CN101869184A - Microbial feed additive and preparation method thereof - Google Patents
Microbial feed additive and preparation method thereof Download PDFInfo
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- CN101869184A CN101869184A CN 201010199569 CN201010199569A CN101869184A CN 101869184 A CN101869184 A CN 101869184A CN 201010199569 CN201010199569 CN 201010199569 CN 201010199569 A CN201010199569 A CN 201010199569A CN 101869184 A CN101869184 A CN 101869184A
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- 238000002360 preparation method Methods 0.000 title claims description 33
- 239000003674 animal food additive Substances 0.000 title abstract description 6
- 230000000813 microbial effect Effects 0.000 title abstract description 5
- 238000000855 fermentation Methods 0.000 claims abstract description 61
- 230000004151 fermentation Effects 0.000 claims abstract description 61
- 210000003746 feather Anatomy 0.000 claims abstract description 51
- 235000012054 meals Nutrition 0.000 claims abstract description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 20
- 238000001035 drying Methods 0.000 claims abstract description 19
- 239000011780 sodium chloride Substances 0.000 claims abstract 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 abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 7
- 239000000654 additive Substances 0.000 claims description 36
- 230000000996 additive effect Effects 0.000 claims description 36
- 244000063299 Bacillus subtilis Species 0.000 claims description 33
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 33
- 239000007788 liquid Substances 0.000 claims description 23
- 239000012530 fluid Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 238000010899 nucleation Methods 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 238000013019 agitation Methods 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 8
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 8
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 235000009973 maize Nutrition 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 208000011580 syndromic disease Diseases 0.000 claims description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 235000009508 confectionery Nutrition 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- CEYULKASIQJZGP-UHFFFAOYSA-L disodium;2-(carboxymethyl)-2-hydroxybutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O CEYULKASIQJZGP-UHFFFAOYSA-L 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 206010010254 Concussion Diseases 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 abstract description 8
- 241001465754 Metazoa Species 0.000 abstract description 7
- 108010059345 keratinase Proteins 0.000 abstract description 4
- 239000002207 metabolite Substances 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 230000029087 digestion Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 abstract 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 abstract 1
- 229930195725 Mannitol Natural products 0.000 abstract 1
- 239000004480 active ingredient Substances 0.000 abstract 1
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 230000000593 degrading effect Effects 0.000 abstract 1
- 235000001727 glucose Nutrition 0.000 abstract 1
- 230000000968 intestinal effect Effects 0.000 abstract 1
- 239000000594 mannitol Substances 0.000 abstract 1
- 235000010355 mannitol Nutrition 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 235000002639 sodium chloride Nutrition 0.000 abstract 1
- 239000001509 sodium citrate Substances 0.000 abstract 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 abstract 1
- 235000011083 sodium citrates Nutrition 0.000 abstract 1
- 241000287828 Gallus gallus Species 0.000 description 15
- 238000004519 manufacturing process Methods 0.000 description 15
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
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- 230000000694 effects Effects 0.000 description 4
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
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- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 2
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 2
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- Fodder In General (AREA)
Abstract
The invention relates to a method for preparing a microbial feed additive. Feather meal, mannitol, glucose, sodium citrate, sodium chloride and the like are taken as raw materials, and inoculated with Bacullus subtilis YYW-1 with the number of CGMCC NO.2396 to prepare the microbial feed additive by submerged fermentation and spin flash drying technology. The active ingredients of the microbial feed additive are the Bacullus subtilis and cultures thereof, and the cultures contain metabolites such as keratinase capable of efficiently degrading feather. The product can be used in the breeding industry and the feed industry, and has the advantages of improving intestinal flora, improving digestion and absorption capacity of animals and the feed conversion and utilization rate, particularly promoting the digestion, absorption and utilization of protein materials such as the feather meal, and reducing cost.
Description
Technical field
The present invention relates to a kind of additive for microbe feedstuff and preparation method thereof, belong to agricultural biological technical field.
