CN101580799A - Micro-ecological preparation and application thereof - Google Patents
Micro-ecological preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a micro-ecological preparation and the application thereof, in particular to a micro-ecological preparation containing a variety of probiotics and the application thereof. The micro-ecological preparation contains any three or four of the components including CGMCC No. 2383 bacillus licheniformis powder, bacillus subtilis powder, CGMCC No. 2386 enterococcus faecalis powder, lactobacillus acidophilus powder, and CGMCC No. 2388 saccharomyces cerevisiae. The micro-ecological preparation has content of live bacteria, 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 carrier can be determined according to different kinds of animals and different animal growth phase. The micro-ecological preparation can improve the feed utilization efficiency, increase the yield of meat, eggs and milk, promote the growth of the animal, improve the immunity and the disease resistance of the animal, replace the antibiotic and improve the quality of the animal product.
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 probiotic bacterium, belong to the probiotics technical field.
Background technology
At present, prevention and medicine to Animal diseases substantially still adopt microbiotic, but owing to use microbiotic for a long time, in a large number, all drawbacks such as resistance, flora imbalance, immunity degradation, palindromia rate height, drug 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 microbiotic separately, as: European Union completely forbade from 2006 and uses microbiotic; The World Health Organization (WTO) has proposed " reducing the global principle that microbiotic 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 fodder additives of no drug residue is an inexorable trend.
Probiotics is commonly used at present and studies the fodder additives of more a kind of green, safety, this type of preparation is obtained through industrial fermentation production by the animal probiotics, include the active bacteria formulation that a lot of beneficial microorganisms and meta-bolites thereof constitute, directly feeding animals.The balance of little ecology plays a role probiotics in the enteron aisle by keeping, multiple function such as have diseases prevention, enhancing body immunizing power, promote to grow, put on weight, and pollution-free, noresidue, not developing immunity to drugs, is the fodder additives 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 meta-bolites of this bacterium generation are not enough to influence the host, are difficult to satisfy the treatment needs; (2) moisture content is higher, and this is one of important factor that influences the probiotics quality; (3) antibiotics 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 widespread use of probiotics.
Summary of the invention
The object of the present invention is to provide a kind of viable bacteria content height, moisture content 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 pathogenic bacteria is given birth to the multiple probiotic bacterium 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, subtilis bacterium powder, enterococcus faecalis (Enterococcus faecium) CGMCC No.2386 bacterium powder, Lactobacterium acidophilum bacterium powder, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the CGMCC No.2388 bacterium powder.
Wherein, described Bacillus licheniformis, enterococcus faecalis, yeast 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 pathogenic bacteria, for the applicant separates from healthy animal enteron aisle or ight soil, seed selection obtains, identify through Chinese agriculture microbial strains 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.
Lactobacterium acidophilum, 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 Lactobacterium acidophilum 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, 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 Lactobacterium acidophilum 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 corn 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 probiotic bacterium 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 Lactobacterium acidophilum 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 probiotics number reaches 2～5 * 10
9Cfu/g; Compound micro-ecological preparation comprises: Lactobacterium acidophilum 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 probiotics content 5～9 * 10
8Cfu/g; Compound micro-ecological preparation comprises: Bacillus licheniformis bacterium powder, 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 probiotics content 2～5 * 10
9Cfu/g; Compound micro-ecological preparation comprises: subtilis bacterium powder, Bacillus licheniformis bacterium powder, S. cervisiae lackey and corn 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 probiotic bacterium 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 probiotics content 5～9 * 10
8Cfu/g; Compound micro-ecological preparation comprises: Lactobacterium acidophilum 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 probiotics 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 probiotics number reaches 2～5 * 10
9Cfu/g; Compound micro-ecological preparation comprises subtilis bacterium powder, Bacillus licheniformis bacterium powder, S. cervisiae powder and corn 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 probiotic bacterium can reach 2～5 * 10
10Cfu/g.
Bacillus licheniformis, enterococcus faecalis, yeast saccharomyces cerevisiae and the subtilis of institute's seed selection and Lactobacterium acidophilum adopt deep liquid high density fermentation technology and spray to do in preparation or a series of aftertreatment technology of freeze-drying is made the bacterium powder among the present invention, viable bacteria content height in the preparation, moisture content 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 pathogenic bacteria and give birth to functions.
