CN104480052A - Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding - Google Patents

Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding Download PDF

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CN104480052A
CN104480052A CN201510001428.2A CN201510001428A CN104480052A CN 104480052 A CN104480052 A CN 104480052A CN 201510001428 A CN201510001428 A CN 201510001428A CN 104480052 A CN104480052 A CN 104480052A
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bacillus
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tank
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张一平
华洵璐
匡群
邹三元
孙梅
钱仁界
张宪中
叶进
秦海萍
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TIANSHI FODDER CO Ltd YIXING CITY
WUXI CITY ANIMAL HUSBANDRY AND FISHERIES TECHNOLOGY EXTENSION STATION
WUXI NANYANG AGRICULTURE AND LIVESTOCK Co Ltd
JIANGSU INSTITUTE OF SUWEI MICROBIOLOGY RESEARCH
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TIANSHI FODDER CO Ltd YIXING CITY
WUXI CITY ANIMAL HUSBANDRY AND FISHERIES TECHNOLOGY EXTENSION STATION
WUXI NANYANG AGRICULTURE AND LIVESTOCK Co Ltd
JIANGSU INSTITUTE OF SUWEI MICROBIOLOGY RESEARCH
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Abstract

The invention provides a composite bacillus preparation containing three strains, a preparation method of the composite bacillus preparation and application of the composite bacillus preparation to ecological breeding, and belongs to the technical field of microorganism and feed additives. Bacillus coagulans, butyric acid clostridia and bacillus megaterium are used as fermentative strains respectively, and large-scale, low-cost and high-density fermentation production of spores is conducted through step-by-step magnification; three live bacterium preparations are prepared by mixing bacterial sludge with the spores and dry starch; the three live bacterium preparations are matched according to the equal weight ratio, the composite bacillus preparation is obtained, and the concentration of the spores reaches more than 1*108-109 cfu/g; the composite bacillus preparation serves as a microorganism and feed additive to be added to farmed animal feed according to the mass ratio ranging from 0.02% to 0.5%. The composite bacillus preparation is simple and stable in process, low in cost, high in spore content and environment tolerance, easy to store and high in survival rate; the purposes of composition and synergism are achieved through the collaborative supplementary effects of different bacilli in the aspect of micro-ecology adjustment, growth of farmed animals is promoted, the feed utilization rate and feed rewards are increased, diarrhea is prevented and reduced, and the breeding ecological environment is improved.

Description

A kind ofly contain composite bacillus preparation of three strain bacterium and preparation method thereof and the application in ecologic breeding
Technical field
The present invention relates to the composite bacillus preparation of a kind of high viable bacteria content, high spore content, high preservation period, and preparation method and as microorganism feed addictive animal ecology cultivation in application, belong to microorganism and field of feed additive technology.
Background technology
In recent years, along with applying of the intensive artificial culture technology of high-density, the breeding production scale of China obtains fast development, has become the production and trade big country of world poultry, fowl, fishery products.But, high-density intensive culture mode makes Animal diseases be situation occurred frequently, people have to use antibiotic etc widely in a large number, resistance and endurance strain is caused to increase, destroy the normal microecological balance of animal intestinal, reduce Immune Function In Animals, body resistance against diseases is declined, be more vulnerable to exogenous and infringement that is autogenous infection.In addition, antibiotic etc can remain in animal product, brings immunity and the health of the direct harm humans of secondary pollution.Therefore, countries in the world particularly developed country take official's strong measures one after another, have carried out strict restriction to the kind of feeding medicine, using method, dosage and compatibility etc.
The various microbe additives be based upon in microecological regulation and control theoretical basis have safety, efficiently feature, development in recent years is rapid, the substitute becoming antibiotic etc just is gradually widely used in animal cultivation, achieve comparatively considerable ecological benefits, Social benefit and economic benefit, effectively facilitate the development of Green Ecological Health culture model.
2009, the Ministry of Agriculture of China announced No. 1231 official approval clostridium butylicum (also known as clostridium butyricum) and Bacillus licheniformis is new feed additive, and it is raw raw with ground intestines that trade(brand)name is respectively junket intestines.Announce in No. 2045 " the fodder additives kind catalogue (2013) " issued in the Ministry of Agriculture of China, formally Bacillus coagulans is classified as feed microbe additive kind.Though bacillus megaterium is not at present also by the official approval of the Ministry of Agriculture of China, existing manyly to report about the research of this bacterial classification for animal cultivation.Up to now, the microorganism fodder kind of China's official approval has reached 34 kinds, includes lactic acid bacteria class, bacillus category, mould fungi and yeast class etc.
Bacillus coagulans belongs to enteron aisle milk-acid bacteria, is also called " having spore milk-acid bacteria ", is the bacillus class milk-acid bacteria of a kind of " generally believing safety ".The functional characteristics of Bacillus coagulans is: the growth 1. effectively suppressing harmful bacteria, and improvement intestine microenvironment, promotes intestinal growth, strengthens intestinal function; 2. improve feed quality, promote digesting and assimilating of feed, reduce feed coefficient; 3. the diarrhoea with dislocation of bacillary groups owing to stress cause with anaphylaxis is effectively improved; 4. without any side effects, use safety; 5. can reduce the obnoxious flavour such as ammonia, hydrogen sulfide in environment, improve breeding environment.
Clostridium butyricum (also known as Butylic acid bacteria, clostridium butylicum or clostridium butyricum) is Gram-positive anaerobic bacillus(cillus anaerobicus), it is the probiotics of regulating intestinal canal microecological balance, its primary biological characteristic shows as: the propagation 1. promoting intestinal beneficial flora (bifidus bacillus, lactobacillus), suppress the growth of harmful bacteria and spoilage organism in enteron aisle, correct enteric flora disturbance, reduce the generation of enterotoxin; 2. in enteron aisle, the material such as vitamin B group and vitamin K is produced, with health role to body; 3. main metabolites butyric acid is the Major Nutrient material of the regeneration of gut epithelium histocyte and reparation, improves feed digestion specific absorption.
Bacillus megaterium is sporiferous Gram positive aerobic bacterium, agriculturally for the manufacture of phosphorous bacterial fertilizer, industrial for the production of glucose isomerase, for making water body treatment agent in environmental protection.As probiotics, feature and the mechanism of action of bacillus megaterium are: 1. to take oxygen ability by force strong for biology, first breed after entering animal intestinal, oxygen in rapid consumption enteron aisle, for the growth of anerobe useful in enteron aisle provides good anaerobic environment, by supporting the growth of useful anerobe, indirectly suppressing the propagation of aerobic pathogenic bacterium, recovering intestinal function; 2. there is abundant extracellular enzyme, in enteron aisle, can produce the multiple digestive ferments such as amylase, proteolytic enzyme, cellulase, lipase in reproductive process, especially cellulase activity is comparatively strong, effectively can promote digesting and assimilating of feed ingredient, improve efficiency of feed utilization, reduce feed coefficient; 3. effectively can reduce the generation of the odorous gas such as ammonia and hydrogen sulfide in breeding process, play the effect improving breeding ecological environment.
Current research shows, different bacterial classifications mechanism of action is in animal body different; Coenocorrelation in animal body is in continuous change, and the optimum environment that different mechanism of action adapts to is also different.Multi-cultur es product can make full use of synergistic mechanism between different strain and Competitive assays effect in the coenocorrelation of change, reaches effect more better than single bacterial classification product.Therefore the compound formulation be made up of two or more different strain becomes the exploitation focus of current Tiny ecosystem product.
