CN110699281A - Special compound bacterium preparation for hybrid mutton sheep and preparation method thereof - Google Patents
Special compound bacterium preparation for hybrid mutton sheep and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 59
- 241000894006 Bacteria Species 0.000 title claims abstract description 36
- 150000001875 compounds Chemical class 0.000 title claims abstract description 18
- 241001494479 Pecora Species 0.000 title claims abstract 16
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 69
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 69
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 50
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 50
- 240000006439 Aspergillus oryzae Species 0.000 claims abstract description 36
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims abstract description 36
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 35
- 241000235646 Cyberlindnera jadinii Species 0.000 claims abstract description 35
- 244000057717 Streptococcus lactis Species 0.000 claims abstract description 35
- 235000014897 Streptococcus lactis Nutrition 0.000 claims abstract description 35
- 241000186016 Bifidobacterium bifidum Species 0.000 claims abstract description 34
- 240000001046 Lactobacillus acidophilus Species 0.000 claims abstract description 34
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims abstract description 34
- 229940002008 bifidobacterium bifidum Drugs 0.000 claims abstract description 34
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims abstract description 34
- 244000005700 microbiome Species 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims description 179
- 230000004151 fermentation Effects 0.000 claims description 179
- 239000000843 powder Substances 0.000 claims description 104
- 238000011081 inoculation Methods 0.000 claims description 43
- 238000012258 culturing Methods 0.000 claims description 36
- 230000004913 activation Effects 0.000 claims description 35
- 238000003756 stirring Methods 0.000 claims description 32
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 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 21
- 239000008103 glucose Substances 0.000 claims description 21
- 239000001888 Peptone Substances 0.000 claims description 19
- 108010080698 Peptones Proteins 0.000 claims description 19
- 235000019319 peptone Nutrition 0.000 claims description 19
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 16
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 16
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 14
- 239000002131 composite material Substances 0.000 claims description 13
- 229920000136 polysorbate Polymers 0.000 claims description 13
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000003223 protective agent Substances 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- 239000001110 calcium chloride Substances 0.000 claims description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- 108010009004 proteose-peptone Proteins 0.000 claims description 5
- 235000015193 tomato juice Nutrition 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- -1 0.1-0.5 g/L Chemical compound 0.000 claims description 3
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 claims description 3
- 239000005862 Whey Substances 0.000 claims description 3
- 108010046377 Whey Proteins Proteins 0.000 claims description 3
- 102000007544 Whey Proteins Human genes 0.000 claims description 3
- 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 claims description 3
- 235000008476 powdered milk Nutrition 0.000 claims description 3
- 235000020183 skimmed milk Nutrition 0.000 claims description 3
- 238000012136 culture method Methods 0.000 claims 2
- 235000013336 milk Nutrition 0.000 claims 1
- 239000008267 milk Substances 0.000 claims 1
- 210000004080 milk Anatomy 0.000 claims 1
- 235000013372 meat Nutrition 0.000 abstract description 13
- 150000002576 ketones Chemical class 0.000 abstract description 5
- 206010059866 Drug resistance Diseases 0.000 abstract description 3
- 238000009396 hybridization Methods 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 111
- 241000283898 Ovis Species 0.000 description 69
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- 241001052560 Thallis Species 0.000 description 12
- 230000000644 propagated effect Effects 0.000 description 12
- 241000283903 Ovis aries Species 0.000 description 11
- 238000003307 slaughter Methods 0.000 description 9
- 235000019764 Soybean Meal Nutrition 0.000 description 8
- 229940099596 manganese sulfate Drugs 0.000 description 8
- 239000011702 manganese sulphate Substances 0.000 description 8
- 235000007079 manganese sulphate Nutrition 0.000 description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 8
- 239000004455 soybean meal Substances 0.000 description 8
- 235000019687 Lamb Nutrition 0.000 description 7
- 235000015278 beef Nutrition 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 6
- 238000001694 spray drying Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 230000003068 static effect Effects 0.000 description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 235000013312 flour Nutrition 0.000 description 4
- 235000013379 molasses Nutrition 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
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- 238000004519 manufacturing process Methods 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 239000001965 potato dextrose agar Substances 0.000 description 3
- 229920002261 Corn starch Polymers 0.000 description 2
- 239000004201 L-cysteine Substances 0.000 description 2
- 235000013878 L-cysteine Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910000365 copper sulfate Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 235000013325 dietary fiber Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000006872 mrs medium Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- 238000011049 filling Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
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- 239000010985 leather Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- 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
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
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- C12N1/18—Baker's yeast; Brewer's yeast
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/517—Bifidum
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Abstract
The invention provides a special compound bacteria preparation for hybrid mutton sheep, which comprises the following microorganisms: bacillus subtilis, Bacillus amyloliquefaciens, lactococcus lactis, Bifidobacterium bifidum, Lactobacillus acidophilus, Candida utilis, Saccharomyces cerevisiae and Aspergillus oryzae. The special compound bacteria preparation for the hybrid mutton sheep is non-toxic, free of drug resistance and free of residue; the quality of F1 generation hybrid mutton sheep obtained by hybridization of pure black-head Dorper sheep and small tailed Han sheep is obviously improved, and the dressing percentage and the ketone body meat yield are improved.
Description
Technical Field
The invention belongs to a microecological preparation for hybrid mutton sheep, and particularly relates to a special composite bacterial preparation for hybrid mutton sheep and a preparation method thereof.
Background
The Dorper sheep has stable heredity, and shows excellent production performance and adaptability regardless of pure breeding offspring or improved offspring, particularly meat production performance, uniform fat distribution in meat and high-quality carcass. The meat sheep breeds introduced and produced in China are not comparable. The carcass quality of the lambs with good growth can reach the excellent standard no matter in the aspect of shape or fat distribution. At present, the domestic introduced variety is mainly white, few black heads are introduced in pure breeds, and the good population of the pure black-head Dorper sheep is obtained by introducing the embryo of the Australia pure breed black-head Dorper sheep, taking small tailed Han sheep and Hu sheep as recipient ewes and adopting an embryo transplantation technology. The obtained pure black-head DuPo sheep and small-tail Han sheep are hybridized through techniques of estrus synchronization, uterine horn fertilization and the like to establish and expand high-quality F1 generation hybrid meat sheep flocks.
The F1 generation mutton sheep obtained by hybridization of the pure black-head Dorper sheep and the small tailed Han sheep has the advantages that the lambs grow rapidly, the weight of 6 months old lambs is as high as more than 52 kilograms, the lamb has the capability of early feeding, the meat yield is as high as 46%, the slaughtering rate of fat lambs is as high as 55%, the fat among muscles is rich, the meat quality is delicious, the additional value of the veneer is high, and the lamb is an internationally popular high-end leather raw material. However, the hybrid mutton sheep grows rapidly, the requirements on the feeding conditions of the lambs are high, the intestinal flora is easy to unbalance, the intestinal diseases of the lambs are caused, and the utilization rate of the feed is reduced. The prior art also has a microbial preparation for conditioning the intestinal flora of mutton sheep, but the effect is not ideal when the microbial preparation is used for the F1 generation mutton sheep obtained by hybridizing pure black-head Dorper sheep and small-tail Han sheep.
