CN101392223A - Breeding method of microbial feed additive strain - Google Patents
Breeding method of microbial feed additive strain Download PDFInfo
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Abstract
The invention pertains to the fields of microbiology and science of animal nutrition and feedstuff and relates to a breeding method of microbial feedstuff additive strains, which comprises: separation and purification of strains, seed selection of stress resistance, seed selection of probiotic functionality, identification of strains and/or safety research. The method is characterized by scientificalness, reasonableness, complete system and strong operation. By the adoption of the method, new products of the microbial feedstuff additive strains with excellent resistance, strong function and outstanding efficiency can be cultured, thus providing guarantee of important strain resources for the development of microbial feedstuff additive products.
Description
Technical field
What the present invention relates to is the selection of microbial feed additive strain, belongs to microbiology and animal nutrition and feed and learns the field.
Background technology
Fodder additivess such as microbiotic have played huge pushing effect to prevention Animal diseases, raising breeding performonce fo animals and economic benefit, but also caused serious negative effect simultaneously, resistance such as pathogenic micro-organism, animal autoimmunity reduces, residual HUMAN HEALTH injury that causes of medicine and manure contamination environment etc.Along with growth in the living standard, people more and more pay attention to the quality of the consumer's goods, and the safety problem of livestock product becomes the focus that people pay close attention to day by day.At present, European Union has banned use of microbiotic, and in recent years, China all has livestock product to exceed standard because of medicine is residual to be returned goods or destroy every year, all caused enormous economic loss for country and enterprise, this shows exploitation is efficient, noresidue, non-harmful safe green fodder additives are social developments requirement and inevitable outcome.
Microorganism feed addictive is " animal microecological formulation ", " probiotics ", " active bacteria formulation " again, be through containing of industrial fermentation production a lot of beneficial microorganisms and the active bacteria formulation that constitutes of meta-bolites, directly feeding animals by the animal probiotics.The balance of little ecology plays a role this type of preparation in the enteron aisle by keeping, multiple function such as have diseases prevention, enhancing body immunizing power, promote grow, put on weight, and pollution-free, and noresidue is not developed immunity to drugs, and is a kind of green, safe fodder additives.And wherein, the seed selection of excellent species is again the key of microorganism feed addictive exploitation and effect.
At present, the research to the probiotic bacterium bacterial classification both at home and abroad focuses mostly in development product and effect, comes the bacterial strain of seed selection excellent property by the particular requirement of probiotic bacterium, and key index aspect reports such as research bacterial strain biological nature are few; In field of animal feed, both at home and abroad still less about the research report of the systematic breeding method of excellent property bacterial strain, and existing report about selection has its limitation more, the screening criteria with animal probiotic bacterium bacterial classification does not organically combine with concrete test method, form the microbial feed additive strain selection of a cover system, perfect, actual operability, this also is an innovation part of the present invention.
Summary of the invention
The object of the present invention is to provide a kind of scientific and reasonable, system perfecting, at different animals kind, age and have the selection of the strain excellent of certain specific function.Use present method, can select good stress resistance, strong, the significant microbial feed additive strain product innovation of effect of function, guarantee for exploitation microorganism feed addictive product provides important microorganism resource.
Purpose of the present invention is achieved through the following technical solutions:
The invention provides a kind of selection of microbial feed additive strain, it is characterized in that comprising that isolation and purification, resistance seed selection, the benefit of bacterial strain given birth to functional seed selection, identification of strains and/or safety research.
The detailed process of seed selection is as follows:
1, the isolation and purification of bacterial strain
At first determine that according to the application target of microbial feed additive strain institute's probiotics bacterial kind kind of wanting and strain separating originate.If in use do not need to utilize it to stick and the field planting characteristic, the source of bacterial classification just is not the problem of a particularly important, in principle, the bacterial strain that screen is preferably the most desirable with the bacterial classification that former host, former habitat, former microecosystem, former little group even former little groupy phase adapt to.
(1) increasing bacterium cultivates: a little sample kind in liquid nutrient medium, is carried out aerobic or anaerobic 28-40 ℃ of constant temperature culture and increased bacterium and cultivate;
(2) separation and purification: on plate culture medium, carry out streak culture to increasing the bacterium culture, therefrom the bacterium colony that each form of picking is different streak culture again with separate, till being pure bacterium colony, will breed in the isolating pure colony inoculation test tube slant, be stored in then in 4 ℃ of refrigerators, stand-by.
