CN102409015B - Composite micro-ecological preparation as well as premixed material and application of preparation in feed additive - Google Patents
Composite micro-ecological preparation as well as premixed material and application of preparation in feed additive Download PDFInfo
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Abstract
The invention belongs to the technical field of feed micro-ecology and relates to a composite micro-ecological preparation as well as a premixed material and an application of the preparation in a feed additive. The preparation contains Lactobacillus acidophilus powder and any two, three or four bacterium powders selected from Bacillus licheniformis, Bcillus pumilus, enteroccus and Bcillus subtilis; the preparation has high live bacteria content and low moisture content, has stress tolerance properties such as gastric acid resistance, cholate resistance, high temperature resistance, common antibiotics tolerance and the like and has the probiotic functions of producing acid and enzyme, inhibiting pathogenic bacteria and the like. By using the composite micro-ecological preparation of the invention, the utilization rate of the feed can be improved, the yields of meat, eggs, milk and the like are increased, the growth of animals is promoted, the immunity and disease-resistant capability of an animal organism are improved, and partial preparation can be used for replacing antibiotics, thereby changing the quality of an animal product.
Description
Technical field
The invention belongs to feeding micro-ecological technical field, relate to a kind of compound micro-ecological preparation and the application in fodder additives and Preblend.
Background technology
At present, prevention to Animal diseases and medicine substantially still adopt microbiotic, but due to for a long time, use microbiotic in a large number, all drawbacks such as the enhancing of pathogenic bacterium resistance, flora imbalance, immunity degradation, recurrence rate are high, drug residue are day by day serious, simultaneously also serious harm animal body and human health.Under this situation, change cultivation idea, prevent to overweight treatment, and Application and Development is nontoxic, the novel green fodder additives of no drug residue is inexorable trend.
Probiotics chamber commonly uses and studies the fodder additives of more a kind of green, safety at present, this type of preparation is produced and is obtained through industrialization high density fermentation with animal probiotics, include the active bacteria formulation that a lot of beneficial microorganisms and meta-bolites thereof form, can Direct-fed animal.Probiotics plays a role by maintaining microecological balance in enteron aisle, the several functions such as there is diseases prevention, enhancing body immunizing power, promote grow, put on weight, and pollution-free, and noresidue, does not develop immunity to drugs, and is a kind of green, safe fodder additives.
Micro-ecological product on market is of a great variety, and quality is uneven, mainly has following problem: (1) viable bacteria content is low, and research shows, if the concentration of a kind of bacterium in cecal content is less than 10
7cfu/g, enzyme and meta-bolites thereof that this bacterium produces are not enough to affect host, are difficult to meet treatment needs; (2) moisture content is higher, makes like this quality guaranteed period of probiotics greatly shorten, and is also one of major reason affecting probiotics poor quality; (3) antibiotics resistant not; (4) unstable to hydrochloric acid in gastric juice and cholate, cause after only having minority bacterial strain to enter enteron aisle and still there is activity, do not reach the required number of viable that plays a role.Due to these quality problems of probiotics existence, thereby affect its effect in detoxification, limited the widespread use of probiotics.
Summary of the invention
The object of the present invention is to provide a kind of compound micro-ecological preparation that comprises multiple brand-new probiotic bacterium, this preparation comprise there is stomach juice-resistant, bile tolerance, the high temperature resistant and tolerance anti-adversity such as common antibiotics and produce acid, produce enzyme and suppress the multiple probiotic bacterium of the prebiotic functions such as pathogenic bacteria, in preparation, each probiotics viable bacteria content is high, moisture content is low.
Another object of the present invention is to provide this compound micro-ecological preparation in the application of preparing in poultry and livestock feed additive.
The present invention also provides the Preblend that comprises this compound micro-ecological preparation.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of compound micro-ecological preparation, said preparation contains wantonly two kinds, three kinds or the bacterium powder of four kinds in Lactobacterium acidophilum (Lactobacillus acidophilus) CGMCC No.5093 bacterium powder and Bacillus licheniformis (Bacillus licheniformis) CGMCC No.5094, bacillus pumilus (Bacillus pumilus) CGMCC No.4756, enterococcus faecalis (Enterococcus faecalis) CGMCC No.5092, tetra-kinds of bacterium of subtilis (Bacillus subtilis) CGMCC No.4628.
Wherein, in compound micro-ecological preparation of the present invention, contain Lactobacterium acidophilum CGMCC No.5093 bacterium powder, Bacillus licheniformis CGMCC No.5094 bacterium powder, bacillus pumilus CGMCC No.4756 bacterium powder and enterococcus faecalis CGMCC No.5092 bacterium powder, the weight proportion of above-mentioned four kinds of bacterium powder is 0.8-2.5: 1: 0.8-1.2: 1.5-2.5, can be made into piglet tailored version compound micro-ecological preparation, total probiotics content is 6-10 × 10
9cfu/g.
Wherein, in compound micro-ecological preparation of the present invention, contain Lactobacterium acidophilum CGMCC No.5093 bacterium powder, Bacillus licheniformis CGMCC No.5094 bacterium powder and subtilis CGMCC No.4628 bacterium powder, the weight proportion of above-mentioned three kinds of bacterium powder is 0.8-3.5: 1-3.5: 0.8-2.5, can be made into feed factory sow product specific complex type probiotics, probiotic bacterium quantity is in 3-6 × 10
10cfu/g.
Wherein, in compound micro-ecological preparation of the present invention, contain Lactobacterium acidophilum CGMCC No.5093 bacterium powder, Bacillus licheniformis CGMCC No.5094 bacterium powder and enterococcus faecalis CGMCC No.5092 bacterium powder, the weight proportion of above-mentioned three kinds of bacterium powder is 1.5-2.5: 0.8-1.2: 1, can be made into laying hen specific complex type probiotics, probiotic bacterium quantity is in 2-5 × 10
9cfu/g.
Wherein, in compound micro-ecological preparation of the present invention, contain Lactobacterium acidophilum CGMCC No.5093 bacterium powder, bacillus pumilus CGMCC No.4756 bacterium powder and enterococcus faecalis CGMCC No.5092 bacterium powder, the weight proportion of above-mentioned three kinds of bacterium powder is 2-2.5: 2-2.5: 1-1.5.
