CN101978849A - Composite micro-ecological preparation and application thereof - Google Patents
Composite micro-ecological preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a composite micro-ecological preparation and application thereof, in particular to a composite micro-ecological preparation containing multiple probiotics and application thereof. The preparation contains any three or four of bacillus licheniformis CGMCC No. 2383 powder, bacillus subtilis powder, enterococcus faecalis CGMCC No. 2386 powder, lactobacillus acidophilus powder and saccharomyces cerevisiae CGMCC No. 2388 powder. The preparation has high content of live bacteria, and has the adversity resistance such as gastric acid resistance, bile salt resistance, high-temperature resistance, common antibiotic resistance and the like and the probiotic functions of producing acid and enzyme and resisting pathogenic bacteria. The variety and the proportion of the probiotics and carriers can be determined according to different animals and different animal growth phases. The micro-ecological preparation can improve the feed utilization efficiency, increase the yield of meat, eggs and milk, promote the growth of animals, improve the immunity and the disease resistance of the animals, replace the antibiotic and improve the quality of animal products.
Description
Technical field
The present invention relates to a kind of compound micro-ecological preparation, specifically relate to comprise compound micro-ecological preparation and the application thereof of multiple probio, belong to the probiotics technical field.
Background technology
At present, prevention and medicine to Animal diseases substantially still adopt antibiotic, but owing to use antibiotic for a long time, in a large number, all drawbacks such as drug resistance, flora imbalance, immunity degradation, palindromia rate height, medicament residue are serious day by day, and the while is serious harm animal body and human health also.The measure current, that countries in the world have taked forbidding or restriction to use to antibiotic separately, as: European Union completely forbade from 2006 and uses antibiotic; The World Health Organization (WTO) has proposed " reducing the global principle that antibiotic uses in edible animal "; China starts " feed safety engineering ", and has forbidden a collection of toxicity and residual bigger medicine.Under this form, change the breed idea, prevent to overweight treatment, and Application and Development is nontoxic, the novel green feed addictive of no drug residue is an inexorable trend.
Probiotics is commonly used at present and studies the feed addictive of more a kind of green, safety, this type of preparation is obtained through industrial fermentation production by the animal beneficial bacterium, include the active bacteria formulation that a lot of beneficial microbes and metabolite thereof constitute, directly feeding animals.The balance of little ecology plays a role probiotics in the enteron aisle by keeping, have diseases prevention, strengthen immunity of organisms, multiple function such as promote to grow, put on weight, and pollution-free, noresidue, not developing immunity to drugs, is the feed addictive of a kind of green, safety.
At present, there is the main Quality of the following aspects in probiotics on the market: (1) viable bacteria content is low, and studies show that, if the low few 107/g of the concentration of a kind of bacterium in cecal content, then the enzyme and the metabolite of this bacterium generation are not enough to influence the host, are difficult to satisfy the treatment needs; (2) moisture is higher, and this is one of key factor that influences the probiotics quality; (3) antibiotic-resistant not; (4) to hydrochloric acid in gastric juice and cholate instability, cause still to have activity after having only the minority bacterial strain to enter enteron aisle, do not reach the required number of viable that plays a role.Since these quality problems that probiotics exists, thus its effect on animal influenced, limited the extensive use of probiotics.
Summary of the invention
The object of the present invention is to provide a kind of viable bacteria content height, moisture low, comprise have stomach juice-resistant, anti-adversity such as bile tolerance, high temperature resistant and tolerance common antibiotics and produce acid, produce enzyme and suppress the probioticses that benefit such as pathogen is given birth to the multiple probio of functions.
Another object of the present invention is to provide the purposes of this probiotics.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of compound micro-ecological preparation, said preparation comprises wantonly three kinds or four kinds in bacillus licheniformis (Bacilluslicheniformis) CGMCC No.2383 bacterium powder, bacillus subtilis bacterium powder, enterococcus faecalis (Enterococcus faecium) CGMCC No.2386 bacterium powder, lactobacillus acidophilus bacterium powder, saccharomyces cerevisiae (Saccharomyces cerevisiae) the CGMCC No.2388 bacterium powder.
Wherein, described bacillus licheniformis, enterococcus faecalis, saccharomyces cerevisiae has good stomach juice-resistant, bile tolerance, high temperature resistant, anti-adversity and product acid such as tolerance common antibiotics, produce enzyme, suppress the living functions of benefit such as pathogen, for the applicant separates from healthy animal enteron aisle or ight soil, seed selection obtains, identify through Chinese agriculture microorganism fungus kind preservation administrative center, and being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (be called for short CGMCC) on March 3rd, 2008, preserving number is respectively: CGMCC No.2383, CGMCC No.2386, CGMCC No.2388.
Lactobacillus acidophilus, bacillus subtilis can separate according to a conventional method and obtain, and also can buy from the market to obtain, and the present invention obtains for buying from the market.
Compound micro-ecological preparation of the present invention comprises lactobacillus acidophilus bacterium powder, enterococcus faecalis bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder, and the weight proportion of above-mentioned each composition is 1: 0.8～1.2: 0.8～2.5: 1.5～2.5.
Compound micro-ecological preparation of the present invention comprises bacillus licheniformis bacterium powder, bacillus subtilis bacterium powder, S. cervisiae powder, and the weight proportion of above-mentioned each composition is: 1～3.5: 0.8～3.5: 0.8～3.5.
Compound micro-ecological preparation of the present invention comprises enterococcus faecalis bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder, and the weight proportion of above-mentioned each composition is: 1: 0.8～1.2: 1.5～2.5.
Compound micro-ecological preparation of the present invention comprises lactobacillus acidophilus bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder, and the weight proportion of above-mentioned each composition is: 1～1.5: 2～2.5: 2～2.5.
Compound micro-ecological preparation of the present invention also can further comprise carrier, and carrier is any one or two kinds of in stone flour, glucose, aluminum potassium sulfate, the maize cob meal.
