CN105211497A - A kind of compound probiotic novel fodder additive and application thereof - Google Patents
A kind of compound probiotic novel fodder additive and application thereof Download PDFInfo
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- CN105211497A CN105211497A CN201510691192.XA CN201510691192A CN105211497A CN 105211497 A CN105211497 A CN 105211497A CN 201510691192 A CN201510691192 A CN 201510691192A CN 105211497 A CN105211497 A CN 105211497A
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Abstract
The invention discloses a kind of compound probiotic novel fodder additive and application thereof.Does is the active component of composite probiotics feed additive provided by the present invention bacillus subtilis (Bacillus? subtilis) 20151014sub-1, Potsdam bacillus brevis (Brevibacillus? borstelensis) 20151014bor-1 and Yunnan micrococcus luteus (Micrococcus? yunnanensis) 20151014yunnan-1 is a kind of microbial forage additive of environmental type.The feed of feeding containing composite probiotics feed additive of the present invention reduces meat feed ratio and the bacterial diarrhea rate of delactational piglets, has not only saved production cost, and the security of pork is guaranteed.
Description
Technical field
The present invention relates to a kind of compound probiotic novel fodder additive and application thereof in feed industry and aquaculture field.
Background technology
China is that the first in the world is raised pigs big country and pork country of consumption, the live pig amount of delivering for sale 700,000,000 bull in 2014, and pork consumption accounts for more than 60% of consumption of meat, and annual live pig and the relevant output value reach 2 trillion yuan people Yu-.Pig-breeding has basic strategic position in the development of national economy, and country also clearly emphasizes " comprehensive regulation agricultural chemicals residue of veterinary drug problem improves agricultural product quality and food safety standard comprehensively ".But China's pig-breeding security situation makes people worried, live pig industry feed safety, breeding process safety, breeding environment safety has become the bottleneck of restriction industry sustainable and healthy development, how to solve pig-breeding safety problem by Integration ofTechnology and has its own strategic significance to national economy.
Add antibiotic in feed and promote that growth of animal has the history of more than 50 year, major contribution has been made to the development of intensive animal husbandry.Raising along with people's living standard and the attention degree to health are constantly strengthened, people start to find antibiotic feed additive bring the behind of great economic benefit the negative effect become increasingly conspicuous hidden, the autogenous infection caused as antibiotic and suprainfection, the generation of drug resistance, the problem such as residual in the destruction of normal intestinal flora and livestock products and environment, these all will bring serious threat to aquaculture, feed industry and animal, and then jeopardize the health of the mankind by food chain.Therefore, forbid that the cry that Antibiotic Additive uses in animal husbandry is more and more higher, the work of researching and developing new Substitutes For Antibiotic is extremely urgent.
Summary of the invention
Technical problem to be solved by this invention how to reduce the feedstuff-meat ratio of delactational piglets and/or reduces the bacterial diarrhea rate of delactational piglets.
For solving the problems of the technologies described above, the present invention provide firstly a kind of composite probiotics feed additive (microbial forage additive).
Composite probiotics feed additive provided by the present invention, its active component is made up of bacillus subtilis, Potsdam bacillus brevis and Yunnan micrococcus luteus.
Described composite probiotics feed additive specifically can be pig feed additive.
In above-mentioned composite probiotics feed additive, described bacillus subtilis is bacillus subtilis (Bacillussubtilis) 20151014sub-1, and it is numbered CGMCCNo.11504 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Potsdam bacillus brevis is Potsdam bacillus brevis (Brevibacillusborstelensis) 20151014bor-1, and it is numbered CGMCCNo.11503 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Yunnan micrococcus luteus is Yunnan micrococcus luteus (Micrococcusyunnanensis) 20151014yunnan-1, and it is numbered CGMCCNo.11505 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
In above-mentioned composite probiotics feed additive, in described active component, the micrococcal CFU ratio in described bacillus subtilis, described Potsdam bacillus brevis and described Yunnan can be (1000-1500): (400-450): (100-150), specifically can be 1491:426:150; Active component content in described composite probiotics feed additive is bacillus subtilis described in every gram of described composite probiotics feed additive, the content sum of described Potsdam bacillus brevis and this three strains bacterial strain of described Yunnan micrococcus luteus, can be (2.0-2.5) × 10
10cfu/g, specifically can be 2.07 × 10
10cfu/g.
Above-mentioned composite probiotics feed additive is made up of described active component and auxiliary material.
For solving the problems of the technologies described above, present invention also offers the preparation method of described composite probiotics feed additive.
The preparation method of described composite probiotics feed additive provided by the present invention, comprising: bacillus subtilis pre-mixing agent, Potsdam bacillus brevis pre-mixing agent and Yunnan micrococcus luteus pre-mixing agent are carried out being mixed to get described composite probiotics feed additive; The active component of described bacillus subtilis pre-mixing agent is described bacillus subtilis; The active component of described Potsdam bacillus brevis pre-mixing agent is described Potsdam bacillus brevis; The active component of described Yunnan micrococcus luteus pre-mixing agent is described Yunnan micrococcus luteus; The mixed proportion of described bacillus subtilis pre-mixing agent, described Potsdam bacillus brevis pre-mixing agent and described Yunnan micrococcus luteus pre-mixing agent meets described bacillus subtilis in described composite probiotics feed additive, the micrococcal CFU ratio of described Potsdam bacillus brevis and described Yunnan can be (1000-1500): (400-450): (100-150), specifically can be 1491:426:150.