Current domestic existing multiple patent being used as fodder additives about complex microorganism preparations, compositely to be formed by genus bacillus wherein specially, the patent close with the present invention is as follows: Chinese patent CN102389032 discloses a kind of fodder additives of the substitute antibiotics be made up of lactic acid bacillus and subtilis; CN101816378 discloses a kind of breeding feed additive be made up of lactic acid bacillus, bacillus natto and subtilis; CN1089442 discloses and to be made up of Bacillus licheniformis and bacillus megaterium
Microbe additive is used for the raising of meat duck; CN103461650 discloses the fodder additives that is made up of subtilis and clostridium butylicum for growing-finishing pig of feeding; CN102429096 discloses a kind of fodder additives be made up of Bacillus licheniformis, subtilis and Butylic acid bacteria for suckling piglet of feeding; CN103349153 discloses a kind of by any two or three composite thalline mixture in Bacillus coagulans, clostridium butylicum and Bacillus licheniformis, for protection and the reparation of animal intestinal.
According to above domestic published patent documentation, up to the present, patent about composite bacillus preparation is still on the low side, the microorganism kind used is single, mainly concentrates on a few microorganisms such as lactic acid bacillus (also known as Bacillus coagulans), clostridium butyricum, subtilis, bacillus natto and Bacillus licheniformis.Especially the patent about bacillus megaterium compound preparation only has 1, and only for the raising of meat duck.As everyone knows, the microorganisms such as milk-acid bacteria, yeast and mould fungi, because not forming gemma, cause formulation products poor stability, not storage endurance, not stomach juice-resistant, easy in inactivation in animal gastrointestinal tract and reduce its beneficial function; And genus bacillus can produce statospore, environment strong stress resistance, can acid-and base-resisting and high temperature, high pressure, and not easy in inactivation in feed manufacturing and storage process, in animal intestinal, easy field planting, keeps higher viable count and give full play to its physiological function.Therefore be more prone in the market use genus bacillus and compound formulation product thereof.
Summary of the invention
Main purpose of the present invention is the deficiency using antibiotic drawback and prior art and product for Present Domestic aquaculture in a large number, the NEW TYPE OF COMPOSITE bacillus preparation product providing a kind of and more efficiently promote growth of animal, prevent animal alimentary canal diseases, improve feed digestibility, improve breeding ecological environment.
A further object of the present invention is to provide and a kind ofly contains NEW TYPE OF COMPOSITE bacillus preparation of three strain bacterium and preparation method thereof, and the method has the advantage simplifying production technique, high-density total viable count, high spore content and lower production cost.
Further object of the present invention is to provide a kind of application method of composite bacillus preparation in animal ecology cultivation containing three strain bacterium, comprises addition manner, additive capacity, feeding cycle, cultural technique pattern and aquaculture management working specification etc.
For reaching the object of the invention, the technical solution used in the present invention is:
Containing a composite bacillus preparation for three strain bacterium, forming by producing the coagulated bacillus living formulation of Pfansteihl, the clostridium butyricum active bacteria formulation producing butyric acid and aerobic bacillus megaterium active bacteria formulation compatibility.Wherein Bacillus coagulans selects that deposit number is the bacterial strain of CGMCC No.2602, clostridium butyricum selects that deposit number is the bacterial strain of CGMCC No.1647, bacillus megaterium selects deposit number to be the bacterial strain of CGMCC No.8045.Each bacterial strain adopts independent fermentation mode, respectively through shake-flask culture, seed tank culture, fermentor cultivation, collected by centrifugation bacterium mud, mixes with dry starch, cryodrying, pulverizes, sieves, then mix with auxiliary material, obtain three kinds of active bacteria formulations.Finally mix with the part by weight of 1 ︰ 1 ︰ 1 and prepare the composite bacillus formulation products containing three strain bacterium, spore content reaches 1 × 10 8-10 9more than cfu/g.
The described preparation method containing the composite bacillus preparation of three strain bacterium, its preparation process is as follows:
(1) preparation of coagulated bacillus living formulation
1. thalline activation
Aseptic unlatching coagulating bacillus strain ( bacillus coagulans) the freeze-drying preservation of bacteria strain of CGMCC No.2602, streak inoculation is in wheat bran nutrient agar medium test tube slant, and slant medium composition is counted with g/L: peptone 10, extractum carnis 3, NaCL5, wheat bran 10, agar 15-20, with tap water preparation, and pH7.0-7.2; Cultivate 24-48h for 30-36 DEG C; Then line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane, cultivates 24-48h, microscopy for 30-36 DEG C, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
2. shaking flask and seed tank culture
Prepare the aseptic triangular flask of an in-built granulated glass sphere, scraped by the ripe bacterium mud on eggplant bottle inclined-plane with sterilized water, load triangular flask, vibrating dispersion bacterium mud, obtains uniform bacteria suspension.Bacteria suspension is heated 10 min in 80 DEG C of water-baths, and with the inoculum size of volume ratio 1%-10% access 250mL triangular flask, liquid amount is 15mL; Or access 500mL triangular flask, liquid amount is 30mL; Shake-flask culture.
Shaking flask liquid medium is heated 10 min in 80 DEG C of water-baths, transfers into seed tank culture with the inoculum size of volume ratio 1%-10%, seeding tank volume selects 10L, 20L, 50L or 100L, and seeding tank loading amount coefficient volume ratio is 60%-70%.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Substratum composition is in g/L: wheat bran 5-20, yeast extract paste 5-10, bean cake powder 5-10, K 2hPO 43, NaCl 5, MnSO 4h 2o 0.3, with tap water preparation, pH7.0.
Culture condition is: shaking flask rotating speed 200r/min, seeding tank mixing speed>=150r/min, air flow 1-2m 3/ h, culture temperature are 30-60 DEG C, incubation time is 18-48h.
3. fermentor cultivation
Seed culture fluid is with the inoculum size of volume ratio 1%-10% access fermentor tank, and the fermentor tank of 1 T, 2T or 3T volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is 60%-70%.
Fermention medium is made up of carbon source, nitrogenous source and inorganic salt, in g/L: carbon source 5-20, nitrogenous source 5-20, K 2hPO 43, NaCl 5, MnSO 4h 2o 0.3, calcium carbonate 2-10, with tap water preparation, pH6.0-8.0.Carbon source selects one or more of wheat bran, glucose, sucrose, lactose, maltose, fructose, pectinose, primverose, rice candy, Zulkovsky starch, W-Gum, tapioca (flour), wheat starch, sweet potato starch or yam starch; Nitrogenous source selects one or more in peptone, yeast extract paste, extractum carnis, corn starch, bean cake powder, soybean peptides, cottonseed protein, soybean cake powder or groundnut meal.
Fermentor cultivation condition is: mixing speed>=150r/min, air flow 1-2m 3/ h, culture temperature are 30-60 DEG C, incubation time is 18-48h.In culturing process, add soya-bean oil according to the surging situation of foam or bubble enemy carries out froth breaking, prevent from escaping out fermentor tank and cause living contaminants because of foam.Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, can put tank and terminate to cultivate, total viable count and gemma number are surveyed in sampling.
Viable bacteria and gemma detection tomato juice agar count substratum, in g/L: yeast extract paste 10, peptone 10, tomato juice 200mL, CaCO 35, agar 15-20, distilled water 800mL, pH7.0-7.2; Record fermented liquid Number of spores and reach 1 × 10 9more than cfu/mL.
4. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 1-5 with dry starch, at 40-50 DEG C of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, add one or more in auxiliary material stone flour, wheat bran or corn cob meal, auxiliary material add-on is the 60%-80% of gross weight, and make Tiny ecosystem coagulated bacillus living formulation after mixing, spore content reaches 1 × 10 9more than cfu/g.