For better reaching invention effect of the present invention, the preparation method of the Lactobacterium acidophilum CGMCC No.5093 bacterium powder described in compound micro-ecological preparation of the present invention is as follows: by the inoculum size of 1-2% to the Lactobacterium acidophilum seed liquor that accesses 8h-12h cell age in fermention medium, fermentation condition is: 30-40 ℃, rotating speed is 60-100rpm, cultivate 12-16h, obtain fermented liquid, fermented liquid is centrifugal, obtain bacterium mud, the lyophilized vaccine that to add with the percent weight in volume of bacterium mud be 15-20%, mix, in-25 ℃--45 ℃ of lyophilizes, obtain, composition and the mass volume ratio thereof of described fermention medium are as follows: glucose 1-2%, soy peptone 1-2%, yeast extract paste 1-2%, ammonium sulfate 0.5-1.0%, dipotassium hydrogen phosphate 0.2-0.5%, sodium-chlor 0.2-0.8%, magnesium sulfate 0.01-0.05%, the preparation method of described Bacillus licheniformis CGMCC No.5094 bacterium powder is as follows: by the inoculum size of mass volume ratio 0.5-2% to the Bacillus licheniformis seed liquor that accesses 10-14h cell age in fermention medium, fermentation condition is: 30-40 ℃, rotating speed is 200-300rpm, cultivate 15-22h, obtain fermented liquid, in fermented liquid, adding mass volume ratio is the weighting material maltodextrin of 20%-25%, mix, spray dry, inlet temperature 140-160 ℃, temperature of outgoing air 40-50 ℃, spraying gun rotating speed 15000-18000rpm, obtain, composition and the mass volume ratio thereof of described fermention medium are as follows: wheat bran 1-2%, Semen Maydis powder 0.5-1.5%, dregs of beans 1-2%, ammonium sulfate 0.5-2%, magnesium sulfate 0.01-0.05%, ammonium citrate 0.3-1.2%, defoamer 0.05-0.1%, pH is 6.2-7.4, the preparation method of described bacillus pumilus CGMCC No.4756 bacterium powder is as follows: by the inoculum size of mass volume ratio 1-2% to the bacillus pumilus seed liquor that accesses 12h-16h cell age in fermention medium, fermentation condition is: 30-40 ℃, rotating speed is 100-300rpm, cultivate 16-24h, obtain fermented liquid, in fermented liquid, adding mass volume ratio is the weighting material W-Gum of 20-25%, mix, spray dry, inlet temperature 150-170 ℃, temperature of outgoing air 40-70 ℃, spraying gun rotating speed 15000-20000rpm, obtain, composition and the mass volume ratio thereof of described fermention medium are as follows: glucose 1-2%, soy peptone 1-2%, dregs of beans 1-2%, ammonium sulfate 0.5-1.0%, sodium-chlor 0.2-0.8%, magnesium sulfate 0.01-0.05%.
Further, probiotics of the present invention also comprises carrier, and carrier is any or two kinds in maltodextrin, aluminum potassium sulfate, corn cob meal, glucose, oily chaff powder.
Compound micro-ecological preparation of the present invention can be used for preparing poultry and livestock feed additive.
The present invention also provides the Preblend that comprises this compound micro-ecological preparation, and wherein, the mass volume ratio that in Preblend, compound micro-ecological preparation adds is 0.5-10 ‰.
Bacillus licheniformis of the present invention (Bacillus licheniformis) separates from Roll road for applicant, through directed primary dcreening operation and multiple sieve, obtains.Its vegetative cell is shaft-like, and its gemma is oval or long tubular, is middle life or the life of inferior end, and sporangiocyst slightly expands.Isolated or be short chain, rod end semicircle.In 12～16h, can form bacterium colony, bacterium colony is circular, or irregular, and edge is hair-like, and bacteria colony white is opaque, corrugationless.Gram-positive.Applicant has carried out the experiments such as acid resistance, bile tolerance, high thermal resistance, fermentation character and bacteriostatic activity to this bacillus licheniformis, this bacillus licheniformis has the features such as strong to contrary environmental resistance, adhesivity strong, growth fast, biomass is large, bacteriostatic activity is good.Bacillus licheniformis of the present invention (Bacillus licheniformis) applicant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2011, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.5094.Enterococcus faecalis of the present invention (Enterococcus faecalis) obtains for applicant separates from Radix Polygalae Crotalarioidis, and its biological property is as follows: bacterium colony rounding, and smooth surface, opaque, oyster white, the smooth of the edge, gram-positive microorganism; Single thalline is spherical or ellipticity.Enterococcus faecalis of the present invention is through simulated gastric fluid, bile fluid and stable on heating screening; Again through enzymatic productivity screening and bacteriostasis screening, can tolerate pH2.0,1% pepsic simulated gastric fluid, can tolerate 0.3% artificial bile fluid, can tolerate the pelleting temperature of 85 ℃, also can suppress pathogenic colon bacillus K88, K99 and streptococcus aureus, have stronger product acid and the ability that suppresses mould in dregs of beans, cotton dregs, maize straw.Enterococcus faecalis of the present invention (Enterococcus faecalis) applicant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2011, be called for short CGMCC, address is the same, and deposit number is CGMCC No.5092.Lactobacterium acidophilum of the present invention (Lactobacillus acidophilus) obtains for applicant separates from swine excrement, and its biological property is as follows: bacterium colony rounding, and smooth surface, opaque, oyster white, the smooth of the edge, gram-positive microorganism; Single thalline is shaft-like.Lactobacterium acidophilum of the present invention is through the screening of simulated gastric fluid, bile fluid and resistance; Can tolerate pH2.0,1% pepsic simulated gastric fluid, can tolerate 0.3% artificial bile fluid, also can suppress pathogenic colon bacillus K88, and dysentery bacterium and streptococcus aureus have stronger product acid and bacteriostasis.Lactobacterium acidophilum of the present invention (Lactobacillus acidophilus) applicant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2011, be called for short CGMCC, address is the same, and deposit number is CGMCC No.5093.Bacillus pumilus of the present invention (Bacillus pumilus) strain being separated at home first for applicant derives from the new bacillus pumilus of animal digestive tract, for applicant separates the bacillus dominant strain obtaining from the cud of ox, through Physiology and biochemistry and 16s RNA Analysis and Identification, show that this Pseudomonas is in bacillus pumilus, its Classification And Nomenclature is bacillus pumilus (Bacillus pumilus), on April 8th, 2011, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is the same, be called for short CGMCC, deposit number is CGMCC No.4756.The present invention subtilis (Bacillus subtilis) used obtains for applicant separates from traditional zymotic fermented soya bean, and its biological property is as follows: bacterium colony surface irregularity is opaque, dirty white, bacterium colony circle, edge indentation, gram-positive microorganism; Gemma form is oval to column, is positioned at thalline central authorities or slightly inclined to one side, and after sporulation, thalline does not expand.Subtilis of the present invention is significantly different from existing subtilis, can tolerate pH2.0,1% pepsic simulated gastric fluid, can tolerate 0.3% artificial cholate, can tolerate the pelleting temperature of 80 ℃, can suppress pathogenic colon bacillus K88, K99 and streptococcus aureus, there is the ability of stronger cellulase-producing, can degraded cellulose.Subtilis of the present invention (Bacillus subtilis) has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 2nd, 2011, be called for short CGMCC, and address is the same, and deposit number is CGMCC No.4628.