Background technology
Additive for microbe feedstuff is a kind of safe, green novel fodder additive, have and promote growth, disease resistance, improve product quality, be of value to effect such as environment, be applied to modern animal husbandry as production safety livestock products important safeguards technique and Antibiotic Additive substitute products.
China has been livestock products production and big export country at present, but in recent years livestock products particularly the outlet of meat is limited repeatedly, mainly be exactly reasons such as antibiotic and medicament residue, epidemic prevention and control system imperfection.Use antibiotic to make the history of growth promoter and existing over half a century of disease preventing and treating in its feeding, over nearly 30 years, the prolonged application antibiotic is caused the generation of animal autogenous infection or suprainfection, antibody-resistant bacterium, livestock and poultry cellular immunity, humoral immune function are descended and antibiotic residual in livestock products meat, egg, milk, directly threatening side effect such as human body health that deep understanding has been arranged.National governments pay close attention, and many developed countries promulgate a decree, and limit or ban use of antibiotic as feed addictive.European Union's legislation forbids that from January 1st, 2006 all antibiotic use as feed addictive.Meanwhile, to overcoming all drawbacks of antibiotic, nontoxic, have no side effect, the microbe additive of noresidue paid attention to day by day, some developed countries have realized industrialization.
China is relative with industrialization development later to the research of additive for microbe feedstuff, begin animal microecology research the eighties in 20th century, the end of the nineties, additive for microbe feedstuff entered industrialization development and application, and aspects such as product yield, quality, industrialization level are compared with international most advanced level and also had big gap at present.Main cause is owing to lack efficient bacterial strain and corresponding production technology preparation method.CN1682599 (200410031196.7) discloses a kind of additive for microbe feedstuff, specifically relate to a kind of additive for microbe feedstuff of being made up of multiple beneficial microorganism and its metabolite (as protease, lipase, amylase, cellulase etc.), this additive for microbe feedstuff is that bacillus subtilis, Bacillus cercus, Bacillus acidi lactici and S. cervisiae are carried out the zymotic fluid of deep liquid high density fermentation cultivation or the mixture of dry powder respectively.CN 101638627A (200910034843) discloses a bacillus subtilis and the application in biology feed additive thereof, and related bacillus subtilis HF-A1 (CGMCCNo3241) obtains near screening in the content of mucous membrane from the intestines and stomach that pick up from healthy sucking pig; This feed addictive contains bacillus subtilis (HF-A1), lactic acid bacteria and saccharomyces cerevisiae.CN1934989 (200610037429.3) discloses a kind of additive for microbe feedstuff, is to contain bacterial population 5.0～6.0 * 10 for bacterial classification through what preparation processes such as purebred expansion cultivation, liquid fermentation and culture, the cultivation of solid second order fermentation obtained with Trichoderma, Aspergillus, actinomyces, bacillus, S. cervisiae Li 1012 (Saccharomyces cerevisiae Li1012)
7Cfu/g, yeast count 4.0～5.0 * 10
4Cfu/g, fungi count 2.0～3.0 * 10
4The additive of cfu/g, the water content of additive≤10%; The product quality height is sneaked into the good stability behind the feed.The bacillus that utilizes that above patent document has produces enzyme performance, as protease, lipase etc., or utilize the composite production feed addictive of bacillus subtilis and other bacterium, fermented and cultured based raw material and drying mode all are extensive uses in conventional the production, are not the preparation methods at specific function bacterial classification (as decomposing feather keratin matter function) and special drying mode (both having guaranteed that high number of viable also can keep metabolite activity).
Feather has become the important forage protein raw material resources of China, the waste feathers resource in China every year has surpassed 1,000,000 tons (China's annual forage protein raw material breach is above 1,000 ten thousand tons), mainly make an addition in the feed with feather meal, because its complex structure utmost point difficult degradation, digestive utilization ratio is low, and the digestive utilization ratio that improves the feather meal raw material is that one of difficult problem of solution is needed in China's animal husbandry badly.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of additive for microbe feedstuff and preparation method thereof is provided.
