CN101982089B - Micro-ecological preparation for livestock, preparation method and application thereof - Google Patents
Micro-ecological preparation for livestock, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a micro-ecological preparation for livestock, a preparation method and application thereof, belonging to the technical field of a micro-ecological preparation. The active ingredient of the micro-ecological preparation is powder of clostridium butyricum CGMCC No.1925. The micro-ecological preparation for livestock has a high content of living bacteria, has antibiotic tolerance and is of no or low sensitivity to some antibiotics; and the micro-ecological preparation can replace some antibiotics and is resistant to gastric acid, bile salt, high temperature and the like. Besides, the preparation method of the micro-ecological preparation is simple, and the micro-ecological preparation prepared by the preparation method has a high content of living clostridium butyricum. Therefore, the micro-ecological preparation for livestock has good market prospects.
Description
Technical field
The present invention relates to a kind of probiotics for animals and preparation method thereof and purposes, belong to the probiotics technical field.
Background technology
Probiotics for animals is the feed addictive of a kind of green, safety, has diseases prevention, strengthens immunity of organisms, the several functions such as promote to grow, put on weight, and pollution-free, noresidue is not developed immunity to drugs.
At present, there is the problem of the following aspects in the probiotics on the market: (1) viable bacteria content is low, and studies show that, if the concentration of a kind of bacterium in cecal content is lower than 10
7Individual/g, then enzyme and the metabolite of this bacterium generation are not enough to affect the host, are difficult to satisfy the treatment needs; (2) antibiotic-resistant not; (3) unstable to high temperature, hydrochloric acid in gastric juice and cholate, cause still to have activity after only having the minority bacterial strain to enter enteron aisle, do not reach the required number of viable that plays a role; (4) unstable, storage life is short.Since these problems that probiotics exists, thus its effect on animal affected, limited the extensive use of probiotics.
Summary of the invention
The object of the present invention is to provide and a kind ofly antibiosis is have tolerance, viable bacteria content are high, stable, the probiotics for animals of long shelf-life.Main active is clostridium butyricum CGMCCNo.1925 bacterium powder in this probiotics for animals.
Another object of the present invention provides preparation method and the purposes of this probiotics for animals.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of probiotics for animals, the active component of said preparation is clostridium butyricum CGMCC No.1925 bacterium powder.
Wherein, CGMCC No.1925 separates from animal intestinal for the applicant described clostridium butyricum (Clostridium butyricum), seed selection obtains, identify through Chinese industrial microorganism fungus kind preservation administrative center, and being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (be called for short CGMCC) on January 22nd, 2007, preserving number is: CGMCC No.1925.Clostridium butyricum CGMCC No.1925 of the present invention is very high to the tolerance of bile, and bile is processed the almost not impact of activity on clostridium butyricum; Can anti-strong acid in short time, such as pH 1.0 or pH 2.0, can be acidproof for a long time under pH 3.0,4.0 conditions, the activity of clostridium butyricum is substantially unaffected; High temperature resistant, have preferably hot shelf stability; Insensitive to some antibiotic, as insensitive to colistine sulfate, a little less than Norfloxacin sensitiveness.
In the probiotics for animals of the present invention, clostridium butyricum CGMCC No.1925 bacteria concentration is 1 * 10
6CFU/g～1 * 10
10CFU/g.
Further, clostridium butyricum CGMCC No.1925 bacteria concentration is 1 * 10
9CFU/g.
Probiotics for animals of the present invention also can further comprise carrier, and carrier is any one or two kinds of in stone flour, aluminum potassium sulfate, the maize cob meal.