In said method, the mixing quality ratio of described bacillus subtilis pre-mixing agent, described Potsdam bacillus brevis pre-mixing agent and described Yunnan micrococcus luteus pre-mixing agent can be (7-7.2): (1.8-2): (1-1.5), specifically can be 7:2:1.
In said method, described bacillus subtilis pre-mixing agent is mixed to get by bacillus subtilis bacterium liquid and described auxiliary material, and the proportioning of bacillus subtilis and described auxiliary material described in described bacillus subtilis pre-mixing agent meets (7.0 × 10
10-7.5 × 10
10) cfu:(2.0-2.5) g, specifically can be 7.46 × 10
10cfu:2.5g; Described Potsdam bacillus brevis pre-mixing agent is mixed to get by Potsdam bacillus brevis bacterium liquid and described auxiliary material, and the proportioning of Potsdam bacillus brevis and described auxiliary material described in the bacillus brevis pre-mixing agent of described Potsdam meets (7.0 × 10
10-7.5 × 10
10) cfu:(2.0-2.5) g, specifically can be 7.46 × 10
10cfu:2.5g; Described Yunnan micrococcus luteus pre-mixing agent is mixed to get by Yunnan micrococcus luteus bacterium liquid and described auxiliary material, and the proportioning of Yunnan micrococcus luteus and described auxiliary material described in the micrococcus luteus pre-mixing agent of described Yunnan meets (5.0 × 10
10-5.5 × 10
10) cfu:(2.0-2.5) g, specifically can be 5.25 × 10
10cfu:2.5g.
Above, described auxiliary material specifically can be zeolite powder.
In said method, described in described bacillus subtilis bacterium liquid, the content of bacillus subtilis is 6.0 × 10
10cfu/mL; Described in the bacillus brevis bacterium liquid of described Potsdam, the content of Potsdam bacillus brevis is 6.0 × 10
10cfu/mL; Described in the micrococcus luteus bacterium liquid of described Yunnan, the micrococcal content in Yunnan is 5.0 × 10
10cfu/mL.
Feed containing described composite probiotics feed additive provided by the present invention also belongs to the scope of protection of the invention.
The above-mentioned feed containing described composite probiotics feed additive specifically can be pig feed.
Above-mentioned feed by pig basal diet and described composite probiotics feed additive according to 100:(1-1.5) mass ratio mix, specifically can be the mass ratio of 100:1.
In above-mentioned feed, the formula of described pig basal diet, in table 4, specifically can be the product of the beautiful bioengineering Co., Ltd in Dalian, and its catalog number is compound feed for piglets 435.
Present invention also offers the product for reducing the feedstuff-meat ratio of delactational piglets and/or the bacterial diarrhea rate of reduction delactational piglets, its active component is described composite probiotics feed additive, or described feed.
Present invention also offers described composite probiotics feed additive, the preparation method of described composite probiotics feed additive, or following 1 of described feed)-4) in any one application:
1) application in the feedstuff-meat ratio of delactational piglets is reduced;
2) application in the product of the feedstuff-meat ratio for the preparation of reduction delactational piglets;
3) application in the bacterial diarrhea rate of delactational piglets is reduced;
4) application in the product of the bacterial diarrhea rate for the preparation of reduction delactational piglets.
In the present invention, the feedstuff-meat ratio of described reduction delactational piglets is embodied as: the feedstuff-meat ratio of delactational piglets as described in the feed (as above-mentioned feed) of feeding containing described composite probiotics feed additive, compared with the feedstuff-meat ratio of the described delactational piglets of normal diet of feeding, significantly reduce (P<0.05).
The bacterial diarrhea rate of described reduction delactational piglets is embodied as: the bacterial diarrhea rate of delactational piglets as described in the feed (as above-mentioned feed) of feeding containing described composite probiotics feed additive, compared with the bacterial diarrhea rate of the described delactational piglets of normal diet of feeding, significantly reduce (P<0.05).
Wherein, described normal diet is mixed by described pig basal diet and the Antibiotic Additive mass ratio according to 1 ton: 300 grams; Described Antibiotic Additive is mixed (namely colistin sulfate and the mass percentage of flavomycoin in described Antibiotic Additive are 4%) according to 1:1:23 mass ratio by colistin sulfate, flavomycoin and starch (carrier).
Present invention also offers following 1)-3) in any one bacterial strain:
1) described bacillus subtilis (Bacillussubtilis) 20151014sub-1;
2) described Potsdam bacillus brevis (Brevibacillusborstelensis) 20151014bor-1;
3) described Yunnan micrococcus luteus (Micrococcusyunnanensis) 20151014yunnan-1.