Strain separating, the used substratum of purifying can be a kind of in BPY substratum, MRS substratum, No. 98 substratum, and the prescription of above-mentioned each substratum gets final product by its conventional formulation preparation.
2, to step 1 the bacterial strain of separation, purifying carry out the resistance seed selection, can to separate, the bacterial classification of purifying directly carries out anti-adversity and measures, and 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; At last the bacterial strain that selects is carried out the resistance performance measurement.Consider the security and the heritability stability of bacterial classification, the present invention tends to directly filter out the natural excellent species of strong stress resistance.
Resistance seed selection of the present invention is meant the seed selection that comprises habitat in the anti-animal body, tolerance drying conditions and formulation process conditions, feed substrate is had tolerance and/or drug susceptibility.
Wherein, the interior habitat seed selection of anti-animal body comprises the seed selection of stomach juice-resistant, bile tolerance, anti-pancreatic juice and/or the liquid of anti-the enzyme; The seed selection of tolerance drying conditions and formulation process conditions is meant the seed selection of tolerance lyophilize, spray-dired drying conditions and tolerance particulate material, tablet complete processing operational condition; The seed selection that feed substrate is had a tolerance is meant the high-copper in the tolerance feed and/or the seed selection of high zinc mineral salt; The drug susceptibility seed selection be meant bacterial classification to the normal microbiotic that adds in the feed, in breeding process, prevent and treat the drug susceptibility seed selection of the antibacterials of usefulness.
Described stomach juice-resistant seed selection is the simulation test by external monofactor, i.e. the quantitative assay screening that manual simulation's gastric juice growth property test qualitatively and survival rate are measured, and the screening survival rate is at the strain excellent more than 1%; The bile tolerance seed selection is the screening of measuring by external artificial simulated intestinal fluid test growth property test qualitatively and survival rate, is that bacterial classification inoculation is handled in manual simulation's intestinal juice of various biliary salt concn, and the screening survival rate is at the strain excellent more than 1 ‰; The seed selection of tolerance particulate material, tablet complete processing operational condition is meant in the tolerance formulation course of processing and under high temperature, high humidity, the mechanical presses process conditions, detects the survival rate of each bacterial strain that the screening survival rate is at the strain excellent more than 90%.
The present invention of stomach juice-resistant seed selection process is preferably as follows method: the preservation slant strains is inoculated in the test tube that the 10ml liquid nutrient medium is housed, 28～40 ℃ leave standstill cultivation 20～24h, be inoculated in respectively by 5～10% inoculum sizes in manual simulation's gastric juice of 9.5～9.0mLpH 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, the screening survival rate is at the strain excellent more than 1%.The present invention of bile tolerance seed selection process is preferably as follows method: the inoculation of preservation inclined-plane is equipped with in the test tube of 10mL liquid nutrient medium, 37 ℃ leave standstill cultivation 20-24h, be inoculated in respectively by 10% inoculum size in the pig cholate solution of 9.0mL0.03%, 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, the screening survival rate is at the strain excellent more than 1 ‰.Seed selection the present invention of tolerance particulate material, tablet complete processing operational condition strain excellent is preferably as follows method: bacterial strain is made the bacterium powder add to and carry out pelletization treatment in Preblend or the complete diet pellet, detect the survival rate of each bacterial strain, screening viable bacteria survival rate is at the strain excellent more than 90%.Drug susceptibility seed selection the present invention is preferably as follows method: adopt agar dilution to measure bacterial classification to the microbiotic that often adds in the feed, the drug susceptibility of preventing and treating the animal health medicine antibacterial medicine of usefulness in breeding process, the normal microbiotic that adds can be in the feed: Webel Tylan Premix, sulfuric acid colomycin, terramycin, neomycinsulphate, sulphamethazine, sulfaquinoxaline, bambermycin, olaquindox, U 10149a, monensin, kitasamycin, nosiheptide, organoarsenic; The animal health medicine can be: duomycin, terramycin; The concrete grammar step of drug susceptibility seed selection is as follows:
(1) preparation of medicine flat board
The preparation of aseptic antibiotic solution: microbiotic to be measured is mixed with different concentration with distilled water, filters with the bacterium filtering membrane and promptly obtain aseptic antibiotic solution;
The preparation of medicine flat board: according to test design, the antibiotic solution of different concns is added respectively in the BPY substratum that is cooled to 45-50 ℃, shake up fully that to topple over sterilization dull and stereotyped, agar thickness 3-4mm, solidifying the back, to be inverted empty training 24h stand-by.