(2) preparation of clostridium butyricum active bacteria formulation
1. thalline activation
Aseptic unlatching clostridium butyricum bacterial strain ( clostridium butyricum) the freeze-drying preservation of bacteria strain of CGMCC No.1647, access is equipped with in the test tube of activation medium, carries out standing Anaerobic culturel, cultivates 18-24h for 30-37 DEG C.Then transfer in the test tube that fresh activation medium is housed with the inoculum size of volume ratio 1%-10%, 30-37 DEG C of standing Anaerobic culturel 20-24h, microscopy, when forming gemma more than 90% thalline, namely ripe.Repeatedly activate 2-3 time.
Activation medium is made up of following raw material and weight proportion, in g/L: yeast extract paste 3-5, extractum carnis 5-20, peptone 5-20, glucose 5-10, Zulkovsky starch 1-2, NaCl 5, sodium acetate 3-5, cysteine hydrochloride 0.5-1, CaCO 33, with tap water preparation, pH 6.0-7.5.
2. shaking flask and seed tank culture
Bacteria suspension after activation is heated 10min in 80 DEG C of water-baths, and transfer with the inoculum size of volume ratio 1%-10% and be equipped with in the 250mL triangular flask of seed culture medium, liquid amount is the 80%-90% of volume ratio, 30-37 DEG C
Anaerobic culturel 20-24h is left standstill in constant incubator.
Shaking flask liquid medium is heated 10min in 80 DEG C of water-baths, transfer into seed tank culture with the inoculum size of volume ratio 1%-10%, seeding tank volume selects 10L, 20L or 50L, and the loading amount coefficient volume ratio of seeding tank is that 60%-70%, 30-37 DEG C of constant temperature leaves standstill Anaerobic culturel 20-48h.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Seed culture medium composition is in g/L: yeast extract paste 3, extractum carnis 10, Tryptones 10, glucose 5, Zulkovsky starch 1, NaCl 5, sodium acetate 3, cysteine hydrochloride 0.5, with tap water preparation, and pH7.0.
3. fermentor cultivation
Seed culture fluid is with the inoculum size of volume ratio 1%-10% access fermentor tank, and the fermentor tank of 100L, 200L or 500L volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is that 60%-70%, 30-37 DEG C of constant temperature leaves standstill Anaerobic culturel 20-48h.Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, can put tank and terminate to cultivate, total viable count and gemma number are surveyed in sampling.
Fermention medium composition is in g/L: glucose 10, beef peptone 10, yeast extract paste 5, NaCl 5, sodium acetate 3, K 2hPO 42, MgS0 47H 20 0.5, CaC0 31, with tap water preparation, pH 7.0.
The mensuration of total viable count adopts MPN method.At 80 DEG C, after water-bath thermal treatment 10min, the viable count of MPN method mensuration is adopted to be gemma number bacterium liquid.The biochemical reagents level thioglycollate solution medium that counting substratum adopts Chemical Reagent Co., Ltd., Sinopharm Group to produce.Record fermented liquid Number of spores and reach 1 × 10 8more than cfu/mL.
4. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtains gemma, will
Gemma mixes with weight ratio 1 ︰ 1-5 with dry starch, at 40-50 DEG C of dry 20-24h, pulverizer is pulverized, cross 40 mesh sieves, add one or more in auxiliary material stone flour, wheat bran or corn cob meal, auxiliary material add-on is the 60%-80% of gross weight, and make Tiny ecosystem clostridium butyricum active bacteria formulation after mixing, spore content reaches 1 × 10 8more than cfu/g.
(3) preparation of bacillus megaterium active bacteria formulation
1. thalline activation
Aseptic unlatching bacillus megaterium bacterial strain ( bacillus megaterium) the freeze-drying preservation of bacteria strain of CGMCC No.8045, streak inoculation, on LB agar test tubes inclined-plane, cultivates 36-48h for 28-37 DEG C.Then line is transferred on LB agar eggplant bottle inclined-plane, cultivates 36-48h, microscopy for 28-37 DEG C, when more than 90% thalline forms gemma, is maturation.
2. shaking flask and seed tank culture
Scraped by ripe bacterium mud on eggplant bottle inclined-plane with sterilized water, put into the sterilizing triangular flask of in-built sterile glass beads, vibrating dispersion bacterium mud, makes uniform bacteria suspension.Bacteria suspension is heated 10min in 80 DEG C of water-baths, and be equipped with in 250mL or the 500mL triangular flask of seed culture medium with the access of the inoculum size of volume ratio 1%-10%, liquid amount is 10% of volume ratio, shake-flask culture.
Shaking flask liquid medium is heated 10 min in 80 DEG C of water-baths, transfers into seed tank culture with the inoculum size of volume ratio 1%-10%, seeding tank volume selects 10L, 20L, 50L or 100L, and the loading amount coefficient volume ratio of seeding tank is 60%-70%.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Seed culture medium composition is in g/L: W-Gum 10, peptone 5, yeast extract paste 5, NaC1 5, CaCO 31, MgSO 47H 2o 0.5, with tap water preparation, pH7.0.
Culture condition is: shaking flask rotating speed 200r/min, seeding tank mixing speed>=200r/min, air flow 1-2m 3/ h, dissolved oxygen control more than 30%, and culture temperature is 28-37 DEG C, incubation time is 36-48h.
3. fermentor cultivation
By the seed culture fluid of maturation after 80 DEG C of water-bath 10min, with the inoculum size of volume ratio 1%-10% access fermentor tank, guarantee rear initial spore concentration>=10 of inoculation 6cfu/mL, the fermentor tank of 1 T, 2T or 3T volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is 70%-80%.
Fermention medium composition is in g/L: W-Gum 10, peptone 5, yeast extract paste 5, NaC1 5, CaCO 31, MgSO 47H 2o 0.5, with tap water preparation, pH7.0.
Fermentor cultivation condition is: mixing speed>=200r/min, air flow 1-2m 3/ h, dissolved oxygen control more than 30%, culture temperature is 28-37 DEG C, incubation time is 36-48h.In culturing process, add soya-bean oil according to the surging situation of foam or bubble enemy carries out froth breaking, prevent from escaping out fermentor tank and cause living contaminants because of foam.Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, can put tank and terminate to cultivate, total viable count and gemma number are surveyed in sampling.
The mensuration of total viable count adopts serial dilutions.At 80 DEG C, after water-bath thermal treatment 10min, the viable count of serial dilutions mensuration is adopted to be gemma number bacterium liquid.Record fermented liquid Number of spores and reach 1 × 10 9more than cfu/mL.
4. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 1-5 with dry starch, at 40-50 DEG C of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, add one or more in auxiliary material stone flour, wheat bran or corn cob meal, auxiliary material add-on is the 60%-80% of gross weight, and make Tiny ecosystem bacillus megaterium active bacteria formulation after mixing, spore content reaches 1 × 10 9more than cfu/g.
(4) preparation of composite bacillus preparation
Clostridium butyricum active bacteria formulation coagulated bacillus living formulation obtained for step (1), step (2) obtained mixes with the part by weight of 1 ︰ 1 ︰ 1 with the bacillus megaterium active bacteria formulation that step (3) obtains, namely obtain the composite bacillus preparation of product containing three strain bacterium, spore content reaches 1 × 10 8-10 9more than cfu/g.