Experiment proves, the active component of composite probiotics feed additive of the present invention is bacillus subtilis (Bacillussubtilis) 20151014sub-1, Potsdam bacillus brevis (Brevibacillusborstelensis) 20151014bor-1 and Yunnan micrococcus luteus (Micrococcusyunnanensis) 20151014yunnan-1, is a kind of microbial forage additive of environmental type.Use composite probiotics feed additive of the present invention can the bacterial diarrhea that causes of prevention and therapy domestic animal bacterial infection effectively, be conducive to stimulating animal immunity of organisms, improve animal gastrointestinal tract digestion power, promote the positive effect of growth of animal.Experimental group feeds the feedstuff-meat ratio of delactational piglets of the feed containing composite probiotics feed additive for (1.63 ± 0.05) are significantly lower than control group (1.87 ± 0.05, P<0.05); The bacterial diarrhea rate of experimental group delactational piglets is (1.5 ± 0.02) %, significantly lower than control group [(7.5 ± 0.05) %, P<0.05]; The clinical sign scoring of the delactational piglets of experimental group is (8.53 ± 0.02), is significantly higher than control group (5.46 ± 0.02, P<0.05).Production cost not only saved by the feed of feeding containing composite probiotics feed additive, and the security of pork is guaranteed.
preservation explanation
Strain name: bacillus subtilis
Latin name: Bacillussubtilis
Strain number: 20151014sub-1
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on October 14th, 2015
Register on the books numbering: CGMCCNo.11504 at preservation center
Strain name: Potsdam bacillus brevis
Latin name: Brevibacillusborstelensis
Strain number: 20151014bor-1
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on October 14th, 2015
Register on the books numbering: CGMCCNo.11503 at preservation center
Strain name: Yunnan micrococcus luteus
Latin name: Micrococcusyunnanensis
Strain number: 20151014yunnan-1
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on October 14th, 2015
Register on the books numbering: CGMCCNo.11505 at preservation center
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Pig basal diet in following embodiment is the beautiful bioengineering Co., Ltd in Dalian product, and its catalog number is compound feed for piglets 435.
Culture medium prescription in following embodiment:
Nutrient agar: peptone 10g, beef extract 3g, sodium chloride 5g, agar 15g (heating is boiled, and agar is dissolved), deionized water 1L, 121 DEG C of autoclaving 15min, pH7.2.
Nutrient broth: peptone 10g, beef extract 3g, sodium chloride 5g, deionized water 1L, 121 DEG C of autoclaving 15min, pH7.4.
Beef-protein medium: beef extract (1-3) g, peptone (5-15) g, sodium chloride (3-8) g, agar (15-20) g, deionized water 1L, 121 DEG C of autoclaving 20min.
The preparation method of embodiment 1, composite probiotics feed additive
One, the preparation of bacillus subtilis pre-mixing agent
1, the Isolation and ldentification of bacillus subtilis (Bacillussubtilis) 20151014sub-1
Gather the fecal specimens of " DLY " tri-crossbreeding, adopt plate streaking isolated culture, obtain each bacterial strain list bacterium colony, Restored in test tube also carries out molecular biology identification, screening obtains bacterial strain 20151014sub-1, carries out molecular biology identification as follows.
1) cellular morphology and biochemical indicator qualification
A, with sterile working by bacterial strain 20151014sub-1 sample 25g/mL sterile saline or PBS dilution 10-10
10doubly, obtain diluting 10-10
10bacterial strain 20151014sub-1 dilution doubly, fully mixes, selects the bacterium liquid 1mL of 2-3 the serial dilution degree be suitable for respectively in sterilizing plate, 40 DEG C of-50 DEG C of nutrient agars will be cooled to pour in plate, mix gently, make mixed plate, each dilution factor does two Duplicate Samples.Be inverted in after culture medium solidifying in (30 ± 1) DEG C incubator and cultivate 24-48h.After cultivation terminates, on picking flat board, 3-5 water white transparency bacterium colony carries out Morphological Identification and chooses single bacterium colony turning in nutrient broth, carries out biochemical identification after cultivating 18-24h in (30 ± 1) DEG C incubator.
Bacterium colony on B, flat board makees smear and carries out gram and spore staining, microscopy.
Cellular morphology and biochemical indicator qualification result as shown in table 1, bacterial strain 20151014sub-1 is Gram-positive bacillus, cell dia < 1 μm, little chaining, and flagellum side is raw, and endospore, gemma does not expand, and does not form parasporal crystal.
The cellular morphology of table 1, bacterial strain 20151014sub-1 and biochemical indicator qualification result
2) Molecular Identification
The pcr amplification product of the 16SrDNA sequence of bacterial strain 20151014sub-1 is carried out agarose gel electrophoresis, obtain the DNA fragmentation of about 1331bp, after order-checking, utilize correlated series known in Blast instrument and database to carry out online comparison, the 16SrDNA sequence similarity of bacterial strain 20151014sub-1 and bacillus subtilis (Bacillussubtilis) is 100%.The pcr amplification product of the gyrB gene of bacterial strain 20151014sub-1 is carried out agarose gel electrophoresis, obtain the DNA fragmentation of about 681bp, according to the morphological feature of bacterial strain 20151014sub-1, gyrB gene order and 16SrDNA sequence, be accredited as bacillus subtilis (Bacillussubtilis).The 16SrDNA sequence of bacterial strain 20151014sub-1 is as shown in SEQIDNo.1, and nucleotide sequence length is 1331bp; GyrB gene order is as shown in SEQIDNo.2, and nucleotide sequence length is 681bp.
