CN103243047B - Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis - Google Patents

Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis Download PDF

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CN103243047B
CN103243047B CN201310169636.4A CN201310169636A CN103243047B CN 103243047 B CN103243047 B CN 103243047B CN 201310169636 A CN201310169636 A CN 201310169636A CN 103243047 B CN103243047 B CN 103243047B
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vomitoxin
subtilis
ansb471
feed
activated protein
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CN103243047A (en
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计成
马秋刚
赵丽红
李笑樱
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Henan billions of Zhongyuan Biotechnology Co., Ltd.
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China Agricultural University
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Abstract

The invention provides Bacillus subtilis ANSB471 capable of effectively degrading vomitoxin in feed and an application of the Bacillus subtilis. The invention also relates to a cultural method of the Bacillus subtilis strain and a method for applying fermentation liquor of the strain to degradation on vomitoxin in the feed. After reacting for 72 hours, degradation rate of the bacillus subtilis ANSB471 on the vomitoxin in the feed is 95%, and the degradation rate on the vomitoxin is still about 90% after subculture. The strain also can secrete amylase and is high in enzyme activity, and utilization rate of animals on starch in the feed can be increased. The Bacillus subtilis ANSB471 has high vomitoxin degradation activity, strong specificity and mild effect and can not damage nutritional ingredients in the feed.

Description

The subtilis of one high-efficiency degradation vomitoxin and application thereof
Technical field
The present invention relates to microorganism field, specifically, relate to subtilis and the application thereof of a high-efficiency degradation vomitoxin.
Background technology
That studies mycotoxin the common concern of food sanitation and security and investigator along with human consumer deepens continuously, and the mycotoxin contamination situation in raw material and feed also causes the attention of Animal nutrition circle and feed circle day by day.The ratio having the investigation Chinese feed of display and raw material mycotoxin contamination to exceed standard is 60% ~ 70%, and wherein the ratio that exceeds standard of vomitoxin is close to 70%.(Chinese feed and feedstuff raw material are subject to the report of survey of mycotoxin contamination to Wang Ruojun etc., fodder industry, 2003,24 (7): 53 ~ 54) to the investigation result display of 28 provinces, cities and autonomous regions of China, 194 parts of feeds and raw material corn, wheat bran, the pollution rate of vomitoxin, T-2 toxin and zearalenone is respectively 50.0%, 18.1% and 26%.Wang Jin bravely waits (Chinese feeds in 2012 and raw material mycotoxin examining report, " Chinese herding magazine ", 4th phase in 2013,29 ~ 34 pages) 841 parts, China various places feeds in 2012 and material sample are detected, find that in current feed and raw material, the main mycotoxin existed is vomitoxin, zearalenone, FT and aflatoxin.Wherein vomitoxin positive rate and mean value the highest.
Vomitoxin (vomitoxin) is extensively present in the Fodder and food raw materials such as barley, wheat, corn and oat.After people and animals have taken in the food polluted by vomitoxin, apocleisis, vomiting, diarrhoea, fever, astasia and the acute poisoning symptom such as slow in reacting can be caused, damage hemopoietic system time serious and cause death.Different animals is different to the sensitivity of vomitoxin, and pig is the animal the most responsive to vomitoxin, and weanling pig is especially responsive.In feed, the micro-toxin of 0.3 ~ 0.5mg/kg will cause pig food refusal, growth declines and decline to the resistibility of transmissible disease, can cause pig food refusal, drowsiness, growth is seriously obstructed, body weight gains slows down, immunity function goes down, muscle coordination is lost and the symptom such as vomiting when containing the vomitoxin of more than 1mg/kg in feed.In addition, vomitoxin at humans and animals body accumulation, can bring out acute or chronic disease, has teratogenecity, neurotoxicity, embryotoxicity and immunosuppressive action.In the resident's diet of esophageal carcinoma high incidence area of gastric cancer, Hebei province of China pollution rate and content all quite high.