The present invention also provides the preparation method of this probiotics for animals, and its preparation process is as follows:
(1) rejuvenation of spawn of clostridium butyricum CGMCC No.1925;
The rejuvenation algebraically of bacterial classification can carry out according to actual needs, but the applicant finds in the invention process, and clostridium butyricum CGMCC No.1925 is through the rejuvenation of 4 generations, and the high and dull and stereotyped numeration of the 4th generation zymotic fluid gemma rate is higher, so applicant preferred the 4th generation is as the rejuvenation bacterial classification;
(2) cultivation of clostridium butyricum CGMCC No.1925, fermentation: with the rejuvenation bacterial classification inoculation in the CBM fluid nutrient medium, cultivated 24 hours in 37 ℃ of insulating boxs, the gemma rate is after more than 75%, be inoculated in the fermentation tank, inoculum concentration 10%, 37 ℃ of constant temperature culture 24 hours, gemma rate reach 75% when above, make zymotic fluid be cooled to 20 ℃;
(3) continuous centrifugal is collected thalline, and bacterium mud is allocated to solid concentration 5-10% with protection liquid and carrier, and with 115 ℃ of inlet temperatures, the condition that outlet temperature is 80 ℃ is carried out spray-drying, collects the bacterium powder, and get final product;
The collection method of bacterium powder can be other method, as with 45 ℃ of left and right sides pneumatic conveying dryings, makes the bacterium powder after the drying, but the applicant finds that the bacterium powder viable bacteria content of spray-drying preparation is high, so preferably spray drying of the present invention.
Wherein, CBM culture medium prescription: glucose 1.0%, casein peptone 1.0%, dusty yeast 0.5%, beef extract 0.5%, (NH
4)
2SO
40.3%, NaCl 0.3%, K
2HPO
4H
2O 0.4%, MnSO
4H
2O 0.02%, MgSO
47H
2O 0.05%, CaCO
30.2%, agar 2.0% (solid medium is used), the pH value: 7.2, sterilising conditions: 121 ℃, 30 minutes.
Wherein, described protection liquid is 10% defatted milk.
The present invention also provides the purposes of this probiotics for animals for the preparation of feed addictive.
Probiotics viable bacteria content for animals of the present invention is high, and clostridium butyricum CGMCC No.1925 bacteria concentration can be up to 1 * 10
10CFU/g; Antibiosis is have tolerance, in the growth course of animal, inevitably to use antibiotic that Animal diseases are treated, for generation and the promotion growth that prevents disease, some domestic Feedstuff Enterprises and breeding enterprise add the antibiotic of sub-doses in feed, this cultivation behavior can have a huge impact the little ecology of animal intestinal, a lot of probioticses can not brought into play its original effect in such cases, but probiotics of the present invention is to some antibiotic a little less than the insensitive or sensitiveness, as insensitive to colistine sulfate, a little less than Norfloxacin sensitiveness, therefore probiotics of the present invention is in preparation and use procedure, just can not be subjected to these antibiotic impacts, even these antibiotic exist, also can bring into play probiotics of the present invention active component---clostridium butyricum CGMCC No.1925 strengthens immunity of organisms, promote growth, the prebiotic effect such as put on weight; And probiotics is subjected to human favor because of its green, the alternative antibiotic of environmental protection, and probiotics of the present invention finds also that under study for action probiotics of the present invention can really substitute some antibiotic, and such as flavomycoin, the two resistance against diseases is suitable; Probiotics stomach juice-resistant of the present invention, cholate, high temperature etc., bacterial strain still has activity after entering enteron aisle, thereby brings into play its effect; And said preparation is active basicly stable under the preservation condition of 25 ℃, 40 ℃ temperature, and with the prolongation of resting period, though active have decline, loss of activity is little; In addition, the preparation method of probiotics of the present invention is simple, various experiment conditions have all passed through applicant's experimental study, the probiotics of preparing with preparation method of the present invention, the clostridium butyricum viable bacteria content is high, and clostridium butyricum CGMCC No.1925 shows preferably anti-machining property in preparation process, after the granulation processing, activity does not have destroyed substantially, stable better etc. in feed.
, it should be understood that described embodiment only is for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
The specific embodiment
Separation, the evaluation of embodiment 1 clostridium butyricum
Sample collection: the sample of strain separating is from the animal intestinal.
Thalline is straight or crooked, the end circle, and single or paired, the accidental long filament shape of short chain thalline, peritrichous can move; Gram-positive can become feminine gender in old culture; Circle or oval gemma are often arranged in the thalline, make thalline middle part enlarge into fusiformis, the eccentric or inferior end of spore gives birth to, without epispore or appendage; SC is circular or irregular, and diameter 1-3 millimeter is slightly protruding, and white is to cream-colored, and the surface is glossy to matt; This bacterium is anaerobic bacteria, well-grown in suitable culture medium, and aerogenesis.Be accredited as clostridium butyricum (Clostridium Butyricum) through Chinese industrial microorganism fungus kind preservation administrative center.