Additive for microbe feedstuff of the present invention is to utilize raw materials such as feather meal, sweet mellow wine, glucose, natrium citricum, sodium chloride, microbe inoculation bacterial classification bacillus subtilis Bacullus subtilis YYW-1, bacterium numbering CGMCC NO.2396, (referring to CN101240302A, 200810014395.5), prepare the additive for microbe feedstuff product by liquid deep layer fermenting and Rotary Flash Drying.This product activity composition is above-mentioned bacillus subtilis viable bacteria and culture thereof, contains the metabolism products such as keratinase of efficient degradation feather in the culture.
Additive for microbe feedstuff preparation method of the present invention, step is as follows:
(1) preparation of seed fermentation liquid
Bacterial classification is selected: select bacillus subtilis (Bacillus subtilis) YYW-1 for use, CGMCC NO.2396; Under aseptic condition with the slant tube bacterial classification inoculation in feather fermentation seed culture medium, 500mL triangular flask culture medium loading amount is 100～150mL, 35～40 ℃ of concussions were cultivated 16～24 hours, shake-flask seed liquid;
Seed tank culture: the inoculum concentration of volume ratio with 2～5%, shake-flask seed liquid is inoculated in the seed fermentation jar that tank volume 65～70% feather fermentation seed culture mediums are housed, 35～40 ℃ are carried out the air agitation cultivation, and wherein speed of agitator is 220～260r/min, and throughput is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 1.1～1.2, jar internal pressure 0.1～0.02MPa ferment 12～16 hours, must the seeding tank zymotic fluid;
The above-mentioned bottle that shakes is identical with the used culture medium of seeding tank, described feather fermentation seed culture medium is: by weight, 0.1～5.0 part of feather meal, 0.01～0.2 part in sodium chloride, 0.005～0.2 part in magnesium chloride, 0.001～0.2 part in calcium chloride, 0.01～0.2 part of potassium dihydrogen phosphate, 0.02～0.5 part of dipotassium hydrogen phosphate, 0.01～0.5 part of manganese sulfate, 0.01～0.5 part of dusty yeast, be dissolved in 99～93 parts of pure water, regulate pH value to 6～8, standby behind the steam sterilizing;
(2) fermentation tank culture
The inoculum concentration of volume ratio with 7～10%, the seeding tank zymotic fluid is inoculated in the fermentation tank that tank volume 60%～70% feather fermentation medium is housed, 35～40 ℃ are carried out the air agitation cultivation, and wherein speed of agitator is 180～220r/min, and throughput is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 1.1～1.2, jar internal pressure 0.1 ± 0.02MPa fermented fermentation ends 32～48 hours;
Above-mentioned feather fermentation medium is: by weight, 1～10 part of feather meal, 0.5～5 part in sweet mellow wine, 0.5～5 part of glucose, 0.5～5 part of natrium citricum, 0.01～0.5 part in sodium chloride, 0.01～0.5 part in magnesium sulfate, 0.01～0.5 part in calcium chloride and dipotassium hydrogen phosphate are dissolved in 97.5～74.5 parts of water for 0.01～1 part, regulate pH to 6～8, the heat treatment method sterilization.
(3) additive for microbe feedstuff preparation
Adding maize cob meal to water content in step (2) gained zymotic fluid is 70-80wt%, utilizes the Rotatingandflashstreamingdrier drying, and drying machine is to 110-125 ℃ of wind-warm syndrome degree, temperature of outgoing air 65-75 ℃; Get additive for microbe feedstuff.
Carry out aseptic subpackaged by prior art to step (3) products obtained therefrom.
Said method of the present invention uses feather meal as culture media nitrogen source in the described culture medium, feather meal comprises the powder product that various waste feathers are made, and can buy by market.