Vomitoxin produces so serious harm to livestock industry and the mankind, and therefore since eighties of last century vomitoxin is found, scientific worker just constantly studies this anatoxic prevention and removing toxic substances, detoxicating method.In removal feedstuff raw material known up to now, the method for vomitoxin comprises physics, chemistry and biology method.Physics, chemical detoxication, all inapplicable for current feed industry, aquaculture in actual applications.Because of its operational difficulty, and large batch of feed and these class methods of raw material cannot be carried out, and after chemical detoxication process, often reduce nutritional quality and the palatability of feed, also do not have the effective method preventing mycotoxins in feed from polluting at present.Accuracy, validity and the feature of environmental protection that microorganism or biological enzyme react because of it and become people pay close attention to focus.
Current screening is few with the genus bacillus quantity being separated the vomitoxin of degrading obtained, and the relevant report of the vomitoxin had no in genus bacillus degraded feed and the characteristic research to bacterial strain resistance function.
Summary of the invention
The object of this invention is to provide subtilis and the application thereof of a high-efficiency degradation vomitoxin.
In order to realize the object of the invention, the invention provides subtilis (Bacillus subtilis) ANSB471 of a high-efficiency degradation vomitoxin, this bacterium is the subtilis that the strain filtered out from mouldy grain has vomitoxin in efficient degradation feed, now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101, preservation date on March 21st, 2013, preserving number CGMCC No.7344.
The cultural method of above-mentioned subtilis ANSB471: getting viable bacteria concentration is 10 9the preferred 1mL of subtilis ANSB4710.5-2.5mL(of CFU/mL), be inoculated in the preferred 50mL of 50-100mL() carry out shake flask fermentation cultivation in substratum, obtain the fermented liquid of subtilis ANSB471.
Described fermention medium is made up of following component: Tryptones 8-12g, yeast extract 1.5-3g, glucose 1.5-4g, extractum carnis 2-5g, sodium-chlor 3-6g, Sodium phosphate dibasic 2.5-4g, magnesium sulfate heptahydrate 0.5-1.5g, adding distil water to 800-1200mL, pH value 7.0-7.4.Preferably, described fermention medium is made up of following component: Tryptones 10g, yeast extract 2g, glucose 2g, extractum carnis 3g, sodium-chlor 4g, Sodium phosphate dibasic 3g, magnesium sulfate heptahydrate 1.0g, distilled water 1000mL, pH value 7.2.
Described conditions of flask fermentation is: leavening temperature 30-40 DEG C, fermentation time 16-24h, rotating speed 180-220r/min, pH value 7.0-7.4.Preferably, described fermentation condition is: leavening temperature 37 DEG C, pH value 7.0, rotating speed 200r/min, fermentation time 20h.
The Microbiological Characteristics of subtilis ANSB471 is in table 1.
The form of table 1ANSB471 and physicochemical characteristics
Test subject Result Test subject Result
Gramstaining + Utilize glucose +
Cell shape Shaft-like Utilize wood sugar +
Cell dia > 1 μm + Utilize L-arabinose +
Form gemma + Utilize N.F,USP MANNITOL +
Gemma expands - Fat hydrolysis -
Gemma is circular - Utilize Citrate trianion +
Catalase + Starch Hydrolysis +
Oxydase + Decompose casein +
Anaerobic growth - Nitrate reduction +
VP tests + Utilize tween 80 +
VP＜pH6 + 10 DEG C of growths -
VP＞pH7 - 50 DEG C of growths -
Methyl red test - 5%NaCl grows +
The 16srDNA sequencing result of subtilis ANSB471 is as shown in Seq ID No.1.
The present invention also provides a kind of preparation method of activated protein of vomitoxin of degrading: the fermented liquid getting above-mentioned subtilis ANSB471, centrifugal, get supernatant liquor, the dehydrated alcohol adding 2-4 times of (preferably 3 times) volume carries out precipitation 0.5-2h, centrifugal (5000r/min, 10min), by the precipitation 0.01mol/L PBS buffer solution obtained, centrifugal, the precipitation obtained is dissolved in 0.01mol/L PBS damping fluid, freeze-drying concentrates the activated protein obtaining degraded vomitoxin.