The cultivation of embodiment 2 clostridium butyricums
1. materials and methods
(1) bacterial classification: clostridium butyricum CGMCC No.1925
(2) equipment: light microscope, anaerobic jar, constant incubator etc.
(3) medicine: crystal violet dye liquor, carbolfuchsin dye liquor.
(4) training method: the triangular flask deep liquid leaves standstill cultivation (non-strictly anaerobic condition).The plate solid culture need place anaerobic jar to carry out.
(5) detection method: the bacterium number is measured available dull and stereotyped tilt-pour process and blood cell notation.Thalli morphology is observed in microscopically after dyeing with crystal violet dye liquor and carbolfuchsin dye liquor.
2. result
(1) carbon nitrogen source proportioning test
Take glucose as carbon source, nitrogenous source selects peptone, dusty yeast and beef extract to form compound nitrogen source provides thalli growth required growth factor.Other amounts of components of film solid media compare cultivation take carbon nitrogen source as variable respectively in the experiment, below are the cultivation results of different carbon nitrogen source consumptions and ratio.
1. carbon source consumption test: take glucose as carbon source, be divided into 6 consumption levels and cultivate, the results are shown in Table 1.
Table 1. glucose consumption comparative test result
The result shows carbon source when 1.0% level, and thalli growth is better, and the bacterium number is higher.
2. nitrogenous source proportioning test: in view of the growth requirement of clostridium butyricum bacterial strain, with reference to the composition of several existing culture mediums, test is done respectively the culture experiment of different proportionings take casein peptone, yeast extract powder and beef extract as compound nitrogen source, the results are shown in Table 2.
Table 2. nitrogenous source proportioning test result
The result shows that nitrogenous source forms and ratio has a significant effect to the bacterium number, and wherein 6,7 groups cultivation results is better.
3. carbon nitrogen compbined test: in conjunction with above two result of the tests, carry out the test of different carbon-nitrogen ratio hypothallus yield and gemma production rate.The results are shown in Table 3.
Table 3. carbon nitrogen composite score
The result shows that 2 groups of bacterium numbers and gemma conversion ratio are higher.
Comprehensive above result of the test is taken into account bacterium amount and gemma rate, finally determines the carbon nitrogen source proportioning: glucose 1.0%, casein peptone 1.0%, dusty yeast 0.5%, beef extract 0.5%.
(2) proportion research of inorganic salts and trace element
According to clostridium butyricum nutritional utilization characteristic, done respectively the culture experiment of several groups of different inorganic salts and microelement match, adopt (NH in the test
4)
2SO
4As inorganic nitrogen-sourced; Use K
2HPO
4As cushion and K
+The source; Add MgSO
4And MnSO
4Result of the test sees Table 4.
Table 4. inorganic salts and microelement match result of the test
The result shows that proportioning and the sporulation of inorganic salts and trace element have close relationship, especially Mg
2+Raising to the gemma conversion ratio has significant effect.Wherein the 5th group cultivation results is more satisfactory, can determine the proportioning of inorganic salts and trace element by this.
(3) add antioxidant to the research of thalli growth impact
This experiment adds culture medium with Vc and cysteine as antioxidant, does the cultivation contrast test with the blank group, the results are shown in Table 5.
The test of table 5. anaerobism
As can be seen from Table 5, whether antioxidant adds cultivation results and has no significant effect, and antioxidant easy inactivation of when sterilization, and can affect medium pH.Therefore can add antioxidant in the culture medium.Comprehensive above result of the test is finally determined culture medium prescription, sees Table 6.
Table 6. Miyarisan culture medium prescription
PH=7.0 sterilising conditions: 121 ℃ of 20min.
(4) inoculation pH
This experiment is carried out inoculated and cultured with several different inoculation pH, and cultivation results sees Table 7.