Preferably, described feather meal is a powder particle diameter average out to 1mm degreasing chicken feather powder.
Preferably, the described culture medium heat treated sterilization of step (2) is to carry out under 116-121 ℃ temperature, and sterilization time is 30-60 minute.
Preferably, the described cultivation temperature of step (2) is 38 ℃ ± 1 ℃, and throughput is 1: 1.1.
Preferably, the described Rotatingandflashstreamingdrier of step (3) is to 115 ℃ of wind-warm syndrome degree, 70 ℃ of temperature of outgoing airs.
Preferably, the product water content≤10wt% of step (3) gained drying.
The number of viable of the prepared additive for microbe feedstuff of the present invention is 8.0 * 10
9CFU/ gram～1.5 * 10
10The CFU/ gram belongs to high bacterium life birth product.Can be mixed with viable bacteria content by the maize cob meal that added 80 mesh sieves when using is 1 * 10
9CFU/ gram～4 * 10
9The goods of CFU/ gram use.
The present invention utilizes the waste resource feather to be fermentation raw material first, with the bacillus subtilis YYW-1 (bacterium numbering CGMCC No.2396) that produces keratinase, pass through liquid deep layer fermenting, utilize Rotary Flash Drying, preparation viable bacteria and metabolite keratinase are the novel microbial feed additive of main component.The product of being produced can use in aquaculture and feed industry, have the gut flora of improvement, improve the animal digestion absorbability, improve the food conversion utilization rate, particularly promoted protein raw materials such as feather meal are digested and absorbed, reduced cost, increase economic efficiency.
The specific embodiment
The present invention will be further described below in conjunction with embodiment.
Throughput unit among the embodiment is: fermentating liquid volume m
3/ volume of air m
3Minute.The flash dryer that uses among the embodiment is one step of a Jiangsu drying equipment Co., Ltd product, and model is XSG-12.Said water content %, product moisture content % are mass percent among the embodiment.Feather meal among the embodiment is the degreasing type feather meal (chicken feather powder) that Shandong six and Pingyi feather meal factory of group produce, powder particle diameter average out to 1mm.
Embodiment 1: the preparation method of additive for microbe feedstuff, and step is as follows:
(1) preparation of seed fermentation liquid
Bacterial classification is selected: select bacillus subtilis (Bacillus subtilis) YYW-1 for use, CGMCC NO.2396; With in the slant tube bacterial classification inoculation liquid feather fermentation seed culture medium, 500mL triangular flask culture medium loading amount is 100～150mL under aseptic condition, and 37 ℃ of concussions were cultivated 18 hours, got shake-flask seed liquid;
The inoculum concentration of volume ratio with 3%, shake-flask seed liquid is inoculated in 2 tons of fermentation tanks that 1.3 tons of feather fermentation mediums are housed, 37 ℃ of cultivation temperature, carrying out air agitation cultivates, wherein speed of agitator is that 220r/ adds in, throughput 1: 1.1, jar internal pressure 0.1 ± 0.02MPa, fermented 13 hours, and got the seeding tank zymotic fluid; Cultivate, speed of agitator is 250r/min, and throughput is 1: 1.1, jar internal pressure 0.1 ± 0.02MPa, and fermenting got the seeding tank zymotic fluid in 12 hours;
Above-mentioned feather fermentation seed culture medium is: by weight, 1 part of feather meal, 0.1 part in sodium chloride, 0.1 part in magnesium chloride, 0.06 part in calcium chloride, 0.02 part of potassium dihydrogen phosphate, 0.02 part of dipotassium hydrogen phosphate, 0.05 part of manganese sulfate, 0.02 part of dusty yeast, be added on one by one in 97 parts of pure water and dissolve, regulate pH value to 7.2, standby behind the steam sterilizing;
(2) fermentation tank culture
The inoculum concentration of volume ratio with 10%, the seeding tank zymotic fluid is inoculated in 20 tons of fermentation tanks that 13 tons of feather fermentation mediums are housed, 38 ℃ of cultivation temperature, air agitation is cultivated, wherein speed of agitator is 220r/min, and throughput is 1: 1.1, jar internal pressure 0.1 ± 0.02MPa, treat that thalline product gemma rate reaches at 90% o'clock and stops to cultivate fermentation period 38 hours;
Above-mentioned feather fermentation medium and preparation method are: with weight portion, 3 parts of feather meals, 1 part in sweet mellow wine, 1 part of glucose, 2 parts of natrium citricums, 0.3 part in sodium chloride, 0.02 part in magnesium sulfate, 0.02 part in calcium chloride, 0.03 part of dipotassium hydrogen phosphate, one by one or mix and to be dissolved in 91 parts of water, regulate pH to 7.2,121 ℃ of sterilizations 50 minutes are standby.