In the present invention, Bacillus licheniformis, enterococcus faecalis, subtilis, Lactobacterium acidophilum and the bacillus pumilus of institute's seed selection adopt deep liquid high density fermentation technology and spray a series of aftertreatment technologies such as dry or lyophilize and make bacterium powder in preparation, viable bacteria content in preparation is high, moisture content is low, has the anti-adversities such as stomach juice-resistant, bile tolerance, high temperature resistant and tolerance common antibiotics and produces acid, produces enzyme and suppress the prebiotic functions such as pathogenic bacteria.Compound micro-ecological preparation of the present invention can improve efficiency of feed utilization, increases meat, eggs and milk equal yield line, promotes growth of animal, improves animal body immunizing power and resistance against diseases, part substitute antibiotics, thereby the quality of change animal product.
By the following example, by more specific description the present invention, it should be understood that described embodiment is only for the present invention is described, rather than limit the scope of the invention in any form.
Embodiment
Separation, screening, seed selection and the evaluation of embodiment 1, excellent species
1) increasing bacterium cultivates: the fresh excreta of healthy animal (pig or poultry), intestinal contents sample are inoculated in to BPY liquid culture and concentrate, at 37 ℃ of constant temperature culture propagation 48h;
2) separation and purification of Bacillus licheniformis and bacillus pumilus: to step 1) 100 ℃ of water-bath 5min of increasing bacterium culture, then on BPY culture medium flat plate, carry out streak culture, therefrom each bacterium colony with characteristic morphology of picking is streak culture and Fen Li again, until be pure bacterium colony, gram stain microscopy, by breeding in the doubtful pure colony inoculation test tube slant separating, be then stored in 4 ℃ of refrigerators, stand-by;
Colonial morphology: bacterium colony surface irregularity, tarnish, oyster white is opaque, and edge is irregular, and diameter 2-3mm, along with the prolongation of incubation time, becomes sorrel; Microscopy: cell is bacillus 1.6-3.8 μ m × 0.6-1.2 μ m, Gram-positive, raw in gemma, ellipse, does not expand, and is tentatively defined as genus bacillus;
By the bacterium of the doubtful genus bacillus of these several strains is carried out to 16SrRNA evaluation, determine two kinds of Bacillus licheniformis and bacillus pumilus.
3) separation and purification of Lactobacterium acidophilum: to step 1) enrichment medium on MRS culture medium flat plate, carry out streak culture, by separate doubtful pure colony inoculation inclined-plane in breed, be then stored in 4 ℃ of refrigerators, stand-by;
Colonial morphology: bacterium colony rounding, smooth surface, glossy, oyster white is opaque, diameter 0.5-1.2mm, along with time lengthening, colour-darkening jaundice, bacterium Gram-positive, single thalline elongated rod-shaped.Through identifying, it is Lactobacterium acidophilum.
4) screening of enterococcus faecalis CGMCC No.5092:
A, primary dcreening operation
The each 5g of sample such as sample thief pig manure, cow dung and chicken manure, are soaked in aseptic 0.85% physiological saline of 20ml, and sample is broken up in concussion, make sample fully and contact with normal saline, standing 3 minutes of 70 ℃ of thermostat water baths, get supernatant, centrifugal 10 minutes of 5000rpm, supernatant discarded.Bacterial sediment after centrifugal is added in 10ml simulated gastric fluid, 37 ℃ of standing 2h, then centrifugal, collect after thalline, bacterial sediment is added in the artificial cholate of 10ml, 37 ℃ of standing 4h, collect thalline, and dilution spread is in LB substratum.37 ℃ of incubators are cultivated after 12-24h, and bacterium colony is carried out to purifies and separates.The bacterium obtaining is preserved, and further sieve again.
The compound method of physiological saline is: 0.85% sodium chloride solution, and standby after autoclaving.
The compound method of simulated gastric fluid is: 1% stomach en-, and 0.85% sodium-chlor, with concentrated hydrochloric acid adjust pH to 2.0, standby after biofilter filtration sterilization.
The compound method of artificial cholate is: in liquid meat soup, add 0.3% pig cholate (analytical pure), and standby after autoclaving.
The making method of LB solid medium is: peptone 10g, and yeast extract paste 5g, sodium-chlor 10g, agar 1.5%, pH7.0, standby after autoclaving.
B, multiple sieve
(1) the LB substratum temperature of the bacterium that configures, gone out is cooled to 45 ℃, add intestinal bacteria K88, K99 and the streptococcus aureus (every milliliter of substratum adds 1 microlitre bacterium liquid) of incubated overnight, concussion mixes, pour aseptic flat board into, after level is solidified, each flat board utilizes four Oxford cups to punch, in every Oxford cup, add 200 μ l bacterium liquid to be measured, build ware lid, carefully move to 37 ℃ of incubators, plate is just being put standing cultivation.Cultivate after 20h, open ware lid, remove Oxford cup, use kind of calliper antibacterial circle diameter.
5) screening of subtilis CGMCC No.4628
A, primary dcreening operation
Each 5 grams of the samples such as natto, fermented soya bean, fermented bean curd, pig manure, chicken manure, cow dung, are soaked in 20ml sterilizing 0.85% physiological saline, and sample is broken up in concussion, make sample fully and contact with normal saline, standing 10 minutes of 90 ℃ of thermostat water baths, get supernatant 5ml, centrifugal 10 minutes of 5000rpm, supernatant discarded.Bacterial sediment adds in 10ml simulated gastric fluid, and 37 ℃ standing 2 hours.Centrifugal 10 minutes of 5000rpm, supernatant discarded, adds bacterial sediment in the artificial cholate of 10ml, and 37 ℃ are standing 4 hours.After concussion mixes, gradient dilution is coated with LB substratum.30 ℃ of incubators were cultivated after 12-24 hour, purifying bacterium colony.Obtain isolated N-1 in natto sample, N-2 bacterium, isolated C-1 in fermented soya bean sample (CGMCC No.4628), C-2 bacterium, in fermented bean curd sample, be not separated to bacterium colony, in pig manure, isolate Z-1, Z-2 bacterium, in chicken manure, isolate J-1, J-2 bacterium, in cow dung, isolate F-1, F-2 bacterium.The bacterial strain that primary dcreening operation obtains carries out further multiple sieve again.