Wherein, the method preparing 0.01mol/L PBS damping fluid is: take 8g NaCl, 0.2gKCl, 1.44g Na 2hPO 4with 0.24g KH 2pO 4, be dissolved in 800mL distilled water, by the pH value to 7.5 of NaOH regulator solution, adding distil water is settled to 1000mL.
The present invention also provides the activated protein of the degraded vomitoxin prepared by aforesaid method.
The present invention also provides described subtilis ANSB471 or the application of described activated protein in degraded feed in vomitoxin.
Particularly, the fermented liquid of described subtilis ANSB471 is mixed with the feed polluted by vomitoxin, the pH value of reaction system is adjusted to 7.0-7.4,36-38 DEG C, rotating speed 180-220r/min, reaction 2-72h.Described fermented liquid is 50mL, by the feed 5g that vomitoxin pollutes.
Or, described activated protein is dissolved in 0.01mol/L PBS damping fluid, then activated protein solution is mixed with the feed polluted by vomitoxin, 36-38 DEG C, rotating speed 180-220r/min, reaction 2-72h.
The present invention also provides a kind of animal feedstuff additive, and its effective constituent is the activated protein of subtilis ANSB471 microbial inoculum or described degraded vomitoxin.
The present invention further provides the application of described subtilis ANSB471 in anti-simulated gastric fluid and/or simulation cholate environment: the fermented liquid getting described subtilis ANSB471, join in the environment of simulated gastric fluid and/or simulation cholate, 36-38 DEG C, cultivate 24h, the pure motility rate of viable bacteria can reach 79%-82%.
The present invention also provides the amylase produced by described subtilis ANSB471 fermentation, enzyme producing method is: get the fermentation of bacillus subtilis liquid 0.5-2mL obtained according to above-mentioned fermentation condition, be inoculated in specific culture medium and ferment, leavening temperature is 36-38 DEG C, pH value 7.0-7.4, rotating speed 180-220r/min, fermentation time 20-30h, obtain supernatant crude enzyme liquid after fermented liquid bactofugation body, in crude enzyme liquid, comprise amylase.
Described specific culture medium formula is: Tryptones 10g/L, yeast leaching powder 5g/L, Zulkovsky starch 5g/L, KH 2pO 42g/L, MgSO 47H 2o0.5g/L, CaCl 22H 2o0.2g/L, prepares with water.
The present invention has following beneficial effect:
1) subtilis ANSB471 of the present invention can secrete the activated protein of degradable vomitoxin, and its degradation efficiency is high, high specificity, can reach 90% to the degradation rate of vomitoxin, and this removing toxic substances reaction is irreversible biological degradation.
2) detoxicating activity of subtilis ANSB471 of the present invention to vomitoxin is high, acts on single-minded, and action effect is gentle, can not destroy the nutritive ingredient in feed, not affect the organoleptic quality of feed.
3) method of subtilis ANSB471 degraded vomitoxin of the present invention is simple to operate, does not pollute, efficiently solve traditional detoxicating method Problems existing to feed and environment.
4) vomitoxin that can also degrade in feed of subtilis ANSB471 of the present invention, has wide market application foreground.
5) subtilis ANSB471 of the present invention has the ability of opposing hydrochloric acid in gastric juice and cholate, is conducive to playing degradation function in animal intestinal.
6) subtilis ANSB471 of the present invention can also secreting amylase, and enzyme activity is higher, can improve the utilization ratio of animal to starch in feed.
7) subtilis ANSB471 of the present invention has vomitoxin function in simultaneously degrade vomitoxin and feedstuff raw material, removing toxic substances efficiency is high, to animal, there is anti-adversity, nontoxic, this bacillus can secreting amylase, can directly be added in feed, be highly suitable for as animal feedstuff additive.
8) subtilis ANSB471 of the present invention has vomitoxin function in simultaneously degrade vomitoxin and feedstuff raw material, removing toxic substances efficiency is high, has anti-adversity to animal, nontoxic, strains A NSB471 can directly be added in feed, is highly suitable for as animal feedstuff additive.
Accompanying drawing explanation
Fig. 1 is the time dependent curve of subtilis ANSB471 degraded vomitoxin in the embodiment of the present invention 6.