(3) dry and additive for microbe feedstuff preparation
After treating the described fermentation ends of step (2), with maize cob meal to the water content that added 80 mesh sieves in the zymotic fluid is 75%, utilize the Rotatingandflashstreamingdrier drying, drying machine is to 115 ℃ of wind-warm syndrome degree, 70 ℃ of temperature of outgoing airs, making product moisture content is 10%, gets additive for microbe feedstuff, carries out aseptic subpackaged then; Bacillus subtilis YYW-1 number of viable is 1.05 * 10 in the product
10The CFU/ gram.
Embodiment 2: the preparation method of additive for microbe feedstuff: the preparation of seed fermentation liquid is with embodiment 1.Different is:
(2) fermentation tank culture:
The inoculum concentration of volume ratio with 10%, the seeding tank zymotic fluid is inoculated in 20 tons of fermentation tanks that 13 tons of feather fermentation mediums are housed, 38 ℃ of cultivation temperature, air agitation is cultivated, wherein speed of agitator is 220r/min, and throughput is 1: 1.1, jar internal pressure 0.1 ± 0.02MPa, treat that thalline product gemma rate reaches at 90% o'clock and stops to cultivate fermentation period 33 hours;
Above-mentioned feather fermentation medium and preparation method are: with weight portion, 1 part of feather meal, 1 part in sweet mellow wine, 1 part of glucose, 1 part of natrium citricum, 0.2 part in sodium chloride, 0.01 part in magnesium sulfate, 0.01 part in calcium chloride, 0.02 part of dipotassium hydrogen phosphate, be dissolved in 95.8 parts of water, regulate pH to 7.2,116 ℃ of sterilizations 60 minutes are standby.
(3) dry and additive for microbe feedstuff preparation
After treating the described fermentation ends of step (2), maize cob meal to the water content that added 80 mesh sieves in zymotic fluid is 75wt%, utilizes the Rotatingandflashstreamingdrier drying, and drying machine is to 110 ℃ of wind-warm syndrome degree, 70 ℃ of temperature of outgoing airs; Product moisture content is 11wt%, and bacillus subtilis YYW-1 number of viable is 9.3 * 10
9The CFU/ gram.
Embodiment 3: the preparation of additive for microbe feedstuff, the preparation of seed fermentation liquid is with embodiment 1.Different is:
(2) fermentation tank culture:
The inoculum concentration of volume ratio with 10%, the seeding tank zymotic fluid is inoculated in 20 tons of fermentation tanks that 13 tons of feather fermentation mediums are housed, 38 ℃ of cultivation temperature, air agitation is cultivated, wherein speed of agitator is 220r/min, and throughput is 1: 1.1, jar internal pressure 0.1 ± 0.02MPa, treat that thalline product gemma rate reaches at 90% o'clock and stops to cultivate fermentation period 44 hours;
Fermentation tank with culture medium and preparation method is: with weight portion, 5 parts of feather meals, 2 parts in sweet mellow wine, 2 parts of glucose, 3 parts of natrium citricums, 0.3 part in sodium chloride, 0.04 part in magnesium sulfate, 0.04 part in calcium chloride, 0.04 part of dipotassium hydrogen phosphate, be dissolved in one by one in (or being mixed in) 87.7 parts of water, regulate pH to 7.2,116 ℃ of sterilizations 60 minutes are standby.
