CN110172426A - There is the bacillus subtilis of high-efficiency detoxicating to T-2 toxin - Google Patents

Abstract

The invention belongs to microbe additive technical fields for animals, and in particular to have the bacillus subtilis of high-efficiency detoxicating to T-2 toxin.T-2 toxin is mainly one of the trichothecene that fusarium tricinctum generates, main to pollute the cereal crops such as wheat and corn, also results in larger harm to animal and fowl fodder.The present invention screens one plant to the detoxicated type probiotics of T-2 from reservation bacterial strain, that is bacillus subtilis (Bacillus subtilis) WXBs-05, the bacterial strain is deposited in China typical culture collection center, and deposit number is CCTCC NO:M2019177.The present invention has carried out Physiology and biochemistry and taxonomic identification to the bacterial strain, is verified to its biological characteristics, detoxification characteristic, detoxification product and detoxification efficiency.WXBs-05 strain growth speed is fast, and resistance and fungistatic effect are stronger, can apply in the microorganism fodder additive as efficient removal T-2 toxin.

Description

There is the bacillus subtilis of high-efficiency detoxicating to T-2 toxin

Technical field

It is related with Antibiotic Additive field the invention belongs to microbe additive technical field for animals, the present invention relates to A kind of pair of T-2 toxin has the screening of bacillus subtilis (Bacillus subtilis) bacterial strain of high-efficiency detoxicating effect, mirror Verifying in fixed, bacteriostasis property, anti-adversity, detoxification characteristic, detoxification substance and detoxification efficiency body.

Background technique

T-2 toxin is mainly the trichothecene (trichothecenes, TS) generated by fusarium tricinctum One of cereal such as pollution barley, wheat all over the world extensively, mycotoxin contamination is serious in current agricultural production, pollutes model It encloses extensively, causes toxin existing for animal derived food to remain after the feed of feed intake endotoxin contamination, the health of the mankind can be made At significant damage, therefore the harm of T-2 toxin bring and influence have caused the concern of decision-making level, various countries and scientific circles.Commonly Poison-removing method is mainly physics detoxicity method, chemical detoxication method and biological detoxication method, since biological detoxication method is removed in mycotoxin In the process have detoxification is high-efficient, has no toxic side effect, free of contamination unique advantage, receive the whole world and widely pay close attention to, therefore Finding the strain excellent of T-2 toxin and can finally develop high-efficiency detoxicating agent and be of great significance in efficient removal feed, be The removing of mycotoxin provides technical support in fermented feed, and for animal husbandry green, developing in a healthy way provides help.

Summary of the invention

It is an object of the invention to overcome the prior art, a kind of pair of T-2 poison is obtained by screening Element has bacillus subtilis (Bacillus subtilis) bacterial strain of high-efficiency detoxicating effect.Filtering out has good removing T- 2 toxin and it can be used as the probiotics strain that feed addictive uses.The probiotics strain that the present invention filters out is a kind of right T-2 toxin has the bacillus subtilis (Bacillus subtilis) of high-efficiency detoxicating effect, and the present invention provides withered grass buds Screening, identification, bacteriostasis property, anti-adversity, detoxification characteristic, the identification of detoxification substance and the verifying of detoxification efficiency of born of the same parents bacillus Using.

It is described that technical scheme is as follows:

The present invention is using T-2 toxin analog phenyl ethylene oxide and corn pulp as sole carbon source and nitrogen source, to laboratory Microorganism in bacterium library has carried out primary dcreening operation, carries out 2 primary dcreening operations using T-2 toxin sterling, is combined with the two result and filter out 7 plants The strain excellent survived under T-2 toxin environment.Further secondary screening is done to this 7 plants of bacterial strains, using T-2 toxin as substrate, with competition Property the detection of ELISA method, finishing screen selects virus elimination rate highest and most stable of purpose bacterial strain WXBs-05.To resulting WXBs-05 Bacterial strain has carried out relevant Physiology and biochemistry and taxonomic identification, and candidate strain WXBs-05 is accredited as bacillus subtilis (Bacillus subtilis)。

The resulting Strain Designation to T-2 toxin with high-efficiency detoxicating is bacillus subtilis WXBs-05 by applicant, Bacillus subtilis WXBs-05 delivers the Chinese Typical Representative culture of the Chinese Wuhan Wuhan University on March 20th, 2019 Object collection preservation, deposit number are CCTCC NO:M2019177.

