increase the synergy between each microorganism, improve culture benefit, for Native buffalo milk
Physical condition improves the feeding efficiency of buffalo milk.
【The content of the invention】
It is each to increase in view of the above, it is necessary to provide the feed addictive that a kind of technique is simple, is convenient for carrying, stores
Synergy between microorganism, improves culture benefit, and the feeding efficiency of buffalo milk is improved for Native buffalo milk physical condition.
To reach above-mentioned purpose, the technical solution adopted in the present invention is：
A kind of probiotic feed additive, the additive is 2.6 × 10 by every gram of viable count8CFU/g~9.3 ×
108CFU/g probiotics fermention is made, and the probiotics is by Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium, thermophilic
Wine candidiasis are constituted.
Further, the Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Y12, preservation
Numbering is CCTCC NO:M 2016469；The fold candida bacterium is fold candida (Candida rugosa) Y7,
Deposit number is CCTCC M 2016468；It is described to be addicted to drink candidiasis for the Candida (Candida that is addicted to drink
Ethanolica) Y1, deposit number is CCTCC M 2016467；It is isolated from fresh buffalo's milk.
Further, methods described comprises the following steps：
(1) Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium and candidiasis of being addicted to drink are seeded in MRS respectively
Cultivated on slant medium, cultivation temperature is 37 DEG C, and incubation time is 24h-48h, respectively obtain the training of Lactococcus garvieae inclined-plane
Bacteria, Lactobacillus plantarum inclined-plane culture bacterium, fold candida bacterium inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink；
(2) by the false silk ferment of the lattice Lactococcus garvieae inclined-plane culture bacterium, Lactobacillus plantarum inclined-plane culture bacterium, gauffer of step (1)
Female bacterium inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink are transferred to equipped with the MRS broth bouillons sterilized respectively
Shaking table culture is carried out in test tube, cultivation temperature is 37 DEG C, and incubation time is 24h, respectively obtain Lactococcus garvieae seed liquor, plant
Thing lactobacillus seed liquor, fold candida bacterium seed liquor and candidiasis seed liquid of being addicted to drink；
(3) by the Lactococcus garvieae seed liquor of step (2), Lactobacillus plantarum seed liquor, fold candida bacterium seed liquor
With candidiasis seed liquid of being addicted to drink by 1：2:2:2 mass ratio carries out being mixed to get seed mixture liquid, then according to hybrid
The mass ratio of sub- liquid and zymotic fluid is 2:25 inoculum concentration is inoculated into the fermentation tank equipped with fermentation substrate, and fermentation temperature is 37
DEG C, fermentation time is 24h, the seed bacterium solution after being expanded；
(4) it is 1-2 that the seed bacterium solution after step (3) is expanded, which presses seed bacterium solution and the mass ratio of zymotic fluid,:10 inoculation
Amount is inoculated into the fermentation tank equipped with fermentation substrate, and fermentation temperature is 37 DEG C, and fermentation time is 24h, and the pH of fermentation is 3.2-
4.8, it is cooled to room temperature and obtains fermented by mixed bacterium liquid；
(5) it is that 3000-8000rpm centrifuges are centrifuged with rotating speed by the fermented by mixed bacterium liquid of step (4), during centrifugation
Between be 15-20min, centrifugation remove supernatant, obtain semi-solid bacterium mud, by bacterium mud be placed in refrigerator freeze 4h after be put into vacuum
It is dried in freeze drier, then takes out and crushed, pack and obtain probiotic feed additive.
Further, the MRS inclined-plane culture based components of the step (1) are：Peptone 10.0g/L, powdered beef 8.0g/L,
Dusty yeast 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L,
Magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, agar 14g/L, Tween-80 1g/L, surplus is distilled water.
Further, the MRS broth bouillon compositions of the step (2) are：Peptone 10.0g/L, meat extract 10.0g/L,
Yeast extract 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/
L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, cysteine 0.5g/L, Tween-80 1.0g/L, surplus are distilled water, pH
It is worth for 6.2-6.4.
Further, the step (3), the mass percent of (4) fermentation substrate are：Buffalo's milk 25%~41%, sucrose 5%
~10%, yeast extract 0.02%~0.14%, agar 5%~8%, calcium carbonate 0.07%~1%, distilled water 40%~60%.