Another object of the present invention is to provide the application of this compound micro-ecological preparation in additive for farm animal feed.This compound micro-ecological preparation can be determined the probio kind that comprises according to livestock and poultry and growth phase difference of living in thereof, kind of carrier and ratio thereof, to make the probiotics of the universal or tailored version of each livestock and poultry more targetedly, thereby better bring into play the function of probiotics, as: compound micro-ecological preparation of the present invention comprises lactobacillus acidophilus bacterium powder, enterococcus faecalis bacterium powder, bacillus licheniformis bacterium powder, the S. cervisiae powder, carrier is a stone flour, the weight proportion of above-mentioned each composition is 1: 0.8～1.2: 1.5～2.5: 1.5～2.5: 3.5～4.5, promptly can be made into the universal compound micro-ecological preparation of pig, total beneficial bacterium number reaches 2～5 * 10
9Cfu/g; Compound micro-ecological preparation comprises: lactobacillus acidophilus bacterium powder, enterococcus faecalis bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder and glucose, the weight proportion of above-mentioned each composition is: 1: 0.8～1.2: 0.8～1.2: 1.5～2.5: 15～20, promptly make piglet tailored version compound micro-ecological preparation, total beneficial bacterium content 5～9 * 10
8Cfu/g; Compound micro-ecological preparation comprises: bacillus licheniformis bacterium powder, bacillus subtilis bacterium powder, S. cervisiae powder, aluminum potassium sulfate and stone flour, the weight proportion of above-mentioned each composition is: 1: 0.8～1.2: 0.8～1.2: 4.5～5.5: 1.5～2.5, promptly make sow tailored version compound micro-ecological preparation, total beneficial bacterium content 2～5 * 10
9Cfu/g; Compound micro-ecological preparation comprises: bacillus subtilis bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae lackey and maize cob meal, the weight proportion of above-mentioned each composition is: 3～3.5: 3～3.5: 2.5～3.5: 0.5～1.5, promptly make feed factory pig product tailored version compound micro-ecological preparation, the total bacterial content of probio can reach 2～5 * 10
10Cfu/g; Compound micro-ecological preparation comprises: enterococcus faecalis bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder and glucose, the weight proportion of above-mentioned each composition is: 1: 0.8～1.2: 1.5～2.5: 15～20, promptly make young fowl tailored version compound micro-ecological preparation, total beneficial bacterium content 5～9 * 10
8Cfu/g; Compound micro-ecological preparation comprises: lactobacillus acidophilus bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder and stone flour, the weight proportion of above-mentioned each composition is: 1～1.5: 2～2.5: 2～2.5: 5～5.5, promptly make poultry tailored version compound micro-ecological preparation, total beneficial bacterium number reaches 2 * 10
9Cfu/g; Compound micro-ecological preparation comprises bacillus licheniformis bacterium powder, Bacillus subtillis bacterium powder, S. cervisiae powder and stone flour, the weight proportion of above-mentioned each composition is: 1: 1～1.2: 1～1.2: 7～8, promptly make egg fowl tailored version compound micro-ecological preparation, total beneficial bacterium number reaches 2～5 * 10
9Cfu/g; Compound micro-ecological preparation comprises bacillus subtilis bacterium powder, bacillus licheniformis bacterium powder, S. cervisiae powder and maize cob meal, the weight proportion of above-mentioned each composition is: 3: 3～3.5: 3～3.5: 0.5～1.5, promptly make feed factory chicken product tailored version compound micro-ecological preparation, the total bacterial content of probio can reach 2～5 * 10
10Cfu/g.
Bacillus licheniformis, enterococcus faecalis, saccharomyces cerevisiae and the bacillus subtilis of institute's seed selection and lactobacillus acidophilus adopt deep liquid high density fermentation technology and spray to do in preparation or a series of post processing technology of freeze-drying is made the bacterium powder among the present invention, viable bacteria content height in the preparation, moisture are low, have anti-adversity such as stomach juice-resistant, bile tolerance, high temperature resistant and tolerance common antibiotics and produce acid, produce enzyme and suppress benefit such as pathogen and give birth to functions.
Compound micro-ecological preparation of the present invention can improve food utilization efficiency, increases the meat, eggs and milk Isoquant, promotes growth of animal, improves immunity and premunition, substitutes antibiotic, thereby improves the animal product quality.
The preparation process of separation, seed selection and the bacterium powder thereof of bacillus licheniformis of the present invention (Bacillus licheniformis) CGMCC No.2383, enterococcus faecalis (Enterococcus faecium) CGMCC No.2386 bacterium powder, saccharomyces cerevisiae (Saccharomycescerevisiae) CGMCC No.2388 bacterial classification is as follows:
1, increasing bacterium cultivates: the fresh excreta or the intestinal contents sample of healthy animal (pig or poultry) are inoculated in the propagation fluid nutrient medium, 30～37 ℃ of constant temperature culture propagation;
2, separation and purification: step 1 is increased the cultivation of ruling of bacterium culture on the proliferated culture medium flat board, therefrom each bacterium colony with characteristic morphology of picking is rule and is cultivated and separate, till being pure bacterium colony, gram stain microscopy, to breed in the doubtful pure colony inoculation test tube slant that separate, then slant strains is preserved in 4 ℃ of refrigerators, stand-by;
Wherein said proliferated culture medium is a kind of in BPY culture medium, MRS culture medium, No. 98 culture mediums;
3, the seed selection of the excellent species of strong stress resistance: to step 2 the bacterial strain of separation, purifying carry out the resistance screening, can separate institute, the bacterial classification of purifying directly carries out anti-adversity mensuration, therefrom filters out the natural excellent species of strong stress resistance; Or by gradient cultivate the acidity increase solution gradually (to pH be about 1.5) and bile salt concentration, select bacterial strain with acidproof and anti-bile characteristic; Or by traditional physics, mutagenesis method, modern genetic engineering technology, directive breeding goes out the bacterial classification of strong stress resistance; Doing bacterial classification evaluation and biology performance again measures:
1) acid resistance is measured: slant strains is inoculated in the seed culture medium activates, 30～37 ℃ leave standstill cultivation 8～20h, be inoculated in respectively by 5～10% inoculum concentrations in manual simulation's gastric juice of pH value 2.0,3.0,4.0, the 0h counting compares, 2h, 6h sampling is pressed 10 times of serial dilutions with phosphate buffer, carry out dull and stereotyped count plate, calculate survival rate;
Wherein said seed culture medium is a kind of in BPY culture medium, MRS culture medium, No. 98 culture mediums;
2) bile tolerance is measured: slant strains is inoculated in the culture medium, 30～37 ℃ leave standstill cultivation 8-24h activation, be inoculated in respectively by 5～10% inoculum concentrations in the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% variable concentrations, the 0h counting compares, 2h, 6h sampling is counted by 10 times of serial dilutions with physiological saline, carry out dull and stereotyped count plate, calculate survival rate;
3) high temperature resistant mensuration: slant strains is inoculated in the seed culture medium, cultivates the 16-24h activation for 30～37 ℃,, handle the preceding contrast of doing, calculate survival rate respectively at 50 ℃～80 ℃ water bath processing 15～30min;
4) drug resistance is measured: adopt the drug sensitivity paper disk method;
5) produce enzymatic determination: adopt People's Republic of China's light industry industry standard, industrial alpha Amylase preparation (QB/T1805.1-1993), the general test method of industrial enzyme preparation (QB/T1803-1993) is carried out amylase activity to bacillus licheniformis and is measured; Adopt industry standard: industrial protease preparation (QB/T1805.3-1993), carry out prolease activity to bacillus licheniformis and measure;
6) produce acidity test: adopt the chromatography of ions that enterococcus faecalis CGMCC No.2386 is carried out production of organic acids and measure;
7) bacteriostatic test: adopt Oxford agar diffusion method (claiming cylinder-plate method again) that common pathogen is carried out bacteria inhibition assay;
The bacillus licheniformis CGMCC No.2383 of institute of the present invention seed selection handles 6h in manual simulation's gastric juice (pH2.0～4.0), survival rate is more than 49.5%; Handle 6h at pig cholate solution (concentration 0.3-3g/kg), survival rate is 100%; Handle 15～30min for 50～80 ℃, survival rate is 100%; Benzyl penicillin, Amoxicillin, spectinomycin, erythromycin, kitasamycin, Bacitracin Zinc, 7 kinds of antibiosis of lincomycin are have drug resistance; Escherichia coli K88, staphylococcus aureus, three kinds of pathogenic bacteria of white diarrhea salmonella are all had good inhibitory effect, and antibacterial circle diameter is at 12～20mm; The white enzyme activity of laying eggs is 1000u/ml; The product amylase activity is: 800u/ml.