Table 7. inoculation pH test
The result shows that the suitable inoculation of clostridium butyricum pH for neutral, determines that pH7.0 is best inoculation condition.
(5) heat treatment method research
Clostridium butyricum is the endogenous spore bacterium, and the preferred and rejuvenation of this type of bacterium generally all can be adopted heat-treating methods.This experiment is heat-treated when strain transfer accordingly.Concrete operations are that culture medium is warming up to 80 ℃ of inoculations, kept 10 minutes in 80 ℃ after the inoculation, then be cooled to cultivation temperature and continue cultivate, process to play by this step and optimize bacterial classification, activate gemma and impel it to sprout as early as possible, facilitate synchronous cultivation and further prevent from polluting texts.Below be transfer continuously control experiment result after 3 times of heat treatment group and normal temperature inoculation group.
Table 8. heat treatment control experiment result
(6) cultivation temperature research
The suitable cultivation temperature of clostridium butyricum is 32-40 ℃, and this experiment is got respectively different temperatures and cultivated, and the 24hr cultivation results sees Table 9.
The test of table 9. cultivation temperature
According to table 9 result, can determine that the suitableeest cultivation temperature is 36 ℃.
(7) incubation time research
Incubation time is the key factor that affects the dense and gemma maturing rate of bacterium, and this experiment is got different incubation times and cultivated, and the results are shown in Table 10.
The test of table 10. incubation time
The result shows that the short bacterium number of incubation time is high, but gemma conversion ratio and maturity are all lower; Overlong time gemma conversion ratio and maturity there is no raising, the situation that thalline is aging, the bacterium number descends occurs on the contrary.Result of the test shows that 24hr is the suitableeest incubation time.
The tolerance experiment of embodiment 3 clostridium butyricums
1, experiment material
Bacterial classification: clostridium butyricum CGMCC No.1925; Lactobacillus acidophilus, fungi engineering experiment chamber, Beijing provides;
Bile: animal bile, Institute of Animal Husbandry, China Academy of Agriculture Scinces provides;
Culture medium: clostridium butyricum and lactobacillus acidophilus are cultivated, and all use the MRS culture medium; MRS culture medium prescription: glucose 2%, casein peptone 0.5%, dusty yeast 0.3%, beef extract 0.5%, (NH
4)
2SO
40.2%, NaCl 0.2%, K
2HSO
4H
2O 0.2%, KH
2SO
40.2%, CaCO
30.5%, agar 2.0% (solid medium with), pH value: 7.2, sterilising conditions: 121 ℃ 30 minutes.
2, experimental technique
2.1 cultivated 24 hours behind the clostridium butyricum culture medium inoculated, gemma production rate 〉=85%, centrifugation uses distilled water to make bacteria suspension; Cultivated 24 hours behind the lactobacillus acidophilus culture medium inoculated, centrifugation uses distilled water to make bacteria suspension;
2.2 the liquid of preparation various biliary juice concentration, the bacteria suspension of adding 1/5, the ultimate density that makes bile is 0.0,0.3,0.6,1.0%, processes 1-6 hour, to finite concentration, carries out the clostridium butyricum plate count with the distilled water stepwise dilution;
2.3 the clostridium butyricum bacteria suspension is transferred pH to 1,2,3,4 with hydrochloric acid, to process after 1-4 hour, the sterile phosphate cushioning liquid of usefulness 0.2M pH7.0 to finite concentration, carries out the clostridium butyricum plate count with the sample stepwise dilution;
2.4 clostridium butyricum bacterium powder processed 5,10,15,20 minutes in normal temperature (25 ℃), 80,90,100 ℃ of physiological saline after, to finite concentration, carry out the clostridium butyricum plate count with the distilled water stepwise dilution.Clostridium butyricum bacterium powder is packed with thin aluminum bag, be put in normal temperature (25 ℃) and 60 ℃ of incubators and deposited 10 days, in the 5th, 10 day, sampling to finite concentration, was carried out the clostridium butyricum plate count with the distilled water stepwise dilution.