3. finished product index
Product gemma number: Bacillus coagulans>=1 × 10 9cfu/g; Clostridium butyricum>=1 × 10 8cfu/g; Bacillus megaterium>=1 × 10 9cfu/g.Sense index: product is canescence pulvis, and color and luster is homogeneous.Granularity: preparation is by 40 mesh sieves; Moisture: water content≤8%.
4. the usage of composite bacillus preparation in animal cultivation is as follows:
(1) for animal feed, addition is the 0.02%-0.5% of feed dry weight.For bird feed, addition is the 0.02%-0.2% of feed dry weight.For aquatic feeds, addition is the 0.02%-0.2% of feed dry weight.
(2) described livestock comprises large-scale herbivore and the non-herbivorous animals such as pig, ox, sheep, rabbit.Described bird comprises the animals such as chicken, duck, goose.Aquatic product comprises the crustaceans hydrocoles such as soft-shelled turtle, shrimp, crab and other various fresh-water fishes, sea water fish etc.
Beneficial effect of the present invention:
1. the Bacillus coagulans that the present invention adopts is amphimicrobian genus bacillus, and clostridium butyricum is the genus bacillus of strictly anaerobic.Bacillus coagulans can produce a large amount of Pfansteihl, antibacterial coagulin, amino acid, VITAMIN and multiple digestive ferment in animal intestinal on the one hand, and improvement intestinal microecology balance, promotes intestinal growth and strengthen intestinal function, promotes that feed digestion absorbs; Clostridium butyricum can produce materials such as body butyric acid, vitamin B group and vitamin Ks with health role on the other hand, promotes the regeneration of gut epithelium histocyte and repairs, and reduces enterotoxin and occurs, improve efficiency of feed utilization.In addition, the Pfansteihl that Bacillus coagulans produces has stronger acidity, the butyric acid that strong acidic environment makes clostridium butyricum produce not easily dissociation, only has non-dissociated molecular state butyric acid could provide energy to intestinal epithelial cells tissue better, promotes the reparation of damaged cell.Therefore, these two kinds of bacterium enter after in animal body simultaneously, can carry out microecosystem regulation balance respectively by different approach and mechanism, and both are mutually collaborative supplements, and produces force action to raising probiotic effects.
2. the present invention have employed the genus bacillus that two kinds are produced acid simultaneously, namely the Pfansteihl of Bacillus coagulans generation and the butyric acid of clostridium butyricum generation can make animal intestinal pH value lower, and complementation between them in other physiological function and additive effect, thus more effectively can suppress the growth of the pathogenic bacterias such as intestinal bacteria, reduce the propagation of diarrhea rate and promotion probiotics.
3. composite bacillus preparation of the present invention further comprises bacillus megaterium.This bacterium is strictly aerobic genus bacillus, enter after in animal body and first breed, play its stronger biology and take oxygen ability by force, consume oxygen residual in enteron aisle rapidly, for Bacillus coagulans and clostridium butyricum provide good micro-ecological environment, promote their field planting and growth.In addition, bacillus megaterium effectively can reduce the discharge of ight soil ammonia and hydrogen sulfide in breeding process, plays deodorizing and improves the effect of breeding ecological environment.
4. composite bacillus preparation of the present invention have employed three kinds of genus bacillus simultaneously, and they can produce multiple enzyme, organic acid, VITAMIN and large number of biological active substance in enteron aisle growth and breeding process.Especially bacillus megaterium has abundant enzyme system, can produce amylase, proteolytic enzyme, cellulase and lipase etc.Three kinds of bacterium are mutually worked in coordination with in animal intestinal, complement one another, and together constitute efficient, a perfect enzyme system, thus play prevent and cure diseases, the effect of promoting digestion and raising efficiency of feed utilization.
5. the preparation method of three kinds of bacillus living formulations of the present invention's employing, is special high-density gemma fermentation manufacturing technique.The substratum prices of raw and semifnished materials that this technique adopts are cheap, and culture condition is extensive, simple to operate, and incubation time short (no longer than 48 hours), energy efficient, production cost is low, and total count and spore forming rate are all higher.
6. what the present invention adopted is all sporiferous microorganisms, and the composite bacillus preparation spore content of preparation reaches 1 × 10 8-10 9more than cfu/g, thus environmental resistance is strong, and not easy in inactivation in feed manufacturing process, product stability is high, long quality-guarantee period, active by the impact of hydrochloric acid in gastric juice, digestive ferment, bile acide in animal body, can give full play to the probiotic effects to animal.
Biological material specimens preservation: a kind of Bacillus coagulans ( bacillus coagulans) JSSW-LA-07, deposit number is CGMCC No.2602, preservation date on July 28th, 2008; A kind of clostridium butyricum ( clostridium butyricum) JSIM-MCB20040312, deposit number is CGMCC No.1647, preservation date on March 10th, 2006; A kind of bacillus megaterium ( bacillus megaterium) JSSW-ID-2F5, deposit number is CGMCC No.8045, preservation date on August 19th, 2013; Three strain bacterium have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center all, are called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained, but embodiments of the present invention is not limited in any way.
Embodiment 1
The described preparation method containing the composite bacillus preparation of three strain bacterium, preparation process is as follows:
(1) preparation of coagulated bacillus living formulation
1. thalline activation
Aseptic unlatching coagulating bacillus strain ( bacillus coagulans) the freeze-drying preservation of bacteria strain of CGMCC No.2602, streak inoculation, in wheat bran nutrient agar medium test tube slant, cultivates 24h for 30 DEG C; Then line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane, cultivates 24h for 30 DEG C.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Slant medium composition is counted with g/L: peptone 10, extractum carnis 3, NaCl 5, wheat bran 10, agar 20, with tap water preparation, and pH7.0.
2. shake-flask seed is cultivated
Prepare the aseptic triangular flask of an in-built granulated glass sphere, scraped by the ripe bacterium mud on eggplant bottle inclined-plane with sterilized water, load triangular flask, vibrating dispersion bacterium mud, obtains uniform bacteria suspension.Bacteria suspension is heated 10 min in 80 DEG C of water-baths, accesses 25 500mL triangular flasks with the inoculum size of volume ratio 10%, liquid amount is 30mL, shake-flask culture.Shaking flask rotating speed 200r/min, culture temperature are 30 DEG C, incubation time is 18h.
3. seed tank culture
Merge shake-flask seed nutrient solution, heat 10 min in 80 DEG C of water-baths, with the inoculum size of volume ratio 1% access 100L seeding tank, the loading amount coefficient volume ratio of seeding tank is 70%.Seeding tank mixing speed 150r/min, air flow 2m 3/ h, culture temperature are 30 DEG C, incubation time is 18h.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Seed culture medium composition is in g/L: wheat bran 5, yeast extract paste 5, bean cake powder 5, K 2hPO 43, NaCl 5, MnSO 4h 2o 0.3, with tap water preparation, pH7.0.
4. fermentor cultivation
Seed culture fluid is with the inoculum size of volume ratio 3.5% access fermentor tank, and the fermentor tank of 3T volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is 65%.Fermentor tank mixing speed 150r/min, air flow 2m 3/ h, culture temperature are 30 DEG C, incubation time is 48h.In culturing process, add bubble enemy according to the surging situation of foam carry out froth breaking, prevent from escaping out fermentor tank and cause living contaminants because of foam.Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, tank can be put and terminate to cultivate.The total viable count of sampling and measuring reaches 6.0 × 10 9cfu/mL, Number of spores reaches 5.8 × 10 9cfu/mL, spore forming rate is 96.7%.
Fermention medium composition is in g/L: W-Gum 5, corn starch 5, K 2hPO 43, NaCl 5, MnSO 4h 2o 0.3, calcium carbonate 2, with tap water preparation, pH7.0.