The prior art has the following technical defects: the F1 generation mutton sheep for the hybridization of the pure breed black-head Dorper sheep and the small tailed Han sheep has poor disease prevention effect on the lambs, can not well condition the intestinal flora of the lambs, and causes the low utilization rate of the feed for the lambs, and the quality of the hybrid mutton sheep is poor.
Disclosure of Invention
Aiming at the technical problems, the invention provides the special compound bacteria preparation for the hybrid mutton sheep and the preparation method thereof, aiming at the F1 generation mutton sheep hybridized by the pure breed of the black-head Dorper sheep and the small tailed Han sheep, the intestinal flora balance of the mutton sheep can be well improved, the disease of the hybrid mutton lamb is prevented, the feed utilization rate is improved, and the quality of the hybrid mutton sheep is improved; meanwhile, the paint has no toxicity, drug resistance and residue.
The preparation method of the special composite bacteria preparation for the hybrid mutton sheep has low production cost and simple and convenient operation.
In order to solve the technical problems, the invention adopts the technical scheme that:
a special compound bacteria preparation for hybrid mutton sheep is prepared from the following raw materials in parts by weight:
bacillus subtilis CICC 100905-15 parts
10-20 parts of bacillus subtilis CICC 2044510
Bacillus amyloliquefaciens CICC 230435-10 parts
Lactococcus lactis CICC 2360910-20 parts
Bifidobacterium bifidum CICC 616810-20 parts
110735-15 parts of Lactobacillus acidophilus ACCC
Candida utilis CICC 180710-20 parts
14215-10 parts of saccharomyces cerevisiae CICC
Aspergillus oryzae CICC 414785-10 parts.
A preparation method of a special compound bacteria preparation for hybrid mutton sheep comprises the following steps:
step 1, culturing bacillus subtilis CICC10090
Inoculating bacillus subtilis CICC10090 strain on a slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 15-30%; the activation culture medium is an LB culture medium, and is cultured for 10-12 h at the culture temperature of 28-30 ℃ and the shaking rotation speed of 140-160 rpm;
inoculating the activated and cultured bacillus subtilis strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 0.1-0.5%, the culture temperature is 28-30 ℃, the stirring speed of a seeding tank is 220-240 rpm, the culture is carried out for 10-12 h, and the strain reaches the logarithmic phase;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 5-10%, the culture temperature is 28-30 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the fermentation is carried out for 36-42 h, the conversion rate of bacillus subtilis spores in the fermentation tank is more than 95%, and the content is more than or equal to 1010Each/ml, then spray drying the single bacillus subtilis obtained by fermentation to prepare dormant microbe dry powder, wherein the content of bacillus subtilis spores in the dry powder is more than or equal to 1011One per gram.
The culture medium formula of the seed tank is the same as that of the fermentation tank, and the culture medium formula comprises 20-25 g/L of corn flour, 5-10 g/L of glucose, 30-40 g/L of soybean meal, 1-3 g/L of calcium chloride, 1-3 g/L of magnesium sulfate and 0.5-0.8 g/L of manganese sulfate.
Step 2, culturing bacillus subtilis CICC20445
Inoculating bacillus subtilis CICC20445 strain on a slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 15-30%; the formula of the activation medium is a beef extract peptone medium, and the activation medium is cultured for 8-12 h at the culture temperature of 36-39 ℃ and the shaking rotation speed of 140-180 rpm;
inoculating the activated and cultured bacillus subtilis strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 0.2-1%, the culture temperature is 36-39 ℃, the stirring speed of a seeding tank is 220-240 rpm, the culture is carried out for 8-10 h, and the strain reaches the logarithmic phase;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 5-10%, the culture temperature is 36-39 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the fermentation tank is cultured for 26-36 h, the conversion rate of bacillus subtilis spores in the fermentation tank is more than 95%, and the content is more than or equal to 1010Each/ml, then spray drying the single bacillus subtilis obtained by fermentation to prepare dormant microbe dry powder, wherein the content of bacillus subtilis spores in the dry powder is more than or equal to 1011One per gram.
The culture medium formula of the seed tank is the same as that of the fermentation tank, and the culture medium formula comprises 15-25 g/L of corn starch, 20-30 g/L of soybean meal, 1-5 g/L of sodium chloride, 2-6 g/L of dipotassium phosphate, 0.5-2 g/L of magnesium sulfate and 0.1-0.5 g/L of manganese sulfate.
Step 3, culturing bacillus amyloliquefaciens CICC23043
Inoculating bacillus amyloliquefaciens CICC23043 strain on a slant culture medium subjected to purification culture into a conical flask, wherein the conical flask is filled with an activated culture medium with the volume percentage of 15-30%; the formula of the activation medium is a beef extract peptone medium, the culture temperature is 36-38 ℃, and the shaking rotation speed is 130-150 rpm for culture for 10-12 h;
inoculating activated and cultured bacillus amyloliquefaciens strains into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 0.1-0.5%, the culture temperature is 36-38 ℃, the stirring speed of a seeding tank is 220-240 rpm, the culture is carried out for 10-12 h, and the strain reaches the logarithmic phase;
inoculating cultured seed tank strain into a fermentation tank, and filling the fermentation tankA fermentation medium with the volume percentage of 50-70 percent is provided; the volume inoculation amount is 5-10%, the culture temperature is 36-38 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the culture is carried out for 35-40 h, the conversion rate of bacillus amyloliquefaciens spores in the fermentation tank is more than 95%, and the content is more than or equal to 1.6 multiplied by 1010Each/ml, then spray drying the bacillus amyloliquefaciens obtained by fermentation to prepare dormant microbe dry powder, wherein the content of bacillus amyloliquefaciens spores in the dry powder is more than or equal to 1.8 multiplied by 1011One per gram.
The formula of the seed tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 25-35 g/L of corn flour, 30-40 g/L of soybean meal, 1-5 g/L of sodium chloride, 1-3 g/L of potassium dihydrogen phosphate, 1-3 g/L of dipotassium hydrogen phosphate and 0.1-0.5 g/L of manganese sulfate.
Step 4, culturing lactococcus lactis CICC23609
Inoculating the lactococcus lactis CICC23609 strain on the slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage of 15-30%; the formula of the activation medium is as follows: yeast extract 7.5g, glucose 10g, tomato juice 100ml, peptone 7.5g, potassium dihydrogen phosphate 2.0g, tween 800.5 ml, distilled water 900ml, pH 7.0; standing and culturing for 26-30 h at the culture temperature of 26-29 ℃;
inoculating activated and cultured lactococcus lactis strains into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 1-3%, the culture temperature is 26-29 ℃, the stirring speed of a seeding tank is 150-180 rpm, the culture is carried out for 24-26 h, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank filled with a fermentation culture medium with the volume percentage content of 50-70%, wherein the volume inoculation amount is 5-10%, culturing at the temperature of 26-29 ℃ and the stirring rotation speed of the fermentation tank of 150-180 rpm for 40-48 h, and the content of lactococcus lactis in the fermentation tank is not less than 109Adding protective agent into the lactococcus lactis single bacterium obtained by fermentation, and freeze-drying to prepare dormant microorganism dry powder, wherein the content of lactococcus lactis in the dry powder is more than or equal to 1010One per gram.