3, experimental result
3.1 anti-bile test
The bacteria suspension bile solution-treated for preparing in advance with clostridium butyricum and lactobacillus acidophilus, processing time was respectively 1,1.5,3,4.5,6 hour, with the distilled water stepwise dilution to finite concentration, then measure the clostridium butyricum viable count, the sample of processing take acholia calculates survival rate as 100%.The results are shown in Table 11.
The anti-bile test of table 11. clostridium butyricum
The result shows that the activity that bile is processed clostridium butyricum does not almost affect, and lactobacillus acidophilus activity after bile is processed has obvious downward trend, and clostridium butyricum is very high to the tolerance of bile.
3.2 acid resistance test
Use hydrochloric acid simulation gastric acid environment, process clostridium butyricum and lactobacillus acidophilus, different time is measured the activity of clostridium butyricum and lactobacillus acidophilus, take the viable count of pH7 sample as 100%, calculates the survival rate of test specimen, and measurement result sees Table 12.
Table 12. clostridium butyricum acid resistance test
The result shows, in 2 hours, clostridium butyricum is insensitive to acid, active substantially without degenerating, even also very stable in the environment of pH 1.0, but with the prolongation of its time in acid solution, active obviously reduction under pH 1.0,2.0 conditions, the activity of clostridium butyricum is substantially unaffected under pH 3.0,4.0 conditions.The result shows that the acid resistance of clostridium butyricum obviously is better than lactobacillus acidophilus.
3.3 heatproof test
Clostridium butyricum bacterium powder is processed 5,10,15,20 minutes in normal temperature (25 ℃), 80,90,100 ℃ of physiological saline after, with the distilled water stepwise dilution to finite concentration, carry out the clostridium butyricum plate count, take the viable count of 25 ℃ of conditions as 100%, calculate survival rate.The results are shown in Table 13.
Clostridium butyricum bacterium powder is preserved in normal temperature (25 ℃) and 60 ℃ of incubators, sampling in the 5th, 10 day, to finite concentration, carries out the clostridium butyricum plate count with the distilled water stepwise dilution, take the viable count of 25 ℃ of conditions as 100%, calculates survival rate.The results are shown in Table 14.
High temperature resistant test in table 13. liquid
The result shows that clostridium butyricum has certain tolerance in high-temp liquid, but the survival rate reduction is very fast under 100 ℃ of conditions, and the feed process is to reach a high temperature moment, judges that therefore clostridium butyricum has the ability of the instantaneous heat destruction of tolerance feed processing.The whole deactivations of lactobacillus acidophilus under the similarity condition.
Preservation that table 14. is high temperature resistant test
The result shows that clostridium butyricum has preferably hot shelf stability.
The resistance of embodiment 4 clostridium butyricums
1, materials and methods
1.1 bacterial classification: clostridium butyricum CGMCC No.1925
1.2 culture medium: MRS culture medium
1.3 stainless steel tubule: internal diameter 6mm, high 10mm, external diameter 7.8mm.
1.4 antibiotic:
(1) kanamycins: content 98%, Bailinkangyuan Biotechnology Co., Ltd., Beijing provides.
(2) Bacitracin Zinc: content 15%, the emerging animal pharmaceutical factory in Tianjin provides.
(3) flavomycoin: content 5%, Beijing Da Bei farming feed technology company provides.
(4) colistine sulfate: content 15%, the emerging animal pharmaceutical factory in Tianjin provides.
(5) Norfloxacin: content 95%, Beijing all moral great achievement feed company provide.
1.5 method
(1) bacteria suspension configuration: get clostridium butyricum and be inoculated in the MRS cultivation of 250ml sterilization, cultivated 24 hours for 37 ℃, behind the counting, being diluted to concentration with sterilized water is 1 * 10
7The bacteria suspension of individual bacterium/ml is as inoculation liquid.
(2) preparation of two dish: the MRS solid medium after the 20ml sterilization is melted rear the adding in the culture dish, and the ground floor culture medium is made in cooling.Then remove the MRS culture medium 5ml and the bacteria suspension 1ml that melt, in 50-60 ℃ of adding culture dish, shake up rear cooling and make second layer culture medium (as the bacterium layer).