The enterococcus faecalis CGMCC No.2386 of institute of the present invention seed selection handles 6h in manual simulation's gastric juice (pH2.0～4.0), survival rate is more than 18.0%; Handle 6h at pig cholate solution (concentration 0.3-3g/kg), survival rate is more than 12.6%; Handle 15～30min for 50～80 ℃, survival rate is 0.43%; Benzyl penicillin, CTX, gentamicin, spectinomycin, neomycin, erythromycin, kitasamycin, Bacitracin Zinc, lincomycin, Norfloxacin, Enrofloxacin, Ciprofloxacin, 13 kinds of antibiosis of Co-trimoxazole are have drug resistance; Escherichia coli K88, staphylococcus aureus, three kinds of pathogenic bacteria of white diarrhea salmonella are all had the good restraining effect, and antibacterial circle diameter is at 16～21mm; Lactic acid, acetate, the total acid yield of isobutyric acid are 4.2g/L, and lactic acid yield reaches 89.4%.
The saccharomyces cerevisiae CGMCC No.2388 of institute of the present invention seed selection handles 6h in manual simulation's gastric juice (pH2.0～4.0), survival rate is more than 15.4%; Handle 6h at pig cholate solution (concentration 0.3-3g/kg), survival rate is more than 11.5%; Handle 15～30min for 50～80 ℃, survival rate is 0.19%;
4, the preparation of bacterium powder
The preparation method of bacillus licheniformis CGMCC No.2383 bacterium powder comprises following step:
1) dull and stereotypedly cultivate rejuvenation: with the bacillus licheniformis bacterial classification inoculation on the BPY plating medium, in 30-37 ℃ of cultivation 12-24h, make the bacillus licheniformis rejuvenation, and form single bacterium colony, picking list bacterium colony is cultivated 24-36h in being inoculated on the slant medium in 30-37 ℃;
2) preparation of first order seed: on the Bacillus subtilis strain switching eggplant bottle BPY slant medium with the step 1) cultivation,, make it be in the logarithm middle and later periods, get first order seed in 30～37 ℃ of cultivation 12～16h;
3) preparation of secondary seed: with step 2) Zhi Bei first order seed is made bacteria suspension with sterilized water, is inoculated into 1-1.6M is housed
3The 2M of BPY seed culture medium
3In the seeding tank, 30～37 ℃ of temperature, rotating speed 200～250rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8 (14.4～19.2M
3/ h), cultivate 10～14h, be secondary seed solution;
4) preparation of the lichen bacillus ferments liquid: the secondary seed solution of step 3) preparation is inoculated into according to 3～10% inoculum concentration 12～16M is housed
3In the fermentation tank of fermentation medium, 30～37 ℃ of temperature, rotating speed 220～300rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8, cultivate 16～20h, to gemma formation rate more than 90%, viable count is 1～2 * 10
10Cfu/ml, then stuck fermentation gets the lichen bacillus ferments liquid;
Described fermentation medium is: wheat bran 1～2%, dregs of beans 1～2%, sodium chloride 0.2～0.8%, magnesium sulfate 0.01～0.05%;
5) preparation of bacillus licheniformis bacterium powder: in the zymotic fluid of step 4) preparation, add 20～25% filler, mixing, carry out spray-drying, 120～130 ℃ of EATs, 40～50 ℃ of temperature of outgoing airs, atomizer rotating speed 15000～18000rpm obtains the bacillus licheniformis bacterium powder of moisture＜5%.
Described filler is: any in rice bran, zeolite powder, the powdered rice hulls.
The preparation method of B, enterococcus faecalis CGMCC No.2386 bacterium powder comprises following step:
1) dull and stereotyped cultivation rejuvenation: the enterococcus faecalis bacterial classification inoculation on the MRS plating medium, in 32～37 ℃ of cultivation 12～24h, is made the rejuvenation of enterococcus faecalis bacterium, and forms single bacterium colony; Picking list colony inoculation is cultivated 24～36h in 32～37 ℃ on slant medium;
2) preparation of first order seed: the enterococcus faecalis slant strains that step 1) is cultivated is transferred in the 500mL triangular flask that 250ml～300ml MRS culture medium is housed, cultivate 12～16h in 32～37 ℃, rotating speed 100～150rpm makes it be in the logarithm middle and later periods, is first order seed;
3) preparation of secondary seed: with step 2) the Bacillus acidi lactici first order seed of Pei Yanging is transferred in the 2.0L triangular flask that 1.2～1.6L MRS culture medium is housed, and in 32～37 ℃ of static cultivation 12～16h, is secondary seed solution;
4) preparation of fermentation liquid: the secondary seed solution of step 3) preparation is inoculated in the fermentation tank that 12～16M3 fermentation medium is housed according to 3～6% inoculum concentration, 35～37 ℃ of temperature, rotating speed 120～160rpm, tank pressure 0.05Mpa, cultivate 16～20h, to viable count be 2～5 * 10
9Cfu/ml, stuck fermentation;
Described fermentation medium is: glucose 1～2%, soy peptone 1～2%, ammonium sulfate 0.5～1.0%, sodium chloride 0.2～0.8%, magnesium sulfate 0.01～0.05%;
5) preparation of enterococcus faecalis bacterium powder: with the zymotic fluid of step 4) preparation; 3500～5000rpm is centrifugal; obtain bacterium mud; the weight/volume percent of adding and bacterium mud is 15～20% freeze drying protectant; mixing; in-25 ℃～-45 ℃ freeze dryings, obtain the enterococcus faecalis bacterium powder of moisture＜5%.
Described freeze drying protectant is: the mixture of skimmed milk power, glycerine, sucrose, maltodextrin and sodium glutamate, according to: skimmed milk power: glycerine: sucrose: maltodextrin: the ratio of sodium glutamate=2: 0.5～1.0: 0.5～1.0: 0.5～1.5: 0.5～1.0 mixes.