The collocation method of physiological saline is: 0.85% sodium-chlor, and standby after autoclaving.
The compound method of simulated gastric fluid is: 1% stomach en-, and 0.85% sodium-chlor, with concentrated hydrochloric acid adjust pH to 2.0, biofilter filters standby.
The collocation method of artificial cholate is: in liquid meat soup, add 0.3% pig cholate (analytical pure), and standby after autoclaving.
The making method of LB solid medium is: peptone 10g, and yeast extract paste 5g, sodium-chlor 10g, agar 1.5%, pH7.0, standby after autoclaving.
B, multiple sieve
(1) screening of cellulase-producing ability
Bacterial strain separation and purification being gone out with aseptic toothpick carries out a bacterium on the LB-CMC of 0.5% carboxymethyl cellulose substratum, cultivate 24h for 30 ℃, with 0.1% congo red staining 1 hour, discard dyestuff, use again 1M NaCl solution washing 1 hour, according to periphery of bacterial colonies, have or not and occur hydrolysis transparent circle, measure transparent circle and colony diameter, calculate transparent circle diameter and colony diameter ratio (H/C), the hydrolysis transparent circle of finding N-1, C-1 (CGMCC No.4628), C-2 is larger, the wherein hydrolysis transparent circle maximum (table 1) of C-1.
The collocation method of LB-CMC substratum is: carboxymethyl cellulose 0.5%, tryptone 10g, yeast extract 5g, sodium-chlor 10g, autoclaving 20min.
Table 1 is hydrolyzed transparent circle and colony diameter ratio (M/C)
The resistance of embodiment 2, excellent species and the mensuration of biology performance
1, the biology performance of Bacillus licheniformis, bacillus pumilus and Lactobacterium acidophilum and resistance are measured:
1) preparation of the culture of Bacillus licheniformis: the slant strains of refrigerator preservation is inoculated in BPY seed culture medium and is activated, and 37 ℃, 200rpm are cultivated 18h, obtain the culture of gemma rate more than 95%.
2) preparation of the culture of bacillus pumilus: the slant strains of preservation in refrigerator is inoculated in BPY seed culture medium and is activated, and 35 ℃, 100rpm are cultivated 16h, obtain;
3) lactobacillus acidophilus cultures's preparation: the slant strains of preservation in refrigerator is inoculated in MRS seed culture medium and is activated, and 37 ℃, standing cultivation 16h, obtain;
4) acid resistance is measured: the culture of above-mentioned preparation is inoculated in respectively by 5% inoculum size in the artificial simulation gastric juices of pH value 2.0,3.0,4.0,0h counting compares, 2h, 6h sampling by 10 times of serial dilutions, is carried out dull and stereotyped viable bacteria technology with phosphoric acid buffer, calculates survival rate.
3 strain bacterial classifications of seed selection of the present invention: Bacillus licheniformis, bacillus pumilus, Lactobacterium acidophilum in hydrochloric acid in gastric juice survival results in Table 1.
The preparation of artificial simulation gastric juices: measure 9.5%-10.5% concentrated hydrochloric acid 16.4mL, 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 test tube, steam sterilizing 15 minutes at 100 ℃, under aseptic condition, in every 10ml liquid, add 0.100g stomach en-, its test-results is as shown in table 2.
The survival rate of table 2 three strain bacterial classifications in artificial simulation gastric juices
5) bile tolerance is measured: by the culture of 3 strain bacterial classifications of above-mentioned preparation, by 5% inoculum size, be inoculated in respectively in the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% different concns, 0h counting compares, 2h, 6h sampling is counted by 10 times of serial dilutions with physiological saline, carry out viable plate count, calculate survival rate.
3 strain bacterial classifications of institute of the present invention seed selection in cholate survival results in Table 3.
The preparation of cholate: in 0.85 physiological saline each 9 milliliters, be sub-packed in test tube, steam sterilizing 30 minutes at 121 ℃, under aseptic condition, make 0.03%, 0.1%, 0.2%, the pig cholate solution of 0.3 different concns, 3 kinds of bacterial classifications are processed survival results after 6h in Table 3 in different concns pig cholate.
Table 33 strain bacterial classifications are processed the survival rate of 6h in different concns pig cholate
6) high temperature resistant mensuration: by the culture of 3 strain bacterial classifications of above-mentioned preparation, respectively at 50 ℃, 60 ℃, 70 ℃, 80 ℃ water bath processing 15min, 30min, calculate survival rate, result is as shown in table 4.
Three kinds of bacterial classifications of table 4 are processed the survival rate (%) of different time under different high temperature
7) bacteriostatic test: adopt Oxford agar diffusion method to carry out bacteria inhibition assay to common pathogen.
A, in the test tube that 10ml nutrition bouillon media is housed, activate 4 strain pathogenic bacterium: K88, 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 sterilizing, horizontal positioned makes it to solidify, as bottom, separately getting the indicator liquid appropriate (50ml substratum adds 8ml left and right) that nutrient agar medium (being chilled to 50 ℃ of left and right) and 37 ℃, 24h cultivate mixes, draw 10ml and water on bottom substratum, horizontal positioned makes it to solidify, as bacterium layer;
C, add sample: with the sterilized Oxford of aseptic nipper gripping cup, open ware lid, be placed on 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, cultivate after 16-18h, take out and measure inhibition zone size, result is as shown in table 5.
The fungistatic effect of table 53 strain bacterial classifications to common pathogen
2, the checking of enterococcus faecalis resistance
(1) high thermal resistance
By cultivating the enterococcus faecalis of 12h, carry out centrifugal collection, insert in physiological saline, carry out resuspended after, sampling coating, meter viable count; Again physiological saline is heated in the water-bath of 70 ℃ after ten minutes, cooling rapidly, then carry out plate count, remaining viable count is 85.3%.
(2) tolerance of simulated gastric fluid
Get the bacterium liquid that 0.5ml cultivates 12h enterococcus faecalis, be dissolved in 4.5ml physiological saline, with colony counting method meter viable count.Get again quantitative above-mentioned bacterium liquid and add in the artificial bile salt culture-medium of 4.5ml, 37 ℃ of standing 2h, the remaining viable count of colony counting method meter, remaining viable count is 92.2%.