(3) detect: adopt three-dose method, in each culture dish, put into 6 stainless steel tubules, wherein drip in 3 of one group of interval pipes high, medium and low concentration with reference to antibiotic solution.Other 3 pipe addings are diluted to the high, medium and low concentration sample of certain density test (using feed to recommend consumption with antibiotic), use kanamycins as reference antibiotic.Test sees Table 15 with the antibiotic solution allocation.
Table 15. antibiotic sample allocation list
2, result of the test
To be added drop-wise in the steel pipe of culture dish by the antibiotic solution of table 15 preparation, in 37 ℃, cultivate in the anaerobic jar after 24 hours, observe the inhibition zone size, with reference to antibiotic identical or approaching be judged as sensitivity.Result of study shows that clostridium butyricum CGMCC No.1925 is insensitive to colistine sulfate, a little less than Norfloxacin sensitiveness.
The preparation of embodiment 5 probioticses
(1) rejuvenation of spawn of clostridium butyricum CGMCC No.1925;
The bacterial classification inoculation of clostridium butyricum CGMCC No.1925 was cultivated 24 hours in 37 ℃ of insulating boxs in 250mlMRS culture medium (500ml triangular flask), inoculated successively 5 generations (inoculum concentration is 10%) by this condition.Experimental result such as table 16.
The test of going down to posterity of table 16. clostridium butyricum
High and the dull and stereotyped numeration of the 4th generation zymotic fluid gemma rate is higher as seen from the above table, but fermentation parameter in the process that goes down to posterity, selected for the 4th generation carried out plate isolation without very large gap, and picking list bacterium colony is made the test tube slant, and it is kept in 4 ℃ of refrigerators.
(2) cultivation of clostridium butyricum CGMCC No.1925, fermentation: with the rejuvenation bacterial classification inoculation in the CBM fluid nutrient medium, cultivated 24 hours in 37 ℃ of insulating boxs, the gemma rate is after more than 75%, be inoculated in the fermentation tank, inoculum concentration 10%, 37 ℃ of constant temperature culture 24 hours, gemma rate reach 75% when above, make zymotic fluid be cooled to 20 ℃;
(3) continuous centrifugal is collected thalline, and bacterium mud is allocated to solid concentration 10% with 10% defatted milk and maize cob meal, and with 115 ℃ of inlet temperatures, the condition that outlet temperature is 80 ℃ is carried out spray-drying, collects bacterium powder or air-dry collection bacterium powder;
CBM culture medium prescription: glucose 1.0%, casein peptone 1.0%, dusty yeast 0.5%, beef extract 0.5%, (NH
4)
2SO
40.3%, NaCl 0.3%, K
2HPO
4H
2O 0.4%, MnSO
4H
2O 0.02%, MgSO
47H
2O0.05%, CaCO
30.2%, agar 2.0% (solid medium is used), the pH value: 7.2, sterilising conditions: 121 ℃, 30 minutes.The results are shown in Table 17
Table 17. probiotics testing result (centrifugal airing separation)
Table 18. probiotics testing result (spray-drying)
The result shows that the bacterium powder that the spray-drying mode prepares is higher than the bacterium powder bacterial content of pneumatic conveying drying preparation, and gained bacterium powder seals with aluminium foil bag, is stored in 4 ℃ of refrigerators.
The stability test of embodiment 6 probioticses
One, materials and methods
1, the probiotics of embodiment 5 spray-dryings preparation, bacteria concentration 7.4 * 10
9Cfu/g
2. culture medium: glucose 15g, casein peptone 5g, yeast extract 5g, sodium chloride 2g, dipotassium hydrogen phosphate 2g, potassium dihydrogen phosphate 2g, ammonium sulfate 2g, agar 20g, distilled water 1000ml, pH 7.2.With 121 ℃ of autoclavings after above-mentioned culture medium packing 20-30 minute.
3. sampling dilution: with sterile working sample thief 1g, put into the triangular flask that 99ml sterilized liquid culture medium and an amount of bead are housed, be put in 37 ℃ of insulating boxs and activate 1 hour, made 1: 100 the equal liquid of sample in 30 minutes with the mechnical oscillator vibration.Use the 1ml aseptic straw, draw 1: 100 equal liquid 1ml of sample, inject the test tube that contains 9ml sterilized water physiological water along tube wall.Other gets the 1ml aseptic straw, presses the aforesaid operations order, does 10 times and increases progressively dilution.So whenever, increase progressively one, change 1 1ml aseptic straw.