Fig. 2 is that the activated protein of the degradable vomitoxin secreted by subtilis ANSB471 in the embodiment of the present invention 8 reacts the degradation rate of 72h to vomitoxin after thermal treatment and Proteinase K process.
Fig. 3 be in the embodiment of the present invention 10 three kinds of different subtilises to the degradation rate of vomitoxin standard substance.
Fig. 4 be in the embodiment of the present invention 10 three kinds of different subtilises to the degradation rate of vomitoxin in feed.
Fig. 5 is that the amylase activity of three kinds of different bacillus subtilis secretions in the embodiment of the present invention 10 compares.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The screening of embodiment 1 bacterial strain
Concrete steps are as follows:
1) bacterial strain screening: carry out bacteria selection from animal intestinal contents, the feed that goes mouldy, go mouldy food and physical environment.
Prepared by seed liquor: each intestinal contents sample and the sample that obtains from environment are mixed with the sterilized water of two volumes respectively, be placed in sterilizing 18mm × 180mm test tube, shaken overnight; Aspirate supernatant 0.1mL, mixes with the screening culture medium of 0.8mL respectively, cultivates 12h, obtain seed liquor in 15 DEG C of thermostat container concussions.
Wherein, the formula of screening culture medium is: NaH 2pO 40.25g, NH 4nO 31.0g, MgSO 4.7H 2o0.25g, FeSO 40.001g, agar 17g, glucose 2.0g, adding distil water is to 1000mL, pH7.0; 121 DEG C of steam sterilizing 20min.
Sample primary dcreening operation: respectively 100 μ l vomitoxins (100 μ g/mL) are added in each seed liquor, vomitoxin is added as blank using the screening culture medium not connecing bacterium, whirlpool concussion mixing, be placed in 37 DEG C of incubator quiescent culture 72h, use enzyme linked immunological kit it to be carried out to the analysis of toxin degradation rate.
Strains separation: select the seed liquor that toxin degradation efficiency is the highest, draws seed liquor 0.1mL, with fermention medium gradient dilution 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6, 10 -7get each dilute sample solution of 0.2mL respectively, coat the flat board of seed culture medium, 15 DEG C of thermostat containers cultivate 3 days, observations, according to form and the feature of different bacterium colony, chooses one by one by representational bacterium colony, at the flat lining out purifying of fresh seed culture medium, number respectively and be forwarded to Storaged media inclined-plane and preserve.
Wherein, seed culture based formulas is: extractum carnis 3g, Tryptones 5g, yeast extract 2g, glucose 2g, NaCl10g, adding distil water to 1000mL, 121 DEG C of steam sterilizing 20min.
Fermentative medium formula is: Tryptones 10g, yeast extract 2g, glucose 2g, extractum carnis 3g, sodium-chlor 4g, Sodium phosphate dibasic 3g, magnesium sulfate heptahydrate 1.0g, adding distil water to 1000mL, pH value 7.2,121 DEG C of steam sterilizing 20min.
The formula of strain store medium is: Tryptones 10g, yeast extract 2g, glucose 2g, extractum carnis 3g, sodium-chlor 4g, Sodium phosphate dibasic 3g, magnesium sulfate heptahydrate 1.0g, agar 18g, pH value 7.2,121 DEG C of steam sterilizing 20min.
Bacterial strain sieves again: by the inoculation of above-mentioned separation and purification in fermention medium, after inoculation, concentration is 2%, 15 DEG C of fermentation 24h, get 900 μ l fermented liquids respectively, be placed in 18mm × 180mm test tube with 100 μ l vomitoxins (100 μ g/mL), add toxin as blank using the fermention medium not connecing bacterium, be placed in 37 DEG C of incubator quiescent culture 72h, measure the residual content of vomitoxin, calculate vomitoxin degradation rate.
The extraction of vomitoxin and meta-bolites measure with reference to (Young, J.C., Zhu, H.H. such as Young, Zhou, T., et al.Degradation of trichothecene mycotoxins byaqueous ozone.Food Chem.Toxicol, 2006,44,417-424.) method.Get 500 μ l reaction mixtures, with equal-volume methanol mixed, after leaving standstill 2h, the centrifugal 10min of 6000 × g, supernatant liquor 0.2 μm of membrane filtration, for LC-MS analysis.