The protective agent is whey powder and skim milk powder which are compounded according to the mass ratio of 3-5: 2-4.
The culture medium formula of the seeding tank and the fermentation tank is the same, and the culture medium formula comprises 5-15 g/L of yeast powder, 10-20 g/L of glucose, 10-20 g/L of peptone, 1-5 g/L of potassium dihydrogen phosphate, 800.5-1 ml/L of tween, 1-5 g/L of sodium acetate and 100-150 ml/L of tomato juice.
Step 5, culturing Bifidobacterium bifidum CICC6168
Inoculating bifidobacterium bifidum CICC6168 strain on the slant culture medium which is subjected to purification culture into an anaerobic bottle, wherein the anaerobic bottle is filled with an activated culture medium with the volume percentage of 60-80%; the formula of the activation medium comprises casein peptone 10-20 g/L, glucose 10-20 g/L, soybean peptone 5-10 g/L, yeast powder 5-10 g/L, dipotassium hydrogen phosphate 1-5 g/L, magnesium sulfate 0.1-0.5 g/L, L-cysteine 0.1-0.5 g/L, Tween 800.5-1 ml/L, sodium chloride 1-5 g/L and calcium chloride 0.01-0.05 g/L; standing and culturing for 42-46 h at the culture temperature of 36-38 ℃;
inoculating the activated and cultured bifidobacterium bifidum strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 60-80%; the volume inoculation amount is 1-3%, the culture temperature is 36-38 ℃, standing culture is carried out for 22-25 h, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 60-80%; the volume inoculation amount is 5-10%, the culture temperature is 36-38 ℃, the static culture is carried out for 44-48 h, and the content of bifidobacterium bifidum in a fermentation tank is more than or equal to 6 multiplied by 108Adding protective agent into the fermented Bifidobacterium bifidum single bacterium, and freeze drying to obtain dormant microorganism dry powder with Bifidobacterium bifidum content not less than 2 × 1010One per gram.
The formula of the seed tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 15-25 g/L of casein peptone, 15-25 g/L of glucose, 10-15 g/L of soybean peptone, 5-10 g/L of yeast powder, 3-8 g/L of dipotassium hydrogen phosphate, 0.1-0.5 g/L, L-cysteine, 0.1-0.5 g/L of magnesium sulfate, 800.5-1 ml/L of tween, 5-10 g/L of sodium chloride and 0.01-0.05 g/L of calcium chloride.
Step 6, culturing lactobacillus acidophilus ACCC11073
Inoculating lactobacillus acidophilus ACCC11073 strain on a slant culture medium subjected to purification culture into a triangular flask, wherein the triangular flask is filled with an activation culture medium with the volume percentage of 60-80%; the formula of the activation medium is MRS medium, and the activation medium is subjected to static culture for 22-26 hours at the culture temperature of 35-38 ℃;
inoculating the lactobacillus acidophilus strain subjected to activated culture into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 60-80%; the volume inoculation amount is 1-3%, the culture temperature is 35-38 ℃, the seeding tank is kept still for 18-22 hours, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 60-80%; the volume inoculation amount is 5-10%, the fermentation tank is kept still for 24-30 h at the culture temperature of 35-38 ℃, and the content of lactobacillus acidophilus in the fermentation tank is more than or equal to 3 multiplied by 109Adding protective agent into lactobacillus acidophilus obtained by fermentation, and freeze drying to obtain dormant microorganism dry powder containing lactobacillus acidophilus at a content of 5 × 10 or more10One per gram.
The culture medium formula of the seeding tank and the fermentation tank is the same, and the culture medium formula comprises 20-25 g/L of glucose, 15-25 g/L of peptone, 5-15 g/L of yeast powder, 1-5 g/L of dipotassium phosphate, 1-5 g/L of diammonium hydrogen citrate, 800.5-1 ml/L of tween, 1-10 g/L of sodium acetate and 0.01-0.05 g/L of magnesium sulfate.
Step 7, culturing Candida utilis CICC1807
Inoculating the candida utilis strain on the slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activated culture medium with the volume percentage of 15-30%; the formula of the activation medium is a glucose potato culture medium, the culture temperature is 28-30 ℃, and the shaking rotation speed is 130-150 rpm for 18-20 h;
inoculating the Candida utilis strain subjected to activated culture into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 1-3%, the culture temperature is 28-30 ℃, the stirring speed of a seeding tank is 220-240 rpm, the culture is carried out for 18-20 h, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 5-10%, the culture temperature is 28-30 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the culture is carried out for 32-36 h, and the content of the candida utilis in the fermentation tank is not less than 3 multiplied by 109Per ml, then the candida utilis obtained by fermentation is fluidized bed boiling dried to prepare dormant microorganism dry powder, and the content of the candida utilis in the dry powder is more than or equal to 2 multiplied by 1010One per gram.
The formula of the seeding tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 10-20 g/L of peptone, 20-30 g/L of molasses, 5-15 g/L of beef powder, 1-5 g/L of monopotassium phosphate, 1-5 g/L of dipotassium phosphate and 10-20 g/L of ammonium sulfate.
Step 8, culturing Saccharomyces cerevisiae CICC1421
And inoculating the saccharomyces cerevisiae strain on the slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activated culture medium with the volume percentage of 15-30%. The formula of the activation culture medium is a wort culture medium, the culture temperature is 28-30 ℃, and the shaking rotation speed is 130-150 rpm for 20-24 h;
inoculating the saccharomyces cerevisiae strain subjected to activation culture into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 50-70%; the volume inoculation amount is 1-3%, the culture temperature is 28-30 ℃, the stirring speed of a seeding tank is 200-220 rpm, the culture is carried out for 20-22 h, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 5-10%, the culture temperature is 28-30 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the culture is carried out for 28-32 hours, and the content of saccharomyces cerevisiae in the fermentation tank is more than or equal to 4 multiplied by 109Drying the fermented Saccharomyces cerevisiae by fluidized bed boiling to obtain dormant microorganism dry powder with Saccharomyces cerevisiae content not less than 5 × 1010One per gram.
The culture medium formula of the seeding tank is the same as that of the fermentation tank, and the culture medium formula comprises 15-25 g/L of glucose, 20-30 g/L of molasses, 5-10 g/L of yeast powder, 1-5 g/L of magnesium sulfate, 1-5 g/L of dipotassium hydrogen phosphate and 10-20 g/L of ammonium sulfate.
Step 8, culturing Aspergillus oryzae CICC41478
Inoculating an Aspergillus oryzae CICC41478 strain on a slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activated culture medium with the volume percentage content of 15-30%; the formula of the activation medium is a PDA (potato dextrose agar) culture medium, the culture temperature is 25-28 ℃, and the shaking flask is cultured for 36-40 h at the rotating speed of 130-150 rpm.