Bacterial strain 20151014sub-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on October 14th, 2015, and it is numbered CGMCCNo.11504 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2, the preparation of bacillus subtilis pre-mixing agent
1) actication of culture
Aseptically open bacterial strain 20151014sub-1 freeze-drying lactobacillus bottle, (solvent of nutrient medium is water to be inoculated in nutrient medium, solute and concentration as follows: sugar 1%, peptone 4%, sodium chloride 1%, potassium dihydrogen phosphate 0.5%, % represents g/100mL) middle dilution activation culture, cultivation temperature is 35 DEG C-38 DEG C, and incubation time is 24-36h, obtain bacterial strain 20151014sub-1 bacterium liquid after cultivation terminates and carry out strain idenfication whether meet characteristic, result display is without making a variation without miscellaneous bacteria.
2) inclined-plane cultivation is expanded numerous
By through step 1) bacterial strain 20151014sub-1 bacterium liquid that activation assay is qualified is inoculated into compared with the formula of the nutrient medium in slant medium (formula and step 1), increase by 1.5% agar) on, 24-36h is cultivated under 35 DEG C of-38 DEG C of conditions, obtain the bacterial strain 20151014sub-1 thalline expanding numerous cultivation, and examine and determine.
3) seed liquor preparation
By step 2) in expand the bacterial strain 20151014sub-1 thalline of numerous cultivation and be inoculated in fluid nutrient medium (solvent of fluid nutrient medium is water, and solute and concentration are: sugar 1%, peptone 4%, sodium chloride 1%, the potassium dihydrogen 0.5% of phosphorus; % represents g/L) in, under 35 DEG C of-38 DEG C of conditions, carry out shaking table cultivate 24-36h, rotating speed is 140rpm, obtains bacterial strain 20151014sub-1 seed liquor, and carries out strain properties in qualification seed liquor.
4) fermentation tank culture
By 1% (volume ratio) by step 3) bacterial strain 20151014sub-1 seed liquor in the fermentation culture (fluid nutrient medium with step 3)) of sterilizing, at 30 DEG C-31 DEG C, tank pressure 0.05-0.08Mpa, 30-40h is cultivated under pH7.0-7.2 condition, obtain bacterial strain 20151014sub-1 zymotic fluid, and examine and determine.
Result shows: in bacterial strain 20151014sub-1 zymotic fluid, bacterial strain 20151014sub-1 viable count is 2.0 × 10
9more than cfu/mL, gemma rate is more than 90%.
5) thalline is collected
By step 4) the bacterial strain 20151014sub-1 zymotic fluid that obtains enters bacterium liquid valve by ultrafiltration apparatus, carries out ultrafiltration concentration.After above-mentioned steps ultrafiltration concentration, side mouth gets rid of the water of thickening filtration, and the delivery port through ultrafiltration apparatus top discharges the bacterial strain 20151014sub-1 concentrate after ultrafiltration.Repeat above operating procedure, the bacterium liquid after ultrafiltration concentration carries out count plate inspection, until viable count reaches requirement, ultrafiltration terminates, and obtain the dense bacterium liquid of bacterial strain 20151014sub-1, wherein bacterial strain 20151014sub-1 viable count reaches 6.0 × 10
10cfu/mL.
6) dry
By step 5) after the dense bacterium liquid of bacterial strain 20151014sub-1 that obtains and zeolite powder mix according to the ratio that weight ratio is 1:2.5, vacuum drying, obtains bacillus subtilis pre-mixing agent dry powder.In bacillus subtilis pre-mixing agent dry powder, bacterial strain 20151014sub-1 viable count is>=2.0 × 10
10cfu/g, is specially 2.13 × 10
10cfu/g.
By filling for bacillus subtilis pre-mixing agent dry powder Bian aluminium pool bag obtained above, after sealing, be bacillus subtilis pre-mixing agent.
Two, the preparation of Potsdam bacillus brevis pre-mixing agent
1, the Isolation and ldentification of Potsdam bacillus brevis (Brevibacillusborstelensis) 20151014bor-1
Gather the fecal specimens of " DLY " tri-crossbreeding, adopt plate streaking isolated culture, obtain each bacterial strain list bacterium colony, Restored in test tube also carries out molecular biology identification, screening obtains bacterial strain 20151014bor-1, carries out molecular biology identification as follows.
1) cellular morphology and biochemical indicator qualification
A, with sterile working by bacterial strain 20151014bor-1 sample 25g/mL sterile saline or PBS dilution 10-10
10doubly, obtain diluting 10-10
10bacterial strain 20151014bor-1 dilution doubly, fully mixes, selects the bacterium liquid 1mL of 2-3 the serial dilution degree be suitable for respectively in sterilizing plate, 42 DEG C of-46 DEG C of nutrient agars will be cooled to pour in plate, mix gently, make mixed plate, each dilution factor does two Duplicate Samples.Be inverted in after culture medium solidifying in (30 ± 1) DEG C incubator and cultivate 24-36h.After cultivation terminates, on picking flat board, 3-5 water white transparency bacterium colony carries out Morphological Identification and chooses single bacterium colony turning in nutrient broth, carries out biochemical identification after cultivating 18-24h in (30 ± 1) DEG C incubator.