Compound micro-ecological preparation of the present invention can improve food utilization efficiency, increases the meat, eggs and milk equal yield line, promotes growth of animal, improves immunizing power and disease resistance, substitutes microbiotic, 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, yeast 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 liquid nutrient medium, 30～37 ℃ of constant temperature culture propagation;
2, separation and purification: step 1 is increased the bacterium culture on the proliferated culture medium flat board, carry out streak culture, therefrom picking each have the bacterium colony of characteristic morphology streak culture again with separate, till being pure bacterium colony, gram stain microscopy, to breed in the isolating doubtful pure colony inoculation test tube slant, then slant strains is preserved in 4 ℃ of refrigerators, stand-by;
Wherein said proliferated culture medium is a kind of in BPY substratum, MRS substratum, No. 98 substratum;
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, chemomorphosis method, modern genetic engineering technology, directive breeding goes out the bacterial classification of strong stress resistance; Doing strain identification 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 sizes 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 phosphoric acid buffer, carry out dull and stereotyped live bacterial count, calculate survival rate;
Wherein said seed culture medium is a kind of in BPY substratum, MRS substratum, No. 98 substratum;
2) bile tolerance is measured: slant strains is inoculated in the substratum, 30～37 ℃ leave standstill cultivation 8-24h activation, be inoculated in respectively by 5～10% inoculum sizes in the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% different concns, the 0h counting compares, 2h, 6h sampling is counted by 10 times of serial dilutions with physiological saline, carry out dull and stereotyped live bacterial count, 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) resistance is measured: adopt the drug susceptibility 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 proteinase activity to Bacillus licheniformis and measure;
6) produce acidity test: adopt ion chromatography that enterococcus faecalis CGMCC No.2386 is carried out production of organic acids and measure;
7) bacteriostatic test: adopt Oxford agar diffusion method (claiming cup-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%; Penicillin G, amoxycilline Trihydrate bp, spectinomycin, erythromycin, kitasamycin, Zinc-bacitracin, 7 kinds of antibiosis of lincomycin are have resistance; Intestinal bacteria K88, streptococcus aureus, three kinds of pathogenic bacterium of white dysentery Salmonellas 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%; Penicillin G, cefotaxime, gentamicin, spectinomycin, Xin Meisu, erythromycin, kitasamycin, Zinc-bacitracin, lincomycin, norfloxicin, Enrofloxacin, Ciprofloxacin, 13 kinds of antibiosis of compound sulfamethoxazole are have resistance; Intestinal bacteria K88, streptococcus aureus, three kinds of pathogenic bacterium of white dysentery Salmonellas are all had the good restraining effect, and antibacterial circle diameter is at 16～21mm; Lactic acid, acetate, the total acid yield of isopropylformic acid are 4.2g/L, and lactic acid yield reaches 89.4%.
The yeast 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 plate culture 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 size 12～16M is housed
3In the fermentor tank of fermention 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 rate of formation more than 90%, viable count is 1～2 * 10
10Cfu/ml, then stuck fermentation gets the lichen bacillus ferments liquid;
Described fermention medium is: wheat bran 1～2%, dregs of beans 1～2%, sodium-chlor 0.2～0.8%, sal epsom 0.01～0.05%;
5) preparation of Bacillus licheniformis bacterium powder: in the fermented liquid of step 4) preparation, add 20～25% weighting material, mixing, carry out spraying drying, 120～130 ℃ of inlet temperature, 40～50 ℃ of temperature of outgoing airs, spraying gun rotating speed 15000～18000rpm obtains the Bacillus licheniformis bacterium powder of moisture content＜5%.
Described weighting material 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 plate culture 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 substratum 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 lactobacillus first order seed of Pei Yanging is transferred in the 2.0L triangular flask that 1.2～1.6L MRS substratum 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 fermentor tank that 12～16M3 fermention medium is housed according to 3～6% inoculum size, 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 fermention medium is: glucose 1～2%, soy peptone 1～2%, ammonium sulfate 0.5～1.0%, sodium-chlor 0.2～0.8%, sal epsom 0.01～0.05%;
5) preparation of enterococcus faecalis bacterium powder: with the fermented liquid of step 4) preparation; 3500～5000rpm is centrifugal; obtain bacterium mud; the weight/volume percent of adding and bacterium mud is 15～20% lyophilized vaccine; mixing; in-25 ℃～-45 ℃ lyophilizes, obtain the enterococcus faecalis bacterium powder of moisture content＜5%.
Described lyophilized vaccine is: the mixture of skim-milk, glycerine, sucrose, maltodextrin and Sodium Glutamate, according to: skim-milk: glycerine: sucrose: maltodextrin: the mixed of Sodium Glutamate=2: 0.5～1.0: 0.5～1.0: 0.5～1.5: 0.5～1.0 forms.