Inoculating the activated and cultured aspergillus oryzae strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 1-3%, the culture temperature is 25-28 ℃, the stirring speed of a seeding tank is 200-220 rpm, the culture is carried out for 40-48 hours, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 50-70%; the volume inoculation amount is 5-10%, the culture temperature is 25-28 ℃, the stirring speed of a fermentation tank is 180-200 rpm, the culture is carried out for 3-4 days, and the content of aspergillus oryzae spores in the fermentation tank is more than or equal to 108Drying fermented Aspergillus oryzae with fluidized bed to obtain dormant microorganism powder with Aspergillus oryzae content not less than 109One per gram.
The formula of the seed tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 10-20 g/L of bran, 20-30 g/L of glucose, 25-35 g/L of soybean meal, 1-5 g/L of sodium chloride, 0.5-1 g/L of calcium chloride, 5-10 g/L of monopotassium phosphate, 1-3 mg/L of ferrous sulfate, 0.5-1 mg/L of copper sulfate and 0.1-5 mg/L of manganese sulfate.
Step 9, obtaining a finished product
Respectively mixing the prepared dormant microorganism dry powder of bacillus subtilis CICC10090, bacillus subtilis CICC20445, bacillus amyloliquefaciens CICC23043, lactococcus lactis CICC23609, bifidobacterium bifidum CICC6168, lactobacillus acidophilus ACCC11073, candida utilis CICC1807, saccharomyces cerevisiae CICC1421 and aspergillus oryzae CICC41478 according to the weight ratio of 5-15 parts of bacillus subtilis (CICC 10096), 10-20 parts of bacillus subtilis (CICC 20445), 5-10 parts of bacillus amyloliquefaciens (CICC 23043), 10-20 parts of lactococcus lactis (CICC 23609), 10-20 parts of bifidobacterium bifidum (CICC 6168), 5-15 parts of bifidobacterium acidophilus (ACCC 11073), 10-20 parts of candida utilis (CICC 1807), 5-10 parts of saccharomyces cerevisiae (CICC 1421) and 5-10 parts of aspergillus oryzae (CICC 41478) to obtain a special hybrid mutton sheep composite preparation.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) the special compound bacteria preparation for the hybrid mutton sheep is non-toxic, free of drug resistance and free of residue;
(2) the special compound bacterium preparation for the hybrid mutton sheep obviously improves the quality of F1 generation hybrid mutton sheep obtained by hybridizing pure black-head Dorper sheep and small-tail Han sheep, the slaughter rate is improved by more than 2.5 percent, and the meat yield of ketone bodies is improved by more than 4 percent;
(3) the special compound bacterium preparation for the hybrid mutton sheep obviously improves the intestinal performance of the F1 generation hybrid mutton sheep hybridized with the pure black-head Dorper sheep and the small tailed Han sheep, and improves the feed utilization rate by over 8 percent;
(4) the special compound bacteria preparation for the hybrid mutton sheep can obviously prevent the lamb diseases of F1 generation hybrid mutton sheep obtained by hybridizing pure black-head Dorper sheep and small-tail Han sheep, and particularly has good curative effect on lamb diarrhea.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1A Special Compound bacteria preparation for hybrid mutton sheep
The feed is prepared by mixing the following raw materials in parts by weight:
bacillus subtilis CICC 100905 parts
Bacillus subtilis CICC 2044520 parts
Bacillus amyloliquefaciens CICC 2304310 parts
Lactococcus lactis CICC 2360910 parts
Bifidobacterium bifidum CICC 616810 parts
Lactobacillus acidophilus ACCC 110735 parts
Candida utilis CICC 180710 parts
Saccharomyces cerevisiae CICC 142110 parts
Aspergillus oryzae CICC 414785 parts.
The bacillus subtilis CICC 10090: dormant microbe dry powder prepared from bacillus subtilis with deposit number of CICC10090, wherein the bacillus subtilis spore content in the dry powder is 1.8 multiplied by 1011Per gram;
the bacillus subtilis CICC 20445: dormant microbe dry powder prepared for bacillus subtilis with deposit number of CICC20445, wherein the bacillus subtilis spore content in the dry powder is 1.5 multiplied by 1011Per gram;
the bacillus amyloliquefaciens CICC 23043: a dormant microbe dry powder prepared from Bacillus amyloliquefaciens with accession number of CICC23043 and containing Bacillus amyloliquefaciens spore content of 1.8 × 1011Per gram;
the lactococcus lactis CICC 23609: a dry powder of dormant microorganism prepared from lactococcus lactis with deposit number CICC23609, wherein the content of lactococcus lactis in the dry powder is 8 × 1010Per gram;
the bifidobacterium bifidum CICC 6168: is dormant microorganism dry powder of Bifidobacterium bifidum with deposit number CICC6168, wherein the content of Bifidobacterium bifidum in the dry powder is 2 × 1010Per gram;
the lactobacillus acidophilus ACCC 11073: is dormant microorganism powder of Lactobacillus acidophilus with accession number ACCC11073, wherein the content of Lactobacillus acidophilus in the dry powder is 5 × 1010Per gram;
the candida utilis CICC 1807: is dormant microorganism dry powder of Candida utilis with deposit number of CICC1807, and the content of Candida utilis in the dry powder is 2 × 1010Per gram;
the saccharomyces cerevisiae CICC 1421: is dormant microorganism dry powder of Saccharomyces cerevisiae with deposit number of CICC1421, wherein the content of Saccharomyces cerevisiae in the dry powder is 5 × 1010Per gram;
the Aspergillus oryzae CICC 41478: aspergillus oryzae dormant microorganism dry powder with deposit number CICC41478, wherein the Aspergillus oryzae content in the dry powder is 8.6 × 109One per gram.
Example 2A composite bacteria preparation for hybrid mutton sheep
The feed is prepared by mixing the following raw materials in parts by weight:
bacillus subtilis CICC 1009010 parts
Bacillus subtilis CICC 2044515 parts
Bacillus amyloliquefaciens CICC 230435 parts
Lactococcus lactis CICC 2360915 parts
Bifidobacterium bifidum CICC 616820 parts
Lactobacillus acidophilus ACCC 1107310 parts
Candida utilis CICC 180715 portions
Saccharomyces cerevisiae CICC 142110 parts
Aspergillus oryzae CICC 414788 parts;
the bacillus subtilis CICC 10090: dormant microbe dry powder prepared from bacillus subtilis with deposit number of CICC10090, wherein the bacillus subtilis spore content in the dry powder is 1.8 multiplied by 1011Per gram;
the bacillus subtilis CICC 20445: dormant microbe dry powder prepared for bacillus subtilis with deposit number of CICC20445, wherein the bacillus subtilis spore content in the dry powder is 1.5 multiplied by 1011Per gram;
the bacillus amyloliquefaciens CICC 23043: a dormant microbe dry powder prepared from Bacillus amyloliquefaciens with accession number of CICC23043 and containing Bacillus amyloliquefaciens spore content of 1.8 × 1011Per gram;
the lactococcus lactis CICC 23609: a dry powder of dormant microorganism prepared from lactococcus lactis with deposit number CICC23609, wherein the content of lactococcus lactis in the dry powder is 8 × 1010Per gram;
the bifidobacterium bifidum CICC 6168: is dormant microorganism dry powder of Bifidobacterium bifidum with deposit number CICC6168, wherein the content of Bifidobacterium bifidum in the dry powder is 2 × 1010Per gram;
the lactobacillus acidophilus ACCC 11073: is dormant body microorganism dry powder of lactobacillus acidophilus with a deposit number of ACCC11073,the content of Lactobacillus acidophilus in the dry powder is 5 × 1010Per gram;
the candida utilis CICC 1807: is dormant microorganism dry powder of Candida utilis with deposit number of CICC1807, and the content of Candida utilis in the dry powder is 2 × 1010Per gram;
the saccharomyces cerevisiae CICC 1421: is dormant microorganism dry powder of Saccharomyces cerevisiae with deposit number of CICC1421, wherein the content of Saccharomyces cerevisiae in the dry powder is 5 × 1010Per gram;
the Aspergillus oryzae CICC 41478: aspergillus oryzae dormant microorganism dry powder with deposit number CICC41478, wherein the Aspergillus oryzae content in the dry powder is 8.6 × 109One per gram.