Bacterium colony on B, flat board makees smear and carries out gram and spore staining, microscopy.
Cellular morphology and biochemical indicator qualification result as shown in table 2, bacterial strain 20151014bor-1 is Gram-positive bacillus, cell dia < 1 μm, form gemma, gemma expands, and non-circular, bacterium colony is creamy white, diameter 0.8-1.2 μ π l.
The cellular morphology of table 2, bacterial strain 20151014bor-1 and biochemical indicator qualification result
2) Molecular Identification
The pcr amplification product of the 16SrDNA sequence of bacterial strain 20151014bor-1 is carried out agarose gel electrophoresis, obtain the DNA fragmentation of about 1412bp, after order-checking, utilize correlated series known in Blast instrument and database to carry out online comparison, the 16SrDNA sequence similarity of bacterial strain 20151014bor-1 and Potsdam bacillus brevis (Brevibacillusborstelensis) is 100%.According to morphological feature and the 16SrDNA sequence of bacterial strain 20151014bor-1, be accredited as Potsdam bacillus brevis (Brevibacillusborstelensis).The 16SrDNA sequence of bacterial strain 20151014bor-1 is as shown in SEQIDNo.3, and nucleotide sequence length is 1412bp.
Bacterial strain 20151014bor-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on October 14th, 2015, and it is numbered CGMCCNo.11503 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2, the preparation of Potsdam bacillus brevis pre-mixing agent
1) actication of culture
Aseptically open bacterial strain 20151014bor-1 freeze-drying lactobacillus bottle, be inoculated in nutrient medium (solvent of nutrient medium is water, solute and concentration as follows: sugar 1%, peptone 4%, sodium chloride 1%, % represents g/100mL.) middle dilution activation culture, cultivation temperature is 42 DEG C-46 DEG C, incubation time is 24-36h, this bacterium grows under amphimicrobian condition, growth acid-base value scope: pH=6.4-7.8, optimal pH=7.0, cultivate after terminating to obtain bacterial strain 20151014bor-1 bacterium liquid and carry out strain idenfication whether meet characteristic, and result display is without making a variation without miscellaneous bacteria.
2) inclined-plane cultivation is expanded numerous
By through step 1) bacterial strain 20151014bor-1 bacterium liquid that activation assay is qualified is inoculated into compared with the formula of the nutrient medium in slant medium (formula and step 1), increase by 1.5% agar) on, 24-36h is cultivated under 42 DEG C of-46 DEG C of conditions, obtain the bacterial strain 20151014bor-1 thalline expanding numerous cultivation, and examine and determine.
3) seed liquor preparation
By step 2) the bacterial strain 20151014bor-1 thalline that expands numerous cultivation is inoculated in fluid nutrient medium (solvent of fluid nutrient medium is water, and solute and concentration are: sugar 1%, peptone 4%, sodium chloride 1%; % represents g/L) in, under 42 DEG C of-46 DEG C of conditions, carry out shaking table cultivate 24-36h, rotating speed is 140rpm, obtains bacterial strain 20151014bor-1 seed liquor, and carries out strain properties in qualification seed liquor.
4) fermentation tank culture
By 1% (volume ratio) by step 3) bacterial strain 20151014bor-1 seed liquor (fluid nutrient medium with step 3)) in the fermentation culture of sterilizing, at 42 DEG C-46 DEG C, tank pressure 0.05-0.08Mpa, 30-40h is cultivated under pH7.0-7.2 condition, obtain bacterial strain 20151014bor-1 zymotic fluid, and examine and determine.
Result shows: in bacterial strain 20151014bor-1 zymotic fluid, bacterial strain 20151014bor-1 viable count is 2.0 × 10
9more than cfu/mL, gemma rate is more than 90%.
5) thalline is collected
By step 4) the bacterial strain 20151014bor-1 zymotic fluid that obtains enters bacterium liquid valve by ultrafiltration apparatus, carries out ultrafiltration concentration.After above-mentioned steps ultrafiltration concentration, side mouth gets rid of the water of thickening filtration, and the delivery port through ultrafiltration apparatus top discharges the bacterial strain 20151014bor-1 concentrate after ultrafiltration.Repeat above operating procedure, the bacterium liquid after ultrafiltration concentration carries out count plate inspection, until viable count reaches requirement, ultrafiltration terminates, and obtain the dense bacterium liquid of bacterial strain 20151014bor-1, wherein bacterial strain 20151014bor-1 viable count reaches 6.0 × 10
10cfu/mL.
6) dry
By step 5) after the bacterial strain 20151014bor-1 concentrate that obtains and zeolite powder mix according to the ratio that weight ratio is 1:2.5, vacuum drying, obtains Potsdam bacillus brevis pre-mixing agent dry powder.In the bacillus brevis pre-mixing agent dry powder of Potsdam, bacterial strain 20151014bor-1 viable count is>=2.0 × 10
10cfu/g, is specially 2.13 × 10
10cfu/g.
