CN103087964A - Bacillus subtilis, microecological preparation thereof and application of bacillus subtilis in animal feed - Google Patents
Bacillus subtilis, microecological preparation thereof and application of bacillus subtilis in animal feed Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 8
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- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 7
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- Fodder In General (AREA)
Abstract
The invention relates to a bacillus subtilis, a microecological preparation thereof and application of the bacillus subtilis in animal feed, which is characterized in that: the bacillus subtilis is the bacillus subtilis preserved in China general microbiological culture Collection center in 12 months and 17 days in 2009BacillussubtilisAnd the registration number of the preservation center: CGMCC No. 3532. The method comprises the steps of fully culturing and fermenting bacillus subtilis in a culture medium to obtain a metabolite, mixing the metabolite with an adsorbent and a diluent according to the ratio of 1:1:2, drying and crushing to obtain the microecological preparation, wherein the adsorbent is specially-prepared silicon dioxide, and the diluent is any one or the combination of more than one of light calcium carbonate, bran, wheat middling, corn cob materials and rice bran. The addition amount of the bacillus subtilis microecological preparation in the animal feed is 0.05-0.15% of the weight of the animal feed, the animal immune function is improved, and the bacillus subtilis microecological preparation can effectively replace antibiotics.
Description
Technical field
The present invention relates to a kind of subtilis, particularly disclose a kind of the contain probiotics of this subtilis and the application in animal-feed, belong to biological technical field.
Background technology
Fodder additives to the fortified feed nutritive value, improve feed quality, save feed cost and promote the animal health growth that very important effect is arranged.After microbiotic is used as fodder additives, provide certain Epidemic prevention guarantee for animal produces, also brought huge economic benefit to livestock industry.But along with the development of society and the progress of science and technology, utilize microbiotic also progressively to expose as the drawback of fodder additives, the resistance that shows as pathogenic micro-organism strengthens gradually, the animal product drug residue
[1], the mankind's health and the safety of ecotope have been brought potential threat.Therefore, research and the application of Substitutes For Antibiotic probiotics preparation, acid supplement, zymin and some complex ecological preparations etc. become the focus that the herding worker pays close attention to gradually
[2-5]
Subtilis is the intestinal beneficial bacterium of generally acknowledging
[6], its metabolic process produces various bioactivators, and effect shows as:
1, the subtilyne that produces in subtilis thalli growth process, polymyxin, nystatin, linear gramicidins isoreactivity material, these active substances have obvious restraining effect to the conditioned pathogen of pathogenic bacterium or autogenous infection;
2, subtilis consumes rapidly the free oxygen in environment, causes the enteron aisle hypoxemia, promotes the growth of useful anerobe, and produces the organic acid such as lactic acid, reduces the enteron aisle pH value, indirectly suppresses other pathogenic bacterium growth;
3, growing of stimulating animal immune organ activated T, bone-marrow-derived lymphocyte, improves immunoglobulin (Ig) and antibody horizontal, strengthens cellular immunization and humoral immune function, improves herd immunity;
4, the enzymes such as the synthetic α-amylase of subtilis thalline self, proteolytic enzyme, lipase, cellulase, in digestive tube with animal body in digestive enzymes together play a role;
5, the multiple vitamin B group such as energy synthesise vitamins B1, B2, B6, nicotinic acid, the activity of raising animal body internal interference element and scavenger cell.
Subtilis has high temperature high voltage resistant, is beneficial to feed processing, the characteristics that are easy to store, and the application in animal produces is the emphasis of livestock industry scientific research personnel research always.The production bacterial classification of China's feedstuff industry probiotics comparatively lags behind at present, and the production of probiotics is comparatively single
[7-8], be badly in need of filtering out more more outstanding bacterial classifications to satisfy the demand of animal husbandry development.