Viable bacteria and gemma detection tomato juice agar count substratum, in g/L: yeast extract paste 10, peptone 10, tomato juice 200mL, CaCO 35, agar 15, distilled water 800mL, pH7.0.
5. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 2 with dry starch, at 50 DEG C of dry 20h, pulverizer is pulverized, and crosses 40 mesh sieves, adds the corn cob meal auxiliary material of weight percent 70%, mix, make spore content reach 1 × 10 9more than cfu/g, namely obtains coagulated bacillus living formulation.
(2) preparation of clostridium butyricum active bacteria formulation
1. thalline activation
Aseptic unlatching clostridium butyricum bacterial strain ( clostridium butyricum) the freeze-drying preservation of bacteria strain of CGMCC No.1647, access is equipped with in the test tube of activation medium, carries out standing Anaerobic culturel, cultivates 18h for 30 DEG C.Then transfer in the test tube that fresh activation medium is housed with the inoculum size of volume ratio 10%, 30 DEG C of standing Anaerobic culturel 20h, microscopy, when forming gemma more than 90% thalline, namely ripe.So repeatedly activate 3 times.
Activation medium composition is in g/L: yeast extract paste 3, extractum carnis 5, peptone 5, glucose 5, Zulkovsky starch 1, NaCl 5, sodium acetate 3, cysteine hydrochloride 0.5, CaCO 33, with tap water preparation, pH 7.0.
2. shake-flask seed is cultivated
Bacteria suspension after activation is heated 10 min in 80 DEG C of water-baths, is equipped with in 15 250mL triangular flasks of seed culture medium with the access of the inoculum size of volume ratio 10%, liquid amount is leave standstill Anaerobic culturel 20h in 80% of volume ratio, 30 DEG C of constant incubators.
3. seed tank culture
Merge shake-flask seed nutrient solution, heat 10 min in 80 DEG C of water-baths, with the inoculum size of volume ratio 10% access seed tank culture, seeding tank volume selects 50L, and the loading amount coefficient volume ratio of seeding tank is that 60%, 30 DEG C of constant temperature leave standstill Anaerobic culturel 20h.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Seed culture medium composition is in g/L: yeast extract paste 3, extractum carnis 10, Tryptones 10, glucose 5, Zulkovsky starch 1, NaCl 5, sodium acetate 3, cysteine hydrochloride 0.5, with tap water preparation, and pH7.0.
4. fermentor cultivation
Seed culture fluid is with the inoculum size of volume ratio 10% access fermentor tank, and the fermentor tank of 500L volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is that 60%, 30 DEG C of constant temperature leave standstill Anaerobic culturel 48h.Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, tank can be put and terminate to cultivate.The total viable count of sampling and measuring reaches 8.5 × 10 8cfu/mL, Number of spores reach 8.1 × 10 8cfu/mL, spore forming rate is 95.3%.
Fermention medium composition is in g/L: glucose 10, beef peptone 10, yeast extract paste 5, NaCl 5, sodium acetate 3, K 2hPO 42, MgS0 47H 20 0.5, CaC0 31, with tap water preparation, pH 7.0.
The mensuration of total viable count adopts MPN method.At 80 DEG C, after water-bath thermal treatment 10min, the viable count of MPN method mensuration is adopted to be gemma number bacterium liquid.The biochemical reagents level thioglycollate solution medium that counting substratum adopts Chemical Reagent Co., Ltd., Sinopharm Group to produce.
5. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 3 with dry starch, at 45 DEG C of dry 24h, pulverizer is pulverized, and crosses 40 mesh sieves, adds the corn cob meal auxiliary material of weight percent 70%, mix, make spore content reach 1 × 10 8more than cfu/g, namely obtains clostridium butyricum active bacteria formulation.
(3) preparation of bacillus megaterium active bacteria formulation
1. thalline activation
Aseptic unlatching bacillus megaterium bacterial strain ( bacillus megaterium) the freeze-drying preservation of bacteria strain of CGMCC No.8045, streak inoculation, on LB agar test tubes inclined-plane, cultivates 36h for 28 DEG C.Then line is transferred on LB agar eggplant bottle inclined-plane, cultivates 36h, microscopy for 28 DEG C, when more than 90% thalline forms gemma, is maturation.
2. shake-flask seed is cultivated
Scraped by ripe bacterium mud on eggplant bottle inclined-plane with sterilized water, put into the sterilizing triangular flask of in-built sterile glass beads, vibrating dispersion bacterium mud, makes uniform bacteria suspension.Bacteria suspension is heated 10min in 80 DEG C of water-baths, and be equipped with in 12 500mL triangular flasks of seed culture medium with the access of the inoculum size of volume ratio 10%, liquid amount is 10% of volume ratio, shake-flask culture.Shaking flask rotating speed 200r/min, culture temperature are 28 DEG C, incubation time is 36h.
3. seed tank culture
Merge shake-flask seed nutrient solution, heat 10 min in 80 DEG C of water-baths, with the inoculum size of volume ratio 1% access seed tank culture, seeding tank volume selects 100L, and the loading amount coefficient volume ratio of seeding tank is 60%.Seeding tank mixing speed 200r/min, air flow 2m 3/ h, dissolved oxygen control more than 30%, and culture temperature is 28 DEG C, incubation time is 36h.Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring.
Seed culture medium composition is in g/L: W-Gum 10, peptone 5, yeast extract paste 5, NaC1 5, CaCO 31, MgSO 47H 2o 0.5, with tap water preparation, pH7.0.
4. fermentor cultivation
By the seed culture fluid of maturation after 80 DEG C of water-bath 10min, with the inoculum size of volume ratio 2.5% access fermentor tank, guarantee rear initial spore concentration>=10 of inoculation 6cfu/mL, the fermentor tank of 3T volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is 80%.Fermentor tank mixing speed 200r/min, air flow 2m 3/ h, dissolved oxygen control more than 30%, culture temperature is 28 DEG C, incubation time is 48h.In culturing process, add bubble enemy according to the surging situation of foam carry out froth breaking, prevent from escaping out fermentor tank and cause living contaminants because of foam.Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, tank can be put and terminate to cultivate.The total viable count of sampling and measuring reaches 3.5 × 10 9cfu/mL, Number of spores reaches 3.4 × 10 9cfu/mL, spore forming rate is 97.1%.
Fermention medium composition is in g/L: W-Gum 10, peptone 5, yeast extract paste 5, NaC1 5, CaCO 31, MgSO 47H 2o 0.5, with tap water preparation, pH7.0.
The mensuration of total viable count adopts serial dilutions.At 80 DEG C, after water-bath thermal treatment 10min, the viable count of serial dilutions mensuration is adopted to be gemma number bacterium liquid.
5. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 1 with dry starch, at 50 DEG C of dry 20h, pulverizer is pulverized, and crosses 40 mesh sieves, adds the corn cob meal auxiliary material of weight percent 70%, mix, make spore content reach 1 × 10 9more than cfu/g, namely obtains bacillus megaterium active bacteria formulation.
(4) preparation of composite bacillus preparation: clostridium butyricum active bacteria formulation coagulated bacillus living formulation obtained for step (1), step (2) obtained mixes with the part by weight of 1 ︰ 1 ︰ 1 with the bacillus megaterium active bacteria formulation that step (3) obtains, namely obtain the composite bacillus preparation of product containing three strain bacterium, spore content reaches 1 × 10 8-10 9more than cfu/g, wherein Bacillus coagulans>=1 × 10 9cfu/g; Clostridium butyricum>=1 × 10 8cfu/g; Bacillus megaterium>=1 × 10 9cfu/g.Product is canescence pulvis, and color and luster is homogeneous, granularity by 40 mesh sieves, water content≤8%.