(2) preparation of inoculum
Actication of culture: bacterial classification one ring of hiding on the inclined-plane of going bail for is inoculated in the test tube that the 5mLBPY liquid nutrient medium is housed, and 37 ℃ static is cultured to the logarithm middle and later periods, makes biomass reach 10
8CFU/mL.
The preparation of inoculum and inoculation: with cultured bacteria suspension, with stroke-physiological saline solution 1:10 dilution, shake up, 4uL is in the medicine agar plate surface in inoculation, and every some bacterium number is about 10
4CFU/mL, forming diameter is the bacterial plaque of 5-8mm.Inoculation is cultivated observations for good rearmounted 37 ℃, is inverted behind the positive 5h of cultivation again and cultivates.
The result judges: flat board being placed on dead color, the no-reflection body surface judge test endpoint, is MIC with the lowest concentration of drug of bacteria growing inhibiting.
3, benefit is given birth to functional seed selection
In the strain improvement process, can directly carry out anti-adversity to bacterial strain to be measured and measure, therefrom filter out the natural excellent species of strong stress resistance; Or by traditional physics, chemomorphosis method, modern genetic engineering technology, directive breeding goes out benefit and gives birth to the strong bacterial strain of function; At last the bacterial strain that selects is carried out the resistance performance measurement.Consider the security and the heritability stability of bacterial classification, the present invention tends to directly filter out benefit and gives birth to the strong natural excellent species of function.
Benefit of the present invention is given birth to functional seed selection and is comprised fungistatic effect, produces acidity test, zymogram is measured and/or stick performance measurement.
Wherein, the fungistatic effect test is to measure the fungistatic effect of bacterial strain to common pathogen with two dish methods, and the good strain excellent of screening fungistatic effect; Producing acidity test is to adopt ion chromatography analysis to measure organic acid content in the fermented liquid, and screening produces the strain excellent of lactic acid, butyric acid, acetate or citric acid; It is to adopt the amylase plate culture medium to carry out the primary dcreening operation of high yield amylase strain excellent and/or the primary dcreening operation that employing casein hydrolysis flat board carries out the high proteinase yield strain excellent that zymogram is measured; Sticking performance measurement is that bacterium is used labelled with radioisotope, then again with cell or mucus incubation after, calculate the percentage that sticks of this bacterium.
Wherein, specifically measure, comprise the mensuration of amylase, proteolytic enzyme, cellulase, phytase or lipase, can determine to produce enzyme class bacterial classification according to the purpose effect of institute's seed selection bacterial strain at the zymogram of gemma class bacterium.The present invention of diastatic primary dcreening operation process is preferably as follows method: in the amylase test substratum, each bacterial classification connects 2 plates with the bacterial classification streak inoculation, strives for must occurring on dull and stereotyped single bacterium colony more than 5, to be easy to observe the measurements and calculations transparent circle; The above-mentioned flat board that inoculation is good is put in the thermostat container 37 ℃ and is cultivated your iodine solution of glug that drips existing preparation behind 24h-48h respectively, until all filling the air flat board, stop a moment slightly, observe the transparent circle that its bacterium colony forms, according to the transparent circle of periphery of bacterial colonies generation or ratio (H/C) size of variable color loop diameter and colony diameter, to judge the enzymatic productivity of each bacterial strain.Primary dcreening operation process the present invention of proteolytic enzyme is preferably as follows method: get suitable dilution bacteria suspension 0.1mL on the casein hydrolyzing culture medium, coating evenly, cultivate 1-3d for 37 ℃ then, observe the size of formation, growth and the casein hydrolysis circle of bacterium colony, the person is positive the transparent circle, no transparent circle person is negative, according to the transparent circle of periphery of bacterial colonies generation or ratio (H/C) size of variable color loop diameter and colony diameter, to judge the enzymatic productivity of each bacterial strain.