Summary of the invention
The objective of the invention is the problem for above-mentioned prior art existence, a kind of subtilis and probiotics thereof and the application in animal-feed are provided, subtilis of the present invention is acidproof, alkaline-resisting, but the long period is born High Temperature High Pressure to a certain degree, can suppress colibacillary growth and breeding.The probiotics that the present invention contains above-mentioned subtilis can substitute antibiotics be used for animal-feed, in order to keeping the normal micro-flora of animal intestinal, thereby promotes growth of animal, improve the price of deed.。
The present invention is achieved in that a kind of subtilis, it is characterized in that: this subtilis is to be deposited in the subtilis at " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on December 17th, 2009
Bacillus subtilis, the preservation center numbering of registering on the books: CGMCC No.3532.Described subtilis (
Bacillus subtilis) CGMCC No.3532 is gram-positive microorganism, the bacterium colony on the LB substratum is that white, flat, surface irregularity, edge are irregular, cell dia is shaft-like less than 1 micron, cellular form, forms gemma but does not produce parasporal crystal.Be well-grown between 5.0 ~ 8.5 at pH, in pH is 3.5 environment, cultivate survival rate in 5h more than 90%, in pH is 9.0 environment, cultivate 5h, survival rate is also more than 85%.After 90 ℃ of waters bath with thermostatic control were processed 30 minutes, survival rate was more than 70%.This subtilis can be good at adapting to the animal digestive tract environment, and is insensitive to the thermal treatment of the feed course of processing, and the technique such as be conducive to granulate and expanded.
A kind of probiotics that contains subtilis, the meta-bolites that obtains after adopting above-mentioned subtilis fully to cultivate in substratum, ferment and sorbent material, mixing diluents evenly after, obtain after dried, crushed probiotics.
The preparation method of described subtilis:
The separation and Culture of bacterial strain original seed: 1. the solid plate culture medium prescription is peptone 10g, yeast extract 5g, sucrose 25g, agar powder 15-20g, sodium-chlor 5g, adds deionized water to cumulative volume 1000ml, and culture condition is that pH6-8, temperature are 35 ℃, cultivation 24-48h; 2. liquid nutrient medium is peptone 10g, sucrose 25g, yeast extract 10g, urea 1g, dipotassium hydrogen phosphate 3g, sal epsom 0.5g, calcium carbonate 0.1g, adds deionized water to cumulative volume 1000ml, picking 1. De carries out fluid enlargement culture Dao De Unit bacterium colony, and culture condition is that pH6-8, temperature are 37 ℃, constant-temperature table 160rpm/min, cultivation 15-18h; 3. fermentative medium formula is the manganese sulfate solution 0.1% of W-Gum 0.2%, dregs of beans 0.1%, sucrose 0.5%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.45%, sal epsom 0.05%, iron(ic) chloride 0.01%, 2.8% concentration, the substratum bacterium liquid mixed solution 3-5% that 2. inoculation obtains carries out fermentation culture, culture condition is that pH6-8, temperature are 35 ℃, fermenter stirrer rotating speed 120rpm/min, and fermentation time is 30-84h.、
Described sorbent material is special silicon-dioxide, and described thinner is any in light calcium carbonate, wheat bran, inferior powder, corn core material and rice bran or more than one combination.
The meta-bolites that obtains after described cultivation, fermentation mixes with mass ratio 1:1:2 ratio with sorbent material, thinner.
The application of described subtilis probiotics in animal-feed comprises animal diets and subtilis probiotics, and the addition of described subtilis probiotics in animal-feed is the 0.05%-0.15% of animal-feed weight.Feeding animal comprises monogastric animal, ruminating animal and aquatic animal.
The invention has the beneficial effects as follows: subtilis of the present invention (
Bacillus subtilis) CGMCC No.3532 has acidproof, alkaline-resisting, heat-resisting, as to suppress harmful intestinal tract flora (as intestinal bacteria) Growth and reproduction, this bacillus subtilis formulation is as animal feedstuff additive, microbiotic in alternative existing animal-feed, can improve the animal gastrointestinal tract micro-flora, promote the animal health growth, strengthen the animal immune function, nutritional factor is provided, improve food conversion ratio, promote nutritive substance absorption, reduce the animal diarrhea rate, can improve the production performance of piglet, chick.Subtilis of the present invention (Bacillus subtilis) CGMCC No.3532 and probiotics thereof have no drug resistance, and can not damage environment, also can the mankind's health not worked the mischief, and are a kind of environmentally friendly microbial forage additives.
The preservation explanation
Bacterial strain: subtilis
Bacillus subtilis
Preservation date: on December 17th, 2009
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Unit address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Deposit number: CGMCC No.3532
Embodiment
Below in conjunction with embodiment introduce in detail subtilis of the present invention (
Bacillus subtilis), preserving number: the effect of CGMCC No.3532 probiotics in actual production.