By filling for Potsdam obtained above bacillus brevis pre-mixing agent dry powder Bian aluminium pool bag, after sealing, be Potsdam bacillus brevis pre-mixing agent.
Three, the preparation of Yunnan micrococcus luteus pre-mixing agent
1, the Isolation and ldentification of Yunnan micrococcus luteus (Micrococcusyunnanensis) 20151014yunnan-1
Gather the fecal specimens of " DLY " tri-crossbreeding, adopt plate streaking isolated culture, obtain each bacterial strain list bacterium colony, Restored in test tube also carries out molecular biology identification, screening obtains bacterial strain 20151014yunnan-1, carries out molecular biology identification as follows.
1) cellular morphology and biochemical indicator qualification
A, with sterile working by bacterial strain 20151014yunnan-1 sample 25g/mL sterile saline or PBS dilution 10-10
10doubly, obtain diluting 10-10
10bacterial strain 20151014yunnan-1 dilution doubly, fully mixes, selects the bacterium liquid 1mL of 2-3 the serial dilution degree be suitable for respectively in sterilizing plate, 30 DEG C of-31 DEG C of beef-protein mediums will be cooled to pour in plate, mix gently, make mixed plate, each dilution factor does two Duplicate Samples.Be inverted in after culture medium solidifying in (30 ± 1) DEG C incubator and cultivate 24-48h.After cultivation terminates, on picking flat board, 3-5 water white transparency bacterium colony carries out Morphological Identification and chooses single bacterium colony turning in nutrient broth, carries out biochemical identification after cultivating 18-24h in (30 ± 1) DEG C incubator.
Bacterium colony on B, flat board makees smear and carries out gram and spore staining, microscopy.
Cellular morphology and biochemical indicator qualification result as shown in table 3, bacterial strain 20151014yunnan-1 is Gram-positive bacillus, cell dia < 0.8 μm.
The cellular morphology of table 3, bacterial strain 20151014yunnan-1 and biochemical indicator qualification result
2) Molecular Identification
The pcr amplification product of the 16SrDNA sequence of bacterial strain 20151014yunnan-1 is carried out agarose gel electrophoresis, obtain the DNA fragmentation of about 1384bp, after order-checking, utilize correlated series known in Blast instrument and database to carry out online comparison, the 16SrDNA sequence similarity of bacterial strain 20151014yunnan-1 and Yunnan micrococcus luteus (Micrococcusyunnanensis) is 100%.The pcr amplification product of the recA gene of bacterial strain 20151014yunnan-1 is carried out agarose gel electrophoresis, obtain the DNA fragmentation of about 763bp, according to the morphological feature of bacterial strain 20151014yunnan-1, recA gene order and 16SrDNA sequence, be accredited as Yunnan micrococcus luteus (Micrococcusyunnanensis).The 16SrDNA sequence of bacterial strain 20151014yunnan-1 is as shown in SEQIDNo.4, and nucleotide sequence length is 1384bp; RecA gene order is as shown in SEQIDNo.5, and nucleotide sequence length is 763bp.
Bacterial strain 20151014yunnan-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on October 14th, 2015, and it is numbered CGMCCNo.11505 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2, the preparation of Yunnan micrococcus luteus pre-mixing agent
1) actication of culture
Aseptically open bacterial strain 20151014yunnan-1 freeze-drying lactobacillus bottle, be inoculated in nutrient medium (solvent of nutrient medium is water, solute and concentration as follows: sugar 1%, peptone 4%, sodium chloride 1%, potassium dihydrogen phosphate 0.5%, % represents g/100mL.) middle dilution activation culture, cultivation temperature is 30 DEG C-31 DEG C, and incubation time is 24-36h, cultivates after terminating to obtain bacterial strain 20151014yunnan-1 bacterium liquid and carry out strain idenfication whether meet characteristic, and result display is without making a variation without miscellaneous bacteria.
2) inclined-plane cultivation is expanded numerous
By through step 1) bacterial strain 20151014yunnan-1 bacterium liquid that activation assay is qualified is inoculated into the inclined-plane fermentation medium (ferrous sulfate of the sodium chloride of the bean powder of 40-100g/L, the starch of 40-100g/L, 0.5-8g/L, the calcium carbonate of 0.5-5g/L, the potassium dihydrogen phosphate of 2-10g/L and l-10g/L, increase by 1.5% agar) on, 24-36h is cultivated under 30 DEG C of-31 DEG C of conditions, obtain the bacterial strain 20151014yunnan-1 thalline expanding numerous cultivation, and examine and determine.
3) seed liquor preparation
By step 2) the bacterial strain 20151014yunnan-1 thalline that expands numerous cultivation is inoculated in fluid nutrient medium (solvent of fluid nutrient medium is water, and solute and concentration are: sugar 1%, peptone 4%, sodium chloride 1%, the potassium dihydrogen 0.5% of phosphorus; % represents g/L) in, under 30 DEG C of-31 DEG C of conditions, carry out shaking table cultivate 24-36h, rotating speed is that 150rpm is cultured to strain density OD value for 0.6-0.8, obtains bacterial strain 20151014yunnan-1 seed liquor, and carries out strain properties in qualification seed liquor.