Example 3A Special Compound bacteria preparation for hybrid mutton sheep
The feed is prepared by mixing the following raw materials in parts by weight:
bacillus subtilis CICC 1009015 parts
Bacillus subtilis CICC 2044510 parts
Bacillus amyloliquefaciens CICC 230437 parts
Lactococcus lactis CICC 2360920 parts
Bifidobacterium bifidum CICC 616810 parts
Lactobacillus acidophilus ACCC 1107315 parts
Candida utilis CICC 180720 parts
14215 parts of saccharomyces cerevisiae CICC
Aspergillus oryzae CICC 4147810 parts;
the bacillus subtilis CICC 10090: dormant microbe dry powder prepared from bacillus subtilis with deposit number of CICC10090, wherein the bacillus subtilis spore content in the dry powder is 1.8 multiplied by 1011Per gram;
the bacillus subtilis CICC 20445: dormant microbe dry powder prepared for bacillus subtilis with deposit number of CICC20445, wherein the bacillus subtilis spore content in the dry powder is 1.5 multiplied by 1011Per gram;
the bacillus amyloliquefaciens CICC 23043: preparation of Bacillus amyloliquefaciens with accession number CICC23043The dormant microbe dry powder contains bacillus amyloliquefaciens spore with the content of 1.8 multiplied by 1011Per gram;
the lactococcus lactis CICC 23609: a dry powder of dormant microorganism prepared from lactococcus lactis with deposit number CICC23609, wherein the content of lactococcus lactis in the dry powder is 8 × 1010Per gram;
the bifidobacterium bifidum CICC 6168: is dormant microorganism dry powder of Bifidobacterium bifidum with deposit number CICC6168, wherein the content of Bifidobacterium bifidum in the dry powder is 2 × 1010Per gram;
the lactobacillus acidophilus ACCC 11073: is dormant microorganism powder of Lactobacillus acidophilus with accession number ACCC11073, wherein the content of Lactobacillus acidophilus in the dry powder is 5 × 1010Per gram;
the candida utilis CICC 1807: is dormant microorganism dry powder of Candida utilis with deposit number of CICC1807, and the content of Candida utilis in the dry powder is 2 × 1010Per gram;
the saccharomyces cerevisiae CICC 1421: is dormant microorganism dry powder of Saccharomyces cerevisiae with deposit number of CICC1421, wherein the content of Saccharomyces cerevisiae in the dry powder is 5 × 1010Per gram;
the Aspergillus oryzae CICC 41478: aspergillus oryzae dormant microorganism dry powder with deposit number CICC41478, wherein the Aspergillus oryzae content in the dry powder is 8.6 × 109One per gram.
Example 4 preparation method of a specific complex bacterial preparation for hybrid mutton sheep
The method comprises the following steps:
step 1, culturing bacillus subtilis CICC10090
Inoculating bacillus subtilis CICC10090 strain on a slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 30%; the activation culture medium is an LB culture medium, and is cultured for 12 hours at the culture temperature of 30 ℃ and the rotating speed of a shake flask of 140 rpm;
inoculating the activated and cultured bacillus subtilis strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 70%; the volume inoculation amount is 0.1 percent, the culture temperature is 30 ℃, the stirring speed of a seeding tank is 220rpm, the culture is carried out for 11 hours, and the strain reaches the logarithmic phase;
inoculating cultured strains in a seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation medium with the volume percentage content of 70%; the volume inoculation amount is 10 percent, the bacillus subtilis spore in the fermentation tank is more than 95 percent and the content is 1.5 multiplied by 10 after the bacillus subtilis spore is cultured for 36 hours at the culture temperature of 30 ℃ and the stirring rotating speed of the fermentation tank of 200rpm10Each/ml, then spray drying the single bacillus subtilis obtained by fermentation to prepare dormant microbe dry powder, wherein the content of bacillus subtilis spores in the dry powder is 1.8 multiplied by 1011One per gram.
The culture medium formulas of the seeding tank and the fermentation tank are the same, and the culture medium formulas are 25g/L of corn flour, 10g/L of glucose, 30g/L of soybean meal, 3g/L of calcium chloride, 3g/L of magnesium sulfate and 0.5g/L of manganese sulfate.
Step 2, culturing bacillus subtilis CICC20445
Inoculating bacillus subtilis CICC20445 strain on a slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 30%; the formula of the activation culture medium is a beef extract peptone culture medium, and the culture is carried out for 12h at the culture temperature of 36 ℃ and the shaking rotation speed of 140 rpm;
inoculating the activated and cultured bacillus subtilis strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 70%; the volume inoculation amount is 0.2 percent, the culture temperature is 36 ℃, the stirring speed of a seeding tank is 220rpm, the culture is carried out for 10 hours, and the strain reaches the logarithmic phase;
inoculating cultured strains in a seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation medium with the volume percentage content of 70%; the volume inoculation amount is 10 percent, the culture temperature is 36 ℃, the stirring speed of a fermentation tank is 200rpm, the fermentation tank is used for culturing for 36 hours, the transformation rate of bacillus subtilis spores in the fermentation tank is more than 95 percent, and the content is 1.3 multiplied by 1010Each/ml, then spray drying the single bacillus subtilis obtained by fermentation to prepare dormant microbe dry powder, wherein the content of bacillus subtilis spores in the dry powder is 1.5 multiplied by 1011One per gram.
The culture medium formulas of the seeding tank and the fermentation tank are the same, and the culture medium formulas are 25g/L of corn starch, 30g/L of soybean meal, 5g/L of sodium chloride, 6g/L of dipotassium phosphate, 2g/L of magnesium sulfate and 0.5g/L of manganese sulfate.