Further, Lactobacillus plantarum is 2.6 × 10 containing viable count in the fermented by mixed bacterium liquid of the step (4)8~
5.3× 108CFU/g, Lactococcus garvieae is 3.1 × 10 containing viable count8~3.8 × 108CFU/g。
Further, the probiotic feed additive is cultivated applied to dairy buffalo.
Microorganism fungus kind source is as follows in the probiotic feed additive of the application：
1st, the Lactobacillus plantarum of the application is Lactobacillus plantarum (Lactobacillus plantarum) Y12：
Above-mentioned Lactobacillus plantarum is that Lactobacillus plantarum (Lactobacillus plantarum) Y12 is separated from buffalo's milk
And obtain, buffalo's milk is derived from Guangxi buffalo research institute and plants buffalo, is separated using MRS culture medium flat plates rubbing method, inclined-plane method of scoring
And obtain, 16SrDNA strain idenfications are completed by giving birth to work bioengineering (Shanghai) company.Bacterial Physiological biochemical test utilizes French plum
The API 20A reagent strips of Li Ai companies are completed.It is 37~39 DEG C that strain, which is adapted to growth temperature, is preserved within 8th in September in 2016
China typical culture collection center, deposit number is CCTCC NO:M 2016469.
2nd, fold candida (Candida rugosa) Y7 of the application：
Above-mentioned fold candida (Candida rugosa) Y7 is isolated from buffalo's milk, and buffalo's milk is derived from Guang Xishui
Buffalo is planted by ox research institute, is isolated using MRS culture medium flat plates rubbing method, inclined-plane method of scoring, 16SrDNA strain idenfications
Completed by raw work bioengineering (Shanghai) company.Bacterial Physiological biochemical test is tried using the CH of API 50 of Mei Liai companies of France
Agent bar is completed.It is 30~35 DEG C that strain, which is adapted to growth temperature, is preserved in September in 2016 in China typical culture collection within 8th
The heart, deposit number is CCTCC NO:M 2016468.
3rd, Candida of being addicted to drink (Candida ethanolica) Y1 of the application：
Above-mentioned Candida of being addicted to drink (Candida ethanolica) Y1 is isolated from buffalo's milk, and buffalo's milk is derived from extensively
Buffalo is planted by western buffalo research institute, is isolated using MRS culture medium flat plates rubbing method, inclined-plane method of scoring, 16SrDNA strains
Identification is completed by raw work bioengineering (Shanghai) company.Bacterial Physiological biochemical test utilizes the API 50 of Mei Liai companies of France
CH reagent strips are completed.It is 30~35 DEG C that strain, which is adapted to growth temperature, and being preserved within 8th Chinese Typical Representative culture in September in 2016 protects
Tibetan center, deposit number is CCTCC NO:M 2016467
4th, the Lactococcus garvieae of the application：
Above-mentioned Lactococcus garvieae is by being commercially available, from China typical culture collection center purchase.
The present invention has the advantages that：
1st, probiotics used in feed addictive of the present invention be from the fresh emulsion of Native buffalo milk separate, purify,
Obtained by culture, than commercially available microorganism probiotic to buffalo body with more specificity, can effectively improve probiotics to water
The regulation and control of ox body, can significantly improve animal intestinal tract microorganism species, improve immunity, can reduce making for antibiotics
With elimination medicament residue applied to production, can shorten breeding cycle, save feed, so as to improve cultivation income.
【Embodiment】
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
Any feature disclosed in this specification (including any accessory claim, summary), unless specifically stated otherwise, each
Feature is an example in a series of equivalent or similar characteristics.
Show by experiment, the Lactococcus garvieae of the application is (referred to as：Grignard), Lactobacillus plantarum (Lactobacillus
Plantarum) Y12 (abbreviations：Y12), fold candida bacterium is fold candida (Candida rugosa) Y7 (abbreviations：
Y7), it is addicted to drink Candida (Candida ethanolica) Y1 (referred to as：Y1 bio-chemical characteristics result) is as shown in table 1：
Table 1
It follows that Lactococcus garvieae, Lactobacillus plantarum (Lactobacillus plantarum) Y12, gauffer vacation silk ferment
Female bacterium is fold candida (Candida rugosa) Y7, Candida of being addicted to drink (Candida ethanolica) Y1 training
The condition of supporting, as shown in embodiment 1-3：
Embodiment 1：
A kind of probiotic feed additive, the additive is 2.6 × 10 by every gram of viable count8CFU/g probiotics hair
Ferment is made, and the probiotics is made up of Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium, candidiasis of being addicted to drink.