Application Example 1: the composite bacillus preparation containing three strain bacterium is fed weanling pig
Test completes in cultivation base, Shuan Miao village, farming animals industry company limited Luo She town, Wuxi City Nan Yang.Choose physically well develop, honey stomach, health, hereditary basis similar, initial weight 10-13 ± 0.08 kg, 45 ± 2 ages in days strong × length × Dasanyuan hybridization weanling pig totally 60, be divided into 2 groups at random, often group 30, male and female half and half.Control group is only fed basal diet, and test group adds 0.2%(weight percent in basal diet) the composite bacillus preparation containing three strain bacterium.The feeding and management of whole duration of test, disinfecting and immune vaccine program are all undertaken by pig farm routine; The immunity of each group, medication, ventilation, drinking-water, feeding manner are all consistent with time etc.The official test phase is 43 days, in the 1st day on-test and off-test day, weighs the gross weight of every hurdle test pig, to calculate every growth indexes.Time recording diarrhoea head number, observed the ight soil situation on every hurdle, calculated diarrhea rate and Scours index morning every day.The early morning on the same day of off-test day, select 6 close to the piglet of average body quality from every group, anus gathers fresh excrement sample, measures intestinal bacteria and content of lactic acid bacteria; And with reference to national standard, measure the apparent digestibility of feed main nutrient composition.Testing data adopts the one-factor analysis of variance to carry out significance test of difference, and result is as follows:
Table 1 containing the composite bacillus preparation of three strain bacterium on weanling pig growth and the impact of efficiency of feed utilization
Note: different letter representation significant difference in same column ( p< 0.05), same letter represent difference not significantly ( p>0.05), following table is same.
Table 2 containing the composite bacillus preparation of three strain bacterium on the impact of diarrhea of weaned piglets and fecal microorganism flora
Table 3 contains the composite bacillus preparation of three strain bacterium to the impact of weanling pig feed nutrient apparent digestibility
As seen from the results in Table 1, the average daily gain of test group, rate of body weight gain and specific growth rate improve 29.44%, 35.07% and 19.92% than control group respectively, difference extremely significantly ( p< 0.05); The feed coefficient of test group reduces 23.46% than control group, and protein efficiency ratio control group improves 30.50%, difference also extremely significantly ( p< 0.05).Show in pig starter feed, add the composite bacillus preparation containing three strain bacterium of the present invention, there is the effect of growth promoting effects, raising efficiency of feed utilization and saving feed cost.
As seen from the results in Table 2, the diarrhea rate of test group and Scours index significantly lower than control group ( p< 0.05); Intestinal bacteria quantity in test group ight soil than contrast group be decreased significantly ( p< 0.05), and lactic acid bacterium number than control group be significantly increased ( p< 0.05).Show in pig starter feed, add the composite bacillus preparation containing three strain bacterium of the present invention, there is the effect that the growth suppressing harmful intestinal tract bacteria, the propagation promoting probiotics, prevention and reduction are suffered from diarrhoea.
As seen from the results in Table 3, test group crude protein and coarse-fibred apparent digestibility improve 20.17% and 34.51% than control group respectively, difference extremely significantly ( p< 0.05); But the apparent digestibility of test group crude fat, coarse ash and phosphorus and control group no significant difference ( p> 0.05).Show in pig starter feed, add the composite bacillus preparation containing three strain bacterium of the present invention, there is the effect promoting that feed main nutrient composition is digested and assimilated.
Overall conclusion, the composite bacillus preparation containing three strain bacterium of the present invention is at 0.2%(weight percent) additive capacity under, to weanling pig, there is multiple probiotic effects, can be used as microorganism feed addictive and use.
Application Example 2: containing the composite bacillus preparation of three strain bacterium to the deodorization of breeding environment
Test completes in cultivation base, Shuan Miao village, farming animals industry company limited Luo She town, Wuxi City Nan Yang.120 healthy fattening pigs are divided into 2 groups at random, often organize 60, male and female half and half.Control group is only fed basal diet,
Test group adds 0.2%(weight percent in basal diet) containing the composite bacillus preparation of three strain bacterium.Raise in the different pig houses of same size for each group, be spaced from each other, in pig house, temperature remains on 26 ~ 32 DEG C.The management of duration of test, health, immunity, epidemic prevention, medication, ventilation, drinking-water, feeding manner are all consistent with time etc.Pig house adopts internet of things sensors to detect temperature, stink substance ammonia and hydrogen sulfide, and the detection data analysis getting ︰ 00 every day 8 compares, and testing data adopts the one-factor analysis of variance to carry out significance test of difference, and result is as follows:
Table 4 contains the composite bacillus preparation of three strain bacterium to the impact of pig house odorous gas change in concentration
Note: different letter representation significant difference in colleague ( p< 0.05), same letter represent difference not significantly ( p>0.05).
As seen from the results in Table 4, before on-test, in two houses, odorous gas ammonia is substantially identical with the concentration of hydrogen sulfide; The 1st day on-test and the 2nd day, test house odorous gas concentration decline gradually, contrast house odorous gas concentration substantially unchanged, but two house between there was no significant difference ( p> 0.05).Along with the passing of culturing time, the ammonia of test house and the concentration continuous decrease of hydrogen sulfide, and always with unconverted contrast house also exist significant difference ( p< 0.05).In the feed period of 7 days, the average ammonia concentration of test house and concentration of hydrogen sulfide reduce 24% and 42.6% than contrast house respectively, show that composite bacillus preparation of the present invention has and reduce the effect that pig house odorous gas produced, improved to a certain extent breeding ecological environment.

Claims (9)

1., containing a composite bacillus preparation for three strain bacterium, it is characterized in that described preparation forms by producing the coagulated bacillus living formulation of Pfansteihl, the clostridium butyricum active bacteria formulation producing butyric acid and aerobic bacillus megaterium active bacteria formulation compatibility;
Wherein coagulated bacillus living formulation is that the coagulating bacillus strain of CGMCC No.2602 is through shake-flask culture, seed tank culture, fermentor cultivation, collected by centrifugation bacterium mud by deposit number, mix with dry starch, cryodrying, pulverize, sieve, then mix with auxiliary material and obtain;
Clostridium butyricum active bacteria formulation is that the clostridium butyricum bacterial strain of CGMCC No.1647 is through shake-flask culture, seed tank culture, fermentor cultivation, collected by centrifugation bacterium mud by deposit number, mix with dry starch, cryodrying, pulverize, sieve, then mix with auxiliary material and obtain;
Bacillus megaterium active bacteria formulation is that the bacillus megaterium bacterial strain of CGMCC No.8045 is through shake-flask culture, seed tank culture, fermentor cultivation, collected by centrifugation bacterium mud by deposit number, mix with dry starch, cryodrying, pulverize, sieve, then mix with auxiliary material and obtain.
2. the composite bacillus preparation containing three strain bacterium according to claim 1, it is characterized in that described preparation is mixed with the part by weight of 1 ︰ 1 ︰ 1 by coagulated bacillus living formulation, clostridium butyricum active bacteria formulation and bacillus megaterium active bacteria formulation and obtains, the spore content of preparation reaches 1 × 10 8-10 9more than cfu/g.