4) fermentation tank culture
By 1% (volume ratio) by step 3) bacterial strain 20151014yunnan-1 seed liquor in the fermentation culture (fluid nutrient medium with step 3)) of sterilizing, at 30 DEG C-31 DEG C, tank pressure 0.05-0.08Mpa, 30-40h is cultivated under pH7.0-7.2 condition, obtain bacterial strain 20151014yunnan-1 zymotic fluid, and examine and determine.
Result shows: in bacterial strain 20151014yunnan-1 zymotic fluid, bacterial strain 20151014yunnan-1 viable count is 2.0 × 10
9cfu/mL.
5) thalline is collected
By step 4) the bacterial strain 20151014yunnan-1 zymotic fluid that obtains enters bacterium liquid valve by ultrafiltration apparatus, carries out ultrafiltration concentration.After above-mentioned steps ultrafiltration concentration, side mouth gets rid of the water of thickening filtration, and the delivery port through ultrafiltration apparatus top discharges the bacterial strain 20151014yunnan-1 concentrate after ultrafiltration.Repeat above operating procedure, the bacterium liquid after ultrafiltration concentration carries out count plate inspection, until viable count reaches requirement, ultrafiltration terminates, and obtain the dense bacterium liquid of bacterial strain 20151014yunnan-1, wherein bacterial strain 20151014yunnan-1 viable count reaches 5.0 × 10
10cfu/mL.
6) dry
By step 5) after the dense bacterium liquid of bacterial strain 20151014yunnan-1 that obtains and zeolite powder mix according to the ratio that weight ratio is 1:2.5, vacuum drying, obtains Yunnan micrococcus luteus pre-mixing agent dry powder.In the micrococcus luteus pre-mixing agent of Yunnan, bacterial strain 20151014yunnan-1 viable count is>=1.5 × 10
10cfu/g, is specially 1.5 × 10
10cfu/g.
By filling for Yunnan obtained above micrococcus luteus pre-mixing agent dry powder Bian aluminium pool bag, after sealing, be Yunnan micrococcus luteus pre-mixing agent.
Four, the preparation of composite probiotics feed additive
Potsdam bacillus brevis pre-mixing agent that bacillus subtilis pre-mixing agent step one obtained, step 2 obtain and the Yunnan micrococcus luteus pre-mixing agent that step 3 obtains mix according to the ratio that mass ratio is 7:2:1, obtain composite probiotics feed additive.
In composite probiotics feed additive, the CFU of bacterial strain 20151014sub-1, bacterial strain 20151014bor-1 and bacterial strain 20151014yunnan-1 is than being 1491:426:150, and active component content is 2.07 × 10
10cfu/g, wherein, the content of bacterial strain 20151014sub-1 is 1.49 × 10
10the content of cfu/g, bacterial strain 20151014bor-1 is 4.26 × 10
9the content of cfu/g, bacterial strain 20151014yunnan-1 is 1.50 × 10
9cfu/g.
The growth to delactational piglets of embodiment 2, composite probiotics feed additive and the influence research of health
Select health, body condition good, average weight is the delactational piglets 100 of 7-9kg " DLY " tri-crossbreeding, is divided into two groups at random, often organizes 50.Experimental group is fed the feed containing composite probiotics feed additive, should the feed containing composite probiotics feed additive be the composite probiotics feed additive adding 10kg embodiment 1 in pig basal diet per ton, and obtain after mixing.Control group fed normal diet, this normal diet adds 300 grams of Antibiotic Additive in above-mentioned pig basal diet per ton, obtain after mixing, this Antibiotic Additive is mixed (namely colistin sulfate and the flavomycoin mass percentage in Antibiotic Additive is 4%) according to 1:1:23 mass ratio by colistin sulfate, flavomycoin and starch (carrier).The formula percentage of above-mentioned pig basal diet is in table 4.
Table 4, pig basal diet formula (mass percentage %)
| Corn | Wheat bran | Dregs of beans | Fish meal | Premix |
| 64 | 6 | 22 | 3 | 5 |
The delactational piglets of experimental group and control group except feed different, all the other rearing conditions are all identical, free choice feeding, freely drink water, and conveniently carry out immunity and expelling parasite.
Date of test: on April 1st, 2015 start, May 31 end of day, 60 days by a definite date.Experimental session, records the average feed intake of each group of pig every day, and the bacterial diarrhea situation of observed and recorded pig, and results of regular determination is respectively organized the average weight of pig and observed clinical sign and mark in addition.After experiment terminates, calculate the scoring of the feedstuff-meat ratio of each group of pig, bacterial diarrhea rate and clinical sign, and carry out statistical analysis.
Wherein, feedstuff-meat ratio=average daily ingestion amount/average daily gain;
Bacterial diarrhea rate (%)=total diarrhoea pig's head number of days/overall test pig's head number of days × 100%
In bacterial diarrhea rate computing formula, pig's head number of days is each head pig bacterial Diarrhoea days sum, as 1 pig bacterial is suffered from diarrhoea 3 days, is then 3 pig's head number of days; 2 each bacterial diarrheas of pig 3 days are then 6 pig's head number of days.