Above-mentioned Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Y12, and deposit number is
CCTCC NO:M 2016469；The fold candida bacterium is fold candida (Candida rugosa) Y7, and preservation is compiled
Number be CCTCC M 2016468；It is described to be addicted to drink candidiasis for Candida (Candida ethanolica) Y1 that is addicted to drink, guarantor
It is CCTCC M 2016467 to hide numbering；It is isolated from fresh buffalo's milk.
The present embodiment also provides a kind of preparation method for being used to prepare probiotic feed additive, and methods described includes as follows
Step：
(1) Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium and candidiasis of being addicted to drink are seeded in respectively
Cultivated on MRS slant mediums, wherein, MRS inclined-plane culture based components are：Peptone 10.0g/L, powdered beef 8.0g/L,
Dusty yeast 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L,
Magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, agar 14g/L, Tween-80 1g/L, surplus are distilled water, and cultivation temperature is 37
DEG C, incubation time is 24h-48h, respectively obtains Lactococcus garvieae inclined-plane culture bacterium and Lactobacillus plantarum inclined-plane culture bacterium；
(2) by the false silk ferment of the lattice Lactococcus garvieae inclined-plane culture bacterium, Lactobacillus plantarum inclined-plane culture bacterium, gauffer of step (1)
Female bacterium inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink are transferred to equipped with the MRS broth bouillons sterilized respectively
Shaking table culture is carried out in test tube, wherein, MRS broth bouillon compositions are：Peptone 10.0g/L, meat extract 10.0g/L, yeast leaching
Powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L, sulphur
Sour magnesium 0.58g/L, manganese sulfate 0.25g/L, cysteine 0.5g/L, Tween-80 1.0g/L, surplus are distilled water, and pH values are
6.2, cultivation temperature is 37 DEG C, and incubation time is 24h, respectively obtains Lactococcus garvieae seed liquor and Lactobacillus plantarum seed liquor；
(3) by the Lactococcus garvieae seed liquor of step (2), Lactobacillus plantarum seed liquor, fold candida bacterium seed liquor
With candidiasis seed liquid of being addicted to drink by 1：2:2:2 mass ratio carries out being mixed to get seed mixture liquid, then according to hybrid
The mass ratio of sub- liquid and zymotic fluid is 2:25 inoculum concentration is inoculated into the fermentation tank equipped with fermentation substrate, wherein, fermentation substrate
Mass percent be：Buffalo's milk 30%, sucrose 5%, yeast extract 0.02%, agar 5%, calcium carbonate 0.07%, distilled water
59.91%, fermentation temperature is 37 DEG C, and fermentation time is 24h, the seed bacterium solution after being expanded；
(4) it is 1.5 that the seed bacterium solution after step (3) is expanded, which presses seed bacterium solution and the mass ratio of zymotic fluid,:10 inoculation
Amount is inoculated into the fermentation tank equipped with fermentation substrate, wherein, the mass percent of fermentation substrate is：Buffalo's milk 40%, sucrose
10%, yeast extract 0.14%, agar 8%, calcium carbonate 1%, distilled water 40.86, fermentation temperature are 37 DEG C, and fermentation time is
24h, the pH of fermentation is 3.2, is cooled to room temperature and obtains fermented by mixed bacterium liquid, wherein, plant breast bar in fermented by mixed bacterium liquid
Bacterium is 2.6 × 10 containing viable count8, Lactococcus garvieae is 3.1 × 10 containing viable count8；
(5) it is that 3000 centrifuges are centrifuged with rotating speed by the fermented by mixed bacterium liquid of step (4), centrifugation time is
Supernatant is removed in 15min, centrifugation, obtains semi-solid bacterium mud, bacterium mud is placed on after freezing 4h in refrigerator and is put into vacuum freeze drying
It is dried in machine, then takes out and crushed, pack and obtain probiotic feed additive.
The feed addictive of the present embodiment is cultivated suitable for dairy buffalo.
Embodiment 2：
A kind of probiotic feed additive, the additive is 9.3 × 10 by every gram of viable count8CFU/g probiotics hair
Ferment is made, and the probiotics is made up of Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium, candidiasis of being addicted to drink.