The preparation method of C, saccharomyces cerevisiae CGMCC No.2388 bacterium powder comprises following step:
1) dull and stereotypedly cultivate rejuvenation: the method that the saccharomyces cerevisiae slant strains of refrigerator preservation is adopted streak inoculation streak inoculation on No. 98 plating mediums, cultivate 24～36h in 28～32 ℃, make the saccharomyces cerevisiae rejuvenation, and form single bacterium colony; Picking list bacterium colony is on No. 98 fresh slant mediums, and in 28～32 ℃ of cultivation 24～36h, it is standby to place refrigerator;
2) preparation of first order seed: the fresh slant strains of the cultured saccharomyces cerevisiae of step 1) is transferred in the 500mL triangular flask that No. 98 culture mediums of 200～250ml are housed, cultivate 12～16h in 28～32 ℃, rotating speed 100～150rpm makes it be in the logarithm middle and later periods, is first order seed;
3) preparation of secondary seed: with step 2) cultured saccharomyces cerevisiae seed liquor is transferred in the 2.0L triangular flask that No. 98 culture mediums of 1.0～1.2L are housed, and in 28～32 ℃ of static cultivation 12～16h, is secondary seed solution;
4) preparation of fermentation liquid: the secondary seed of step 3) preparation is inoculated into according to 3～10% inoculum concentration 15～16M is housed
3In the fermentation tank of fermentation medium, 28～32 ℃ of temperature, rotating speed 150～200rpm, tank pressure 0.05Mpa cultivates 12～16h stuck fermentation, and this moment, viable count was 1～3 * 10
9Cfu/ml;
Described fermentation medium is: brown sugar 1～2%, soy peptone 1～2%, yeast extract 0.2～0.5%, sodium chloride 0.5～1.0%, magnesium sulfate 0.01～0.05%, dipotassium hydrogen phosphate 0.2～0.5%;
5) preparation of S. cervisiae powder: with the zymotic fluid of step 4) preparation, 2000～3000rpm is centrifugal, obtain bacterium mud, the weight/volume percent of adding and bacterium mud is 10～15% freeze drying protectant, in-25 ℃～-45 ℃ freeze dryings, obtain the S. cervisiae powder of moisture＜5% behind the mixing;
Described freeze drying protectant be skimmed milk power, glycerine, maltodextrin according to: 2: 0.5～1: 1～2 ratios mix.
, it should be understood that described embodiment only is for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
The specific embodiment
Embodiment 1, excellent species separation, screening, seed selection and evaluation
1) increasing bacterium cultivates: fresh excreta, the intestinal contents sample of healthy animal (pig or poultry) are inoculated in the BPY fluid nutrient medium, at 37 ℃ of constant temperature culture propagation 48h;
2) separation and purification of bacillus licheniformis: step 1) is increased 100 ℃ of water-bath 5min of bacterium culture, the cultivation of on the BPY culture medium flat plate, ruling then, therefrom each bacterium colony with characteristic morphology of picking is rule and is cultivated and separate, till being pure bacterium colony, gram stain microscopy, with breeding in the doubtful pure colony inoculation test tube slant that separates, be stored in then in 4 ℃ of refrigerators, stand-by;
Colonial morphology: the bacterium colony rough surface, gloss not, milky is opaque, and the edge is irregular, diameter 2-3mm, the prolongation along with incubation time becomes bronzing; Microscopy: cell is bacillus 1.6～3.8 μ m * 0.6～1.2 μ m, and Gram-positive is given birth in the gemma, and ellipse does not expand, and tentatively is defined as bacillus licheniformis;
3) separation and purification of enterococcus faecalis: to the cultivation of on the MRS culture medium flat plate, ruling of step 1) enriched medium, therefrom each bacterium colony with characteristic morphology of picking is rule and is cultivated and separate, till being pure bacterium colony, gram stain microscopy, to breed in the doubtful pure colony inoculation test tube slant that separate, be stored in then in 4 ℃ of refrigerators, stand-by;
Colonial morphology: the bacterium colony rounding, smooth surface is creamy white, big or small 1-3mm; Cell circle or oval, diameter 0.5～1.0um, great majority become short chain or right, Gram-positive;
4) separation and purification of saccharomyces cerevisiae: to the cultivation of on No. 98 culture medium flat plates, ruling of step 1) enriched medium, therefrom each bacterium colony with characteristic morphology of picking is rule and is cultivated and separate, till being pure bacterium colony, gram stain microscopy, to breed in the doubtful pure colony inoculation test tube slant that separate, be stored in then in 4 ℃ of refrigerators, stand-by;
Colonial morphology: bacterium colony is soft and moistening, cheese look, glossy, smooth or protruding slightly, neat in edge; Cell is spherical or avette, diameter 5～10um, modes of reproduction budding;
To step 3) or 4) separate, the enterococcus faecalis of purifying or Wine brewing yeast strain carry out the resistance screening, can separate institute, the bacterial classification of purifying directly carries out anti-adversity mensuration, therefrom filters out the natural excellent species of strong stress resistance; Or by gradient cultivate the acidity increase solution gradually (to pH be about 1.5) and bile salt concentration, select bacterial strain with acidproof and anti-bile characteristic; Or by traditional physics, mutagenesis method, modern genetic engineering technology, directive breeding goes out the bacterial classification of strong stress resistance; And it is carried out bacterial classification identify, at last the bacterial strain that selects is carried out the resistance performance measurement.
The resistance of embodiment 2,3 strain excellent species and biology performance are measured
3 strain bacterial classifications: the biology performance of bacillus licheniformis CGMCC No.2383, enterococcus faecalis CGMCC No.2386, saccharomyces cerevisiae CGMCC No.2388 and anti-adversity are measured:
1) preparation of bacillus licheniformis CGMCC No.2383 culture: the slant strains of refrigerator preservation is inoculated in the BPY seed culture medium activates, 37 ℃, 200rpm are cultivated 18h, obtain the gemma rate at the culture more than 95%;
2) preparation of enterococcus faecalis CGMCC No.2386 culture: the slant strains of refrigerator preservation is inoculated in the MRS seed culture medium activates, 35 ℃, 100rpm are cultivated 16h, promptly;
3) preparation of saccharomyces cerevisiae CGMCC No.2388 culture: the slant strains of refrigerator preservation is inoculated in No. 98 seed culture mediums activates, 30 ℃, 150rpm are cultivated 16h, promptly;
4) acid resistance is measured: be inoculated in the culture of above-mentioned preparation in manual simulation's gastric juice of pH value 2.0,3.0,4.0 respectively by 5% inoculum concentration, the 0h counting compares, 2h, 6h sampling by 10 times of serial dilutions, is carried out dull and stereotyped count plate with phosphate buffer, calculates survival rate.
3 strain bacterial classifications of seed selection of the present invention: bacillus licheniformis CGMCC No.2383, enterococcus faecalis CGMCC No.2386, saccharomyces cerevisiae CGMCC No.2388 survival results in hydrochloric acid in gastric juice see Table 1.
The preparation of manual simulation's gastric juice: measure 16.4 milliliters of 9.5%～10.5% concentrated hydrochloric acids, adding distil water to 1000 milliliter, do basic simulated gastric fluid, with hydrochloric acid or NaOH adjust pH 2.0,3.0,4.0, respectively get 10mL (9mL), be sub-packed in the test tube, 100 ℃ of following steam sterilizings 15 minutes, under aseptic condition, add the 0.100g pepsin in every 10mL liquid.