4, identification of strains
Identification of strains is with microbiology classification authentication method form and the physiological and biochemical property of selecting bacterial strain to be studied, simultaneously in conjunction with a kind of authentication method based on molecule.
5, safety research
Safety research comprises hemolytic test, acute toxicity test and chronic toxicity test.
Included step and the included screening content of each step in the strain improvement process of the present invention, on screening one strain bacterial strain and nonessentially all comprise, can be according to the difference of bacterial classification application target, select all or part of screening content, final purpose is to select excellent property, the good probiotic strain of effect.
, 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
(1) separation of gemma class bacterial classification, purifying
Increasing bacterium cultivates: a little content that will from small intestine, large intestine and the caecum of piglet, obtain near mucous membrane, be inoculated in the BPY liquid nutrient medium, and in 37 ℃ of constant temperature culture shaking tables, carry out aerobic and increase the bacterium cultivation;
Separation and purification: the above-mentioned bacterium liquid that increases behind the bacterium is boiled 10min in boiling water, on the BPY culture medium flat plate, carry out streak culture again, therefrom transfer the different bacterium colony of each form streak culture again with separate, whether microscopy has gemma to form, whether be single bacterium colony, with the pure bacterium colony of 32 strains that is separated to and be to rise in value in the sporeformer inoculation BPY substratum test tube slant, be stored in then in 4 ℃ of refrigerators, stand-by.
BPY substratum composed as follows: extractum carnis 0.3～0.5g, yeast extract paste 0.3～0.5g, peptone 0.5～1g, sodium-chlor 0.2～0.5g, glucose 0.3～0.5g is settled to 100ml with tap water, and solid medium adds agar: 2～2.5g again.
(2) separation of lactic acid class bacterial classification, purifying
Increasing bacterium cultivates: a little content that will from small intestine, large intestine and the caecum of piglet, obtain near mucous membrane, be inoculated in the MRS liquid nutrient medium, and 37 ℃ of static anaerobism of constant temperature are cultivated;
Separation and purification: on the MRS culture medium flat plate, carry out streak culture to the above-mentioned bacterium liquid that increases behind the bacterium, therefrom transfer the different bacterium colony of each form streak culture again with separate, till being pure bacterium colony, to cultivate in the isolating pure colony inoculation MRS substratum test tube slant, be stored in then in 4 ℃ of refrigerators, stand-by.Microscopy does not have gemma and forms, and catalase test is negative, tentatively is judged as lactic acid class bacterium, totally 40 strains.
MRS substratum composed as follows: glucose 15～20g; Tryptones 8～10g; Extractum carnis 5～10g; Yeast extract 3～5g; Dibasic ammonium citrate 1～2g; Dipotassium hydrogen phosphate 1～2g; MgSO
47H
2O0.2～0.58g; MnSO
44H
2O 0.1～0.25g; Tween 80 0.5～1mL; Distilled water 1L; Solid medium adds agar 20～25g again.
Compound method: with MgSO
47H
2O, MnSO
44H
2Each composition dissolving beyond O, glucose and the tween 80 is chilled to 50 ℃, is 6.0～6.5 with the vinegar acid for adjusting pH value, then adds MgSO
47H
2O, MnSO
44H
2O adds glucose and tween 80 at last.At 121 ℃, autoclaving 20min.
Embodiment 2
The totally 72 strain bacterial classifications of a kind of seed selection of embodiment are carried out the screening of anti-alimentary tract habitat strain excellent
(1) preservation slant strains is inoculated in the test tube that the 10mL liquid nutrient medium is housed, 37 ℃ leave standstill cultivation 20-24h, be inoculated in respectively by 10% inoculum size in manual simulation's gastric juice of 9.0mLpH value 2.0,3.0,4.0, the 0h counting is done contrast, 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, the screening survival rate is at the strain excellent more than 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 I0mL liquid.
(2) inoculation of preservation inclined-plane is equipped with in the test tube of 10mL liquid nutrient medium, 37 ℃ leave standstill cultivation 20-24h, be inoculated in respectively by 10% inoculum size in the pig cholate solution of 9.0mL0.03%, 0.1%, 0.2%, 0.3% different concns, the 0h counting is done contrast, 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, the screening survival rate is at the strain excellent more than 1 ‰.
The preparation of manual simulation's intestinal juice: 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.