Above-mentioned Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Y12, and deposit number is
CCTCC NO:M 2016469；The fold candida bacterium is fold candida (Candida rugosa) Y7, and preservation is compiled
Number be CCTCC M 2016468；It is described to be addicted to drink candidiasis for Candida (Candida ethanolica) Y1 that is addicted to drink, guarantor
It is CCTCC M 2016467 to hide numbering；It is isolated from fresh buffalo's milk.
The present embodiment also provides a kind of preparation method for being used to prepare probiotic feed additive, and methods described includes as follows
Step：
(1) Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium and candidiasis of being addicted to drink are seeded in respectively
Cultivated on MRS slant mediums, wherein, MRS inclined-plane culture based components are：Peptone 10.0g/L, powdered beef 8.0g/L,
Dusty yeast 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L,
Magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, agar 14g/L, Tween-80 1g/L, surplus are distilled water, and cultivation temperature is 37
DEG C, incubation time is 24h-48h, respectively obtains Lactococcus garvieae inclined-plane culture bacterium and Lactobacillus plantarum inclined-plane culture bacterium；
(2) by the false silk ferment of the lattice Lactococcus garvieae inclined-plane culture bacterium, Lactobacillus plantarum inclined-plane culture bacterium, gauffer of step (1)
Female bacterium inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink are transferred to equipped with the MRS broth bouillons sterilized respectively
Shaking table culture is carried out in test tube, wherein, MRS broth bouillon compositions are：Peptone 10.0g/L, meat extract 10.0g/L, yeast leaching
Powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L, sulfuric acid
Magnesium 0.58g/L, manganese sulfate 0.25g/L, cysteine 0.5g/L, Tween-80 1.0g/L, surplus are distilled water, and pH values are
6.4, cultivation temperature is 37 DEG C, and incubation time is 24h, respectively obtains Lactococcus garvieae seed liquor and Lactobacillus plantarum seed liquor；
(3) by the Lactococcus garvieae seed liquor of step (2), Lactobacillus plantarum seed liquor, fold candida bacterium seed liquor
With candidiasis seed liquid of being addicted to drink by 1：2:2:2 mass ratio carries out being mixed to get seed mixture liquid, then according to hybrid
The mass ratio of sub- liquid and zymotic fluid is 2:25 inoculum concentration is inoculated into the fermentation tank equipped with fermentation substrate, wherein, fermentation substrate
Mass percent be：Buffalo's milk 29.91%, sucrose 5%, yeast extract 0.02%, agar 5%, calcium carbonate 0.07%, steaming
Distilled water 60%, fermentation temperature is 37 DEG C, and fermentation time is 24h, the seed bacterium solution after being expanded；
(4) it is 2 that the seed bacterium solution after step (3) is expanded, which presses seed bacterium solution and the mass ratio of zymotic fluid,:10 inoculum concentration
It is inoculated into the fermentation tank equipped with fermentation substrate, wherein, the mass percent of fermentation substrate is：Buffalo's milk 40.86%, sucrose
10%, yeast extract 0.14%, agar 8%, calcium carbonate 1%, distilled water 40%, fermentation temperature are 37 DEG C, and fermentation time is
24h, the pH of fermentation is 3.2-4.8, is cooled to room temperature and obtains fermented by mixed bacterium liquid, wherein, plant in fermented by mixed bacterium liquid
Lactobacillus is 5.3 × 10 containing viable count8CFU/g, Lactococcus garvieae is 3.8 × 10 containing viable count8CFU/g；
(5) it is that 8000rpm centrifuges are centrifuged with rotating speed by the fermented by mixed bacterium liquid of step (4), centrifugation time is
Supernatant is removed in 20min, centrifugation, obtains semi-solid bacterium mud, bacterium mud is placed on after freezing 4h in refrigerator and is put into vacuum freeze drying
It is dried in machine, then takes out and crushed, pack and obtain probiotic feed additive.
The feed addictive of the present embodiment is cultivated suitable for dairy buffalo.
Embodiment 3：
A kind of probiotic feed additive, the additive is 6.3 × 10 by every gram of viable count8CFU/g probiotics hair
Ferment is made, and the probiotics is made up of Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium, candidiasis of being addicted to drink.
Above-mentioned Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Y12, and deposit number is
CCTCC NO:M 2016469；The fold candida bacterium is fold candida (Candida rugosa) Y7, and preservation is compiled
Number be CCTCC M 2016468；It is described to be addicted to drink candidiasis for Candida (Candida ethanolica) Y1 that is addicted to drink, guarantor
It is CCTCC M 2016467 to hide numbering；It is isolated from fresh buffalo's milk.