2) resistance research: resistance research be by inoculation of the present invention in simulated gastric fluid and simulation cholate, cultivate 24h for 37 DEG C, detect the survival rate of bacterium, thus judge its resistant function to gastrointestinal tract environment.
The cultural method of embodiment 2 subtilis ANSB471 of the present invention
Getting subtilis (Bacillus subtilis) ANSB471 (preserving number CGMCCNo.7344) 1.5mL(viable bacteria concentration is 10 9cFU/mL), be inoculated in 80mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 37 DEG C, pH value 7.0, rotating speed 200r/min, fermentation time 24h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 10g, yeast extract 2g, glucose 2g, extractum carnis 3g, sodium-chlor 4g, Sodium phosphate dibasic 3g, magnesium sulfate heptahydrate 1.0g, distilled water 1000mL, pH value 7.2.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
The cultural method of embodiment 3 subtilis ANSB471 of the present invention
Getting subtilis ANSB4711mL(viable bacteria concentration is 10 9cFU/mL), be inoculated in 50mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 37 DEG C, pH value 7.0, rotating speed 180r/min, fermentation time 20h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 8g, yeast extract 1.5g, glucose 1.5g, extractum carnis 2g, sodium-chlor 3g, Sodium phosphate dibasic 2.5g, magnesium sulfate heptahydrate 0.5g, distilled water 800mL, pH value 7.0.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
The cultural method of embodiment 4 subtilis ANSB471 of the present invention
Getting subtilis ANSB4712.5mL(viable bacteria concentration is 10 9cFU/mL), be inoculated in 100mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 40 DEG C, pH value 7.4, rotating speed 220r/min, fermentation time 30h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 12g, yeast extract 3g, glucose 4g, extractum carnis 5g, sodium-chlor 6g, Sodium phosphate dibasic 4g, magnesium sulfate heptahydrate 1.5g, distilled water 1200mL, pH value 7.4.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
The cultural method of embodiment 5 subtilis ANSB471 of the present invention
Getting subtilis ANSB4711mL(viable bacteria concentration is 10 9cFU/mL), be inoculated in 60mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 35 DEG C, pH value 7.3, rotating speed 190r/min, fermentation time 28h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 9g, yeast extract 1.5g, glucose 1.5g, extractum carnis 3.5g, sodium-chlor 4.5g, Sodium phosphate dibasic 2.5g, magnesium sulfate heptahydrate 1.0g, distilled water 1000mL, pH value 7.3.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
Embodiment 6 subtilis ANSB471 of the present invention is for vomitoxin of degrading
Draw the subtilis ANSB471 fermented liquid of 2 preparations in 900 μ l embodiments, react with 100 μ l vomitoxins (100 μ g/mL); Control group is add 100 μ l vomitoxins (100 μ g/mL) in 900 μ l0.01mol/L PBS damping fluids; The pH value of reaction system is adjusted to 7.2, and treatment group and control group are respectively at 37 DEG C of reactions 2h, 24h, 48h and 72h.
By the centrifugal 5min of each sample obtained after reaction, with 0.22 μm of membrane filtration, enzyme linked immunological kit--vomitoxin ELISA kit (Beijing Clover Technology Co., Ltd.) detects the concentration of vomitoxin in employing.
Detected result: it is 25% that subtilis ANSB471 reacts 2 hours degradation rates to vomitoxin, reacting 24 hours degradation rates to vomitoxin is 56%, reacting 48 hours degradation rates to vomitoxin is that 80%, subtilis ANSB471 to react 72 hours degradation rates to vomitoxin be 90%(Fig. 1).
The preparation method of the activated protein of the degradable vomitoxin that embodiment 7 subtilis ANSB471 of the present invention secretes
The subtilis ANSB471 fermented liquid of preparation in Example 2, obtains supernatant liquor after bactofugation body, adds 3 times of volume dehydrated alcohols and precipitate, after precipitation 1h centrifugal (5000r/min, 10min).The precipitation 0.01mol/L PBS buffer solution obtained, then the ethanol of centrifugal segregation remnants.The precipitation obtained is dissolved in 0.01mol/L PBS damping fluid, and freeze-drying is concentrated obtains activated protein.
The preparation of 0.01mol/L PBS damping fluid: take NaCl8g, KCl0.2g, Na 2hPO 41.44g and KH 2pO 40.24g, is dissolved in 800mL distilled water, and adjust solution ph to 7.5 with NaOH, last adding distil water is settled to 1000mL.
The activated protein degraded vomitoxin that embodiment 8 utilizes subtilis ANSB471 of the present invention to secrete
Activated protein 0.01g prepared by Example 7 is dissolved in 1mL PBS(0.01mol/L) prepare activated protein solution in damping fluid.Get 900 μ l activated protein solution to heat-treat (100 DEG C of heating 10min), then react with 100 μ l vomitoxins (100 μ g/mL); Separately getting 900 μ l activated protein solution adds after Proteinase K (0.01g Proteinase K) reacts 2 hours, adds 200 μ l vomitoxins in mixed solution.
Subtilis ANSB471 fermented liquid and the untreated activated protein solution of 900 μ l of getting preparation in 900 μ l embodiments 2 add 100 μ l vomitoxin reactions respectively; Control group is 900 μ lPBS(0.01mol/L) add 100 μ l vomitoxins (100 μ g/mL) in damping fluid; Detect after 37 DEG C of reaction 72h respectively.
Detected result shows, the activated protein solution degradation rate after Overheating Treatment or Proteinase K process reduces 55-65%(Fig. 2 than untreated fermented liquid and activated protein solution).
The resistance experiment of embodiment 9 subtilis ANSB471 of the present invention
Resistance experiment is cultivated in the environment of simulated gastric fluid and simulation cholate at strain inoculation, detects the survival rate of bacterium, thus judge its resistant function to gastrointestinal tract environment.
1) fermented liquid getting preparation in 4 DEG C of 5mL embodiments 2 of preserving is injected in centrifuge tube, adopts classification dilution, dull and stereotyped coating.The fermented liquid separately getting preparation in 10mL embodiment 2 is injected in centrifuge tube, is placed in 80 DEG C of water-baths and heats 20min.Get the bacterium liquid after heating and carry out stepwise dilution, dull and stereotyped coating.Finally the flat board after not heating and heating all is cultivated 24h under 37 DEG C of conditions, calculate the quantity of subtilis respectively.
Result shows, compares with the bacterium liquid phase do not heated, and the viable count after heating is 90% when not heating, and namely Viable detection reaches 90%.
2) tolerance test of simulated gastric fluid: be dissolved in by stomach en-in 0.5% physiological saline, makes its final concentration be 3g/L, and with concentrated hydrochloric acid or 10%NaOH adjust pH to 2.0.The fermented liquid getting in 0.5mL embodiment 2 preparation join in the simulated gastric fluid of 4.5mL respectively and 4.5mL physiological saline in (i.e. 10 times of dilutions), and rapid fully mixed on the oscillator, be then placed in 37 DEG C of quiescent culture 24h.After process 2h, take out nutrient solution and count remaining viable count immediately.
Result shows, Viable detection is 79%.
The resistance test of simulation cholate adopts pancreatin to make 1g/L solution, and in solution, add 0.3% pig cholate, adjust pH to be 8.0 with 10%NaOH, and the solution of preparation is degerming with 0.45 μm of micro-filtrate membrane filtration.The bacterium liquid of preparation in embodiment 2 is carried out 10 times of classifications with the simulation cholate solution of preparation in physiological saline dilute, be then coated with LB dull and stereotyped, take out after being placed in 37 DEG C of quiescent culture 24h and count remaining viable count immediately.
Result shows, Viable detection is 82%.
The simultaneous test of embodiment 10 different bacillus subtilis strain degraded vomitoxin
Vomitoxin test and secreting amylase vigor simultaneous test in degraded vomitoxin and feedstuff raw material are carried out respectively to three kinds of different bacillus subtilis strains (ANSB471, ANSB719, ANSB921).
1, the simultaneous test of different bacillus subtilis strain degraded vomitoxin
1) to get in 900 μ l embodiments 2 the subtilis ANSB471 fermented liquid of preparation and reacted with 100 μ l vomitoxins (100 μ g/mL) respectively by the other two bacillus ANSB719 fermented liquids of animal nutrition National Key Laboratory of China Agricultural University preservation and ANSB921 fermented liquid; Control group is add 100 μ l vomitoxins (100 μ g/mL) in 900 μ l0.01mol/L PBS damping fluids; The pH value of reaction system is adjusted to 7.2, and treatment group and control group are respectively at 37 DEG C of reaction 72h.
2) the centrifugal 5min of each sample will obtained after reaction, with 0.22 μm of membrane filtration, adopts vomitoxin ELISA kit (Beijing Clover Technology Co., Ltd.) to detect the concentration of vomitoxin.The results are shown in Table 2 and Fig. 3.
2, the simultaneous test of vomitoxin in different bacillus subtilis strain degraded feedstuff raw material
1) to get in 50mL embodiment 2 the subtilis ANSB471 fermented liquid of preparation and ANSB719 fermented liquid and ANSB921 fermented liquid to mix with the feed 5g polluted by vomitoxin respectively, the pH value of reaction system is adjusted to 7.2, and treatment group and control group are respectively at 37 DEG C of reaction 72h.
2) the centrifugal 5min of each sample will obtained after reaction, with 0.22 μm of membrane filtration, adopts vomitoxin ELISA kit (Beijing Clover Technology Co., Ltd.) to detect the concentration of vomitoxin.The results are shown in Table 2 and Fig. 4.
3, different bacillus subtilis strain secreting amylase vigor simultaneous test
1) the subtilis ANSB471 fermented liquid of preparation in Example 2 respectively and ANSB719 fermented liquid and ANSB921 fermented liquid 0.5-2mL are inoculated in specific culture medium and ferment, leavening temperature is 36-38 DEG C, pH value 7.0-7.4, rotating speed 180-220r/min, fermentation time 20-30h.
Described specific culture medium is made up of following component: Tryptones 10g/L, yeast leaching powder 5g/L, Zulkovsky starch 5g/L, KH 2pO 42g/L, MgSO 47H 2o0.5g/L, CaCl 22H 2o0.2g/L, prepares with water.
2) the subtilis ANSB471 fermented liquid of respectively preparation in Example 2 and ANSB719 fermented liquid and ANSB921 fermented liquid, supernatant liquor is obtained after bactofugation body, adopt Yoo improved method amylase activity, get 0.5% Zulkovsky starch solution 5mL, preheating 10min in 37 DEG C of water-baths, adds the supernatant liquor 0.5mL of suitably dilution, 37 DEG C of water-bath concussions, after reaction 5min, add 5mL0.lmol/L H 2s0 4termination reaction.Get 0.5mL reaction solution and the rare iodine liquid of 5mL develops the color, measure optical density value in 620nm place.Replace 0.5mL reaction solution as blank, not manage in contrast with fermented liquid (adding the damping fluid of same volume) using 0.5mL water.Enzyme activity is according to following formulae discovery:
Enzyme activity (U)=(Ro-R) × 50 × D/Ro
In formula: Ro, R represent the optical density value of contrast and reaction solution respectively, and D is the extension rate of enzyme.Adjustment D makes (Ro-R)/Ro 0.2 ~ 0.7.Enzyme is lived and is defined as: at 37 DEG C, under pH6.0 condition, and in 5min, the enzyme amount of hydrolysis 1mg starch is 1 unit of activity.The results are shown in Table 2 and Fig. 5.