The preparation method of C, yeast saccharomyces cerevisiae CGMCC No.2388 bacterium powder comprises following step:
1) dull and stereotypedly cultivate rejuvenation: the method that the yeast saccharomyces cerevisiae slant strains of refrigerator preservation is adopted streak inoculation streak inoculation on No. 98 plate culture mediums, cultivate 24～36h in 28～32 ℃, make the yeast 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 yeast saccharomyces cerevisiae of step 1) is transferred in the 500mL triangular flask that No. 98 substratum 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 yeast saccharomyces cerevisiae seed liquor is transferred in the 2.0L triangular flask that No. 98 substratum 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 size 15～16M is housed
3In the fermentor tank of fermention 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 fermention medium is: brown sugar 1～2%, soy peptone 1～2%, yeast extract paste 0.2～0.5%, sodium-chlor 0.5～1.0%, sal epsom 0.01～0.05%, dipotassium hydrogen phosphate 0.2～0.5%;
5) preparation of S. cervisiae powder: with the fermented liquid of step 4) preparation, 2000～3000rpm is centrifugal, obtain bacterium mud, the weight/volume percent of adding and bacterium mud is 10～15% lyophilized vaccine, in-25 ℃～-45 ℃ lyophilizes, obtain the S. cervisiae powder of moisture content＜5% behind the mixing;
Described lyophilized vaccine be skim-milk, glycerine, maltodextrin according to: 2: 0.5～1: 1～2 mixed form.
, 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.
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 liquid 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, on the BPY culture medium flat plate, carry out streak culture then, therefrom picking each have the bacterium colony of characteristic morphology streak culture again with separate, till being pure bacterium colony, gram stain microscopy, with breeding in the isolating doubtful pure colony inoculation test tube slant, be stored in then in 4 ℃ of refrigerators, stand-by;
Colonial morphology: the bacterium colony surface irregularity, gloss not, oyster white is opaque, and the edge is irregular, diameter 2-3mm, the prolongation along with incubation time becomes sorrel; 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: on the MRS culture medium flat plate, carry out streak culture to the step 1) enrichment medium, therefrom picking each have the bacterium colony of characteristic morphology streak culture again with separate, till being pure bacterium colony, gram stain microscopy, to breed in the isolating doubtful pure colony inoculation test tube slant, 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 yeast saccharomyces cerevisiae: on No. 98 culture medium flat plates, carry out streak culture to the step 1) enrichment medium, therefrom picking each have the bacterium colony of characteristic morphology streak culture again with separate, till being pure bacterium colony, gram stain microscopy, to breed in the isolating doubtful pure colony inoculation test tube slant, 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, chemomorphosis method, modern genetic engineering technology, directive breeding goes out the bacterial classification of strong stress resistance; And it is carried out strain identification, 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 CGM CC No.2386, yeast 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 yeast 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 size, the 0h counting compares, 2h, 6h sampling by 10 times of serial dilutions, is carried out dull and stereotyped live bacterial count with phosphoric acid 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, yeast 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 sodium hydroxide 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 stomach en-in every 10mL liquid.
The survival rate of table 13 strain bacterial classifications 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 size in the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% different concns, the 0h counting compares, 2h, 6h sampling is counted by 10 times of serial dilutions with physiological saline, carry out dull and stereotyped live bacterial count, 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, yeast 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% different concns.
Table 23 strain bacterial classifications are handled the survival rate of 6h in different concns 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 classifications are handled the survival rate (%) of different time under different high temperature
7) resistance is measured: adopt the drug sensitive test paper method.
The resistance measurement result of table 43 strain bacterial classifications
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 proteinase activity to Bacillus licheniformis and measure.
Bacillus licheniformis CGMCC No.2383 amylase activity is: 800u/ml; Proteinase activity is 1000u/ml.
9) produce acidity test: adopt ion chromatography that enterococcus faecalis CGMCC No.2386 fermented liquid 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 isopropylformic 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 bacterium: K99, streptococcus aureus, white dysentery Salmonellas, 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 medium 15～20mL of sterilization, horizontal positioned makes it to solidify, as bottom, other gets nutrient agar medium (being chilled to about 50 ℃) and an amount of (the 50mL substratum adds about the 8mL) mixing of the indicator liquid of 37 ℃ of 240h cultivations, draw 10mL and water on the bottom substratum, 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 substratum.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 thermostat containers, behind the cultivation 16-18h, take out and measure the inhibition zone size.