The present embodiment also provides a kind of preparation method for being used to prepare probiotic feed additive, and methods described includes as follows
Step：
(1) Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium and candidiasis of being addicted to drink are seeded in respectively
Cultivated on MRS slant mediums, wherein, MRS inclined-plane culture based components are：Peptone 10.0g/L, powdered beef 8.0g/L,
Dusty yeast 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L,
Magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, agar 14g/L, Tween-80 1g/L, surplus are distilled water, and cultivation temperature is 37
DEG C, incubation time is 24h-48h, respectively obtains Lactococcus garvieae inclined-plane culture bacterium and Lactobacillus plantarum inclined-plane culture bacterium；
(2) by the false silk ferment of the lattice Lactococcus garvieae inclined-plane culture bacterium, Lactobacillus plantarum inclined-plane culture bacterium, gauffer of step (1)
Female bacterium inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink are transferred to equipped with the MRS broth bouillons sterilized respectively
Shaking table culture is carried out in test tube, wherein, MRS broth bouillon compositions are：Peptone 10.0g/L, meat extract 10.0g/L, yeast leaching
Powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L, sulphur
Sour magnesium 0.58g/L, manganese sulfate 0.25g/L, cysteine 0.5g/L, Tween-80 1.0g/L, surplus are distilled water, and pH values are
6.3, cultivation temperature is 37 DEG C, and incubation time is 24h, respectively obtains Lactococcus garvieae seed liquor and Lactobacillus plantarum seed liquor；
(3) by the Lactococcus garvieae seed liquor of step (2), Lactobacillus plantarum seed liquor, fold candida bacterium seed liquor
With candidiasis seed liquid of being addicted to drink by 1：2:2:2 mass ratio carries out being mixed to get seed mixture liquid, then according to hybrid
The mass ratio of sub- liquid and zymotic fluid is 2:25 inoculum concentration is inoculated into the fermentation tank equipped with fermentation substrate, wherein, fermentation substrate
Mass percent be：Buffalo's milk 40%, sucrose 8%, yeast extract 0.12%, agar 7%, calcium carbonate 0.08%, distilled water
44.8%, fermentation temperature is 37 DEG C, and fermentation time is 24h, the seed bacterium solution after being expanded；
(4) it is 1 that the seed bacterium solution after step (3) is expanded, which presses seed bacterium solution and the mass ratio of zymotic fluid,:10 inoculum concentration
It is inoculated into the fermentation tank equipped with fermentation substrate, wherein, the mass percent of fermentation substrate is：Buffalo's milk 25%, sucrose 9%,
Yeast extract 0.10%, agar 6%, calcium carbonate 0.9%, distilled water 59%, fermentation temperature are 37 DEG C, and fermentation time is 24h,
The pH of fermentation is 4.1, is cooled to room temperature and obtains fermented by mixed bacterium liquid, wherein, Lactobacillus plantarum contains in fermented by mixed bacterium liquid
Viable count is 4.3 × 108CFU/g, Lactococcus garvieae is 3.5 × 10 containing viable count8CFU/g；
(5) it is that 5000rpm centrifuges are centrifuged with rotating speed by the fermented by mixed bacterium liquid of step (4), centrifugation time is
Supernatant is removed in 17min, centrifugation, obtains semi-solid bacterium mud, bacterium mud is placed on after freezing 4h in refrigerator and is put into vacuum freeze drying
It is dried in machine, then takes out and crushed, pack and obtain probiotic feed additive.
The feed addictive of the present embodiment is cultivated suitable for dairy buffalo.
According to group 1：
Other preparation methods, formula are identical with embodiment 1, but substitute the prebiotic of the application with commercially available EM bacterium solutions
Bacterium leavening agent.
Control group 2：
Other preparation methods, formula are identical with embodiment 1, but with commercially available Lactobacillus plantarum, the false silk ferment of gauffer
Female bacterium, candidiasis of being addicted to drink substitute Lactobacillus plantarum (Lactobacillus plantarum) Y12 of the application, and gauffer is false
Silk yeast (Candida rugosa) Y7, Candida of being addicted to drink (Candida ethanolica) Y1.