Embodiment 1:
The present embodiment is that preserving number is the screening process of the subtilis of CGMCC No.3532, and concrete steps are as follows:
(1), the preliminary screening of bacterial classification
Get piglet duodenum, ileum and rectal contents and carry out biological screening.With the intestinal contents of getting thermal treatment 15 minutes in the waters of 80-90 ℃, after doing respectively 1 times of dilution, 5 times of dilutions, 10 times of dilutions, 20 times of dilutions, 100 times of dilutions, 1000 times of dilutions with the sterilization deionized water, the cultivation of LB culture medium flat plate.
The LB substratum is used in screening, and both peptone 10g, yeast extract 5g, sodium-chlor 5g, agar powder 15-20g, added deionized water 1000mL, 115 ℃ of autoclaving 15min.
(2), the multiple sieve of bacterial classification
To cultivate in the subtilis liquid medium within that just sifts out, culture medium prescription is that liquid nutrient medium is peptone 10g, sucrose 25g, yeast extract 10g, urea 1g, dipotassium hydrogen phosphate 3g, sal epsom 0.5g, calcium carbonate 0.1g, adds deionized water to cumulative volume 1000ml, 37 ℃, 210rpm constant-temperature table cultivation 15-18h.The bacterium liquid that obtains is processed multiple sieve after 5min in 80 ℃ of water-baths, sieve again substratum and be: peptone 10g, yeast extract 5g, sodium-chlor 5g, sucrose 25g, agar powder 15-20g add deionized water 1000mL, 115 ℃ of autoclaving 15min.
(3), the experiment of bacterial classification acid and alkali-resistance
The subtilis bacterium liquid that multiple sieve is obtained is 3.0 at pH respectively, 3.5, cultivate 5h in 4.0,4.5,5.0,6.0,6.5,7.0,7.5,8.0,8.5,9.0,10.0 liquid LB substratum after, this subtilis is that in 3.5 treatment group, survival rate is 91.5% at pH, is that in 9.0 treatment group, survival rate is 86.2% at pH.
Embodiment 2:
with the subtilis (Bacillus subtilis) of embodiment 1 gained, preserving number: CGMCC No.3532 is inoculated on the solid slant culture base, and described solid slant culture based formulas is: peptone 10g, yeast extract 5g, sucrose 25g, agar powder 15-20g, sodium-chlor 5g, add deionized water to cumulative volume 1000ml, 35 ℃ of constant temperature culture 36h obtain inoculum and are inoculated in enlarged culturing in liquid nutrient medium, and culture medium prescription is: peptone 10g, sucrose 25g, yeast extract 10g, urea 1g, dipotassium hydrogen phosphate 3g, sal epsom 0.5g, calcium carbonate 0.1g, add deionized water to cumulative volume 1000ml, 37 ℃, 160rpm/min constant temperature culture 15-18h, the bacterium liquid of obtaining 3-5% is by volume cultivated in fermention medium, and fermentation culture and formula are W-Gum 0.2%, dregs of beans 0.1%, sucrose 0.5%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.45%, sal epsom 0.05%, iron(ic) chloride 0.01%, 0.1%, 35 ℃ of the manganese sulfate solution of 2.8% concentration, fermenter stirrer rotating speed 120rpm, fermentation time are 72h, the culture that obtains are mixed drying by the 1:1:2 weight ratio with silicon-dioxide and light calcium carbonate, pulverizing obtains the subtilis probiotics.
Embodiment 3:
with embodiment: the subtilis of 1 gained (Bacillus subtilis), preserving number: CGMCC No.3532 is inoculated on the solid slant culture base, and described solid slant culture based formulas is: peptone 10g, yeast extract 5g, sucrose 25g, agar powder 15-20g, sodium-chlor 5g, add deionized water to cumulative volume 1000ml, 35 ℃ of constant temperature culture 36h obtain inoculum and are inoculated in enlarged culturing in liquid nutrient medium, and culture medium prescription is: peptone 10g, sucrose 25g, yeast extract 10g, urea 1g, dipotassium hydrogen phosphate 3g, sal epsom 0.5g, calcium carbonate 0.1g, add deionized water to cumulative volume 1000ml, 37 ℃, 160rpm/min constant temperature culture 15-18h, the bacterium liquid of obtaining 3-5% is by volume cultivated in fermention medium, and fermentation culture and formula are W-Gum 0.2%, dregs of beans 0.1%, sucrose 0.5%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.45%, sal epsom 0.05%, iron(ic) chloride 0.01%, 0.1%, 35 ℃ of the manganese sulfate solution of 2.8% concentration, fermenter stirrer rotating speed 120rpm, fermentation time are 72h, the culture that obtains are mixed drying by the weight ratio of 1:1:2 with silicon-dioxide and wheat bran, pulverizing obtains the subtilis probiotics.