The strain excellent that the result filters out the anti-alimentary tract habitat has: 4 strain sporeformer, 3 strains of lactic acid bacteria are numbered respectively: DW-AQFB-1, DW-RDYL-46, DW-USA/BT-10, DW-ZKY-55, DW-SGY-49, DW-BLKY-27, DW-CMJ-47.
Embodiment 3
7 strain bacterial classifications of screening among the embodiment 2 are carried out the screening of the good strain excellent of the assay determination of meta-bolites and beneficial natural disposition
Produce acidity test: adopt ion chromatography analysis to measure organic acid content in the fermented liquid, screening produces a certain amount of lactic acid, butyric acid, acetate, citric acid organic acid strain excellent, the result shows that 3 strains of lactic acid bacteria DW-SGY-49, DW-BLKY-27, DW-CMJ-47 lactic acid production reach 9.132g/L, 4.141g/L, 3.758g/L respectively, and lactic acid accounts for total acid yield per-cent and is respectively 95%, 85.5%, 89.4%; 1.230,0.298,1.814,1.695g/L the production of organic acids of 4 strain sporeformer DW-USA/BT-10, DW-RDYL-46, DW-AQFB-1, DW-ZKY-55 is respectively:.
Fungistatic effect is measured: adopt two dish methods to measure the fungistatic effect of bacterial strain to the common bacterium e. coli k99 of curing the disease, streptococcus aureus, white dysentery Salmonellas, the good strain excellent of the screening fungistatic effect of inhibition zone more than 7mm, two dish method concrete steps are as follows:
(1) preparation of indicator bacterium liquid: will in the test tube that 10mL nutrition bouillon media is housed, activate three strain pathogenic bacterium respectively: intestinal bacteria, streptococcus aureus, white dysentery Salmonellas, 37 ℃ of constant temperature culture 20h;
(2) preparation of 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 37 ℃ of an amount of mixings of indicator liquid of cultivating 24h, draw 10mL and water on the bottom substratum, horizontal positioned makes it to solidify, as the bacterium layer;
(3) 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 strain fermentation supernatant liquor (about 200uL) of same amount, 3 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.The result shows: 2 strains of lactic acid bacteria and 1 strain sporeformer have bacteriostatic action preferably, and concrete outcome sees Table 1.
Table 13 is selected good strains in the field for seed and is educated the inhibition effect of bacterial strain to pathogenic bacterium
Zymogram is measured: adopt the amylase plate culture medium to carry out gemma class bacterium is carried out the primary dcreening operation of high yield amylase strain excellent, adopt casein hydrolysis flat board gemma class bacterium to be carried out the primary dcreening operation of high proteinase yield strain excellent, amylase primary dcreening operation plate culture medium composition is: extractum carnis 2～5g, peptone 5～10g, sodium-chlor 2～5g, Zulkovsky starch 1.5～2g, agar 20～25g, distilled water 1000mL, pH 7.2～7.4;
The casein plate culture medium: extractum carnis 6～8g, yeast powder 1.5～2g, polyprotein peptone 2～5g, sodium-chlor 1～2g, casein food grade 2～4g, agar 17～20g, distilled water 1000mL, pH 7.2～7.4.
The result shows: sporeformer has higher proteinase activity, and enzyme activity (H/C) is 11.12 relatively.
2 strains of lactic acid bacteria DW-BLKY-27, DW-SGY-49 and 1 strain sporeformer DW-ZKY-55 are identified in Chinese agriculture microorganism strains preservation administrative center (ACCC), are respectively enterococcus faecalis Enterococcus faecalis (ACCCNo.DW-BLKY-27), plant lactobacillus Lactobacillusplantarum (CICC 6026) and Bacillus coagulans Bacillus coagulans (AS 1.2407).
Embodiment 4
Bacillus coagulans Bacillus coagulans (AS 1.2407) to seed selection among the embodiment 3 carries out anti-feed processing experiment:
Sporeformer among the embodiment 3 is made the bacterium powder add to and carry out pelletization treatment in 5% Preblend and the complete diet pellet, detect survival rate, the result shows its survival rate 92.3%.