The survival rate of table 13 strain bacterial classification in manual simulation's gastric juice
5) bile tolerance is measured: with 3 strain culture of strains things of above-mentioned preparation, be inoculated in respectively by 5% inoculum concentration in the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% variable concentrations, the 0h counting compares, 2h, 6h sampling is counted by 10 times of serial dilutions with physiological saline, carry out dull and stereotyped count plate, calculate survival rate.
3 strain bacterial classifications of institute of the present invention seed selection: bacillus licheniformis CGMCC No.2383, enterococcus faecalis CGMCC No.2386, saccharomyces cerevisiae CGMCC No.2388 survival results in cholate see Table 2.
The preparation of cholate: each 9mL in 0.85% physiological saline, be sub-packed in the test tube, 121 ℃ of following steam sterilizings 30 minutes under aseptic condition, are made the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% variable concentrations.
Table 23 strain bacterial classification is handled the survival rate of 6h in variable concentrations pig cholate
6) high temperature resistant mensuration:,, calculate survival rate respectively at 50 ℃, 60 ℃, 70 ℃, 80 ℃ water bath processing 15min, 30min with 3 strain culture of strains things of above-mentioned preparation.
Table 33 strain bacterial classification is handled the survival rate (%) of different time under different high temperature
7) drug resistance is measured: adopt the drug sensitive test paper method.
The drug resistance measurement result of table 43 strain bacterial classification
8) produce enzymatic determination: adopt People's Republic of China's light industry industry standard, industrial alpha Amylase preparation (QB/T1805.1-1993), the general test method of industrial enzyme preparation (QB/T1803-1993) is carried out amylase activity to bacillus licheniformis and is measured; Adopt industry standard: industrial protease preparation (QB/T1805.3-1993), carry out prolease activity to bacillus licheniformis and measure.
Bacillus licheniformis CGMCC No.2383 amylase activity is: 800u/ml; Prolease activity is 1000u/ml.
9) produce acidity test: adopt the chromatography of ions that enterococcus faecalis CGMCC No.2386 zymotic fluid has been carried out the product acidity test.
Produce sour result and show, enterococcus faecalis CGMCC No.2386 total organic acids amount is 4.2g/L, and wherein lactic acid production is 3.76g/L, and the acetate amount is 0.39g/L, also has a spot of isobutyric acid.
10) bacteriostatic test: adopt the Oxford agar diffusion method that common pathogen is carried out bacteria inhibition assay.
A, in the test tube that 10mL nutrition bouillon media is housed activation three strain pathogenic bacteria: K99, staphylococcus aureus, white diarrhea salmonella, 37 ℃ of constant temperature culture 20h;
The preparation of b, double-layer plate: the flat board of cut-off footpath 90mm, inject the nutrient agar 15～20mL of sterilization, horizontal positioned makes it to solidify, as bottom, other gets nutrient agar (being chilled to about 50 ℃) and an amount of (the 50mL culture medium adds about the 8mL) mixing of the indicator bacteria liquid of 37 ℃ of 240h cultivations, draw 10mL and water on the bottom culture medium, horizontal positioned makes it to solidify, as the bacterium layer;
C, add sample: with the sterilized Oxford of aseptic nipper gripping cup, open the ware lid, be placed on the culture medium.In the cup of Oxford, fill it up with the fermented liquid supernatant liquid (about 200uL) of same amount, 2 repetitions of each sample.The two dish that add sample are carefully put into 37 ℃ of insulating boxs, behind the cultivation 16-18h, take out and measure the inhibition zone size.
Table 53 strain bacterial classification is to the antibacterial result of common pathogen
The preparation of embodiment 3, bacillus licheniformis CGMCC No.2383 bacterium powder
1) dull and stereotyped cultivation rejuvenation: the bacillus licheniformis bacterial classification inoculation on the BPY plating medium, in 37 ℃ of cultivation 24h, is made the bacillus licheniformis rejuvenation, and forms single bacterium colony, and picking list bacterium colony is cultivated 36h in being inoculated on the slant medium in 37 ℃;
2) preparation of first order seed: on the Bacillus subtilis strain switching bottle inclined plane culture medium of eggplant with the step 1) cultivation,, make it be in the logarithm middle and later periods, get first order seed in 37 ℃ of cultivation 12h;
Described slant medium is the BPY solid medium;
3) preparation of secondary seed: with step 2) Zhi Bei first order seed is made bacteria suspension with sterilized water, is inoculated into 1.6M is housed
3The 2M of BPY seed culture medium
3In the seeding tank, 37 ℃ of temperature, rotating speed 200rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8 (14.4～19.2M
3/ h), cultivate 10～14h, be secondary seed solution;
4) preparation of the lichen bacillus ferments liquid: the secondary seed solution of step 3) preparation is inoculated into according to 10% inoculum concentration 16M is housed
3In the fermentation tank of fermentation medium, 37 ℃ of temperature, rotating speed 220rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8, cultivate 20h, to gemma formation rate more than 90%, viable count is 1～2 * 10
10Cfu/ml, then stuck fermentation gets the lichen bacillus ferments liquid;
Described fermentation medium is: wheat bran 2%, dregs of beans 1%, sodium chloride 0.8%, magnesium sulfate 0.01%.
5) preparation of bacillus licheniformis bacterium powder: the filler rice bran of adding 25% in the zymotic fluid of step 4) preparation, mixing, carry out spray-drying, 120～130 ℃ of EATs, 40～50 ℃ of temperature of outgoing airs, atomizer rotating speed 15000～18000rpm obtains the bacillus licheniformis bacterium powder of moisture＜5%.
Described filler is: any in rice bran, zeolite powder, the powdered rice hulls.
The preparation of embodiment 4, enterococcus faecalis CGMCC No.2383 bacterium powder
1) dull and stereotyped cultivation rejuvenation: the enterococcus faecalis bacterial classification inoculation on the MRS plating medium, in 37 ℃ of cultivation 24h, is made the rejuvenation of enterococcus faecalis bacterium, and forms single bacterium colony; Picking list colony inoculation is cultivated 24h in 37 ℃ on slant medium;
2) preparation of first order seed: the enterococcus faecalis slant strains that step 1) is cultivated is transferred in the 500mL triangular flask that the 300mlMRS culture medium is housed, and in 37 ℃ of cultivation 12h, rotating speed 100rpm makes it be in the logarithm middle and later periods, is first order seed;
3) preparation of secondary seed: with step 2) the enterococcus faecalis first order seed of Pei Yanging is transferred in the 2.0L triangular flask that the 1.6LMRS culture medium is housed, and in 37 ℃ of static cultivation 12h, is secondary seed solution;
4) preparation of fermentation liquid: the secondary seed solution of step 3) preparation is inoculated into according to 3% inoculum concentration 16M is housed
3In the fermentation tank of fermentation medium, 37 ℃ of temperature, rotating speed 120rpm, tank pressure 0.05Mpa cultivates 16h, to viable count be 2 * 10
9Cfu/ml, stuck fermentation;
Described fermentation medium is: glucose 2%, soy peptone 1%, ammonium sulfate 0.5%, sodium chloride 0.2%, magnesium sulfate 0.05%.