Table 53 strain bacterial classifications are 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 plate culture 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 size 16M is housed
3In the fermentor tank of fermention medium, 37 ℃ of temperature, rotating speed 220rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8, cultivate 20h, to gemma rate of formation more than 90%, viable count is 1～2 * 10
10Cfu/ml, then stuck fermentation gets the lichen bacillus ferments liquid;
Described fermention medium is: wheat bran 2%, dregs of beans 1%, sodium-chlor 0.8%, sal epsom 0.01%.
5) preparation of Bacillus licheniformis bacterium powder: the weighting material rice bran of adding 25% in the fermented liquid of step 4) preparation, mixing, carry out spraying drying, 120～130 ℃ of inlet temperature, 40～50 ℃ of temperature of outgoing airs, spraying gun rotating speed 15000～18000rpm obtains the Bacillus licheniformis bacterium powder of moisture content＜5%.
Described weighting material 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 plate culture 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 substratum 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 substratum 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 size 16M is housed
3In the fermentor tank of fermention medium, 37 ℃ of temperature, rotating speed 120rpm, tank pressure 0.05Mpa cultivates 16h, to viable count be 2 * 10
9Cfu/ml, stuck fermentation;
Described fermention medium is: glucose 2%, soy peptone 1%, ammonium sulfate 0.5%, sodium-chlor 0.2%, sal epsom 0.05%.
5) preparation of enterococcus faecalis bacterium powder: with the fermented liquid of step 4) preparation, 5000rpm is centrifugal, obtains bacterium mud, the weight/volume percent of adding and bacterium mud is 20% lyophilized vaccine, mixing in-45 ℃ of lyophilizes, obtains the enterococcus faecalis bacterium powder of moisture content＜5%;
Described lyophilized vaccine is: the mixture of skim-milk, glycerine, sucrose, maltodextrin and Sodium Glutamate, according to: skim-milk: glycerine: sucrose: maltodextrin: Sodium Glutamate=2: 1.0: 1.0: 1.5: 1.0 mixed forms.
The preparation of embodiment 5, yeast saccharomyces cerevisiae CGMCC No.2383 bacterium powder
1) dull and stereotypedly cultivate rejuvenation: the method that the yeast saccharomyces cerevisiae slant strains of refrigerator preservation is adopted streak inoculation streak inoculation on No. 98 plate culture mediums, cultivate 46h in 30 ℃, make the yeast 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 yeast saccharomyces cerevisiae of step 1) is transferred in the 500mL triangular flask that No. 98 substratum 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 yeast saccharomyces cerevisiae seed liquor is transferred in the 2.0L triangular flask that the 1.2L98 substratum 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 size 15～16M is housed
3In the fermentor tank of fermention 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 fermention medium is: brown sugar 1%, soy peptone 1%, yeast extract paste 0.2%, sodium-chlor 0.5%, sal epsom 0.01%, dipotassium hydrogen phosphate 0.2%.
5) preparation of S. cervisiae powder: with the fermented liquid of step 4) preparation, 2000pm is centrifugal, obtains bacterium mud, and the weight/volume percent of adding and bacterium mud is 10～15% lyophilized vaccine, in-25 ℃ of lyophilizes, obtain the S. cervisiae powder of moisture content＜5% behind the mixing;
Described lyophilized vaccine be skim-milk, glycerine, maltodextrin according to: mixed formed in 2: 0.5: 1.
The preparation of embodiment 6, compound micro-ecological preparation
The preparation of table 6 compound micro-ecological preparation
| Classification | Lactobacterium acidophilum bacterium powder (%) | Enterococcus faecalis bacterium powder (%) | Bacillus licheniformis bacterium powder (%) | 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 | Corn 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 | Corn 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.