(3) tolerance of artificial bile fluid
Get the bacterium liquid that 0.5ml cultivates 12h enterococcus faecalis, be dissolved in 4.5ml physiological saline, with colony counting method meter viable count.Then separately get the above-mentioned bacterium liquid of 0.5ml and add in the artificial bile fluid substratum of 4.5ml, 37 ℃ of standing 2h, the remaining viable count of the method for plate culture count meter, remaining viable count is 86.2%.
Result: above several experiments show, the resistance of the enterococcus faecalis being separated to from Radix Polygalae Crotalarioidis in the present invention is stronger, can tolerate higher temperature, tolerance simulated gastric fluid, artificial cholate, is therefore used as fodder additives, can guarantee that a large amount of thalline is smoothly by the enteron aisle of animal, in enteron aisle, survive, bring into play its prebiotic function.
3, the prebiotic function of enterococcus faecalis (CMGCC No.5092)
(1) bacteria resistance function checking
The pathogenic colon bacillus K88 of incubated overnight, K99 and streptococcus aureus bacterium liquid are counted with colony counting method.In intestinal bacteria K88, the K99 of 3 pipe 5ml and streptococcus aureus, add 5ml enterococcus faecalis bacterium liquid respectively.Cultivate 4h for 37 ℃, the method for plate culture count calculates remaining intestinal bacteria K88, K99 and the viable count of streptococcus aureus.The viable count of remaining pathogenic colon bacillus K88, K99 and streptococcus aureus is 24.7%, 30.2% and 31.8%.
(2) checking of mould fungus inhibition in dregs of beans or Semen Maydis powder
Get the bacterium liquid 20ml of the enterococcus faecalis of cultivating 12h, evenly mix with 30g Semen Maydis powder or dregs of beans, blank assay is that 20ml sterilized water and 30g Semen Maydis powder or dregs of beans carry out intimate mixing, and then at 3d, 5d, 8d and 10d, the growing state of the corn to two bottles of the insides or dregs of beans and mould is observed respectively.Show by experiment, after 5 days, do not add in the Semen Maydis powder of enterococcus faecalis bacterium liquid and dregs of beans and all have mould filament length to go out, and add the Semen Maydis powder of enterococcus faecalis bacterium liquid and the color of dregs of beans still fresh, decompose to some extent but without the appearance of mould bacterial plaque or mould mycelia.
4, the checking of subtilis CGMCC No.4628 resistance
(1) tolerance of granulation high temperature
0.5g bacterium powder is dissolved in the glass test tube of 4.5mL sterile saline, calculates viable count with the method for plate culture count.Glass test tube is heated after 10 minutes in 90 ℃ of water-baths, cool to room temperature rapidly, the method for plate culture count calculates remaining viable count, and remaining viable count is 95.4%.
(2) tolerance of simulated gastric fluid
0.5g bacterium powder is dissolved in the glass test tube of 4.5mL sterile saline, with the method for plate culture count meter viable count.Get the above-mentioned bacterium liquid of 0.5mL and add in 4.5mL simulated gastric fluid, 37 ℃ standing 2 hours, the remaining viable count of the method for plate culture count meter, remaining viable count is 93.7%.
(3) tolerance of artificial cholate
0.5g bacterium powder is dissolved in the glass test tube of 4.5mL sterile saline, with the method for plate culture count meter viable count.Get the above-mentioned bacterium liquid of 0.5mL and add in the artificial bile salt culture-medium of 4.5mL, 37 ℃ standing 2 hours, the remaining viable count of the method for plate culture count meter, remaining viable count is 97.7%.
Replication experiment shows the strong stress resistance of subtilis CGMCC No.4628 of the present invention, can tolerate granulation high temperature, tolerance simulated gastric fluid, artificial cholate, therefore through feed granulating, after animal gastrointestinal tract environment, also have a large amount of viable bacterias can enter smoothly the enteron aisle of animal, in enteron aisle, survive, bring into play prebiotic function.
5, the prebiotic functional verification of subtilis CGMCC No.4628
(1) bacteria resistance function checking
Pathogenic colon bacillus K88, the K99 of incubated overnight and streptococcus aureus bacterium liquid are counted with the method for plate culture count.In intestinal bacteria K88, the K99 of 3 pipe 4.5ml and streptococcus aureus bacterium liquid, add respectively the bacterium powder of 0.5g subtilis CGMCCNo.4628, cultivate 4 hours for 37 ℃, the method for plate culture count calculates remaining intestinal bacteria K88, K99 and the viable count of streptococcus aureus.The viable count of remaining pathogenic colon bacillus K88, K99 and streptococcus aureus is 24.7%, 28.8% and 30.4%.
(2) checking of cellulase-producing ability
With 2% inoculum size, inoculate subtilis CGMCC No.4628 bacterium powder to 1000ml LB substratum, 28 ℃ ferment 14 hours, dripping 20uL fermented liquid is containing on the LB-CMC culture medium flat plate of 0.5% carboxymethyl cellulose, observe cellulose hydrolysis transparent circle size, calculating hydrolysis transparent circle diameter and colony diameter ratio is 3.0.
The preparation of embodiment 3, bacterium powder
1, enterococcus faecalis (CMGCC No.5092)
(1) high density fermentation of enterococcus faecalis (CMGCC No.5092)
The fermention medium of using during fermentation enterococcus faecalis consists of the following composition: brown sugar 18g/L, and peptone 4g/L, extractum carnis 3g/L, yeast soaks powder, 0.8/L, bitter salt 0.4g/L, manganous sulfate 0.005g/L, pH is 7.2.
Fermentation condition is: leavening temperature is 37 ℃, and fermentation time is 12h, and pH value is 7.2, and rotating speed is 100rpm, and initial inoculum is 2%.After fermentation ends, obtain 7.3 × 10
9the bacterium liquid of cfu/ml.
(2) preparation of enterococcus faecalis (CMGCC No.5092) bacterium powder
Enterococcus faecalis is carried out to high temperature spray-drying; the mass volume ratio of protective material and fermented liquid is the ratio preparation protective material of 1: 3; its protective material composition is: xanthan gum: trehalose: glycerine: Sodium Glutamate: maltodextrin=10: 10: 2.5: 2.5: 75; 180 ℃ of air intakes; 70 ℃ of air-out; obtain free-running property good, soluble in water, viable count is 4 × 10
10the enterococcus faecalis bacterium powder of cfu/g.