5) preparation of enterococcus faecalis bacterium powder: with the zymotic fluid of step 4) preparation, 5000rpm is centrifugal, obtains bacterium mud, the weight/volume percent of adding and bacterium mud is 20% freeze drying protectant, mixing in-45 ℃ of freeze dryings, obtains the enterococcus faecalis bacterium powder of moisture＜5%;
Described freeze drying protectant is: the mixture of skimmed milk power, glycerine, sucrose, maltodextrin and sodium glutamate, according to: skimmed milk power: glycerine: sucrose: maltodextrin: sodium glutamate=2: 1.0: 1.0: 1.5: 1.0 ratio mixes.
The preparation of embodiment 5, saccharomyces cerevisiae CGMCC No.2383 bacterium powder
1) dull and stereotypedly cultivate rejuvenation: the method that the saccharomyces cerevisiae slant strains of refrigerator preservation is adopted streak inoculation streak inoculation on No. 98 plating mediums, cultivate 46h in 30 ℃, make the saccharomyces cerevisiae rejuvenation, and form single bacterium colony; Picking list bacterium colony is on No. 98 fresh slant mediums, and in 30 ℃ of cultivation 36h, it is standby to place refrigerator;
2) preparation of first order seed: the fresh slant strains of the cultured saccharomyces cerevisiae of step 1) is transferred in the 500mL triangular flask that No. 98 culture mediums of 200ml are housed, and in 30 ℃ of cultivation 12～16h, rotating speed 150rpm makes it be in the logarithm middle and later periods, is first order seed;
3) preparation of secondary seed: with step 2) cultured saccharomyces cerevisiae seed liquor is transferred in the 2.0L triangular flask that the 1.2L98 culture medium is housed, and in 30 ℃ of static cultivation 12h, is secondary seed solution;
4) preparation of fermentation liquid: the secondary seed of step 3) preparation is inoculated into according to 10% inoculum concentration 15～16M is housed
3In the fermentation tank of fermentation medium, 30 ℃ of temperature, rotating speed 200rpm, tank pressure 0.05Mpa cultivates the 16h stuck fermentation, and this moment, viable count was 1.5 * 10
9Cfu/ml;
Described fermentation medium is: brown sugar 1%, soy peptone 1%, yeast extract 0.2%, sodium chloride 0.5%, magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.2%.
5) preparation of S. cervisiae powder: with the zymotic fluid of step 4) preparation, 2000pm is centrifugal, obtains bacterium mud, and the weight/volume percent of adding and bacterium mud is 10～15% freeze drying protectant, in-25 ℃ of freeze dryings, obtain the S. cervisiae powder of moisture＜5% behind the mixing;
Described freeze drying protectant be skimmed milk power, glycerine, maltodextrin according to: ratio mixed in 2: 0.5: 1.
The preparation of embodiment 6, compound micro-ecological preparation
The preparation of table 6 compound micro-ecological preparation
| Classification | Lactobacillus acidophilus bacterium powder (%) | Enterococcus faecalis bacterium powder (%) | Bacillus licheniformis bacterium powder (%) | Bacillus subtilis bacterium powder (%) | S. cervisiae powder (%) | Carrier (%) |
| Pig is universal | 10 | 10 | 20 | - | 20 | Stone flour: 40 |
| The piglet tailored version | 5 | 5 | 5 | - | 10 | Glucose: 75 |
| The sow tailored version | - | - | 10 | 10 | 10 | Aluminum potassium sulfate: 50, stone flour 20 |
| Feed factory pig tailored version | - | - | 30 | 30 | 30 | Maize cob meal: 10 |
| Young fowl tailored version | - | 5 | 5 | - | 10 | Glucose: 80 |
| The poultry tailored version | 10 | - | 20 | - | 20 | Stone flour: 50 |
| Egg fowl tailored version | - | - | 10 | 10 | 10 | Stone flour: 70 |
| Feed factory chicken tailored version | - | - | 30 | 30 | 30 | Maize cob meal: 10 |
The piglet tailored version compound micro-ecological preparation of embodiment 7, embodiment 6 preparations is to the efficacy test of piglet
Experimental animal: select the wean of 35 ages in days for use, the white x Da Bai hybridization of the length of body weight about 9kg piglet is as experimental animal, and totally 120, test pig derives from the Quan Limin pig farm, Beijing of feeding and management standard, anti-epidemic measure strictness, a collection of choosing is neat, and the date of birth differs and is no more than 7 days.
Experimental design: adopt single-factor design at random.Test divides four groups, and every group has three repetitions, and each repeats 10 weanling pigs, between group and different 5% of the average weight that is no more than of the repetition mesosome method of double differences.
Test daily ration: adopt corn one dregs of beans type daily ration to make basal diet, the control group fed basal diet, test group I adds 1 ‰ piglet tailored version compound micro-ecological preparations on basal diet, test group II is for adding the antibiotic arsanilic acid that the 100g/T prevention is had loose bowels on basal diet, test group III is for adding the antibiotic arsanilic acid that 1 ‰ piglet tailored version compound micro-ecological preparations+interpolation 100g/T prevention is had loose bowels on basal diet.Basal diet is formed and trophic level sees Table 7.
Feeding and management: adopt ground flat to support and raise, except that the daily ration difference, other conditionally complete unanimity is carried out routinely between each group.Manually feed intake, free choice feeding, freely drink water, keep the pig house cleaning.30 days experimental periods, raise 7 days phases in advance.
Table 7 is fed, and basal diet is formed and trophic level
Test index and method: on an empty stomach pig is only weighed morning in on-test with when finishing, be respectively that unit carries out full group and weighs, calculates indexs such as feed consumption rate, daily gain, feedstuff-meat ratio, diarrhea rate with the repeating groups, and carry out otherness and significantly check.Result of the test is as follows:
(1) production performance
The production performance of test piglet sees Table 8.As can be seen from Table 8, average daily gain piglet test group I, test group II and the comparison of test group III according to component you can well imagine high by 7.3%, 7.5%, 9.5%; Test group I, test group II and the comparison of test group III reduce by 5.5%, 5.2%, 8.0% respectively according to group aspect feedstuff-meat ratio; Test group I, test group II and the comparison of test group III reduce by 67.3%, 47.7%, 70.1% respectively according to group aspect diarrhea rate.The test group I is compared aspect daily gain, feedstuff-meat ratio difference with the test group II not remarkable, but aspect prevention diarrhoea significant difference, illustrate that piglet tailored version compound micro-ecological preparation can substitute the antibiotic that prevention is suffered from diarrhoea in the feed.The test group III all is being better than test group I, test group II and control group aspect daily gain, feedstuff-meat ratio and the diarrhoea, illustrate that piglet tailored version compound micro-ecological preparation and antibiotic have share synergy.Difference is not remarkable between each is organized aspect the average feed consumption.