Embodiment 5
Enterococcus faecalis Enterococcus faecalis (ACCCNo.DW-BLKY-27), plant lactobacillus Lactobacillus plantarum (CICC 6026) and Bacillus coagulans Bacillus coagulans (AS 1.2407) to embodiment 1-4 seed selections carry out security mensuration:
(1) hemolytic test: will be inoculated in liquid nutrient medium after the bacterial strain activation, 37 ℃ of shaking culture 24h get 100 μ l bacterium drops and are added in the blood agar, and observing behind 37 ℃ of cultivation 48h has not transparent circle.Test-results is not seen haemolysis.
(2) acute toxicity test: select 60 of the local thin and small hen chick in 16 age in days Beijing, be divided into two groups (test group and control groups) at random, every group of 30 chickens, every group of three repetitions, each repeats 10 chickens.Every intramuscular injection 1ml of test group chicken test strain fermented liquid (10
8CFU/ml), in addition every every day oral 1ml fermented liquid (10
9CFU/ml), every chicken injection 1ml distilled water of control group chicken and the blank substratum of oral 1.0ml every day, the back dissection of two weeks.Observation index: general performance, body weight, the food utilization of (1) animal.(2) routine blood test and biochemical indicator: red blood cell count(RBC), white corpuscle numeration, gpt, glutamic-oxal(o)acetic transaminase, blood urea nitrogen, creatinine, cholesterol, blood sugar, total protein, albumin.(3) pathological anatomy: organ coefficient, gross examination of skeletal muscle and pathological tissue inspection (liver,kidney,spleen, the fabricius bursa, duodenum).
(3) chronic toxicity test: select 60 of the local thin and small hen chick in 7 age in days Beijing, be divided into two groups (test group and control groups) at random, every group of 30 chickens, every group of three repetitions, each repeats 10 chickens.The oral Bacillus coagulans (1.32 * 10 of test group every chicken every day
8CFU/ml) bacterium liquid 10ml, continuous oral one month, give after 25 days various stress: increase stocking density, sprinkle cold water, every chicken of control group chicken oral 10ml every day cultivates the substratum of Bacillus coagulans, close observation chicken overall health of patients.Dissect after one month.Observation index: general performance, body weight, the food utilization of (1) animal; (2) routine blood test and biochemical indicator: red blood cell count(RBC), white corpuscle numeration, gpt, glutamic-oxal(o)acetic transaminase, blood urea nitrogen, creatinine, cholesterol, blood sugar, total protein, albumin; (3) pathological anatomy: organ coefficient, gross examination of skeletal muscle and pathological tissue inspection (liver,kidney,spleen, the fabricius bursa, duodenum).
Beijing thin and small hen chick acute toxicity test and 30 days feeding studys are in the phase, and biochemical each index of experimental group routine blood test and blood is all in normal range.In liver, kidney, stomach, intestines, spleen and the ovary/testis tissue of each experimental group animal of gross examination of skeletal muscle, show no obvious abnormalities.In the histopathologic examination, liver,spleen,kidney, the fabricius bursa, duodenum show no obvious abnormalities.Originally 2 strains of lactic acid bacteria and the 1 strain gemma bacterial classification that experimental results show that screening among the embodiment 1-4 are safe and reliable.Wherein the test-results of Bacillus coagulans Bacillus coagulans (AS 1.2407) is shown in table 2,3,4.
Table 2 beginning and end of term mean body weight, materials amount
Table 3 organ coefficient
Table 4 routine blood test and blood parameters
Embodiment 6
Adopt isotope-labelling method to carry out the enteron aisle field planting enterococcus faecalis Enterococcus faecalis (ACCCNo.DW-BLKY-27), the plant lactobacillus Lactobacillus plantarum (CICC 6026) of embodiment 1-5 seed selections and the Bacillus coagulans Bacillus coagulans (AS 1.2407) and stick the mensuration of performance:
(1) preparation mark bacterial strain: the inclined-plane of inciting somebody to action is preserved bacterial classification and is inoculated in BPY or MRS liquid nutrient medium respectively, coat BPY or the dull and stereotyped 36h of cultivation of MRS behind 37 ℃ of cultivation 24h, get single bacterium colony 37 ℃ of cultivation 24h in liquid nutrient medium, get bacteria suspension, get fresh BPY of 9.9ml or MRS liquid nutrient medium and add 0.1ml bacterium liquid, add 10ulH again
3-TdR cultivates 24h for 37 ℃.