Test 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 mean body 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 microbiotic Pro-gen 90 that the 100g/T prevention is had loose bowels on basal diet, test group III is for adding the microbiotic Pro-gen 90 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 trial 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, day weight gain, feedstuff-meat ratio, diarrhea rate with the repeating groups, and carry out otherness and significantly check.Test-results 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 test group III comparison according to component you can well imagine high by 7.3%, 7.5%, 9.5%; Test group I, test group II and test group III comparison reduces by 5.5%, 5.2%, 8.0% respectively according to group aspect feedstuff-meat ratio; Test group I, test group II and test group III comparison reduces by 67.3%, 47.7%, 70.1% respectively according to group aspect diarrhea rate.Test group I compares aspect day weight gain, feedstuff-meat ratio difference with test group II not remarkable, but aspect prevention diarrhoea significant difference, illustrate that piglet tailored version compound micro-ecological preparation can substitute the microbiotic that prevention is suffered from diarrhoea in the feed.Test group III is being better than test group I, test group II and control group aspect day weight gain, feedstuff-meat ratio and the diarrhoea, illustrate that piglet tailored version compound micro-ecological preparation and microbiotic 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 day weight 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 |
| 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 |
| 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 |
| 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, test-results and analysis
In trial 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 surviving rate is apparently higher than control group, and its surviving 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, food consumption 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 lactasinum 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 fodder additives, 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 surviving 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 nutritive ingredient 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 nutritive ingredient
| Composition | Content (%) | Nutritive ingredient | |
| 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(Met) (%) | 0.738 |
| Zein powder | 2.0 | Methionine(Met)+Gelucystine (%) | 0.676 |
| The alcohol protein powder | 5.0 | ||
| Stone flour | 5.8 | ||
| Zeolite | 1.0 | ||
| Secondary calcium phosphate | 1.8 | ||
| Preblend | 5 |
Annotate: in every kilogram of daily ration: retinol1 2000IU, Vitamin D3 500,000 I.U/GM 1500IU, vitamin E2 5IU, vitamin K3 1.0mg, VitB1 5.5mg, riboflavin 5.0mg, pantothenic acid 16mg, vitamin B6 8.0mg, vitamin H 0.3mg, choline 500mg, folic acid 1.8mg, vitamins B 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, mortality ratio, food consumption 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
Test-results 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 laying hen mortality ratio 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/ | Daily consumption g/ only | Feedstuff-egg ratio | Laying rate % | Breakage rate % | Mortality ratio % |
| 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 mortality ratio of laying hen;
(3) add the immunological competence 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 vaccination cause.
In sum, probiotics of the present invention adds in the feed, to guarantee the chicken state of health, 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 materials 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 trial periods.
Test design: contrast control group with virginiamycin as microbiotic: 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 |
| Secondary calcium phosphate | 1.4 | 1.4 |
| Salt | 0.3 | 0.3 |
| Soya-bean oil | 3.5 | 3.3 |
| Preblend | 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, test-results
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 cartogram unit: g
| Control group | Test group 1 | 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 food consumption: each organizes total food consumption difference of full phase not remarkable (P＞0.05), sees table 14 for details.
Table 14 food consumption cartogram unit: gram/only
| Control group | Test group 1 | 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, mortality ratio 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 | Mortality ratio (%) |
| Test group 1 | 2073.68±89.83 | 4595.90±149.60 | 2.22±0.04 | 2.35 |
| 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 7.80% and 9.58% than control group respectively; Each test group of mortality ratio 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 broiler chicken 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 broiler chicken 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 broiler chicken daily ration, add 1 ‰ Bacillus coagulanses in the Ai Weiyin broiler chicken daily ration and can make every chicken increase income 1.308 yuan.
Claims (7)
1, a kind of compound micro-ecological preparation is characterized in that comprising wantonly three kinds or four kinds in Bacillus licheniformis CGMCC No.2383 bacterium powder, subtilis bacterium powder, enterococcus faecalis CGMCC No.2386 bacterium powder, Lactobacterium acidophilum bacterium powder, the yeast saccharomyces cerevisiae CGMCC No.2388 bacterium powder.
2, compound micro-ecological preparation as claimed in claim 1, it is characterized in that comprising Lactobacterium acidophilum bacterium powder, enterococcus faecalis bacterium powder, Bacillus licheniformis bacterium powder, S. cervisiae powder, the weight proportion of above-mentioned each composition is 1: 0.8～1.2: 0.8～2.5: 1.5～2.5.
3, compound micro-ecological preparation as claimed in claim 1 is characterized in that comprising Bacillus licheniformis bacterium powder, 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.
4, compound micro-ecological preparation as claimed in claim 1 is characterized in that comprising 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.
5, compound micro-ecological preparation as claimed in claim 1 is characterized in that comprising Lactobacterium acidophilum 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.
6, 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 corn cob meal.
7, the application of each described compound micro-ecological preparation of claim 1～6 in the preparation additive for farm animal feed.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Effective date of registration: 20220825 Address after: 100080, Zhongguancun building, No. 27 Zhongguancun street, Beijing, Haidian District, China, 14 floor Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: Kunming yunzhongmei agriculture and animal husbandry technology Co.,Ltd. Address before: 100083, Zhongguancun building, No. 27 Zhongguancun street, Beijing, Haidian District, China, 14 floor Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |