CN103555640A - Bacillus subtilis and application thereof to livestock breeding - Google Patents
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Abstract
The invention aims to provide bacillus subtilis WL-04 and application thereof to livestock breeding. The preservation number of the bacillus subtilis WL-04 is CCTCC NO:M 2013343. The bacillus subtilis WL-04 can be used for remarkably inhibiting the growth of Escherichia coli, salmonella, clostridium pefringens and other conditional pathogenic bacteria of the intestinal canal of livestock, and particularly, the bacteriostasis rate of the bacillus subtilis WL-04 to Escherichia coli and clostridium perfingens is above 75%; the enzyme production is stable; the average activity of alpha-amylase is 22U/mL, and the average activity of neutral protease is 170U/mL; the reproduction of probiotics of intestinal canal can be promoted; high temperature for pelletizing can be endured; the influence of inverse environments with low-pH gastric fluid and high-concentration cholate is endured; the production performance of breeding animals can be effectively improved; immune organ development can be promoted. The bacillus subtilis WL-04 can be widely applied to livestock feeds as a feed additive.
Description
Technical field
The invention belongs to microbe to screen and applied technical field, be specifically related to a kind of subtilis WL-04 bacterial strain and as the application of fodder additives.
Background technology
Nowadays; mass-producing and intensive culture development are rapidly; the problem that has thereupon occurred some breeding environment Quality Downs such as stocking density is excessive, breeding house environment ventilation is bad; the various disease of alimentary tract of livestock husbandry and birds that caused by conditionality malignant bacteria are also more and more outstanding to the puzzlement of cultivation, the necrotic enteritis causing as colibacillosis, salmonellosis, clostridium perfringens etc.For reaching the object of disease preventing and treating or promotion growth of animal, a large amount of microbiotic are applied to a plurality of links of Production of Livestock and Poultry.Antibiotic life-time service and abuse bring many negative effects and serious consequence, the problem such as exceed standard that as low in fowl poultry immune, bacterial drug resistance, medicine be residual, particularly some pathogenic bacterium are zoonosis type, if produce the superbacteria of superpower resistance, will bring serious public health security problem.Therefore, in breeding production few with or forbid microbiotic and become social consensus.
Through technological development and accumulation for many years, probiotics micro-ecological formulation becomes the first-selection of substitute antibiotics.Probiotics self secretion many kinds of substance, the growth of antagonism pathogenic micro-organism, regulates animal immune, has no drug resistance, noresidue, deeply popular to people.Genus bacillus the most widely used a kind of probiotics especially wherein.
But the prebiotic performance difference of different Bacillus strains is larger.At present, studying more is to take single performance as sieve bacterium index, such as simple bacteriostatic activity or the enzymatic productivity etc. investigated, to bacterial strain overall performance to consider report less.
Summary of the invention
The object of this invention is to provide a bacillus subtilis WL-04 and the application in livestock and poultry cultivation thereof, this bacterium has remarkable restraining effect, and the breeding of probiotic bacterium is had to promoter action animal and bird intestines conditionality pathogenic bacterium colibacillosis, salmonellosis, clostridium perfringens; This bacterium can produce multiple enzyme, gemma form can tolerate the adverse environments such as high temperature in artificial hydrochloric acid in gastric juice, cholate and the feed granulating process of higher concentration, using it as fodder additives, be applied in animal and fowl fodder, the outburst that can effectively take precautions against livestock and poultry intestinal canal diseases also can improve the production performance of cultivated animals.
One aspect of the present invention provides a bacillus subtilis WL-04(Bacillus subtilis WL-04), on July 16th, 2013, be preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University, its deposit number is CCTCC NO:M2013343.
Subtilis WL-04 of the present invention is in the application of feed additive field.
Another aspect of the present invention provides a kind of microbial preparation that comprises above-mentioned subtilis.
Above-mentioned microbial preparation is the bacterium powder of subtilis WL-04, by mass percentage for the ratio of 0.1%-1% is added in animal and fowl fodder.
Subtilis WL-04 of the present invention, has significant restraining effect to animal and bird intestines conditionality pathogenic bacterium as the growth of intestinal bacteria, Salmonellas, clostridium perfringens, wherein higher to intestinal bacteria and clostridium perfringens bacteriostasis rate, reaches more than 75%; Produce enzyme more stable, alpha-amylase activity average out to 22U/mL; Neutral protease vigor average out to 170U/mL; Promote the breeding of beneficial bacteria of intestinal tract; Can tolerate feed granulating high temperature; Tolerate the impact in the inverse ring border of low pH gastric juice, high density cholate, and can effectively improve the production performance of cultivated animals, Promote immunity allelotaxis, can be used as fodder additives and is widely used in animal and fowl fodder.
Embodiment
The invention provides a strain and there is efficient bacteriostatic activity (can effectively suppress colibacillosis, salmonellosis, clostridium perfringens), the stable genus bacillus that produces amylase and proteolytic enzyme, antibiotic additive agent for feeding as an alternative, can in animal intestinal, play a role, balance intestinal microflora, suppresses the outburst of enteron aisle conditionality pathogenic bacterium, secretes multiple enzyme and VITAMIN etc., thereby improve the production performance of animal, safeguard that animal body is healthy.
Below in conjunction with embodiment, bacterial strain of the present invention is described in detail.
Embodiment 1 subtilis WL-04 separation and evaluation
1, the separation screening of genus bacillus
The mud that Pingdu broiler breeding field, Jiang Qingdao City gathers, adds in sterilized water with the ratio of 1:10, vibrates, mixes, and is prepared into suspension, through 80 ℃ of water bath processing 10min, kills most of heat labile bacterial strain, tentatively obtains the genus bacillus of good heat resistance.
2, there is the genus bacillus screening that suppresses pathogenic bacteria and high enzymatic productivity
1) genus bacillus bacterium solution preparation: by separated genus bacillus, be inoculated in respectively in nutrient broth, 37 ℃, 200rpm, shaking culture 24h;
2) In Vitro Bacteriostasis experiment-micro-meat soup method: the bacillus liquid of cultivating 24h is centrifugal, collection supernatant, get aseptic 96 well culture plates, in 1:1 ratio, add genus bacillus supernatant liquor and pathogenic bacteria liquid (testing pathogenic bacteria used is intestinal bacteria (ATCC25922), Salmonellas (ATCC14028), clostridium perfringens (ATCC13124)), at 37 ℃, under 200rpm condition, jointly hatch 6h, calculate bacteriostasis rate, formula is as follows:
A is control group OD
600, A0 is 0h data; A2 is 6h data;
B is genus bacillus supernatant treatment group OD
600, B0 is 0h data; B2 is 6h data;
3) enzymatic productivity is measured: the bacillus liquid of cultivating 24h is centrifugal, collection supernatant, carry out the detection (GB/T24401-2009) of alpha-amylase activity, and the detection of neutral protease vigor (GB/T28715-2012);
4), in conjunction with the situation analysis that suppresses pathogenic bacteria and enzymatic productivity, be finally separated to a bacillus and do further to investigate.
3, Bacillus Determination
1) above-mentioned genus bacillus physiological and biochemical property is as shown in table 1:
Table 1: separated genus bacillus physiological and biochemical analysis result
2) utilize test kit to extract above-mentioned subtilis genomic dna, utilize round pcr amplification 16S rRNA sequence, after order-checking, obtain the fragment that length is 1406bp.By BLAST compare of analysis, this bacterium and subtilis homology are the highest, in conjunction with Physiology and biochemistry result, identify that this genus bacillus is subtilis.By this bacterium called after subtilis WL-04(Bacillus subtilis WL-04), and on July 16th, 2013, be preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2013343.
Embodiment 2: subtilis WL-04 suppresses animal source Analysis of pathogenic bacteria
1, indication Bacteria culturing: random choose separation is from the pathogenic bacteria in sick chicken, chitling road, intestinal bacteria, Salmonellas, clostridium perfringens, totally 5 strains (be so kind as to give in Qingdao Agricultural University's Preventive Veterinary Medicine laboratory), wherein intestinal bacteria, Salmonellas are inoculated in nutrient broth, 37 ℃, 200rpm, cultivates 15h; Clostridium perfringens is inoculated in cooked meat medium, 37 ℃, cultivates 15h;
2, preparation subtilis WL-04 bacterium liquid: picking list bacterium colony, be inoculated in nutrient broth, 37 ℃, 200rpm, cultivates 24h;
3, In Vitro Bacteriostasis experiment-micro-meat soup method: the bacillus liquid of cultivating 24h is centrifugal, collection supernatant, get aseptic 96 well culture plates, in 1:1 ratio, add genus bacillus supernatant liquor and pathogenic bacteria liquid, at 37 ℃, under 200rpm condition, jointly hatch 6h, calculate bacteriostasis rate, formula is as follows:
A is control group OD
600, A0 is 0h data; A2 is 6h data;
B is genus bacillus supernatant treatment group OD
600, B0 is 0h data; B2 is 6h data;
Subtilis WL-04 is as shown in table 2 to the zoogenous intestinal bacteria of 4 strain, Salmonellas, clostridium perfringens growth-inhibiting situation:
Table 2: the subtilis WL-04 bacteriostatic experiment result of separation of the present invention
+++ represent that bacteriostasis rate is greater than 75%; ++ represent that bacteriostasis rate is between 50-75%;
In upper table, In Vitro Bacteriostasis experimental result is known, subtilis WL-04 of the present invention has significantly suppressed the separated growth from zoogenous 5 pathogen strain bacterium, wherein higher to 2 strain intestinal bacteria and 1 strain clostridium perfringens bacteriostasis rate, reach more than 75%, show that subtilis WL-04 has significant restraining effect to animal source intestinal bacteria, Salmonellas, clostridium perfringens.
Embodiment 3: subtilis WL-04 produces enzyme performance analysis
1, preparation subtilis WL-04 bacterium liquid: picking list bacterium colony, be inoculated in nutrient broth, 37 ℃, 200rpm, cultivates 24h;
2, obtain fermented supernatant fluid: by cultivating the bacillus liquid of 24h, at 10000rpm, centrifugal under 10min condition, collect supernatant;
3, produce enzyme situation: carry out the detection (GB/T24401-2009) of alpha-amylase activity, and the detection of neutral protease vigor (GB/T28715-2012), carry out altogether 3 batches of mensuration, result is as shown in table 3;
Table 3: subtilis WL-04 produces enzyme analysis
Through three batches, repeat to test, carry out enzyme activity determination analysis, it is more stable that subtilis WL-04 produces enzyme, alpha-amylase activity average out to 22U/mL; Neutral protease vigor average out to 170U/mL.In feed, macromolecules starch particle, protein are difficult to directly by animal, absorbed and utilize, and α-amylase and neutral protease can be decomposed into these macromole the small molecules nutritive substance of easy absorption effectively.Subtilis WL-04 of the present invention can stably secrete α-amylase and the neutral protease that high enzyme is lived, and therefore, is expected to be developed as fodder additives, for improving the utilization ratio of feed starch, protein.
Embodiment 4: subtilis WL-04 resistance is analyzed
1, subtilis WL-04 bacterium liquid viable bacteria amount counting (control group): by ten times of gradient dilutions of stroke-physiological saline solution for bacterium liquid, select applicable extent of dilution bacterium liquid, coat on nutrient agar plate and cultivate, counting viable bacteria;
2, Analysis of Heat Tolerance: get subtilis WL-04 bacterium liquid 1mL and join in the triangular flask containing 99mL sterilized water, after 75 ℃ of bath temperatures, 80 ℃, 95 ℃ processing 10min, totally 3 treatment group, count viable bacteria respectively;
3, stomach juice-resistant analysis: get in the triangular flask of artificial simulation gastric juices that subtilis WL-04 bacterium liquid 1mL joins respectively the different pH of 99mL, pH is followed successively by 1,2,3, totally 3 treatment group, in 37 ℃ of water bath processing 2h, counting viable bacteria;
4, bile tolerance analysis: get in the triangular flask of artificial simulation intestinal juice that subtilis WL-04 bacterium liquid 1mL joins 99mL various biliary salt concn, gallbladder salinity is followed successively by 0.3%, 0.5%, 1%, totally 3 treatment group, in 37 ℃ of water bath processing 4h, counting viable bacteria;
Table 4: subtilis WL-04 resistance toheat is analyzed
As shown in Table 4, subtilis WL-04 all can keep high survival rate in different high temperature baths is processed, and illustrates that this bacterium is to hot environment better tolerance.
Table 5: subtilis WL-04 tolerance hydrochloric acid in gastric juice, cholate analysis
As shown in Table 5, after the artificial simulation gastric juices of different pH is processed, when pH >=3, subtilis WL-04 survival rate is 100.0%, when pH=2, subtilis WL-04 survival rate slightly reduces, and is 99.2%, grow up cultivated animals if the Gastric pH scope of pig, chicken is about 2～4 fluctuations, and experiment shows that subtilis WL-04 acid resistance is good.
Through the processing of the artificial simulation intestinal juice of various biliary salt concn, along with the increase of gallbladder salinity, subtilis WL-04 survival rate has certain decline, but survival rate is all more than 95%, illustrates that this bacterium has the ability of very strong tolerance cholate.
In feeding animals, active bacteria formulation must experience competence exertion effect after the barrier of hydrochloric acid in gastric juice and enteron aisle cholate.Subtilis WL-04 of the present invention can tolerate the inverse ring border of acid and cholate, contributes to its being widely used in feed additive field.