Step 3, culturing bacillus amyloliquefaciens CICC23043
Inoculating bacillus amyloliquefaciens CICC23043 strain on a slant culture medium which is subjected to purification culture into a conical flask, wherein the conical flask is filled with an activated culture medium with the volume percentage of 15%; the formula of the activation culture medium is a beef extract peptone culture medium, the culture temperature is 37 ℃, and the shaking rotation speed is 150rpm for 10 h;
inoculating activated and cultured bacillus amyloliquefaciens strains into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 50%; the volume inoculation amount is 0.1 percent, the culture temperature is 37 ℃, the stirring speed of a seeding tank is 220rpm, the culture is carried out for 10 hours, and the strain reaches the logarithmic phase;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage content of 50%; the volume inoculation amount is 10 percent, the culture temperature is 37 ℃, the stirring speed of a fermentation tank is 200rpm, the fermentation is carried out for 40 hours, the conversion rate of bacillus amyloliquefaciens spores in the fermentation tank is more than 95 percent, and the content is 1.6 multiplied by 1010Each/ml, then spray drying the bacillus amyloliquefaciens obtained by fermentation to prepare dormant microbe dry powder, wherein the bacillus amyloliquefaciens spore content in the dry powder is 1.8 multiplied by 1011One per gram.
The formula of the seed tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 35g/L of corn flour, 40g/L of soybean meal, 5g/L of sodium chloride, 3g/L of monopotassium phosphate, 3g/L of dipotassium phosphate and 0.5g/L of manganese sulfate.
Step 4, culturing lactococcus lactis CICC23609
Inoculating the lactococcus lactis CICC23609 strain on the slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 30%; the formula of the activation medium is as follows: yeast extract 7.5g, glucose 10g, tomato juice 100ml, peptone 7.5g, potassium dihydrogen phosphate 2.0g, tween 800.5 ml, distilled water 900ml, pH 7.0; standing and culturing at 29 deg.C for 30 h;
inoculating activated and cultured lactococcus lactis strains into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 70%; the volume inoculation amount is 1 percent, the culture temperature is 29 ℃, the stirring speed of a seeding tank is 150rpm, the culture is carried out for 26 hours, and the thalli are propagated in large quantities;
inoculating cultured strain in seeding tank into fermentation tank containing 70% fermentation medium, inoculating 10% by volume, culturing at 29 deg.C and 150rpm for 48 hr, and culturing lactococcus lactis in the fermentation tank at 2.5 × 109Adding protective agent into the lactococcus lactis single bacterium obtained by fermentation, and freeze-drying to prepare dormant microorganism dry powder, wherein the content of lactococcus lactis in the dry powder is 8 multiplied by 1010One per gram.
The protective agent is whey powder and skim milk powder which are compounded according to the mass ratio of 4: 3.
The culture medium formula of the seeding tank and the fermentation tank is the same, and the culture medium formula comprises 15g/L of yeast powder, 20g/L of glucose, 10g/L of peptone, 5g/L of potassium dihydrogen phosphate, 801 ml/L of tween, 5g/L of sodium acetate and 150ml/L of tomato juice.
Step 5, culturing Bifidobacterium bifidum CICC6168
Inoculating bifidobacterium bifidum CICC6168 strain on a slant culture medium which is purified and cultured into an anaerobic bottle, wherein the anaerobic bottle contains an activated culture medium with the volume percentage of 80%; the formula of the activation medium comprises casein peptone 20g/L, glucose 20g/L, soybean peptone 10g/L, yeast powder 5g/L, dipotassium phosphate 5g/L, magnesium sulfate 0.1g/L, L-cysteine 0.1g/L, Tween 800.5 ml/L, sodium chloride 5g/L and calcium chloride 0.05 g/L; standing and culturing for 46h at the culture temperature of 36 ℃;
inoculating activated and cultured bifidobacterium bifidum strains into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage of 80%; the volume inoculation amount is 1 percent, the culture temperature is 36 ℃, the static culture is carried out for 24 hours, and the thalli are propagated in a large amount;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 80%; volume inoculation amount is 10%, and the culture temperature is 36 deg.C, standing culture is performed for 48h, two in fermentation tankBifidobacterium bifidum content 6 × 108Adding protective agent into fermented Bifidobacterium bifidum single bacterium, freeze drying to obtain dormant microorganism dry powder with Bifidobacterium bifidum content of 2 × 1010One per gram.
The formula of the seed tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 25g/L of casein peptone, 25g/L of glucose, 15g/L of soybean peptone, 10g/L of yeast powder, 8g/L of dipotassium phosphate, 0.5g/L, L-cysteine, 0.5g/L of magnesium sulfate, 801 ml/L of tween, 5g/L of sodium chloride and 0.01g/L of calcium chloride.
Step 6, culturing lactobacillus acidophilus ACCC11073
Inoculating lactobacillus acidophilus ACCC11073 strain on a slant culture medium subjected to purification culture into a triangular flask, wherein the triangular flask is filled with an activation culture medium with the volume percentage content of 80%; the formula of the activation medium is MRS medium, and the activation medium is subjected to static culture for 26 hours at the culture temperature of 35 ℃;
inoculating the lactobacillus acidophilus strain subjected to activated culture into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 80%; the volume inoculation amount is 3 percent, the culture temperature is 35 ℃, the seeding tank is kept stand for 22 hours, and the thalli are propagated in a large quantity;
inoculating the cultured strain in the seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation culture medium with the volume percentage of 80%; volume inoculation amount is 10%, the culture temperature is 35 deg.C, the fermentation tank is static cultured for 30h, the content of Lactobacillus acidophilus in the fermentation tank is 3 × 109Adding protective agent into lactobacillus acidophilus obtained by fermentation, and freeze drying to obtain dormant microorganism dry powder with lactobacillus acidophilus content of 5 × 1010One per gram.
The culture medium formula of the seeding tank and the fermentation tank is the same, and the culture medium formula comprises 25g/L of glucose, 15g/L of peptone, 15g/L of yeast powder, 1g/L of dipotassium phosphate, 1g/L of diammonium hydrogen citrate, 801 ml/L of tween, 10g/L of sodium acetate and 0.01g/L of magnesium sulfate.
Step 7, culturing Candida utilis CICC1807
Inoculating the candida utilis strain on the slant culture medium subjected to purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 15%; the formula of the activation culture medium is a glucose potato culture medium, the culture temperature is 28 ℃, and the shaking rotation speed is 130rpm for 20 h;
inoculating the Candida utilis strain subjected to activated culture into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 70%; the volume inoculation amount is 1 percent, the culture temperature is 28 ℃, the stirring speed of a seeding tank is 240rpm, the culture is carried out for 18h, and the thalli are propagated in large quantities;
inoculating cultured strains in a seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation medium with the volume percentage content of 70%; the volume inoculation amount is 5 percent, the culture temperature is 28 ℃, the stirring speed of a fermentation tank is 220rpm, the culture is carried out for 36 hours, and the content of the candida utilis in the fermentation tank is 3 multiplied by 109Per ml, then the candida utilis obtained by fermentation is boiled and dried by a fluidized bed to prepare dormant microorganism dry powder, and the content of the candida utilis in the dry powder is 2 multiplied by 1010One per gram.