2, the preparation of subtilis CGMCC No.4628 bacterium powder
The subtilis CGMCC No.4628 10mL that gets incubated overnight transfers into 500ml fermention medium (initial inoculum is 2%), and 28 ℃ ferment 14 hours, and pH value is 7.0, and rotating speed is 180rpm.After fermentation, thalline is centrifugal, 60 ℃ of oven dry, 4 ℃ of preservations of bacterium powder.
The viable count that the method for plate culture count detects bacterium powder is about 10
10cFU/g.
Consisting of of fermention medium: sucrose 10g/L, peptone 5g/L, extractum carnis 3g/L, yeast soaks powder 1g/L, bitter salt 0.5g/L, manganous sulfate 0.0005g/L, pH 7.0.
The working method of the method for plate culture count is: testing sample is after suitable multiple dilution, microorganism wherein is fully dispersed into individual cells, getting a certain amount of dilute sample is applied on flat board, through cultivating, each unicellular growth and breeding forms macroscopic bacterium colony, single bacterium colony represent one in raw sample unicellular.Statistics colony number, can converse the viable count in sample according to its extension rate and sampling inoculum size.
3, the preparation of Bacillus licheniformis CGMCC No.5094 bacterium powder
A. the preparation method of Bacillus licheniformis powder, comprises following step
1) dull and stereotyped cultivation rejuvenation: Bacillus licheniformis strain is inoculated on BPY plate culture medium, in 37 ℃ of cultivation 16h, makes Bacillus licheniformis rejuvenation, and form single bacterium colony, picking list bacterium colony, on inoculation medium, is cultivated 24h for 37 ℃;
2) preparation of first order seed: by step 1) on the Bacillus subtilis strain switching eggplant bottle BPY slant medium cultivated, cultivate 16h, make, in the logarithm later stage, to obtain first order seed for 37 ℃;
3) preparation of secondary seed: by step 2) the first order seed sterilized water prepared makes bacteria suspension, is inoculated in the 100L seeding tank that 60L BPY seed culture medium is housed 37 ℃ of temperature, rotating speed 220rpm, tank pressure 0.05MPA, ventilating ratio: 1: 0.7, cultivate 12h, obtain secondary seed solution.
4) preparation of the lichen bacillus ferments liquid: by step 3) secondary seed solution prepared is inoculated into 5-8m according to 1% inoculum size
3in the fermentor tank of fermention medium, 37 ℃ of temperature, rotating speed 220rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.7, cultivate 20h, to gemma production rate, more than 90%, viable count is 1.6 × 10
10cfu/ml, stops fermentation, obtains the lichen bacillus ferments liquid;
Described fermention medium is: wheat bran 1.8%, Semen Maydis powder 1.5%, dregs of beans 2%, ammonium sulfate 2%, magnesium sulfate 0.04%; Ammonium citrate 0.8%, defoamer 0.06%, pH is 6.8.
5) preparation of Bacillus licheniformis powder: in step 4) add 25% weighting material maltodextrin in the fermented liquid prepared, mix, spray dry, 160 ℃ of inlet temperature, 50 ℃ of temperature of outgoing airs, spraying gun rotating speed 16000rpm, obtains the Bacillus licheniformis powder of moisture content 4.78%.
4, the preparation of Lactobacterium acidophilum bacterium powder
1) dull and stereotyped cultivation rejuvenation: Lactobacterium acidophilum bacterial classification is inoculated on MRS plate culture medium, in 37 ℃ of cultivation 16h, makes the rejuvenation of Lactobacterium acidophilum bacterium, and form single bacterium colony, picking list bacterium colony, on inoculation medium, is cultivated 14h for 37 ℃;
2) preparation of first order seed: by step 1) cultivate Lactobacterium acidophilum strain transfer be equipped with in 300ml MRS liquid culture medium 2L shaking flask, 37 ℃ of standing cultivation 18h, to the logarithm later stage, obtain first order seed;
3) preparation of secondary seed: by step 2) first order seed prepared is transferred in the 100L seeding tank that 75LMRS seed culture medium is housed, 37 ℃ of temperature, rotating speed 80rpm, cultivates 18h, obtains secondary seed solution.
4) preparation of Lactobacterium acidophilum fermented liquid: by step 3) secondary seed solution prepared is inoculated into 0.75m according to 1.5% inoculum size
3in the fermentor tank of fermention medium, 37 ℃ of temperature, rotating speed 80rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.2, cultivate 18h, obtaining viable count is 4.67 × 10
9cfu/ml Lactobacterium acidophilum fermented liquid.
Described fermention medium is: glucose 2%, soy peptone 1.8%, yeast extract paste 1.5%, ammonium sulfate 0.75%, dipotassium hydrogen phosphate 0.4%, sodium-chlor 0.6%, magnesium sulfate 0.04%;
The preparation of Lactobacterium acidophilum bacterium powder: by step 4) fermented liquid prepared, 4000rpm is centrifugal, obtains bacterium mud; the lyophilized vaccine that to add with the mass/volume per-cent of bacterium mud be 20%; mix, in-40 ℃ of lyophilizes, obtain moisture content and be 4.5% enterococcus faecalis bacterium powder.
Consisting of of lyophilized vaccine: the mixture of skim-milk, glycerine, lactose, W-Gum and Sodium Glutamate, according to skim-milk: glycerine: lactose: W-Gum: Sodium Glutamate=2: the ratio of 1: 1: 0.8 mixes.
5, the preparation of bacillus pumilus bacterium powder
1) dull and stereotyped cultivation rejuvenation: bacillus pumilus bacterial classification is inoculated on BPY plate culture medium, in 37 ℃ of cultivation 18h, makes bacillus pumilus rejuvenation, and form single bacterium colony, picking list bacterium colony, on inoculation medium, is cultivated 30h for 37 ℃;
2) preparation of first order seed: by step 1) cultivate bacillus pumilus strain transfer eggplant bottle BPY slant medium on, 36 ℃ cultivate 14h, make, in the logarithm later stage, to obtain first order seed;
3) preparation of secondary seed: by step 2) the first order seed sterilized water prepared makes bacteria suspension, is inoculated in the 100L seeding tank that 60L BPY seed culture medium is housed 37 ℃ of temperature, rotating speed 300rpm, tank pressure 0.05MPA, ventilating ratio: 1: 0.8, cultivate 12h, obtain secondary seed solution.