Embodiment 5: the impact of subtilis WL-04 on growth of meat chicken performance and Development of Immune Organs
Purchase the white plumage meat cock of the essentially identical Roche 308 of 1 age in days healthy state, be divided at random 3 groups, comprise 1 control group and 2 experimental group, 16 repetitions of each treatment group, each repeats 8 plumage chickens, the production test phase is 35 days, wherein control group is the general goods daily ration of feeding, experimental group is the general goods daily ration of subtilis WL-04 bacterium powder of Different adding amount of feeding, bacterium powder addition is 0.1%(mass ratio), 0.5%(mass ratio), measure 1) changing conditions such as food consumption, material anharmonic ratio, body weight in process of the test; 2) observe broiler chicken Development of Immune Organs situation, experimental result is as shown in table 6:
Table 6: the bacterium powder feeding dorking experiment statistics result of making
The broiler chicken 0-35 days feeding experiment results by table 6, with day weight gain and feedstuff-meat ratio, compare, the experimental group 1 and 2 of adding subtilis WL-04 is all better than control group, but between two experimental group, difference is not remarkable, illustrates that the subtilis WL-04 that feeds can promote growth of meat chicken; And aspect Immune Organs Index, experimental group 2 optimums, are secondly experimental group 1, control group is the poorest, illustrates that subtilis addition is 0.5% to 0.1% more to contribute to the development growth of broiler chicken immune organ.
Comprehensive analysis, feed and add after subtilis WL-04, well improved the production performance of broiler chicken, and addition 0.1% can effectively improve the production efficiency of broiler chicken, increased raiser's economic benefit, when addition reaches 0.5%, more remarkable to the Development of Immune Organs promoter action of broiler chicken.
Embodiment 6: the impact of subtilis WL-04 on Intestine of Broiler Flora dynamics of feeding
Purchase the white plumage meat cock of the essentially identical Roche 308 of 1 age in days healthy state, be divided at random 3 groups, comprise 1 control group and 2 experimental group, 16 repetitions of each treatment group, each repeats 8 plumage chickens, control group is the general goods daily ration of feeding, experimental group is the general goods daily ration of subtilis WL-04 bacterium powder of Different adding amount of feeding, bacterium powder addition is 0.1%(mass ratio), 0.5%(mass ratio), 1-7d is for raising the phase in advance, test period is 14d, 21d, 28d, collect fresh chicken manure sample, measure wherein microorganism species changing conditions, experimental result is as shown in table 7:
Table 7: the intestinal microflora of subtilis WL-04 feeding dorking test changes
From table 7 test-results, in the experiment of feeding, 1) the bacterium amount change detection process of prebiotic lactobacillus, experimental group 1 and 2 bacterium amounts are significantly higher than control group, and between two experimental group, difference is not remarkable; 2) enteric pathogenic bacteria intestinal bacteria, Salmonellas and clostridium perfringens bacterium amount change detection process, experimental group 1 and 2 bacterium amounts are significantly lower than control group, and between two experimental group, difference is not remarkable.Illustrate in the process of feeding the proliferation of probiotics of Intestine of Broiler is had to promoter action after adding subtilis WL-04 of the present invention, and enteron aisle conditionality pathogenic bacterium are played to restraining effect, be obviously better than control group.Add 0.1% or 0.5% subtilis WL-04 microbial inoculum feeding dorking, aspect regulating intestinal canal flora, showing good result.
Claims (6)
1. a subtilis, is characterized in that, described subtilis deposit number is CCTCC NO:M2013343.
2. the application of subtilis claimed in claim 1 in preparing fodder additives.
3. a microbial preparation, described microbial preparation comprises subtilis claimed in claim 1.
4. microbial preparation as claimed in claim 3, is characterized in that, the bacterium powder that described microbial preparation is subtilis claimed in claim 1.
5. microbial preparation claimed in claim 3 is as the application of fodder additives.
6. application as claimed in claim 5, is characterized in that the ratio that described microbial preparation is 0.1%-1% according to mass percent is added in animal and fowl fodder.
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| CN105039223A (en) * | 2015-08-14 | 2015-11-11 | 山东宝来利来生物工程股份有限公司 | Bacillus subtilis with effect of inhibiting clostridium perfringens and application of bacillus subtilis |
| CN109337841A (en) * | 2017-12-27 | 2019-02-15 | 山东农业大学 | One plant of bacillus subtilis BYS2 with Efficient antibacterial performance |
| CN109679879A (en) * | 2019-01-25 | 2019-04-26 | 石河子大学 | A kind of bacterial strain, microbial inoculum and application |
| CN110272854A (en) * | 2019-07-29 | 2019-09-24 | 北京林业大学 | A kind of bacillus subtilis strain and its application |
| CN112760251A (en) * | 2021-01-05 | 2021-05-07 | 安徽农业大学 | Compound bacillus probiotics and preparation and application thereof |
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| CN114032200A (en) * | 2021-12-10 | 2022-02-11 | 安徽天富来生物科技有限公司 | Preparation method of bacillus subtilis feed additive for breeding animals |
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