The centrifugal 10min of 2000rpm gets precipitation NSS liquid centrifuge washing 3 times, and is diluted to 107～108CFU/mL, puts 4 ℃ of refrigerators and preserves standby.
(2) mucous preparation: get 1～2ml mucus, all mucus are measured its protein content with Folin-phenol method, with NSS liquid it are diluted to 0.05mgPr/ml again, and-20 ℃ of preservations are standby.
(3) sticking of bacterial classification: get 24 porocyte culture plates, test holes respectively adds 0.25ml mucus, replace mucus to do negative control with the equivalent bovin serum albumin, 4 ℃ are spent the night fixing, every then hole adds 0.5mlNSS liquid washed twice, remove not adherent mucus, every hole adds equivalent 0.25ml mark bacteria suspension, and each sample is done 3 holes.Do positive control with 0.25ml mark bacteria suspension in addition, 28 hatch 2h.Do not stick bacterium with the 0.5mlNSS flush away again.Every hole adds 0.5Mp150%SDS solution, handles 1h, the mucus on the wash-out culture plate for 60 ℃.
Measure H in the elutriant with liquid scintillation instrument
3-TdR content calculates and sticks percentage.
The result shows that the percentage that sticks of 3 strain bacterial classifications to be measured is respectively: 45.6%, 50.3%, 62.3%, and have and stick performance preferably.
Resistance and the living functional assays result of benefit by the 3 strain bacterial strain enterococcus faecalis Enterococcus faecalis (ACCCNo.DW-BLKY-27) that filter out, plant lactobacillus Lactobacillus plantarum (CICC 6026) and Bacillus coagulans Bacillus coagulans (AS 1.2407) find out, for prevention and treatment baby pig diarrhoea certain effect is arranged, further verify its effect but also need to do the animal feeding experiment.
Embodiment 7
Bacillus coagulans Bacillus coagulans (AS 1.2407) is to the animal efficacy test of control baby pig diarrhoea.
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, 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 ‰ Bacillus coagulans Bacillus coagulans (AS 1.2407) bacterium powder 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 ‰ Bacillus coagulans Bacillus coagulans (AS 1.2407) bacterium powder+interpolation 100g/T prevention is had loose bowels on basal diet.Basal diet is formed and trophic level sees Table 5.
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 5 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 feed consumption rate, day weight gain, feedstuff-meat ratio, diarrhea rate index with the repeating groups, and carry out otherness and significantly check.
Test-results
1.1 production performance
The production performance of test piglet sees Table 6.As seen from Table 6 average daily gain piglet test group I, test group II and test group III comparison according to component you can well imagine high by 6.7%, 7.5%, 9.5%; Test group I, test group II and test group III comparison reduces by 7.5%, 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 Bacillus coagulans 1.2407 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 Bacillus coagulans 1.2407 and microbiotic have share synergy.Difference is not remarkable between each is organized aspect the average feed consumption.
The production performance of table 6 test piglet
| Group | The beginning counterpoise | The end counterpoise | Equal day weight gain | Average feed consumption | Feedstuff-meat ratio | Diarrhea rate |
| (kg) | (kg) | (g/ head) | (the g/ head. day) | (％) | ||
| Control group | 8.8±0.4 2 | 23.6±1.32 | 493±21 | 856±45 | 1.74±0. 08 | 10.7±0.6 |
| Test group I | 8.7±0.3 7 | 24.5±1.47 | 526±35 | 851±39 | 1.61±0. 07 | 3.5±0.3 |
| Test group II | 8.9±0.4 5 | 24.8±1.43 | 530±31 | 873±42 | 1.65±0. 09 | 5.6±0.5 |
| Test group III | 8.8±0.4 0 | 25.0±1.50 | 540±40 | 864±51 | 1.60±0. 10 | 3.2±0.4 |
1.2 economic benefit
The Economic and Efficiency Analysis of test pig sees Table 7.The result shows that the head of test group III all gross profit is the highest, thereby finds out that the economic benefit that has a net increase of of adding Bacillus coagulans 1.2407 in feed is significant, and test group is compared with control group and improved 9.34%, 9.12%, 11.79% respectively.This 1.2407 groups of Bacillus coagulans of test is compared on economic benefit difference with the microbiotic group not remarkable, can substitute microbiotic fully, Bacillus coagulans 1.2407 reduces drug residue at alternative microbiotic, produces environment-protecting feed and has very big advantage.