The production performance of table 8 test piglet
| Group | Beginning counterpoise (kg) | End counterpoise (kg) | Equal daily gain (g/ head) | Average feed consumption (the g/ head. day) | Feedstuff-meat ratio | Diarrhea rate (%) |
| Control group | 8.8±0.4 2 | 23.6±1. 32 | 493±21 | 856±45 | 1.74±0.0 8 | 10.7±0.6 |
| The test group I | 8.7±0.3 7 | 24.5±1. 47 | 526±35 | 851±39 | 1.61±0.0 7 | 3.5±0.3 |
| The test group II | 8.9±0.4 5 | 24.8±1. 43 | 530±31 | 873±42 | 1.65±0.0 9 | 5.6±0.5 |
| The test group III | 8.8±0.4 | 25.0±1. | 540±40 | 864±51 | 1.60±0.1 | 3.2±0.4 |
| 0 | 50 | 0 |
The sow tailored version compound micro-ecological preparation of embodiment 8, embodiment 6 preparations is to the efficacy test of milking sow
1, test method
The selection of test pig: the sow of giving a birth about selecting just before giving birth preceding 1 week from the pig farm, 18 of pigs choosing healthy anosis, age, parity, body weight basically identical are divided into two groups, respectively 9 of test group and control groups at random.
Feed is formed: control group fed basal diet, test group are to add 1 ‰ sow tailored version compound micro-ecological preparations on basal diet.
Feeding and management: test is carried out in this same delivery room, is fed the feeding and management condition unanimity by same keeper, feed is taked siccative to add water to mix and feed after wet, begin the feeding experiment material when the test sow changes obstetric table over to, day feeds 3 times, free choice feeding, material is not a principle cannot have enough surplusly.Test group and control group are added up feed consumption rate, the heavy and 28 days weanling weights of piglet birth respectively, and the way of taking whole day to supply water appoints pig freely to drink water.Clear up ight soil by the poultry raiser every day, sweeps colony house once, and the growth of viewing test group and health condition no matter be milking sow or piglet, disease take place, in time treatment at any time.
2, result of the test and analysis
In experimental period, test group wean in the 28 days counterpoise that adds the 0.1% sow tailored version compound micro-ecological preparation of feeding is 8.27kg, and control group wean in the 28 days counterpoise that does not add sow tailored version compound micro-ecological preparation is 7.54kg, test group than control group many weightening finish 0.73kg, check through t, two groups of significant differences (p＜0.05), and the test group survival rate is apparently higher than control group, and its survival rate is respectively 95.83% and 91.67%.The results are shown in Table 9.
Table 9 sow tailored version compound micro-ecological preparation is to the influence of milking sow production performance
Search for food and health condition: in the entire test, the milking sow performance happiness of adding sow tailored version compound micro-ecological preparation is eaten, grazing speed is fast, feed intake slightly increases than control group, and the ruddy light of hair color.Consumptive material situation: average every milking sow feed consumption 6.42kg of duration of test test group and control group and 6.31kg.The test group sow the newborn phenomenon of significantly overflowing occurs in farrowing after 8 days, show that sow tailored version compound micro-ecological preparation can improve the lactation amount of sow.The test later stage, test group and control group all have the baby pig diarrhea phenomenon, intramuscular injection Enrofloxacin and oral alctasin treatment back defecation are normal, and test piglet hair is rarer, and skin is ruddy, and ight soil reduces and is dry, stink also reduces, illustrate that the sow tailored version compound micro-ecological preparation of feeding for regulating microorganism species balance in the milking sow enteron aisle, prevents that grice diarrhoea from strengthening the resistance against diseases of sow, has certain effect.
3, conclusion
(1) sow tailored version compound micro-ecological preparation can improve the milking sow galactopoiesis as feed addictive, solves the problem of sow hypogalactia;
(2) in feed, add 0.1% sow tailored version compound micro-ecological preparation and can improve the heavy and survival rate of weaned piglet, remarkable in economical benefits.
The egg fowl tailored version compound micro-ecological preparation of embodiment 9, embodiment 6 preparations is to the efficacy test of laying hen
1, test method:
Experimental animal and daily ration: select for use at random 60 ages in week 3000 of short and small laying hens, be divided into 2 groups at random, 1500 every group, test according to the random packet arrangement.
The test daily ration is respectively: I. blank group (basal diet group); II. test group (basal diet+0.1% egg fowl tailored version compound micro-ecological preparation).Its composition of basal diet and nutritional labeling see Table 10.Test chicken is three layers raises in cages, 3 in every cage, and free choice feeding and drinking-water carry out feeding and management routinely, and test is carried out in laying hen field, Miyun fountain village, Beijing.
Table 10 basal diet is formed and nutritional labeling
| Composition | Content (%) | Nutritional labeling | |
| Corn | 56.0 | Metabolizable energy (MJ/kg) | 12.59 |
| Dregs of beans | 14.6 | Crude protein (%) | 17.06 |
| The dish dregs of rice | 3.4 | Calcium (%) | 3.57 |
| Peanut cake | 3.0 | Total phosphorus (%) | 0.55 |
| Fish meal | 2.0 | Methionine (%) | 0.738 |
| Corn protein powder | 2.0 | Methionine+cystine (%) | 0.676 |
| The alcohol albumen powder | 5.0 | ||
| Stone flour | 5.8 | ||
| Zeolite | 1.0 | ||
| Calcium monohydrogen phosphate | 1.8 | ||
| Premix | 5 |
Annotate: in every kilogram of daily ration: retinol1 2000IU, cholecalciferol 1500IU, vitamin E2 5IU, prokeyvit 1.0mg, thiamine 5.5mg, riboflavin 5.0mg, pantothenic acid 16mg, vitamin B6 8.0mg, biotin 0.3mg, choline 500mg, folic acid 1.8mg, Cobastab 120.008mg, iron 90mg, copper 20mg, iodine 0.45mg, manganese 80mg, zinc 80mg, selenium 0.2mg, DL-methionine 1.50g.
Testing index: the every repetition egg number of observed and recorded every day, egg size, defective egg number and chicken are extremely washed in a pan situation; Feed consumption rate is added up at the end weekly; Add up every group of chicken lay eggs laying rate, egg size, breakage rate, the death rate, feed intake and the feedstuff-egg ratio in later stage respectively.
2, result and analysis
2.1, egg fowl tailored version compound micro-ecological preparation is to the influence of short and small laying hen production performance
Result of the test is as shown in table 11, is laying eggs the later stage, adds 0.1% egg fowl tailored version compound micro-ecological preparation group II laying rate and improves 2.53% than blank group respectively, and egg size increases to some extent; Aspect the laying hen death rate and eggshell breakage rate, descend.
Table 11 egg fowl tailored version compound micro-ecological preparation is to the influence of short and small laying hen production performance
| Group | Egg size g/ | Day consumption g/ only | Feedstuff-egg ratio | Laying rate % | Breakage rate % | Death rate % |
| Test group | 55.61 | 124.6 | 2.24 | 72.58 | 0.23 | 0.2 |
| Control group | 55.56 | 126.1 | 2.27 | 70.79 | 0.21 | 0.5 |
3, conclusion
(1) add the laying rate that 0.1% egg fowl tailored version compound micro-ecological preparation can significantly improve laying hen in feed, laying rate improves 2.53%;
(2) adding 0.1% egg fowl tailored version compound micro-ecological preparation in feed can reduce the generation of disease and reduce the death rate of laying hen;
(3) add the immunocompetence that 0.1% egg fowl tailored version compound micro-ecological preparation can be able to improve body in feed, prevention is because the stress reaction that aspects such as heat stress or vaccine inoculation cause.