The mycology feature of bacillus subtilis WXBs-05:

The growth characteristics of WXBs-05 bacterial strain of the invention on LB culture medium are shown in Fig. 1, and Gram's staining characteristic is shown in Fig. 2, Its peacock green dyeing property is shown in Fig. 3.WXBs-05 bacterial strain colonial morphology after LB cultured on solid medium 12h is circle, white Light yellow complexion, edge is irregular, and bacterium colony is wet.It can find that WXBs-05 is gram by Gram's staining and peacock green coloration result Positive bacteria, is elongated rod shape, and no pod membrane is middle life or nearly middle raw type brood cell.

Growth characteristics, bacteriostasis and the resistance of bacillus subtilis WXBs-05 is studies have shown that the bacterial strain fertility Very strong, no lag phase enters logarithmic phase quickly, enters stationary phase after 2h, to E.coli K88, O157, Staphylococcus aureus Bacterium, salmonella have certain bacteriostasis, and high temperature resistant, acid and alkali-resistance, bile tolerance, stomach juice-resistant ability are stronger.

Study bacterial strain WXBs-05 detoxification condition show the bacterial strain to virus elimination rate and the detoxification of T-2 toxin when it is anti- Between seasonable, temperature it is related with pH, the inoculum concentration with WXBs-05 bacterial strain without obvious relation, and for 24 hours, pH and 37 DEG C when detoxification It is rate highest, most stable.

Different component detoxifying agent is made in WXBs-05, as a result viable bacteria suspension detoxification ability is most strong, and supernatant, inactivated bacteria are outstanding Liquid takes second place, and initial guess is based on absorption, and degradation accounts for extremely least a portion of two ways detoxification.The detoxification of Primary Study WXBs-05 Substance, SDS, Proteinase K processing thallus are reacted with T-2 toxin, and verifying degradation material is ectoenzyme.It assesses thallus and toxin is inhaled The complex stabilities of attached formation, detoxification liquid are repeatedly eluted, are extracted, and show the compound of thallus toxin closely, not easy to wash It is de-.

Animal test results show that WXBs-05 bacterial strain can also prevention and treatment T-2 while playing excellent detoxification Damage of the toxin to animal body.

The bacillus subtilis that the present invention screens has the advantages that

(1) bacterial strain screen from the bacterial strain of a large amount of candidate probiotics and is obtained, the speed of growth fastly, to common pathogenic entero becteria (staphylococcus aureus, salmonella have apparent fungistatic effect.

(2) bacterial strain resistance is strong, can be resistant to gastric acid and enteron aisle cholate hyperosmosis environment completely, therefore can be in intestinal colonisation Play prebiotic effect.

(3) bacterial strain produces brood cell, very strong to the tolerance of high temperature, can tolerate 90 DEG C, 10min, therefore the bacterial strain can be through By environment such as high temperature process and granulations.

(4) bacterial strain is absorption, degradation two ways detoxification, the compound pole of high-efficient, substantially irreversible, toxin and thallus To be closely not easy to elute.

(5) in vivo studies proves, bacterial strain WXBs-05 detoxification efficiency is obvious, and also has prevention and treatment T-2 toxin The effect of animal body damage, with more environmental-friendly, the advantages of to safety of human and livestock.

More detailed technical solution is shown in that " specific embodiment " describes.

Detailed description of the invention

Fig. 1: growth conditions of the purpose bacterial strain that the present invention screens on LB culture medium.