(3) treat the described fermentation ends of step (2) after, be 80wt% with maize cob meal to the water content that added 80 mesh sieves in the zymotic fluid, utilize the Rotatingandflashstreamingdrier drying, drying machine is to 115 ℃ of wind-warm syndrome degree, 70 ℃ of temperature of outgoing airs; Product moisture content is 10wt%, and bacillus subtilis YYW-1 number of viable is 1.2 * 10
10The CFU/ gram.
Embodiment 4: the preparation method of additive for microbe feedstuff
(1) preparation of seed fermentation liquid
Bacterial classification selection and shake-flask seed cultural method are with embodiment 1.Different is:
The inoculum concentration of volume ratio with 3%, shake-flask seed liquid is inoculated in 1 ton of fermentation tank that 0.7 ton of feather fermentation medium is housed, 37 ℃ are carried out air agitation and cultivate, wherein speed of agitator is 220r/min, throughput 1: 1.1, jar internal pressure 0.1 ± 0.02MPa fermented 10 hours, got the seeding tank zymotic fluid;
Above-mentioned feather fermentation seed culture medium and preparation method are with embodiment 1.
(2) fermentation tank culture
The inoculum concentration of volume ratio with 10%, the seeding tank zymotic fluid is inoculated in 5 tons of fermentation tanks that 3.2 tons of feather fermentation mediums are housed, 38 ℃ of cultivation temperature, air agitation is cultivated, wherein speed of agitator is 250r/min, and throughput is 1: 1.1, jar internal pressure 0.1 ± 0.02MPa, treat that thalline product gemma rate reaches at 90% o'clock and stops to cultivate fermentation period 40 hours;
Above-mentioned feather fermentation medium and preparation method are with embodiment 1.
(3) dry and additive for microbe feedstuff preparation
After treating the described fermentation ends of step (2), be 75wt% with maize cob meal to the water content that added 80 mesh sieves in the zymotic fluid, utilize the Rotatingandflashstreamingdrier drying, drying machine is to 115 ℃ of wind-warm syndrome degree, 70 ℃ of temperature of outgoing airs; Product moisture content is 9wt%, and bacillus subtilis YYW-1 number of viable is 9.5 * 10
9The CFU/ gram.
Embodiment 5: bacillus subtilis YYW-1 (CGMCC NO.2396) per os acute toxicity test.
This test is undertaken by HSPH of Shandong Medical University toxicologic study chamber.
Feed 60 of Kunming mouses of a nutrient solution before the fermentation tank of the bacillus subtilis YYW-1 (CGMCC NO.2396) that cultivates with embodiment 1 step (1) is put jar, body weight 18-22g, male and female half and half.Mouse is divided into 6 groups at random, 10 every group, male and female half and half, behind the fasting 12h, per os is irritated stomach, and dosage is respectively 40mL/kg, 32mL/kg, 25.6mL/kg, 20.5mL/kg, 16.4mL/kg, 13.1mL/kg (r=0.8).Observed 14 days continuously after the mouse administration, record mouse poisoning symptom and death toll according to each group mouse death rate, are calculated LD with the Bliss method
50With 95% fiducial limit.The results are shown in Table 2.
Table 2 bacillus subtilis YYW-1 (CGMCC NO.2396) acute toxicity test in mice result
The result shows that per os is to the no abnormal phenomenon of mouse behind the animal subject.Per os of mouse gives lactic acid bacteria oral liquor half lethal dose LD
50Greater than 40mL/kg, belong to innocuous substance by acute toxicity grading criteria.