Clinical sign carries out the standard of marking:
Result shows, and the feedstuff-meat ratio of the delactational piglets of experimental group is that (1.63 ± 0.05) are significantly lower than control group (1.87 ± 0.05, P<0.05); The bacterial diarrhea rate of the delactational piglets of experimental group is (1.5 ± 0.02) %, significantly lower than control group [(7.5 ± 0.05) %, P<0.05]; The clinical sign scoring of the delactational piglets of experimental group is (8.53 ± 0.02), is significantly higher than control group (5.46 ± 0.02, P<0.05), the results are shown in Table 5.
The growth of table 5, two groups of delactational piglets and health status statistics
| Average daily ingestion amount (g) | Feedstuff-meat ratio | Clinical sign scoring statistics | Bacterial diarrhea rate (%) | |
| Experimental group | 900±0.02 | 1.63±0.05 | 8.53±0.02 | 1.5±0.02 |
| Control group | 1150±0.03 | 1.87±0.05 | 5.46±0.02 | 7.5±0.05 |
In addition, carried out calculating comparing to the feeding cost of the delactational piglets of experimental group and control group, result display experimental group originally reduces 12.31% than contrast composition.
Computational methods: because two kinds of additive use costs, artificial, charges for water and electricity, management cost are all identical.Original body mass is identical, and it is also identical that experiment terminates body weight, so only compare feed cost: the rate of cost reduction=(control group fed feed relative-experimental group feeding feed stuff weight)/control group fed feed relative × 100%.The feed relative containing composite probiotics feed additive that experimental group is fed is 38.355kg, and the weight of the normal diet of control group fed is 43.740kg, and the rate of cost reduction is 12.31%.
Claims (10)
1. feed addictive, its active component is made up of bacillus subtilis, Potsdam bacillus brevis and Yunnan micrococcus luteus.
2. feed addictive according to claim 1, it is characterized in that: described bacillus subtilis is bacillus subtilis (Bacillussubtilis) 20151014sub-1, and it is numbered CGMCCNo.11504 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Potsdam bacillus brevis is Potsdam bacillus brevis (Brevibacillusborstelensis) 20151014bor-1, and it is numbered CGMCCNo.11503 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Yunnan micrococcus luteus is Yunnan micrococcus luteus (Micrococcusyunnanensis) 20151014yunnan-1, and it is numbered CGMCCNo.11505 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
3. feed addictive according to claim 1 and 2, it is characterized in that: in described active component, the micrococcal CFU in described bacillus subtilis, described Potsdam bacillus brevis and described Yunnan is than being (1000-1500): (400-450): (100-150).
4. according to described feed addictive arbitrary in claim 1-3, it is characterized in that: described feed addictive is according to the method preparation described in claim 5 or 6.
5. the preparation method of arbitrary described feed addictive in claim 1-4, comprising: bacillus subtilis pre-mixing agent, Potsdam bacillus brevis pre-mixing agent and Yunnan micrococcus luteus pre-mixing agent are carried out being mixed to get described feed addictive; The active component of described bacillus subtilis pre-mixing agent is the bacillus subtilis described in claim 1 or 2; The active component of described Potsdam bacillus brevis pre-mixing agent is Potsdam bacillus brevis described in claim 1 or 2; The active component of described Yunnan micrococcus luteus pre-mixing agent is the Yunnan micrococcus luteus described in claim 1 or 2; The mixed proportion of described bacillus subtilis pre-mixing agent, described Potsdam bacillus brevis pre-mixing agent and described Yunnan micrococcus luteus pre-mixing agent meets described bacillus subtilis in claim 1-4 in arbitrary described feed addictive, the micrococcal CFU of described Potsdam bacillus brevis and described Yunnan than being (1000-1500): (400-450): (100-150).
6. method according to claim 5, is characterized in that: described bacillus subtilis pre-mixing agent is mixed to get by bacillus subtilis bacterium liquid and auxiliary material; Described Potsdam bacillus brevis pre-mixing agent is mixed to get by Potsdam bacillus brevis bacterium liquid and described auxiliary material; Described Yunnan micrococcus luteus pre-mixing agent is mixed to get by Yunnan micrococcus luteus bacterium liquid and described auxiliary material.
7. the feed containing arbitrary described feed addictive in claim 1-4.
8. for reducing delactational piglets feedstuff-meat ratio and/or reduce the product of bacterial diarrhea rate of delactational piglets, its active component is arbitrary described feed addictive in claim 1-4, or feed according to claim 7.
9. arbitrary described feed addictive in claim 1-4, the method described in claim 5 or 6, or following 1 of feed according to claim 7)-4) in any one application:
1) application in the feedstuff-meat ratio of delactational piglets is reduced;
2) application in the product of the feedstuff-meat ratio for the preparation of reduction delactational piglets;
3) application in the bacterial diarrhea rate of delactational piglets is reduced;
4) application in the product of the bacterial diarrhea rate for the preparation of reduction delactational piglets.
10. any one bacterial strain following 1)-3):
1) bacillus subtilis according to claim 2 (Bacillussubtilis) 20151014sub-1;
2) Potsdam according to claim 2 bacillus brevis (Brevibacillusborstelensis) 20151014bor-1;
3) Yunnan according to claim 2 micrococcus luteus (Micrococcusyunnanensis) 20151014yunnan-1.
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