The economic benefit of table 7 test piglet
| Test group I | Test group II | Test group III | Control group | |
| Every net weight (kg) | 15.8 | 15.9 | 16.2 | 14.8 |
| Head is feed consumption (kg) all | 25.53 | 26.19 | 25.92 | 25.68 |
| Add cost (unit/head) | 0.76 | 0.80 | 1.56 | 0 |
| Head is gross profit (unit) all | 111.29 | 111.06 | 113.78 | 101.78 |
| Difference (unit) | +9.51 | +9.28 | +12.00 | 0 |
Annotate: the every kg body weight of piglet is 10 yuan, and basal feed is 1.80 yuan/kg.
Claims (8)
1, a kind of selection of microbial feed additive strain is characterized in that comprising that isolation and purification, resistance seed selection, the benefit of bacterial strain given birth to functional seed selection, identification of strains and/or safety research.
2, the selection of microbial feed additive strain according to claim 1 is characterized in that: the resistance seed selection is meant the seed selection that comprises habitat in the anti-animal body, tolerance drying conditions and formulation process conditions, feed substrate is had tolerance and/or drug susceptibility.
3, the selection of microbial feed additive strain according to claim 2 is characterized in that: the habitat seed selection comprises the seed selection of stomach juice-resistant, bile tolerance, anti-pancreatic juice and/or the liquid of anti-the enzyme in the anti-animal body; The seed selection of tolerance drying conditions and formulation process conditions is meant the seed selection of tolerance lyophilize, spray-dired drying conditions and tolerance particulate material, tablet complete processing operational condition; The seed selection that feed substrate is had a tolerance is meant the high-copper in the tolerance feed and/or the seed selection of high zinc mineral salt; The drug susceptibility seed selection be meant bacterial classification to the normal microbiotic that adds in the feed, in breeding process, prevent and treat the drug susceptibility seed selection of the antibacterials of usefulness.
4, the selection of microbial feed additive strain according to claim 3, it is characterized in that: the stomach juice-resistant seed selection is the simulation test by external monofactor, be the quantitative assay screening that growth property test qualitatively of manual simulation's gastric juice and survival rate are measured, the screening survival rate is at the strain excellent more than 1%; The bile tolerance seed selection is the screening of measuring by external artificial simulated intestinal fluid test growth property test qualitatively and survival rate, is that bacterial classification inoculation is handled in manual simulation's intestinal juice of various biliary salt concn, and the screening survival rate is at the strain excellent more than 1 ‰; The seed selection of tolerance particulate material, tablet complete processing operational condition is meant in the tolerance formulation course of processing and under high temperature, high humidity, the mechanical presses process conditions, detects the survival rate of each bacterial strain that the screening survival rate is at the strain excellent more than 90%.
5, the selection of microbial feed additive strain according to claim 1 is characterized in that: benefit is given birth to functional seed selection and is comprised fungistatic effect, produces acidity test, zymogram is measured and/or stick performance measurement.
6, the selection of microbial feed additive strain according to claim 5 is characterized in that: the fungistatic effect test is to measure the fungistatic effect of bacterial strain to common pathogen with two dish methods, and the good strain excellent of screening fungistatic effect; Producing acidity test is to adopt ion chromatography analysis to measure organic acid content in the fermented liquid, and screening produces the strain excellent of lactic acid, butyric acid, acetate or citric acid; It is to adopt the amylase plate culture medium to carry out the primary dcreening operation of high yield amylase strain excellent and/or the primary dcreening operation that employing casein hydrolysis flat board carries out the high proteinase yield strain excellent that zymogram is measured; Sticking performance measurement is that bacterium is used labelled with radioisotope, then again with cell or mucus incubation after, calculate the percentage that sticks of this bacterium.
7, the selection of microbial feed additive strain according to claim 1, it is characterized in that: identification of strains is with microbiology classification authentication method form and the physiological and biochemical property of selecting bacterial strain to be studied, simultaneously in conjunction with a kind of authentication method based on molecule.
8, the selection of microbial feed additive strain according to claim 1 is characterized in that: safety research comprises hemolytic test, acute toxicity test and chronic toxicity test.
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