The formula of the seeding tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 20g/L of peptone, 30g/L of molasses, 15g/L of beef powder, 5g/L of monopotassium phosphate, 5g/L of dipotassium phosphate and 20g/L of ammonium sulfate.
Step 8, culturing Saccharomyces cerevisiae CICC1421
Inoculating the saccharomyces cerevisiae strain on the slant culture medium subjected to the purification culture into a shake flask, wherein the shake flask is filled with an activation culture medium with the volume percentage content of 15%. The formula of the activation culture medium is a wort culture medium, the culture temperature is 30 ℃, and the shaking rotation speed is 150rpm for 20 h;
inoculating the saccharomyces cerevisiae strain subjected to activation culture into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 70%; the volume inoculation amount is 1 percent, the culture temperature is 30 ℃, the stirring speed of a seeding tank is 220rpm, the culture is carried out for 20 hours, and the thalli are propagated in large quantities;
inoculating cultured strains in a seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation medium with the volume percentage content of 70%; the volume inoculation amount is 5 percent, the culture temperature is 30 ℃, the stirring speed of a fermentation tank is 220rpm, the culture is carried out for 32 hours, and the content of the saccharomyces cerevisiae in the fermentation tank is 4 multiplied by 109Per mlThen, the saccharomyces cerevisiae obtained by fermentation is dried by fluidized bed boiling to prepare dormant microbe dry powder, wherein the content of the saccharomyces cerevisiae in the dry powder is 5 multiplied by 1010One per gram.
The culture medium formula of the seeding tank and the fermentation tank is the same, and the culture medium formula comprises 25g/L glucose, 30g/L molasses, 10g/L yeast powder, 5g/L magnesium sulfate, 5g/L dipotassium hydrogen phosphate and 10g/L ammonium sulfate.
Step 8, culturing Aspergillus oryzae CICC41478
Inoculating Aspergillus oryzae CICC41478 strain on a slant culture medium subjected to purification culture into a shake flask filled with an activated culture medium with a volume percentage of 30%; the formula of the activation culture medium is a PDA culture medium, the culture temperature is 28 ℃, and the shaking bottle rotation speed is 150rpm for culture for 40 h.
Inoculating the activated and cultured aspergillus oryzae strain into a seed tank, wherein the seed tank is filled with a fermentation culture medium with the volume percentage content of 70%; the volume inoculation amount is 1 percent, the culture temperature is 28 ℃, the stirring speed of a seeding tank is 220rpm, the culture is carried out for 45 hours, and the thalli are propagated in large quantity;
inoculating cultured strains in a seeding tank into a fermentation tank, wherein the fermentation tank is filled with a fermentation medium with the volume percentage content of 70%; the volume inoculation amount is 10%, the culture temperature is 28 deg.C, the fermentation tank is stirred at 220rpm for 4 days, and the content of Aspergillus oryzae spore in the fermentation tank is 7.5 × 108Drying fermented Aspergillus oryzae with fluidized bed to obtain dormant microorganism powder with Aspergillus oryzae content of 8.6 × 109One per gram.
The formula of the seeding tank is the same as that of the fermentation tank, and the formula of the culture medium comprises 20g/L of bran, 20g/L of glucose, 35g/L of soybean meal, 5g/L of sodium chloride, 1g/L of calcium chloride, 10g/L of potassium dihydrogen phosphate, 3mg/L of ferrous sulfate, 1mg/L of copper sulfate and 5mg/L of manganese sulfate.
Step 9, obtaining a finished product
Respectively compounding the prepared dormant body microorganism dry powder of bacillus subtilis CICC10090, bacillus subtilis CICC20445, bacillus amyloliquefaciens CICC23043, lactococcus lactis CICC23609, bifidobacterium bifidum CICC6168, lactobacillus acidophilus ACCC11073, candida utilis CICC1807, saccharomyces cerevisiae CICC1421 and aspergillus oryzae CICC41478 according to the formula of the special composite bacteria preparation for the hybrid mutton sheep, and mixing to obtain the special composite bacteria preparation for the hybrid mutton sheep.
Example 5 test assay
The experimental mutton sheep are F1 generation mutton sheep obtained by hybridizing pure black Dorper sheep and small tailed Han sheep, are healthy hybridized mutton sheep of 3 months after weaning, are all 30kg in weight, have the experimental time of 4 months, are 60 hybridized mutton sheep, are randomly divided into two groups, each group is 30, and the two groups are respectively a control group and an experimental group; the control group was fed with concentrate and roughage, the experimental group was fed with concentrate and roughage, and 0.1% of the complex bacterial preparation of example 3 (prepared according to the preparation method of example 4) was added to the concentrate, and the other feeding conditions were the same.
During the experiment, the disease condition of the weaned lambs and the odor of the sheep hurdle are observed.
Slaughtering lambs after four months of feeding; and counting various indexes of slaughter performance and meat quality of F1 generation hybrid mutton sheep hybridized by pure black-headed Dorper sheep and small tailed Han sheep, specifically referring to table 1, data processing and analysis of the experiment adopt Excel2007 for data arrangement, SPSS17.0 for single-factor variance analysis, and data are expressed by mean number +/-standard deviation.
TABLE 1 detection indexes of slaughtering performance and meat quality of hybrid mutton sheep
Note: the difference between people marked with different capital letters in the same row is extremely significant (P <0.01), the difference between people marked with different lower letters is significant (P <0.05), and the difference between people marked with the same letter or without the same letter is not significant (P > 0.05).
As can be seen from the above table, the invention has significant influence on the growth of F1 generation mutton sheep hybridized with pure black-head Dorper and small tailed Han sheep; the differences of pre-slaughter live weight, ketone body weight and dorsal longissimus drip loss of 7-month-old lamb experimental group and control group are very obvious (P)<0.01), the live weight of the experimental group before slaughtering reaches 62.8 kg; the carcass weight reaches 34.1kg, the drip loss of longissimus dorsi is 1.25 percent, and the slaughtering rates of the experimental group and the control group areThe difference between the yield of ketone bodies, the area of eye muscles and the difference between the cooked meat percentage is obvious (P)<0.05), the slaughtering rate of the experimental group is 54.3%, the meat yield of ketone bodies is 78.6%, and the eye muscle area is 13.09cm2The cooked meat rate is 58.2 percent, and the quality of the mutton sheep is high; in addition, in the experimental process, the odor of the sheep hurdle of the control group is obviously higher than that of the experimental group, the odor of the sheep hurdle of the experimental group is not abnormal, and the intestinal digestion food of the lamb of the experimental group is better.
Except for special description, the percentages are mass percentages, and the ratios are mass ratios.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A special compound bacteria preparation for hybrid mutton sheep is characterized in that: the compound bacterium preparation comprises the following microorganisms: bacillus subtilis, Bacillus amyloliquefaciens, lactococcus lactis, Bifidobacterium bifidum, Lactobacillus acidophilus, Candida utilis, Saccharomyces cerevisiae and Aspergillus oryzae.