4) preparation of bacillus pumilus fermented liquid: by step 3) secondary seed solution prepared is inoculated into 6m according to 1% inoculum size
3in the fermentor tank of fermention medium, temperature 30-40 ℃, rotating speed 280rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.8, cultivate 24h, obtain gemma production rate more than 90%, viable count is 5.48 × 10
9the bacillus pumilus fermented liquid of cfu/ml;
Described fermention medium is: glucose 1.5%, soy peptone 1.5%, dregs of beans 1.5%, ammonium sulfate 0.75%, sodium-chlor 0.6%, magnesium sulfate 0.05%; PH nature.
5) preparation of bacillus pumilus bacterium powder: in step 4) add 25% weighting material W-Gum in the fermented liquid prepared, mix, spray dry, 165 ℃ of inlet temperature, 60 ℃ of temperature of outgoing airs, spraying gun rotating speed 16000rpm, obtains the bacillus pumilis bacterium powder of moisture content 3.85%.
The preparation of embodiment 4, compound micro-ecological preparation
After several bacterium powder that obtain in embodiment 3 mix with carrier according to different ratios, can obtain being applicable to the specificity compound microecological of different fowl poultrys, wherein piglet is special, sow is special and laying hen tailored version compound microecological is as shown in table 6.
The preparation method of several compound microecologicals of table 6
Piglet tailored version compound micro-ecological preparation prepared by embodiment 5, embodiment 4 efficacy test to piglet
Experimental animal: select 35 age in days wean, the Du Chang-Bai piglet of body weight about 9KG is as 300 of experimental animals, and a collection of choosing is neat, and the date of birth differs and is no more than 7 days.Trial period is 30 days, and the phase of raising is 7 days in advance.Raise and adopt flat the supporting in ground to raise, each assembly is except daily ration difference, and other conditions are in full accord, carries out routinely, Manual material feeding, free choice feeding, freely drinks water, keeps pig house to clean.
Test index and method: when on-test and end, weigh to pig morning on an empty stomach, take repeating groups as unit, carry out respectively full group and weigh, calculate the indexs such as feed consumption rate, day weight gain, feedstuff-meat ratio, diarrhea rate, and carry out otherness and significantly check, test-results is as shown in table 7:
Table 7 piglet tailored version probiotics is to the piglet efficacy test result of feeding
As can be seen from the table, at average weight gain piglets group I, test group II and test group III, than contrast component, you can well imagine high 7.5%, 7.3%, 9.5%; Aspect feedstuff-meat ratio, test I, test group II and test group III have not reduced by 5.5%, 5.2%, 8.0% than contrast component; Aspect diarrhea rate, test I, test group II and test group III have not reduced by 67.3%, 47.7%, 70.1% than contrast component.It is not remarkable that test I group and test II compare in difference aspect day weight gain, feedstuff-meat ratio, but pre-anti-diarrhea be wanting in different significantly, illustrate that piglet tailored version compound micro-ecological preparation can substitute in feed the microbiotic of anti-diarrhea in advance.
The test of the sow tailored version compound micro-ecological preparation of preparation to milking sow in embodiment 6, enforcement 4
1, test method
The selection of test pig: select the just before giving birth sow about the last week from pig farm, choose and healthy anosis, pig age, parity, 18 basically identical of sows of body weight be divided at random two groups, 9 of test group and control groups.
Feedstuff composition: control group fed basal diet, test group is to add 1 ‰ sow tailored version compound micro-ecological preparations on basal diet basis.
Feeding and management: test is carried out in this same delivery room, is fed by same keeper, and feeding and management condition is consistent, feed is taked siccative to add water to mix after wet and feed, start feeding experiment material when test sow proceeds to obstetric table, day feeds 3 times, free choice feeding, take the material of cannot not having enough surplusly as principle.Test group and control group are added up respectively feed consumption rate, the heavy and 28 days weanling weights of piglet birth, and the way of taking whole day to supply water, appoints pig freely to drink water.Clean colony house every day, observe at any time growth and health condition.
2, test result analysis
Within trial period, the 28 days wean counterpoises of test group that add the 0.1% sow tailored version compound micro-ecological preparation of feeding are 8.27kg, and the 28 days wean counterpoises of control group that do not add sow tailored version compound micro-ecological preparation are 7.54, test group is than the many weightening finishes of control group 0.73kg, through check, two groups of significant differences (p < 0.05), and test group surviving rate is apparently higher than control group, and its surviving rate is respectively 85.83% and 91.67%.The results are shown in Table 8.
The impact of table 8 sow tailored version compound micro-ecological preparation on milking sow production performance
| Control group mean value | Test group mean value | |
| Young number goes out to live | 9.11 | 9.44 |
| Birth counterpoise (kg) | 1.60 | 1.56 |
| 3 ages in days are adjusted number | 10.67 | 10.67 |
| 3 ages in days are adjusted counterpoise (kg) | 1.90 | 1.79 |
| Counterpoise (kg) before 7 age in days supplementary feedings | 2.53 | 2.50 |
| 28 days wean numbers | 9.78 | 10.22 |
| Wean counterpoise | 7.54 | 8.27 |
The efficacy test of the egg fowl tailored version compound micro-ecological preparation that embodiment 7 implements in 4 preparation to laying hen
Experimental animal: select at random 3000 of small egg laying hens in 60 week age, be divided at random 2 groups, 1500 every group, test according to random packet arrangement.Test group I is blank group (basal diet); Test group II is test group (basal diet+0.1% egg fowl tailored version compound micro-ecological preparation), and three layers, test chicken tail is raised in cages, 3, every cage, and free choice feeding and drinking-water, raise routinely and manage.
Testing index: the every repetition egg number of observed and recorded every day, egg size, defective egg number and chicken extract are extremely washed in a pan situation; Add up feed consumption rate per weekend; Add up respectively every group of chicken lay eggs laying rate, egg size, breakage rate, mortality ratio, food consumption and the feedstuff-egg ratio in later stage.
The impact of table 9 egg fowl tailored version compound micro-ecological preparation on small egg laying hen production performance
Interpretation of result: 1, add 0.1% egg fowl tailored version compound micro-ecological preparation and can significantly improve the laying rate of laying hen in feed, laying rate improves 2.80%; 2, in feed, adding 0.1% egg fowl tailored version compound micro-ecological preparation can reduce the generation of disease and reduce the mortality ratio of laying hen; 3, in feed, add 0.1% egg fowl tailored version compound micro-ecological preparation and can improve the immunizing power of body, the stress reaction that prevention causes due to aspects such as heat stress or vaccinations.
In sum, probiotics of the present invention is added in feed, and the digestive utilization ratio to assurance chicken monitor state, coordination promotion body abilities of digestive and absorption, raising performance in layers and feed etc. all has good effect.