3. claim 1 and the preparation method containing the composite bacillus preparation of three strain bacterium according to claim 2, is characterized in that preparation process is as follows:
(1) preparation of coagulated bacillus living formulation
1. thalline activation
Aseptic unlatching coagulating bacillus strain ( bacillus coagulans) the freeze-drying preservation of bacteria strain of CGMCC No.2602, streak inoculation is in wheat bran nutrient agar medium test tube slant, and slant medium composition is counted with g/L: peptone 10, extractum carnis 3, NaCl 5, wheat bran 10, agar 15-20, with tap water preparation, and pH 7.0-7.2; Cultivate 24-48h for 30-36 DEG C; Then line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane, cultivates 24-48h, microscopy for 30-36 DEG C, when more than 90% thalline forms gemma, is maturation, prepares culture transferring;
2. shaking flask and seed tank culture
Prepare the aseptic triangular flask of an in-built granulated glass sphere, scraped by the ripe bacterium mud on eggplant bottle inclined-plane with sterilized water, load triangular flask, vibrating dispersion bacterium mud, obtains uniform bacteria suspension; Bacteria suspension is heated 10min in 80 DEG C of water-baths, and with the inoculum size of volume ratio 1%-10% access 250mL triangular flask, liquid amount is 15mL; Or access 500mL triangular flask, liquid amount is 30mL; Shake-flask culture;
Shaking flask liquid medium is heated 10 min in 80 DEG C of water-baths, transfers into seed tank culture with the inoculum size of volume ratio 1%-10%, seeding tank volume selects 10L, 20L, 50L or 100L, seeding tank loading amount coefficient
Volume ratio is 60%-70%, microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring;
Substratum composition is in g/L: wheat bran 5-20, yeast extract paste 5-10, bean cake powder 5-10, K 2hPO 43, NaCl 5, MnSO 4h 2o 0.3, with tap water preparation, pH 7.0;
Culture condition is: shaking flask rotating speed 200r/min, seeding tank mixing speed>=150r/min, air flow 1-2m 3/ h, culture temperature are 30-60 DEG C, incubation time is 18-48h;
3. fermentor cultivation
Seed culture fluid is with the inoculum size of volume ratio 1%-10% access fermentor tank, and the fermentor tank of 1 T, 2T or 3T volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is 60%-70%;
Fermention medium is made up of carbon source, nitrogenous source and inorganic salt, in g/L: carbon source 5-20, nitrogenous source 5-20, K 2hPO 43, NaCl 5, MnSO 4h 2o 0.3, calcium carbonate 2-10, with tap water preparation, pH 6.0-8.0; Carbon source selects one or more of wheat bran, glucose, sucrose, lactose, maltose, fructose, pectinose, primverose, rice candy, Zulkovsky starch, W-Gum, tapioca (flour), wheat starch, sweet potato starch or yam starch; Nitrogenous source selects one or more in peptone, yeast extract paste, extractum carnis, corn starch, bean cake powder, soybean peptides, cottonseed protein, soybean cake powder or groundnut meal;
Fermentor cultivation condition is: mixing speed>=150r/min, air flow 1-2m 3/ h, culture temperature are 30-60 DEG C, incubation time is 18-48h; In culturing process, add soya-bean oil according to the surging situation of foam or bubble enemy carries out froth breaking, prevent from escaping out fermentor tank and cause living contaminants because of foam; Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, can put tank and terminate to cultivate, total viable count and gemma number are surveyed in sampling;
Viable bacteria and gemma detection tomato juice agar count substratum, in g/L: yeast extract paste 10, peptone 10, tomato juice 200mL, CaCO 35, agar 15-20, distilled water 800mL, pH 7.0-7.2; Record fermented liquid Number of spores and reach 1 × 10 9more than cfu/mL;
4. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 1-5 with dry starch, at 40-50 DEG C of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, add one or more in auxiliary material stone flour, wheat bran or corn cob meal, auxiliary material add-on is the 60%-80% of gross weight, and make Tiny ecosystem coagulated bacillus living formulation after mixing, spore content reaches 1 × 10 9more than cfu/g;
(2) preparation of clostridium butyricum active bacteria formulation
1. thalline activation
Aseptic unlatching clostridium butyricum bacterial strain ( clostridium butyricum) the freeze-drying preservation of bacteria strain of CGMCC No.1647, access is equipped with in the test tube of activation medium, carries out standing Anaerobic culturel, cultivates 18-24h for 30-37 DEG C; Then transfer in the test tube that fresh activation medium is housed with the inoculum size of volume ratio 1%-10%, 30-37 DEG C of standing Anaerobic culturel 20-24h, microscopy, when forming gemma more than 90% thalline, namely ripe; Repeatedly activate 2-3 time;
Activation medium is made up of following raw material and weight proportion, in g/L: yeast extract paste 3-5, extractum carnis 5-20, peptone 5-20, glucose 5-10, Zulkovsky starch 1-2, NaCl 5, sodium acetate 3-5, cysteine hydrochloride 0.5-1, CaCO 33, with tap water preparation, pH 6.0-7.5;
2. shaking flask and seed tank culture
Bacteria suspension after activation is heated 10min in 80 DEG C of water-baths, is equipped with in the 250mL triangular flask of seed culture medium with the access of the inoculum size of volume ratio 1%-10%, liquid amount is leave standstill Anaerobic culturel 20-24h in the 80%-90% of volume ratio, 30-37 DEG C constant incubator;
Shaking flask liquid medium is heated 10min in 80 DEG C of water-baths, transfer into seed tank culture with the inoculum size of volume ratio 1%-10%, seeding tank volume selects 10L, 20L or 50L, and the loading amount coefficient volume ratio of seeding tank is that 60%-70%, 30-37 DEG C of constant temperature leaves standstill Anaerobic culturel 20-48h; Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring;
Seed culture medium composition is in g/L: yeast extract paste 3, extractum carnis 10, Tryptones 10, glucose 5, Zulkovsky starch 1, NaCl 5, sodium acetate 3, cysteine hydrochloride 0.5, with tap water preparation, and pH7.0;
3. fermentor cultivation
Seed culture fluid is with the inoculum size of volume ratio 1%-10% access fermentor tank, and the fermentor tank of 100L, 200L or 500L volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is that 60%-70%, 30-37 DEG C of constant temperature leaves standstill Anaerobic culturel 20-48h; Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, can put tank and terminate to cultivate, total viable count and gemma number are surveyed in sampling;
Fermention medium composition is in g/L: glucose 10, beef peptone 10, yeast extract paste 5, NaCl 5, sodium acetate 3, K 2hPO 42, MgS0 47H 20 0.5, CaC0 31, with tap water preparation, pH 7.0;
The mensuration of total viable count adopts MPN method, by bacterium liquid after 80 DEG C of water-bath thermal treatment 10min, the viable count adopting MPN method to measure is gemma number, the biochemical reagents level thioglycollate solution medium that counting substratum adopts Chemical Reagent Co., Ltd., Sinopharm Group to produce, records fermented liquid Number of spores and reaches 1 × 10 8more than cfu/mL;
4. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtains gemma, will
Gemma mixes with weight ratio 1 ︰ 1-5 with dry starch, at 40-50 DEG C of dry 20-24h, pulverizer is pulverized, cross 40 mesh sieves, add one or more in auxiliary material stone flour, wheat bran or corn cob meal, auxiliary material add-on is the 60%-80% of gross weight, and make Tiny ecosystem clostridium butyricum active bacteria formulation after mixing, spore content reaches 1 × 10 8more than cfu/g;
(3) preparation of bacillus megaterium active bacteria formulation
1. thalline activation