2. The special compound bacteria preparation for hybrid mutton sheep as claimed in claim 1, which is characterized in that: the compound bacteria preparation is prepared from the following raw materials in parts by weight:
bacillus subtilis CICC 100905-15 parts
10-20 parts of bacillus subtilis CICC 2044510
Bacillus amyloliquefaciens CICC 230435-10 parts
Lactococcus lactis CICC 2360910-20 parts
Bifidobacterium bifidum CICC 616810-20 parts
110735-15 parts of Lactobacillus acidophilus ACCC
Candida utilis CICC 180710-20 parts
14215-10 parts of saccharomyces cerevisiae CICC
Aspergillus oryzae CICC 414785-10 parts.
3. The special compound bacteria preparation for hybrid mutton sheep as claimed in claim 2, which is characterized in that: the bacillus subtilis CICC 10090: the spore content is more than or equal to 1011Per gram; the bacillus subtilis CICC 20445: the spore content is more than or equal to 1011Per gram; the bacillus amyloliquefaciens CICC 23043: the spore content is more than or equal to 1.8 multiplied by 1011Per gram; the lactococcus lactis CICC 23609: the bacteria content is more than or equal to 1010Per gram; the bifidobacterium bifidum: the bacteria content is not less than 2 × 1010Per gram; the lactobacillus acidophilus ACCC 11073: the bacteria content is not less than 5 × 1010Per gram; the candida utilis CICC 1807: the yeast content is not less than 2 x 1010Per gram; the culture of the saccharomyces cerevisiae CICC 1421: the yeast content is not less than 5 × 1010Per gram; the Aspergillus oryzae CICC 41478: the content of Aspergillus oryzae is not less than 109One per gram.
4. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 1, wherein the preparation method comprises the following steps: comprises the steps of culturing bacillus subtilis CICC 10090; the bacillus subtilis CICC10090 culture method comprises the following steps: the volume inoculation amount of fermentation culture is 5-10%, the fermentation culture temperature is 28-30 ℃, the stirring rotation speed of a fermentation tank is 200-220 rpm, the culture time is 36-42 h, the conversion rate of bacillus subtilis spores in the fermentation tank is more than 95%, and the content is more than or equal to 1010One per ml.
5. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 4, wherein the preparation method comprises the following steps: the method also comprises the step of culturing bacillus subtilis CICC 20445; the bacillus subtilis CICC20445 culture: the volume inoculation amount of fermentation culture is 5-10%, and the fermentation culture temperature isThe temperature is 36-39 ℃, the stirring speed of the fermentation tank is 200-220 rpm, the culture time is 26-36 h, the conversion rate of bacillus subtilis spores in the fermentation tank is more than 95%, and the content is more than or equal to 1010One per ml.
6. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 4, wherein the preparation method comprises the following steps: also comprises a step of culturing bacillus amyloliquefaciens CICC 23043; the culture method comprises the following steps of culturing bacillus amyloliquefaciens CICC 23043: the volume inoculation amount of fermentation culture is 5-10%, the fermentation culture temperature is 36-38 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the fermentation culture lasts for 35-40 h, the conversion rate of bacillus amyloliquefaciens spores in the fermentation tank is more than 95%, and the content is more than or equal to 1.6 multiplied by 1010One per ml.
7. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 4, wherein the preparation method comprises the following steps: also comprises a step of culturing lactococcus lactis CICC 23609; the cultured lactococcus lactis CICC 23609: the volume inoculation amount of fermentation culture is 5-10%, the fermentation culture temperature is 26-29 ℃, the stirring speed of a fermentation tank is 150-180 rpm, the fermentation culture is 40-48 h, and the content of lactococcus lactis in the fermentation tank is not less than 109Protecting the milk powder by adopting a protective agent, wherein the protective agent is whey powder and skim milk powder which are compounded according to the mass ratio of 3-5: 2-4; the formula of the fermentation medium comprises 5-15 g/L of yeast powder, 10-20 g/L of glucose, 10-20 g/L of peptone, 1-5 g/L of monopotassium phosphate, 800.5-1 ml/L of Tween, 1-5 g/L of sodium acetate and 100-150 ml/L of tomato juice.
8. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 4, wherein the preparation method comprises the following steps: also comprises a step of culturing Bifidobacterium bifidum CICC 6168; the culture of bifidobacterium bifidum CICC 6168: the volume inoculation amount of the fermentation culture is 5-10%, the fermentation culture temperature is 36-38 ℃, the fermentation culture is 44-48 h, and the content of bifidobacterium bifidum in the fermentation tank is more than or equal to 6 multiplied by 108Each ml, the formula of the fermentation medium is casein peptone 15-25 g/L, glucose 15-25 g/L, soybean peptone 10-15 g/L, yeast powder 5 ∞10g/L, 3-8 g/L dipotassium phosphate, 0.1-0.5 g/L, L-cysteine, 0.1-0.5 g/L magnesium sulfate, 800.5-1 ml/L Tween, 5-10 g/L sodium chloride and 0.01-0.05 g/L calcium chloride.
9. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 4, wherein the preparation method comprises the following steps: also comprises a step of culturing lactobacillus acidophilus ACCC 11073; the cultured lactobacillus acidophilus ACCC 11073: the culture temperature of the activation culture is 35-38 ℃, and the activation culture time is 22-26 h; the volume inoculation amount of fermentation culture is 5-10%, the fermentation culture temperature is 35-38 ℃, the fermentation culture is 24-30 h, and the content of lactobacillus acidophilus in a fermentation tank is more than or equal to 3 multiplied by 109The fermentation medium comprises, by weight, 20-25 g/L of glucose, 15-25 g/L of peptone, 5-15 g/L of yeast powder, 1-5 g/L of dipotassium phosphate, 1-5 g/L of diammonium hydrogen citrate, 800.5-1 ml/L of tween, 1-10 g/L of sodium acetate and 0.01-0.05 g/L of magnesium sulfate.
10. The preparation method of the special composite bacteria preparation for hybrid mutton sheep as claimed in claim 4, wherein the preparation method comprises the following steps: also comprises the steps of culturing Candida utilis CICC1807, culturing Saccharomyces cerevisiae CICC1421 and culturing Aspergillus oryzae CICC 41478; the culture of the candida utilis CICC 1807: the volume inoculation amount of fermentation culture is 5-10%, the fermentation culture temperature is 28-30 ℃, the stirring speed of a fermentation tank is 200-220 rpm, the fermentation culture is 32-36 h, and the content of candida utilis in the fermentation tank is not less than 3 multiplied by 109Per ml; the culture of the saccharomyces cerevisiae CICC 1421: the fermentation culture time is 28-32 h, and the content of the saccharomyces cerevisiae in the fermentation tank is more than or equal to 4 multiplied by 109Per ml; the culture Aspergillus oryzae CICC 41478: the volume inoculation amount of fermentation culture is 5-10%, the fermentation culture temperature is 25-28 ℃, the stirring speed of a fermentation tank is 180-200 rpm, the fermentation culture is carried out for 3-4 days, and the content of aspergillus oryzae spores in the fermentation tank is more than or equal to 108One per ml.
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