Claims (2)
1. a compound micro-ecological preparation, it is characterized in that said preparation contain Lactobacterium acidophilum (
lactobacillus acidophilus) CGMCC No.5093 bacterium powder, and Bacillus licheniformis (
bacillus licheniformis) CGMCC No.5094, bacillus pumilus (
bacillus pumilus) CGMCC No.4756, enterococcus faecalis (
enterococcus faecalis) CGMCCNo.5092, subtilis (
bacillus subtilis) wantonly two kinds, three kinds or the bacterium powder of four kinds in tetra-kinds of bacterium of CGMCC No.4628.
2. compound micro-ecological preparation as claimed in claim 1, it is characterized in that containing Lactobacterium acidophilum CGMCC No.5093 bacterium powder, Bacillus licheniformis CGMCC No.5094 bacterium powder, bacillus pumilus CGMCC No.4756 bacterium powder and enterococcus faecalis CGMCC No.5092 bacterium powder in preparation, the weight proportion of above-mentioned four kinds of bacterium powder is 0.8-2.5: 1: 0.8-1.2: 1.5-2.5.
3. compound micro-ecological preparation as claimed in claim 1, it is characterized in that containing Lactobacterium acidophilum CGMCC No.5093 bacterium powder, Bacillus licheniformis CGMCC No.5094 bacterium powder and subtilis CGMCC No.4628 bacterium powder in preparation, the weight proportion of above-mentioned three kinds of bacterium powder is 0.8-3.5: 1-3.5: 0.8-2.5.
4. compound micro-ecological preparation as claimed in claim 1, it is characterized in that containing Lactobacterium acidophilum CGMCC No.5093 bacterium powder, Bacillus licheniformis CGMCC No.5094 bacterium powder and enterococcus faecalis CGMCC No.5092 bacterium powder in preparation, the weight proportion of above-mentioned three kinds of bacterium powder is 1.5-2.5: 0.8-1.2: 1.
5. compound micro-ecological preparation as claimed in claim 1, it is characterized in that containing Lactobacterium acidophilum CGMCC No.5093 bacterium powder, bacillus pumilus CGMCC No.4756 bacterium powder and enterococcus faecalis CGMCC No.5092 bacterium powder in preparation, the weight proportion of above-mentioned three kinds of bacterium powder is 2-2.5: 2-2.5: 1-1.5.
6. compound micro-ecological preparation as claimed in claim 1, the preparation method who it is characterized in that described Lactobacterium acidophilum CGMCC No.5093 bacterium powder is as follows: by the inoculum size of 1-2% to the Lactobacterium acidophilum seed liquor that accesses 8h-12h cell age in fermention medium, fermentation condition is: 30-40 ℃, rotating speed is 60-100rpm, cultivate 12-16h, obtain fermented liquid, fermented liquid is centrifugal, obtain bacterium mud, the lyophilized vaccine that to add with the percent weight in volume of bacterium mud be 15-20%, mix, in-25 ℃--45 ℃ of lyophilizes, obtain, composition and the mass volume ratio thereof of described fermention medium are as follows: glucose 1-2%, soy peptone 1-2%, yeast extract paste 1-2%, ammonium sulfate 0.5-1.0%, dipotassium hydrogen phosphate 0.2-0.5%, sodium-chlor 0.2-0.8%, magnesium sulfate 0.01-0.05%, the preparation method of described Bacillus licheniformis CGMCC No.5094 bacterium powder is as follows: by the inoculum size of mass volume ratio 0.5-2% to the Bacillus licheniformis seed liquor that accesses 10-14h cell age in fermention medium, fermentation condition is: 30-40 ℃, rotating speed is 200-300rpm, cultivate 15-22h, obtain fermented liquid, in fermented liquid, adding mass volume ratio is the weighting material maltodextrin of 20%-25%, mix, spray dry, inlet temperature 140-160 ℃, temperature of outgoing air 40-50 ℃, spraying gun rotating speed 15000-18000rpm, obtain, composition and the mass volume ratio thereof of described fermention medium are as follows: wheat bran 1-2%, Semen Maydis powder 0.5-1.5%, dregs of beans 1-2%, ammonium sulfate 0.5-2%, magnesium sulfate 0.01-0.05%, ammonium citrate 0.3-1.2%, defoamer 0.05-0.1%, pH is 6.2-7.4, the preparation method of described bacillus pumilus CGMCC No.4756 bacterium powder is as follows: by the inoculum size of mass volume ratio 1-2% to the bacillus pumilus seed liquor that accesses 12h-16h cell age in fermention medium, fermentation condition is: 30-40 ℃, rotating speed is 100-300rpm, cultivate 16-24h, obtain fermented liquid, in fermented liquid, adding mass volume ratio is the weighting material W-Gum of 20-25%, mix, spray dry, inlet temperature 150-170 ℃, temperature of outgoing air 40-70 ℃, spraying gun rotating speed 15000-20000rpm, obtain, composition and the mass volume ratio thereof of described fermention medium are as follows: glucose 1-2%, soy peptone 1-2%, dregs of beans 1-2%, ammonium sulfate 0.5-1.0%, sodium-chlor 0.2-0.8%, magnesium sulfate 0.01-0.05%.
7. the compound micro-ecological preparation as described in claim 1-6 any one, is characterized in that further comprising carrier, and carrier is any or two kinds in maltodextrin, aluminum potassium sulfate, corn cob meal, glucose, oily chaff powder.
8. the compound micro-ecological preparation described in claim 1-7 any one is in the application of preparing in poultry and livestock feed additive.
9. comprise a livestock and poultry Preblend for the compound micro-ecological preparation described in claim 1-7 any one, it is characterized in that the mass volume ratio that in Preblend, compound micro-ecological preparation adds is 0.5-10 ‰.
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| CN102409015A (en) | 2012-04-11 |
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Address after: 100080 Beijing Haidian District Zhongguancun Street 27 Zhongguancun building 14 level big north agriculture group Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: Beijing Kegao dabainong Biotechnology Co.,Ltd. Patentee after: HUNAN DABEINONG AGRICULTURAL TECHNOLOGY Co.,Ltd. Address before: 100080 Beijing Haidian District Zhongguancun Street 27 Zhongguancun building 14 level big north agriculture group Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee before: Beijing Kegao Dabeinong Feedstuff Co.,Ltd. Patentee before: HUNAN DABEINONG AGRICULTURAL TECHNOLOGY Co.,Ltd. |
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