Fig. 2: the purpose bacterial strain Gram's staining microscope form that the present invention screens.

Fig. 3: the purpose bacterial strain peacock green that the present invention screens dyes microscope form.

Fig. 4: 16S rRNA gene PCR augmentation detection result.Description of symbols:

M:DL 2000DNA molecular weight standard in Fig. 4；Swimming lane 1:WXBs-05 bacterial strain；Swimming lane 2: negative control.

Fig. 5: the growth curve of bacterial strain of the present invention.

Fig. 6: the mouse heart pathological section figure of verification test in bacterial strain detoxification efficiency body of the present invention.Description of symbols:

A figure in Fig. 6: malicious group is attacked；B figure in Fig. 6: detoxification group；C figure in Fig. 6: prevention group；D figure in Fig. 6: pre- Anti- control group；E figure in Fig. 6: treatment group；F figure in Fig. 6: treatment control group.

Fig. 7: the mouse liver pathological section figure of verification test in bacterial strain detoxification efficiency body of the present invention.Description of symbols:

A figure in Fig. 7: malicious group is attacked；B figure in Fig. 7: detoxification group；C figure in Fig. 7: prevention group；D figure in Fig. 7: pre- Anti- control group；E figure in Fig. 7: treatment group；F figure in Fig. 7: treatment control group.

Fig. 8: the mouse intestinal pathological section figure of verification test in bacterial strain detoxification efficiency body of the present invention, description of symbols:

A figure in Fig. 8: malicious group is attacked；B figure in Fig. 8: detoxification group；C figure in Fig. 8: prevention group: the D figure in Fig. 8: pre- Anti- control group；E: treatment group in Fig. 8；F figure in Fig. 8: treatment control group.

Fig. 9: the mouse weight variation of verification test in bacterial strain detoxification efficiency body of the present invention.

Figure 10: the mouse cytokine variation of verification test in bacterial strain detoxification efficiency body of the present invention.Description of symbols:

Description of symbols: the A figure in Figure 10: the B figure in cytokine TNF-α: Figure 10: cell factor IL-6；Figure 10 In C figure: cell factor IL-1 β；D figure in Figure 10: cell factor IL-12；E figure in Figure 10: cell factor IL-13；Figure F figure in 10: cell factor IL-10.

Specific embodiment

To the explanation of sequence table:

Sequence table SEQ ID NO:1 is the part for the bacillus subtilis WXBs-05 16S rDNA gene that the present invention screens Sequence.

Embodiment 1: the screening and identification of bacterial strain

One, the primary dcreening operation of bacterial strain

The analogue primary dcreening operation of T-2 toxin first, the Hormisch culture medium after improvement are added in the ratio of 1% volume Concentration is the phenyl ethylene oxide solution of 10% volume, corn pulp, as sole carbon source, not by routine preservation in laboratory Know that strain inoculated is placed on 37 DEG C of incubator stationary culture 72h, to avoid carbon source interference of the bacterium in former environment, picking is raw Longer single colonie, continuous 3 pure cultures finally save those bacterial strains that can be grown on culture medium, record and analyze bacterial strain Upgrowth situation；The T-2 toxin sterling that concentration after dilution is 250ng/mL is pressed 0.1% by T-2 toxin sterling primary dcreening operation again Improvement Hormisch culture medium is added in ratio, and inoculating strain is placed in 37 DEG C of incubators, carries out continuous 3 pure cultures again, remembers The bacterial strain grown above is recorded, and is compared with toxin analog primary dcreening operation result, 7 plants of dominant strains, strain number are screened to obtain Respectively WXBs-37, WXBs-05, WXBs-01, WXBs-33, WXBs-20, WXBs-06, WXBs-36.