Aseptic unlatching bacillus megaterium bacterial strain ( bacillus megaterium) the freeze-drying preservation of bacteria strain of CGMCC No.8045, streak inoculation, on LB agar test tubes inclined-plane, cultivates 36-48h for 28-37 DEG C, then line is transferred on LB agar eggplant bottle inclined-plane, cultivates 36-48h, microscopy for 28-37 DEG C, when more than 90% thalline forms gemma, be maturation;
2. shaking flask and seed tank culture
Scraped by ripe bacterium mud on eggplant bottle inclined-plane with sterilized water, put into the sterilizing triangular flask of in-built sterile glass beads, vibrating dispersion bacterium mud, makes uniform bacteria suspension; Bacteria suspension is heated 10min in 80 DEG C of water-baths, and be equipped with in 250mL or the 500mL triangular flask of seed culture medium with the access of the inoculum size of volume ratio 1%-10%, liquid amount is 10% of volume ratio, shake-flask culture;
Shaking flask liquid medium is heated 10min in 80 DEG C of water-baths, transfers into seed tank culture with the inoculum size of volume ratio 1%-10%, seeding tank volume selects 10L, 20L, 50L or 100L, and the loading amount coefficient volume ratio of seeding tank is 60%-70%; Microscopy, when more than 90% thalline forms gemma, is maturation, prepares culture transferring;
Seed culture medium composition is in g/L: W-Gum 10, peptone 5, yeast extract paste 5, NaC1 5, CaCO 31, MgSO 47H 2o 0.5, with tap water preparation, pH 7.0;
Culture condition is: shaking flask rotating speed 200r/min, seeding tank mixing speed>=200r/min, air flow 1-2m 3/ h, dissolved oxygen control more than 30%, and culture temperature is 28-37 DEG C, incubation time is 36-48h;
3. fermentor cultivation
By the seed culture fluid of maturation after 80 DEG C of water-bath 10min, with the inoculum size of volume ratio 1%-10% access fermentor tank, guarantee rear initial spore concentration>=10 of inoculation 6cfu/mL, the fermentor tank of 1T, 2T or 3T volume selected by fermentor tank, and fermentor tank loading amount coefficient volume ratio is 70%-80%;
Fermention medium composition is in g/L: W-Gum 10, peptone 5, yeast extract paste 5, NaC1 5, CaCO 31, MgSO 47H 2o 0.5, with tap water preparation, pH 7.0;
Fermentor cultivation condition is: mixing speed>=200r/min, air flow 1-2m 3/ h, dissolved oxygen control more than 30%, culture temperature is 28-37 DEG C, incubation time is 36-48h; In culturing process, add soya-bean oil according to the surging situation of foam or bubble enemy carries out froth breaking, prevent from escaping out fermentor tank and cause living contaminants because of foam; Get fermented liquid and carry out microscopy, when the thalline when more than 90% has formed gemma, can put tank and terminate to cultivate, total viable count and gemma number are surveyed in sampling;
The mensuration of total viable count adopts serial dilutions; By bacterium liquid after 80 DEG C of water-bath thermal treatment 10min, the viable count adopting serial dilutions to measure is gemma number; Record fermented liquid Number of spores and reach 1 × 10 9more than cfu/mL;
4. gemma is collected and the preparation of bacterium powder
Nutrient solution being pumped into rotating speed is that the continuous centrifugal machine of 15000r/min carries out centrifugal, obtain gemma, gemma is mixed with weight ratio 1 ︰ 1-5 with dry starch, at 40-50 DEG C of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, add one or more in auxiliary material stone flour, wheat bran or corn cob meal, auxiliary material add-on is the 60%-80% of gross weight, and make Tiny ecosystem bacillus megaterium active bacteria formulation after mixing, spore content reaches 1 × 10 9more than cfu/g;
(4) preparation of composite bacillus preparation
Clostridium butyricum active bacteria formulation coagulated bacillus living formulation obtained for step (1), step (2) obtained mixes with the part by weight of 1 ︰ 1 ︰ 1 with the bacillus megaterium active bacteria formulation that step (3) obtains, namely obtain the composite bacillus preparation of product containing three strain bacterium, spore content reaches 1 × 10 8-10 9more than cfu/g.
4., with the application containing the composite bacillus preparation of three strain bacterium prepared by method described in claim 3, it is characterized in that for animal feed, addition is the 0.02%-0.5% of feed dry weight.
5., with the application containing the composite bacillus preparation of three strain bacterium prepared by method described in claim 3, it is characterized in that for bird feed, addition is the 0.02%-0.2% of feed dry weight.
6., with the application containing the composite bacillus preparation of three strain bacterium prepared by method described in claim 3, it is characterized in that for aquatic feeds, addition is the 0.02%-0.2% of feed dry weight.
7., according to claim 4 containing the application of the composite bacillus preparation of three strain bacterium, it is characterized in that described livestock comprises pig, ox, sheep, the large-scale herbivore of rabbit and non-herbivorous animal.
8., according to claim 5 containing the application of the composite bacillus preparation of three strain bacterium, it is characterized in that described bird comprises chicken, duck, goose.
9., according to claim 6 containing the application of composite bacillus preparation of three strain bacterium, it is characterized in that described aquatic product comprises soft-shelled turtle, shrimp, crab crustaceans hydrocoles and other various fresh-water fishes, sea water fish.
CN201510001428.2A 2015-01-05 2015-01-05 Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding Pending CN104480052A (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1844364A (en) * 2006-05-11 2006-10-11 江苏省微生物研究所有限责任公司 Microecological preparation-tetragenous viable bacteria preparation and method for preparing same
US20080003207A1 (en) * 2004-12-15 2008-01-03 Qingdao East Sea Pharmaceuticals, Ltd Treating inflammatory bowel disease with live bacteria
CN101412983A (en) * 2008-11-20 2009-04-22 江苏省苏微微生物研究有限公司 Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof
CN103349153A (en) * 2013-08-07 2013-10-16 王春 Thallus compound with animal intestine repairing function and production method of complex
CN103468613A (en) * 2013-09-12 2013-12-25 江苏省苏微微生物研究有限公司 Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080003207A1 (en) * 2004-12-15 2008-01-03 Qingdao East Sea Pharmaceuticals, Ltd Treating inflammatory bowel disease with live bacteria
CN1844364A (en) * 2006-05-11 2006-10-11 江苏省微生物研究所有限责任公司 Microecological preparation-tetragenous viable bacteria preparation and method for preparing same
CN101412983A (en) * 2008-11-20 2009-04-22 江苏省苏微微生物研究有限公司 Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof
CN103349153A (en) * 2013-08-07 2013-10-16 王春 Thallus compound with animal intestine repairing function and production method of complex
CN103468613A (en) * 2013-09-12 2013-12-25 江苏省苏微微生物研究有限公司 Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
C. M. YANG ET AL.: "Effects of probiotic, Clostridium butyricum, on growth performance, immune function, and cecal microflora in broiler chickens", 《POULTRY SCIENCE》 *
Q. W. MENG ET AL.: "Influence of probiotics in different energy and nutrient density diets on growth performance, nutrient digestibility, meat quality, and blood characteristics in growing-finishing pigs", 《JOURNAL OF ANIMAL SCIENCE》 *
何若天: "益生芽孢杆菌在畜禽养殖中的应用效果和使用方法", 《科学种养》 *
徐鹏等: "微生物饲料添加剂的主要功能及其研究进展", 《动物营养学报》 *
洪坚平等主编: "《应用微生物学 第2版》", 28 February 2011, 中国林业出版社 *
洪坚平等著: "《农业微生物资源的开发与利用》", 31 August 2000, 中国林业出版社 *
蔡辉益主编: "《常用饲料添加剂无公害使用技术》", 31 January 2003, 中国农业出版社 *

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