The simultaneous test of the different bacillus subtilis strain of table 2
Bacterial strain Vomitoxin degradation rate in feedstuff raw material Vomitoxin degradation rate Amylase (U/mL)
ANSB471 95% 90% 58.4
ANSB719 70% 68% 12.8
ANSB921 10% 12% 5.7
As shown in Table 2, strains A NSB471 of the present invention far above other two bacillus subtilis, and is also greater than other two strains to vomitoxin degradation rate and amylase activity in feedstuff raw material to the degradation rate of vomitoxin, has the difference of highly significant.As can be seen here, bacterial strain of the present invention can be degraded vomitoxin in vomitoxin and feedstuff raw material simultaneously, is highly suitable for as animal feedstuff additive.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (9)

1. subtilis (Bacillus subtilis) ANSB471 of a high-efficiency degradation vomitoxin, its preserving number is CGMCC No.7344.
2. the cultural method of subtilis ANSB471 described in claim 1, is characterized in that, getting viable bacteria concentration is 10 9the subtilis ANSB4710.5-2.5mL of CFU/mL, is inoculated in 50-100mL substratum and carries out shake flask fermentation cultivation, obtains the fermented liquid of subtilis ANSB471;
The substratum that fermentation uses is made up of following component: Tryptones 8-12g, yeast extract 1.5-3g, glucose 1.5-4g, extractum carnis 2-5g, sodium-chlor 3-6g, Sodium phosphate dibasic 2.5-4g, magnesium sulfate heptahydrate 0.5-1.5g, adding distil water to 800-1200mL, pH value 7.0-7.4;
Described conditions of flask fermentation is: leavening temperature 30-40 DEG C, fermentation time 16-24h, rotating speed 180-220r/min, pH value 7.0-7.4.
3. the preparation method of the activated protein of a vomitoxin of degrading, it is characterized in that, get the fermented liquid of subtilis ANSB471 described in claim 2, centrifugal, get supernatant liquor, the dehydrated alcohol adding 2-4 times of volume carries out precipitation 0.5-2h, centrifugal, by the precipitation 0.01mol/L PBS buffer solution obtained, centrifugal, the precipitation obtained is dissolved in 0.01mol/L PBS damping fluid, freeze-drying concentrates the activated protein obtaining degraded vomitoxin.
4. preparation method according to claim 3, is characterized in that, the method preparing described 0.01mol/L PBS damping fluid is: take 8g NaCl, 0.2g KCl, 1.44gNa 2hPO 4with 0.24g KH 2pO 4, be dissolved in 800mL distilled water, by the pH value to 7.5 of NaOH regulator solution, adding distil water is settled to 1000mL.
5. the activated protein of the degraded vomitoxin prepared by method described in claim 3 or 4.
6. the application of activated protein described in subtilis ANSB471 described in claim 1 or claim 5 in degraded feed in vomitoxin.
7. application according to claim 6, is characterized in that, is mixed by the fermented liquid of subtilis ANSB471 described in claim 2 with the feed polluted by vomitoxin, the pH value of reaction system is adjusted to 7.0-7.4,36-38 DEG C, rotating speed 180-220r/min, reaction 2-72h; Or,
Activated protein described in claim 5 is dissolved in 0.01mol/L PBS damping fluid, then activated protein solution is mixed with the feed polluted by vomitoxin, 36-38 DEG C, rotating speed 180-220r/min, reaction 2-72h.
8. an animal feedstuff additive, is characterized in that, its effective constituent is the activated protein of vomitoxin of degrading described in subtilis ANSB471 microbial inoculum or claim 5.
9. the application of subtilis ANSB471 described in claim 1 in anti-simulated gastric fluid and/or simulation cholate environment, it is characterized in that, get the fermented liquid of subtilis ANSB471 described in claim 2, join in the environment of simulated gastric fluid and/or simulation cholate, 36-38 DEG C, cultivate 24h, the pure motility rate of viable bacteria can reach 79%-82%;
Wherein, the environment of described simulated gastric fluid is: be dissolved in by stomach en-in 0.5% physiological saline, makes its final concentration be 3g/L, and with concentrated hydrochloric acid or 10%NaOH adjust pH to 2.0;
Simulation cholate environment is: adopt pancreatin to make 1g/L solution, and in solution, add 0.3% pig cholate, adjust pH to be 8.0 with 10%NaOH, and the solution of preparation is degerming with 0.45 μm of micro-filtrate membrane filtration.
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