Two, bacterial strain secondary screening

The secondary screening that substrate carries out bacterial strain detoxification ability is done with T-2 toxin, picking isolated strains are in the LB liquid medium of 5mL In, 37 DEG C, cultivate for 24 hours afterwards as seed liquor under the conditions of 200r/min.Seed liquor is inoculated into the inoculum concentration of 1% volume In the LB liquid medium of 10mL, 37 DEG C, cultivate for 24 hours as fermentation liquid under the conditions of 200r/min.The hair of isolated strains is taken respectively Zymotic fluid 2ml, each T-2 toxin that 4 μ l are added react under the conditions of 37 DEG C, 200r/min as storage working solution (250 μ g/ml) For 24 hours, add the T-2 toxin storage working solution of equivalent for control with the LB liquid medium of equivalent.Liquid to be checked is uniformly mixed, 10000r/min is centrifuged 5min, and the supernatant after taking centrifugation is used for the measurement of T-2 content of toxins after being filtered with 0.22 μm of filter. It is remaining to T-2 toxin in liquid to be checked using T-2 toxin enzyme-linked immune quantitative detection reagent box using the method for competitive ELISA Content is measured, and when detection, each sample did multiple holes, is chosen and is carried out in next step to the bacterial strain that T-2 toxin has higher virus elimination rate Test.Primary dcreening operation result is that WXBs-05 bacterial strain detoxification ability is most strong, is up to 90% to the virus elimination rate of T-2 toxin sterling.

Three, bacterial strain is identified

The observation of the bacterium colony of pure culture colonial morphology and Gram's stain are subjected to Preliminary Identification.Of the invention Growth characteristics of the WXBs-05 bacterial strain on LB culture medium are shown in Fig. 1, and Gram's staining characteristic is shown in Fig. 2, and peacock green dyeing is special Property is shown in Fig. 3.WXBs-05 bacterial strain colonial morphology after LB cultured on solid medium 12h is round, white light yellow complexion, and edge is not whole Together, bacterium colony is wet.It can find that WXBs-05 is gram-positive bacteria by Gram's staining and peacock green coloration result, be stock Shape, no pod membrane are middle life or nearly middle raw type brood cell.

By above-mentioned isolated bacterial strain referring to the correlation technique on " primary Jie Shi Bacteria Identification handbook " (the 9th edition), detested Bacterial strain is identified kind by the test result that oxygen, gelatin liquefaction, M.R, V.P, ornithine, lysine, arginine, salt tolerant are grown.Tool Body the results are shown in Table 1.

1 bacillus subtilis WXBs-05 bacterial strain biochemical identification result of table

On the basis of above-mentioned identification, the inspection of 16S rRNA gene order further is carried out to bacterial strain WXBs-05 of the invention It surveys, confirmation identification is carried out to kind belonging to bacterial strain.

Specific step is as follows:

(1) purpose strain gene group rapidly extracting

Purpose bacterial strain (WXBs-05) is inoculated in 5mL LB culture medium with the inoculum concentration of 1% volume, 37 DEG C of 200r/min Culture for 24 hours, takes 2mL bacterium solution, and 12000r/min is centrifuged 5min, discards supernatant liquid, is resuspended with the DEPC water of 50 μ L, 100 DEG C of water Pre-process 10min, ice bath 10min immediately, wink from take supernatant as template.

(2) PCR amplification system of 16S rRNA

Amplimer (16S universal primer):

Forward primer 27F:AGAGTTTGATCCTGGCTCAG,

Reverse primer 1492R:TACGGTTACCTTGTTACGACTT；

PCR reaction condition: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C of extension 90s, 35 Circulation, 72 DEG C of extension 7min.

Under 120V voltage, 10 μ L PCR products, 1% agarose gel electrophoresis 30min is taken, is examined with ultraviolet imagery system Survey record.

(3) 16S rRNA sequencing and analysis:

The PCR product commission one Hui Yuan biotechnology company of Wuhan Tian that amplification obtains is sequenced.By the sequence of sequencing Blast is carried out in NCBI database compares analysis.

16S rRNA PCR amplification result is shown in Fig. 4, retrieval discovery, the 16S rRNA of WXBs-05 and bacillus subtilis Sequence homology highest, similitude 100%.The part 16S rRNA gene order of amplification is shown in sequence table SEQ ID NO:1 institute Show.