Experimental example:
This experimental example be the resulting subtilis probiotics of embodiment 2 on the impact of weaned piglets, 60 of the DLY weanling pigs that, body weight consistent with age in days approaches are observed the subtilis probiotics to the impact of weaned piglets.Piglet is divided three test group, and test I group is control group, and feed basal diet and add 0.05% subtilis probiotics of test II group is processed feed basal diet and add 0.1% subtilis probiotics of III group.
1 materials and methods
1.1 probiotics
The subtilis probiotics, the bacillus subtilis formulation of employing embodiment 2.
1.2 experimental animal
Select 60 of (DLY) weanling pigs that age in days is consistent, body weight approaches, male and female half and half is divided into 3 groups by echelon design fully immediately, every group of 5 repetitions, and each repeats 4 piglets, and male and female is separately fed.
1.3 test daily ration
Test is divided into 3 treatment group, be control group (basal diet of feeding+55mg/kg duomycin), test I group (the subtilis probiotics of the basal diet of feeding+0.05%), test II group (the subtilis probiotics of basal diet+0.1%).The basal diet raw material forms and nutritive ingredient sees Table 1.Table 1 forms and the trophic level comparison sheet for the experimental basis daily ration.
| Daily ration forms | Content (%) | Trophic level | Content (%) |
| Corn | 56 | Digestible energy (MJ/kg) | 13.2 |
| Dregs of beans | 28 | Crude protein (%) | 20.6 |
| Wheat bran | 3 | Calcium (%) | 1.2 |
| Domestic fish meal | 5 | Phosphorus (%) | 0.72 |
| Whey powder | 4 | Methionin (%) | 1.18 |
| Preblend | 4 | Methionine(Met)+Gelucystine (%) | 0.7 |
Remarks: control group adds duomycin 55mg/kg in the basal diet basis; Test I group is added 0.05% subtilis probiotics (every 1000kg basal diet adds 0.5kg subtilis probiotics) in basal diet; Test II group is added 0.1% subtilis probiotics (adding 1kg subtilis probiotics in every 1000kg basal diet) in basal diet.
1.4 feeding and management
Test in our company's raising experiment base and carry out, raised 28 days pig free choice feeding and drinking-water by normal feeding and management.
1.5 lab analysis
The feed routine analysis is edited according to Peng Jian, and the method that provide " forage analysis and detection technique " (in July, 2008) that science tech publishing house publishes is measured.
1.6 the analysis of data and statistics
Testing data is processed with SPSS6.0 analytic statistics software.
2 the results are shown in Table 2.Table 2 is that probiotics is on the comparison sheet that affects of Production Performance of Weaning Pigs.
| Project | Control group | Test I group | Test II group |
| Initial counterpoise (kg) | 9.32±0.73 a | 9.21±0.84 a | 9.41±0.69 a |
| Counterpoise in latter stage (kg) | 17.12±1.12 b | 17.72±0.97 a | 19.39±1.15 a |
| Average daily gain (g) | 275.1±0.04 b | 303.2±0.05 a | 356.4±0.06 a |
| Total feed consumption (kg) | 315.1 a | 318.2 a | 320.1 a |
| Head is feed consumption (kg) all | 15.76 b | 15.91 a | 16.01 a |
| Feedstuff-meat ratio | 2.05 a | 1.80 b | 1.61 b |
| A dead number (head) | 1 | 0 | 0 |
| Mortality ratio (%) | 5 | 0 | 0 |
| Diarrhea rate (%) | 21% | 5% | 0 |
Remarks: the digital right shoulder of going together indicates same letter person and represents difference not significantly (P〉0.05), indicates different alphabetical persons and represents significant difference (P＜0.05)
As can be seen from Table 2, the average daily gain that adds subtilis probiotics test I group and subtilis probiotics test II group weanling pig respectively than control group improved 10.2% and 29.5%(P＜0.05), feedstuff-meat ratio respectively than control group reduced by 7.3% and 16.6%(P＜0.05); Add 0.1% subtilis probiotics in basal diet, significantly reduced the diarrhea rate of piglet.