Feed experiment：
Cattle farm, experimental design are planted by Guangxi buffalo research institute：Test period is on December 7,7 days to 2015 October in 2015,
Random pair principle is taken in this experiment, and test ox is that (﹣ draws luxuriant and rich with fragrance buffalo × mora water to the miscellaneous dairy buffalo of three products in Native buffalo × Buddhist nun
Ox) by 60 dairy buffalos, experimental group and control group are randomly divided into, every group of 10 dairy buffalos are formulated according to following raising and carried out
Feed experiment：
Experimental group 1：The probiotic feed additive of 20g embodiments 1 is added into 4kg basal diet；
Experimental group 2：The probiotic feed additive of 20g embodiments 2 is added into 4kg basal diet；
Experimental group 3：The probiotic feed additive of 20g embodiments 3 is added into 4kg basal diet；
Experimental group 4：The probiotic feed additive of 20g control groups 1 is added into 4kg basal diet；
Experimental group 5：The probiotic feed additive of 20g control groups 2 is added into 4kg basal diet；
Control group：Basal diet
After feeding 10 days, the excrement dry substance raising of each group dairy buffalo is calculated；After feeding 30 days, giving milk for each group is calculated
Amount；After feeding 60 days, the per day gain in weight of each group is calculated；The outward appearance and record of dairy buffalo are observed, specific experiment the results are shown in Table
2：：Excrement dry substance raising calculation formula：
Output of milk calculation formula：
Per day gain in weight calculation formula：
Table 2
As seen from the above table, experimental group 1-3 dry substance raising is significantly lower than experimental group 4-5 and control group, increase of giving milk
Amount is apparently higher than experimental group 4-5 and control group, and per day weightening is apparently higher than test group 4-5 and control group；Diarrhea disease percentage is bright
It is aobvious to be less than experimental group 4-5 and control group；Illustrate, milk cow can be significantly improved by preparing feed addictive using the present processes
The output of milk, per day weightening, the dry matter content reduction of excrement, can improve the application to active principle；While diarrhea disease percentage
Reduction, can effectively improve enteron aisle.
In summary, animal intestinal tract microbial bacteria can be significantly improved by producing feed addictive using the present processes
Group, improves immunity, can reduce the use of antibiotics, eliminates medicament residue, applied to production, can shorten raising week
Phase, feed is saved, so as to improve cultivation income.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore limitation of the scope of the invention can not be interpreted as.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, protection scope of the present invention should be determined by the appended claims.
Claims (8)
1. a kind of probiotic feed additive, it is characterised in that the additive is 2.6 × 10 by every gram of viable count8CFU/g~
9.3×108CFU/g probiotics fermention is made, and the probiotics is by Lactococcus garvieae, Lactobacillus plantarum, fold candida
Bacterium, candidiasis of being addicted to drink composition.
2. a kind of probiotic feed additive according to claim 1, it is characterised in that the Lactobacillus plantarum is plant breast
Bacillus (Lactobacillus plantarum) Y12, deposit number is CCTCC NO:M 2016469；The false silk ferment of the gauffer
Female bacterium is fold candida (Candida rugosa) Y7, and deposit number is CCTCC M 2016468；The false silk ferment of being addicted to drink
Female bacterium is Candida (Candida ethanolica) Y1 that is addicted to drink, and deposit number is CCTCC M 2016467；From fresh water
It is isolated in cow's milk.