Embodiment 6: additive for microbe feedstuff culture experiment of the present invention
Carry out culture experiment with embodiment 3 prepared additive for microbe feedstuff.With the number of viable of embodiment 3 is 1.2 * 10
10It is 3 * 10 that the product of CFU/ gram is mixed with viable bacteria content by the maize cob meal that added 80 mesh sieves
9Bacillus subtilis YYW-1 (CGMCC NO.2396) goods of CFU/ gram, using dosage are that complete diet pellet per ton adds 1000 grams.Test in Shandong six and fryer plant of feed group carry out.Corn-dregs of beans type typical case daily ration of broiler (containing 2% feather meal in the component), basal diet preparation standard is with reference to the fowl raising standard (NY/T 33-2004) of The Ministry of Agriculture of the People's Republic of China, MOA's issue in 2004.The test daily ration is pellet, and the A group is positive control group; The B group is negative control group: promptly fall the 30kcal/kg metabolizable energy in the A group on according to the daily ration basis; The C group is added the bacillus subtilis formulation of 1000g/T on B group (negative control group) daily ration basis.Single-factor random experiment design, 432 1 aa broiler chicken are divided into 3 daily ration processed group at random according to the principle of male and female half and half, body weight unanimity, and 6 repetitions are established in each processing, and each repeats 12 chickens.Duration of test record feed consumption rate every day is according to average individual daily gain of each stage, individual daily material consumption, the feedstuff-meat ratio of measuring 1～3 age in week, 4～5 ages in week and 1～5 age in week.The test chicken adopts the stereo-overlap formula to raise in cages, Artificial Control illumination, temperature and humidity, and free choice feeding and drinking-water, gravity-flow ventilation, other feedings and managements and immunity feeding of broiler way and immune programme for children are routinely carried out.
Result of the test:
1. bacillus subtilis YYW-1 (CGMCC NO.2396) is to the influence of 1～21 age in days meat chicken production performance
The production performance result of the test in 1～21 age in days stage shows, adds the average individual weight of bacillus subtilis test group 21 ages in days and improves 8.2% (P＜0.05), and the test group daily gain improves 7.4% (P＜0.05); Feedstuff-meat ratio reduces by 0.08 (P＜0.05), table 1.
Table 1, bacillus subtilis YYW-1 (CGMCC NO.2396) are to the influence of 1～21 age in days meat chicken production performance
Annotate: same column data shoulder mark lowercase different table differential different significantly (P＜0.05); Shoulder marks the nothing letter or alphabetical identical person represents difference not remarkable (P＞0.05), down together.
2. bacillus subtilis YYW-1 (CGMCC NO.2396) is to the influence of 22～35 age in days meat chicken production performances
22～35 age in days stage production performance result of the tests show, add the average individual weight of bacillus subtilis test group 35 ages in days and improve 11.0% (P＜0.05), and daily gain improves 10.1% (P＜0.05); Feedstuff-meat ratio reduces by 0.13 (P＜0.05), table 2.
Table 2, bacillus subtilis YYW-1 (CGMCC NO.2396) are to the influence of 22～35 age in days meat chicken production performances
3. bacillus subtilis YYW-1 (CGMCC NO.2396) is to the influence of 1～35 age in days meat chicken production performance
The production performance result of the test that 1～35 age in days is tested the full phase shows that the individual daily gain of adding the bacillus subtilis test group improves 12.2% (P＜0.05); Feedstuff-meat ratio has reduced by 0.10 (P＜0.05), table 3.