4. condition of culture: select three suitable serial dilution degree sample liquid to carry out plate count.Each dilution factor is made 6 plates, marks at plate.Adding 12-15ml in each plate has melted and has been cooled to 45-50 ℃ MRS culture medium, after culture medium cooling and dry tack free, the mixed diluting liquid 0.1ml that draws corresponding gradient with aseptic straw adds in the plate, and is uniformly coated on the cooling media surface with spreader immediately.More than whole operation should add culture dish from culture and begin to finish and must in 20 minutes, finish to inoculation.If a certain sample liquid surpasses 2 minutes in the standing time of taking out for before the examination part, application machine oscillator vibration 15 seconds.Liquid to be diluted with Flat plate turnover, places anaerobic jar to cultivate taking-up in 24-48 hour in 37 ± 1 ℃ of incubators after infiltrating in the culture medium fully.
5. anaerobism is cultivated:
(1) utilize the reaction of borohydride sodium or potassium borohydrid and water to produce hydrogen, under the catalysis of palladium, hydrogen generates water with a bag interior combination with oxygen, thereby sets up oxygen-free environment.
(2) carbon dioxide of adding about 10% under oxygen-free environment is conducive to the growth of anaerobic bacteria.Carbon dioxide is obtained by citric acid and reaction of sodium bicarbonate.
(3) take by weighing 2 gram borohydride sodium or potassium borohydrids, 1 gram citric acid and 1 gram sodium acid carbonate are wrapped with the filter paper of suitable size, and are contained in the small plastic bag of three sealings.Drier electric furnace baking about 5 minutes, is installed in below the lid of anaerobic jar.
(4) ready flat-plate inverted is placed in the little shelf of aluminum, be placed in the anaerobic jar, the small plastic bag that medicine is housed is placed on little shelf next door.
(5) lid of anaerobic jar is built, the osculum on the lid is opened, the distilled water with about suction pipe absorption 10ml joins medicine by osculum and wraps, and withdraws from as early as possible suction pipe, and mouth is tightened.
(6) treat that medicine reacts completely, and tank places 37 ± 1 ℃ of incubators to cultivate in tank without gas leak phenomenon.
6. count:
7. sampling test:
(1) preparation shelf test: the probiotics of embodiment 6 spray-dryings preparation is sub-packed in the thin aluminum bag, deposits in respectively in the incubator of 25 ℃, 40 ℃ temperature the bacterium number in the different time working sample.
(2) stability test of preparation in feed: the probiotics of embodiment 6 spray-dryings preparations is made an addition in the feed by 1% addition, carry out cold granulation, particulate material is sub-packed in the polybag, deposit in room temperature (20-30 ℃), the clostridium butyricum number of viable in different time sampling and measuring feed.
Two. result of the test
1. preparation shelf test
(1) 25 ℃ of shelf test sees Table 19.
25 ℃ of shelf test results of table 19
(2) 40 ℃ of shelf tests see Table 20.
40 ℃ of shelf test results of table 20
2. the stability test of probiotics of the present invention in feed sees Table 21.
The stability test of table 21 probiotics of the present invention in feed
Result of the test shows that the probiotics of the embodiment of the invention 5 preparations is active basicly stable under the preservation condition of 25 ℃, 40 ℃ temperature, and with the prolongation of resting period, activity slightly descends, but loss of activity is little within experimental period.
Embodiment 7 clostridium butyricums substitute the experiment of broiler fodder Antibiotic use effect
1 materials and methods
1.1 test material
The probiotics of embodiment 5 spray-dryings preparations, i.e. probiotics, viable count 7.4 * 10
9CFU/g.
1.2 experimental animal and grouping
Test site: test is at Shouguang, Shandong chicken house
Test period: 42 days
Choose 1200 of the AA broiler chicken (male and female mixing) of 10 age in days health, random distribution to 10 processed group, each processes 3 repetitions, and each repeats 40.Feeding time is 42 days.Test grouping situation sees Table 22.