From above-mentioned test as can be known, subtilis probiotics provided by the invention, by regulating the gastral micro-flora of animal, promoted profitable strain growth, improved the immunological competence of animal body, effective substitute antibiotics.Add the subtilis probiotics and can improve the price of deed in daily ration, thereby help to reduce feeding cost.
The subtilis probiotics that the inventor obtains embodiment 3 adds in the daily ration of monogastric animal, aquatic animal to be tested, and test-results shows, equally effectively substitute antibiotics is as fodder additives and improve breeding performonce fo animals.
Claims (10)
1. subtilis, it is characterized in that: this subtilis is to be deposited in the subtilis at " China Committee for Culture Collection of Microorganisms's common micro-organisms center " on December 17th, 2009
Bacillus subtilis, the preservation center numbering of registering on the books: CGMCC No.3532.
2. subtilis according to claim 1 is characterized in that:
1) in pH is 3.5 environment, cultivate survival rate in 5h more than 90%;
2) in pH is 9.0 environment, cultivate 5h, survival rate is also more than 85%;
3) after 90 ℃ of waters bath with thermostatic control were processed 30 minutes, survival rate was more than 70%.
3. probiotics that contains claim 1 or 2 described subtilis, it is characterized in that: resulting meta-bolites mixes in the 1:1:2 ratio with sorbent material/thinner subtilis through fully cultivating, ferment afterwards in substratum, the probiotics that obtains after dried, crushed, described sorbent material is special silicon-dioxide, and described thinner is any one or more than one the combination in light calcium carbonate, wheat bran, inferior powder, corn core material and rice bran.
4. subtilis probiotics according to claim 3, it is characterized in that: described culturing process is inoculated in subtilis carries out enlarged culturing in liquid nutrient medium.
5. according to the probioticses of right 3 or 4 described subtilises, it is characterized in that: described liquid nutrient medium is for being fit to any substratum of bacillus subtilis bacteria growing, and its preferred culture medium formula is: peptone 10g, sucrose 25g, yeast extract 10g, urea 1g, dipotassium hydrogen phosphate 3g, sal epsom 0.5g, calcium carbonate 0.1g, add deionized water to cumulative volume 1000ml.
6. according to the probiotics of right 3 described subtilises, it is characterized in that: described fermention medium is for being fit to any substratum of bacillus subtilis bacteria growing; Its preferred culture medium formula is: the manganese sulfate solution 0.1% of W-Gum 0.2%, dregs of beans 0.1%, sucrose 0.5%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.45%, sal epsom 0.05%, iron(ic) chloride 0.01%, 2.8% concentration.
7. according to the probiotics of the described subtilis of any one in right 3-6, it is characterized in that: described substratum is under temperature is the condition of 35-37 ℃, cultivates 15-72h; Its preferred subtilis is inoculated in the solid slant culture base, in the constant incubator of 35 ℃, cultivates 36h as inoculum, and described inoculum is inoculated in the fluid enlargement culture base, is under 35-37 ℃ in temperature, cultivates 15-18h; The meta-bolites of enlarged culturing is inoculated in fermention medium to be cultivated, and under 35 ℃, cultivates 72h.
8. one kind contains the application of the described subtilis probiotics of any one in animal-feed in claim 3-7, comprise animal diets and subtilis probiotics, the addition of described subtilis probiotics in animal-feed is the 0.05%-0.15% of animal-feed weight.
9. the application of subtilis probiotics according to claim 8 in animal-feed is characterized in that: the addition of described subtilis probiotics in animal-feed is preferably 0.1% of animal-feed weight.
10. the application of subtilis probiotics according to claim 8 in animal-feed is characterized in that: described animal diets comprise pig with daily ration, chicken with daily ration, aquatic animal with daily ration and ruminating animal daily ration.
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