In sum, probiotics of the present invention adds in the feed, to guarantee the chicken health status, coordinate to promote body to digest and assimilate ability, the digestive utilization ratio etc. that improves laying hen production performance and feed all has good effect.
The poultry tailored version compound micro-ecological preparation of embodiment 10, embodiment 6 preparations is to the efficacy test of fryer
1, test material and method
Experimental animal and grouping: select for use 1 age in days commodity to mix strong young 480, adopt the design of completely random single-factor to divide 4 groups and test for the Chinese mugwort denapon.Establish 4 repetitions for every group, 30 of every repetitions (beginning body weight group difference is not remarkable, P＞0.05).49 days experimental periods.
Experimental design: contrast control group with virginiamycin as antibiotic: basal diet, test group 1: basal diet+1 ‰ poultry tailored version compound micro-ecological preparations, test group II: basal diet+5mg/kg virginiamycin.
For trying daily ration and feeding and management: this experimental basis daily ration prescription and trophic level are respectively organized identical (seeing Table 12), and feeding and management condition is respectively organized in full accord.
Table 12 basal diet is formed and trophic level
| Daily ration is formed | 0～21 age in days (%) | 22～42 ages in days (%) |
| Corn | 57.9 | 62.3 |
| Dregs of beans | 32.0 | 28.8 |
| Fish meal | 3.0 | 2.0 |
| Stone flour | 0.9 | 0.9 |
| Calcium monohydrogen phosphate | 1.4 | 1.4 |
| Salt | 0.3 | 0.3 |
| Soya-bean oil | 3.5 | 3.3 |
| Premix | 1.0 | 1.0 |
| ME(MJ/Kg) | 12.47 | 12.59 |
| CP(％) | 20.62 | 19.0 |
| Ca(％) | 0.97 | 0.89 |
| Effective P (%) | 0.50 | 0.46 |
| Lys(％) | 1.05 | 0.94 |
| Met(％) | 0.36 | 0.33 |
2, result of the test
2.1 body weight gains: the body weight gains of each test group all utmost point is significantly higher than control group (P＜0.01), test II group is the highest, but compare for 1 group with test, difference is remarkable (P＞0.05) not, two test group body weight gains improve 10.24% and 12.48% than control group respectively, illustrate that the gaining effect of each test group all is better than control group.Each stage gaining effect sees table 13 for details.
Table 13. is for examination chicken body weight statistical form unit: g
| Control group | Test group 1 | The test group II | ||
| 0～21 age in days | Starting weight | 44.13±1.14 | 44.25±0.57 | 44.13±1.21 |
| The end is heavy | 474.82±11.61 | 515.65±16.29 | 526.31±18.58 | |
| Weightening finish | 430.69±11.98 | 471.40±16.57 | 482.18±17.30 | |
| 22～42 ages in days | The end is heavy | 1654.09±45.18 | 1692.89±29.25 | 1760.41±50.14 |
| Weightening finish | 1179.27±44.75 | 1177.24±30.64 | 1234.10±53.13 |
2.2 feed intake: each organizes total feed intake difference of full phase not remarkable (P＞0.05), sees table 14 for details.
Table 14 feed intake statistical form unit: gram/only
| Control group | Test group 1 | The test group II | |
| 0～21 age in days | 918.78±19.85 | 899.44±14.90 | 750.28±28.83 |
| 22～42 ages in days | 3172.33±47.70 | 2715.76±106.84 | 3055.59±55.57 |
2.3 material anharmonic ratio, the death rate see Table 15
Table 15 material anharmonic ratio, mortality analysis table
| Group | Full phase weightening finish (gram/only) | Full phase feed consumption (gram/only) | The material anharmonic ratio | The death rate (%) |
| Test group 1 | 2073.68±89.83 | 4595.90±149.60 | 2.22±0.04 | 2.35 |
| The test group II | 2115.97±29.57 | 4580.09±47.27 | 2.17±0.04 | 1.65 |
| Control group | 1880.88±33.05 | 4519.75±40.76 | 2.40±0.04 | 9.65 |
Each test group material anharmonic ratio all is starkly lower than control group (P＜0.01), has reduced by 7.50% and 9.58% than control group respectively; Each test group of the death rate all significantly is lower than control group (P＜0.01), descends 75.65% and 82.90% than control group respectively; It is not remarkable to test between 1 group, test II group difference.
2.4 Economic and Efficiency Analysis
Test group 1: many feed consumptions: many feed consumptions (gram/only) (4595.9-4519.75) * feed price (unit/gram) (0.0028)=0.21322 yuan
Bacterium powder cost: full phase consumption bacterium powder (gram/only) (4595.90 * 0.1%) * bacterium powder price (unit/gram) (0.06)=0.2757 yuan
The weightening finish benefit: full phase weightening finish (gram/only) (2073.68-1880.88) * selling price (unit/gram) (0.01)=1.928 yuan
Every fryer can increase income more: weightening finish benefit-many feed consumptions cost-bacterium powder drops into (1.928-0.21322-0.2757)=1.439 yuan
3. conclusion
3.1 adding 1 ‰ poultry tailored version compound micro-ecological preparations in the Chinese mugwort denapon meat chick daily ration can make body weight gains improve 10.24%, expect that anharmonic ratio reduction by 7.50%, death rate of the onset descend 75.65%.
3.2 add 1 ‰ poultry tailored version compound micro-ecological preparation groups in the Chinese mugwort denapon meat chick daily ration and add 5mg/kg Wei Jiniya mycin group equal difference not significantly (P＞0.05) on indexs such as body weight gains, material anharmonic ratio, death rate of the onset.
Can make every chicken increase income 1.439 yuan 3.3 add 1 ‰ poultry tailored version compound micro-ecological preparations in the Chinese mugwort denapon meat chick daily ration, add 1 ‰ bacillus coagulanses in the Ai Weiyin meat chick daily ration and can make every chicken increase income 1.308 yuan.
Claims (3)
1. compound micro-ecological preparation, it is characterized in that comprising bacillus licheniformis CGMCC No.2383 bacterium powder, bacillus subtilis bacterium powder, saccharomyces cerevisiae CGMCC No.2388 bacterium powder, the weight proportion of above-mentioned each composition is: 1～3.5: 0.8～3.5: 0.8～3.5.
2. compound micro-ecological preparation as claimed in claim 1 is characterized in that further comprising carrier, and carrier is any one or two kinds of in stone flour, glucose, aluminum potassium sulfate, the maize cob meal.
3. the application of each described compound micro-ecological preparation of claim 1～2 in the preparation additive for farm animal feed.
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