The grouping of table 22 experimental animal and grouping
1.3 daily ration preparation
Carry out daily ration design and preparation with reference to the feeding standard of NRC (1994) broiler chicken nutrition requirements and this test chicken house.The basal diet prescription sees Table 23.
Table 23 experimental basis daily ration forms
1.4 feeding and management
Routine Management program according to this test chicken house is carried out immunity and daily management, adopts the flat bedding and padding mode of supporting in ground to raise, and free choice feeding and drinking-water feed intake twice every day, namely at 9 in the morning and afternoons 4 point.Weigh as unit carries out empty stomach repeating at 10,22 and 43 ages in days, examine and record health status and the ight soil situation of chicken every day, to repeat as the unit record inventory of every day.
1.5 detection index
Average daily ingestion amount, average daily gain, material anharmonic ratio, diarrhea rate and survival rate
1.6 data analysis
Adopt SPSS13.0 software to carry out data statistic analysis.
2 results and analysis
2.1 different probio processed group to 10-21 age in days production performance relatively
Table 24 10-21 age in days production performance relatively
Remarks: the different letters of same row represent significant difference (p≤0.05) in the form, and alphabetical identical person represents difference not remarkable (p 〉=0.05), and following table together.
The result learns from table 24, the average individual weight of 21d: add the test group of clostridium butyricum, 0.2% group of addition is the highest, improves 2.27% than antibiotic control group.
The 10-21d average daily gain: the clostridium butyricum group is the highest with 0.2% additive, is significantly higher than 0.05% and 0.1% interpolation group (p≤0.05), and each processed group of clostridium butyricum and two control group difference is not significantly (p≤0.05); Each processed group is the highest to add 0.2% group of clostridium butyricum.
10-21d expects anharmonic ratio: the clostridium butyricum group is minimum with 0.2% additive, but each group difference not significantly (p 〉=0.05).
2.2 the different probio process for producing of 10-42 age in days Performance Ratio
Table 25 10-42 age in days production performance relatively
As can be seen from Table 25, all difference is not remarkable between each group of 10-42d average daily gain and material anharmonic ratio and the average individual weight of 42d.The average individual weight of the 42d of clostridium butyricum group and 10-42d average daily gain are best with 0.05% interpolation group of low dosage, and have improved 1.97% than antibiotic control group respectively.
3 conclusions
This result of the test shows that the resultant effect that in earlier stage adds 0.2% clostridium butyricum substitute antibiotics at growth of meat chicken is better; Growth later stage clostridium butyricum is better with 0.05% additive effect of low dosage, adding probiotics group death rate of the present invention significantly reduces, flavomycoin group death rate is suitable with adding, illustrate that probiotics of the present invention and flavomycoin have equal bacteriostasis, the disease of alternative flavomycoin prevention animal occurs.
Claims (5)
1. a probiotics for animals is characterized in that active component is clostridium butyricum (Clostridium butyricum) CGMCC No.1925 bacterium powder.
2. probiotics for animals as claimed in claim 1 is characterized in that clostridium butyricum CGMCC No.1925 bacteria concentration is 1 * 10
6CFU/g～1 * 10
10CFU/g.
3. probiotics for animals as claimed in claim 2 is characterized in that clostridium butyricum CGMCC No.1925 bacteria concentration is 1 * 10
9CFU/g.
4. such as each described probiotics for animals of claim 1～3, it is characterized in that further comprising carrier, carrier is any one or two kinds of in stone flour, aluminum potassium sulfate, the maize cob meal.
5. the application of each described probiotics for animals of claim 1～4 in the preparation feed addictive.
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| 安沙.大北农合作模式多样整合资源推进成果转化.《中关村周刊》.2010, * |
| 林云等.益菌多微生态制剂对雏鸭的饲养试验.《饲料研究》.2001,(第6期),第26-27页. * |
| 黄俊等.一株饲用酪酸菌特性及培养条件的研究.《饲料工业》.2004,第25卷(第2期),第22-25页. * |
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