Table 3, bacillus subtilis YYW-1 (CGMCC NO.2396) are to the influence of 1～35 age in days meat chicken production performance
Conclusion: the production performance result in full growth period shows, add bacillus subtilis YYW-1 (CGMCCNO.2396) in the daily ration of broiler, the test group daily gain improves 11.7%, 12.2% (P＜0.05) respectively than positive and negative control group, feedstuff-meat ratio reduces by 0.10,0.13 (P＜0.05) respectively than positive and negative control group, all reach the significance level of difference, show that adding bacillus subtilis YYW-1 (CGMCC NO.2396) can improve utilization ratio and the growth of meat chicken performance of fryer to daily ration.
Claims (4)
1. additive for microbe feedstuff preparation method of the present invention, step is as follows:
(1) preparation of seed fermentation liquid
Bacterial classification is selected: select bacillus subtilis (Bacillus subtilis) YYW-1 for use, CGMCC NO.2396; Under aseptic condition with the slant tube bacterial classification inoculation in feather fermentation seed culture medium, 500mL triangular flask culture medium loading amount is 100～150mL, 35～40 ℃ of concussions were cultivated 16～24 hours, shake-flask seed liquid;
Seed tank culture: the inoculum concentration of volume ratio with 2～5%, shake-flask seed liquid is inoculated in the seed fermentation jar that tank volume 65～70% feather fermentation seed culture mediums are housed, 35～40 ℃ are carried out the air agitation cultivation, and wherein speed of agitator is 220～260r/min, and throughput is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 1.1～1.2, jar internal pressure 0.1～0.02MPa ferment 12～16 hours, must the seeding tank zymotic fluid;
Above-mentioned shake the bottle and seeding tank with feather fermentation seed culture medium be: by weight, 0.1～5.0 part of feather meal, 0.01～0.2 part in sodium chloride, 0.005～0.2 part in magnesium chloride, 0.001～0.2 part in calcium chloride, 0.01～0.2 part of potassium dihydrogen phosphate, 0.02～0.5 part of dipotassium hydrogen phosphate, 0.01～0.5 part of manganese sulfate, 0.01～0.5 part of dusty yeast, be dissolved in 99～93 parts of pure water, regulate pH value to 6～8, standby behind the steam sterilizing;
(2) fermentation tank culture
The inoculum concentration of volume ratio with 7～10%, the seeding tank zymotic fluid is inoculated in the fermentation tank that tank volume 60%～70% feather fermentation medium is housed, 35～40 ℃ are carried out the air agitation cultivation, and wherein speed of agitator is 180～220r/min, and throughput is with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 1.1～1.2, jar internal pressure 0.1 ± 0.02MPa fermented fermentation ends 32～48 hours;
Above-mentioned feather fermentation medium is: by weight, 1～10 part of feather meal, 0.5～5 part in sweet mellow wine, 0.5～5 part of glucose, 0.5～5 part of natrium citricum, 0.01～0.5 part in sodium chloride, 0.01～0.5 part in magnesium sulfate, 0.01～0.5 part in calcium chloride and dipotassium hydrogen phosphate are dissolved in 97.5～74.5 parts of water for 0.01～1 part, regulate pH to 6～8, the heat treatment method sterilization;
(3) adding maize cob meal to water content in step (2) gained zymotic fluid is 70-80wt%, utilizes the Rotatingandflashstreamingdrier drying, and drying machine is to 110-125 ℃ of wind-warm syndrome degree, temperature of outgoing air 65-75 ℃; Get additive for microbe feedstuff.
2. the preparation method of additive for microbe feedstuff as claimed in claim 1 is characterized in that the described culture medium heat treated sterilization of step (2) is to carry out under 116-121 ℃ temperature, and sterilization time is 30-60 minute.
3. the preparation method of additive for microbe feedstuff as claimed in claim 1 is characterized in that the described cultivation temperature of step (2) is 38 ℃ ± 1 ℃, and throughput is 1: 1.1.
4. the preparation method of additive for microbe feedstuff as claimed in claim 1 is characterized in that the described Rotatingandflashstreamingdrier of step (3) to 115 ℃ of wind-warm syndrome degree, 70 ℃ of temperature of outgoing airs.
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