3. the preparation method for preparing claim 1-2 probiotic feed additives, it is characterised in that methods described is included such as
Lower step：
(1) that Lactococcus garvieae, Lactobacillus plantarum, fold candida bacterium and candidiasis of being addicted to drink are seeded in into MRS respectively is oblique
Cultivated on the culture medium of face, cultivation temperature is 37 DEG C, and incubation time is 24h-48h, respectively obtain the training of Lactococcus garvieae inclined-plane
Bacteria, Lactobacillus plantarum inclined-plane culture bacterium, fold candida bacterium inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink；
(2) by lattice Lactococcus garvieae inclined-plane culture bacterium, Lactobacillus plantarum inclined-plane culture bacterium, the fold candida bacterium of step (1)
Inclined-plane culture bacterium and candidiasis inclined-plane culture bacterium of being addicted to drink are transferred to equipped with the MRS broth bouillon test tubes sterilized respectively
Shaking table culture is carried out, cultivation temperature is 37 DEG C, and incubation time is 24h, respectively obtains Lactococcus garvieae seed liquor, Lactobacillus plantarum
Seed liquor, fold candida bacterium seed liquor and candidiasis seed liquid of being addicted to drink；
(3) by the Lactococcus garvieae seed liquor of step (2), Lactobacillus plantarum seed liquor, fold candida bacterium seed liquor and thermophilic
Wine candidiasis seed liquid presses 1：2:2:2 mass ratio carries out being mixed to get seed mixture liquid, then according to seed mixture liquid
Mass ratio with zymotic fluid is 2:25 inoculum concentration is inoculated into the fermentation tank equipped with fermentation substrate, and fermentation temperature is 37 DEG C, hair
The ferment time is 24h, the seed bacterium solution after being expanded；
(4) it is 1-2 that the seed bacterium solution after step (3) is expanded, which presses seed bacterium solution and the mass ratio of zymotic fluid,:10 inoculum concentration connects
Plant into the fermentation tank equipped with fermentation substrate, fermentation temperature is 37 DEG C, fermentation time is 24h, and the pH of fermentation is 3.2-4.8, cold
But arrive room temperature and obtain fermented by mixed bacterium liquid；
(5) it is that 3000-8000rpm centrifuges are centrifuged with rotating speed by the fermented by mixed bacterium liquid of step (4), centrifugation time is
Supernatant is removed in 15-20min, centrifugation, obtains semi-solid bacterium mud, bacterium mud is placed on after freezing 4h in refrigerator and is put into vacuum refrigeration and does
It is dried in dry machine, then takes out and crushed, pack and obtain probiotic feed additive.
4. preparation method according to claim 3, it is characterised in that the MRS inclined-plane culture based components of the step (1)
For：Peptone 10.0g/L, powdered beef 8.0g/L, dusty yeast 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, lemon
Lemon acid hydrogen two ammonium 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.04g/L, agar 14g/L, Tween-80
1g/L, surplus is distilled water.
5. preparation method according to claim 3, it is characterised in that the MRS broth bouillon compositions of the step (2)
For：Peptone 10.0g/L, meat extract 10.0g/L, yeast extract 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, lemon
Lemon acid hydrogen two ammonium 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.25g/L, cysteine 0.5g/L, tell
- 80 1.0g/L of temperature, surplus is distilled water, and pH value is 6.2-6.4.
6. preparation method according to claim 3, it is characterised in that the step (3), the quality hundred of (4) fermentation substrate
Fraction is：Buffalo's milk 25%~41%, sucrose 5%~10%, yeast extract 0.02%~0.14%, agar 5%~8%, carbon
Sour calcium 0.07%~1%, distilled water 40%~60%.
7. preparation method according to claim 3, it is characterised in that planted in the fermented by mixed bacterium liquid of the step (4)
Thing lactobacillus is 2.6 × 10 containing viable count8~5.3 × 108CFU/g, Lactococcus garvieae is 3.1 × 10 containing viable count8~3.8 ×
108CFU/g。
8. a kind of probiotic feed additive according to claim 1, it is characterised in that the probiotic feed additive
Applied to dairy buffalo cultivation.
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| CN107812018A (en) * | 2017-11-13 | 2018-03-20 | 浙江禾健生营养食品有限公司 | A kind of inactivated probiotic product, preparation method and applications |
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| CN111955742A (en) * | 2020-08-21 | 2020-11-20 | 中民集团生物科技有限公司 | Composite probiotics and preparation method thereof |
| CN112094777A (en) * | 2020-09-24 | 2020-12-18 | 青岛普罗百世生物科技有限公司 | Lactobacillus plantarum and application thereof in Laoshan milk goat feed |
| CN113749179A (en) * | 2021-09-10 | 2021-12-07 | 南京工业大学 | Application of curdlan in preparation of feed additive |
| CN115197885A (en) * | 2022-09-13 | 2022-10-18 | 中国农业科学院北京畜牧兽医研究所 | Lactococcus garvieae LGM15, microbial inoculum and application |
| CN115197885B (en) * | 2022-09-13 | 2022-12-09 | 中国农业科学院北京畜牧兽医研究所 | Lactococcus garvieae LGM15, microbial inoculum and application |
| CN116286556A (en) * | 2023-05-06 | 2023-06-23 | 江门市澳保生物科技有限公司 | Lactococcus garvieae ABML2022003, microbial starter and preparation method thereof |
| CN116286556B (en) * | 2023-05-06 | 2023-07-28 | 江门市澳保生物科技有限公司 | Lactococcus garvieae ABML2022003, microbial starter and preparation method thereof |
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