The growth curve of embodiment 2:WXBs-05 bacterial strain, bacteriostasis property measurement

Picking purpose bacterial strain (WXBs-05) is in the LB liquid medium of 5mL, and 37 DEG C, cultivate for 24 hours under the conditions of 200r/min It is used as seed liquor afterwards, seed liquor is inoculated into the LB liquid medium of 5mL with the inoculum concentration of 1% (volume), 37 DEG C, 200r/ It is cultivated under the conditions of min for 24 hours as fermentation liquid.Fermentation liquid is inoculated into the LB liquid medium of 50mL with 1% inoculum concentration, Respectively in 0h, 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 12h, 16h, 20h, 28h, 32h, 48h by culture solution It takes out, the growth curve of each bacterial strain, 3 repetitions of every group of test will be surveyed after culture solution doubling dilution with tilt-pour process.With incubation time For abscissa, log (CFU/mL) value is ordinate, draws the growth curve of bacterial strain.The result of growth curve is shown in Fig. 5, it is known that WXBs-05 enters logarithmic phase in 2h or so, enters stationary phase after 8h.

It is instruction with pig source staphylococcus aureus, Salmonella choleraesuls, E.coli K88, K99, O139, O157 Indicator bacteria is inoculated in 5mL LB liquid medium by bacterium by 1% volume ratio, and 37 DEG C, cultivate 6h under the conditions of 200r/min, Turbidimetry will indicate that bacteria culture fluid is diluted to after suitable concentration and is uniformly coated on LB solid medium using cotton swab；Use phase Same method preparation purpose bacterial strain and positive control (the pig source enterococcus faecium HDRsEf1 that laboratory separates) fermentation liquid, by purpose Bacterial strain (WXBs-05) and positive control (enterococcus faecium HDRsEf1) fermentation liquid are centrifuged, and are taken supernatant filtration sterilization, are added in Oxford cup Purpose bacterial strain, positive control (enterococcus faecium HDRsEf1) supernatant after entering aseptic filtration are put into after 4 DEG C of refrigerator diffusions overnight Inhibition zone is observed after cultivating 12h in 37 DEG C of constant incubators.The inhibition zone of purpose bacterial strain and positive control is measured, to judge purpose Bacteriostasis of the bacterial strain to indicator bacteria, continuous 3 repetitions.Test result is shown in Table 2.

2 bacillus subtilis WXBs-05 fermented supernatant fluid extracorporeal bacteria inhibitor test of table

Embodiment 3: the adverse-resistant characteristic test of bacillus subtilis WXBs-05

(1) the high temperature resistance test of WXBs-05 bacterial strain

The spore suspension of the WXBs-05 bacterial strain kept of going bail for is injected into centrifuge tube, is respectively placed in 60,70,80,90 DEG C of water 5min is heat-treated in bath, the bacterium solution after taking heat treatment carries out doubling dilution step by step, remaining viable count is surveyed with tilt-pour process, with etc. The spore suspension of amount is placed in room temperature as control, 3 repetitions of every group of test.Bacterial strain WXBs-05 high temperature resistant the results are shown in Table 3, can be with Learn that WXBs-05 bacterial strain still has 86.17%, 71.36% bioactivity after 80 DEG C, 90 DEG C of processing 5min, it is known that the bacterium Brood gemma has higher tolerance to temperature.Therefore confirmation bacillus subtilis WXBs-05 resistant against high temperatures, can be subjected to The environment such as high temperature process and granulation.It the results are shown in Table 3.

3 bacillus subtilis WXBs-05 of table tests the tolerance of high temperature

(2) the Bile salt resistance experimental test of WXBs-05 bacterial strain

The spore solution of 0.5mLWXBs-05 bacterial strain is taken to be added in the 0.30% simulation cholate of the 4.5mL of pH value 8.0, it is fast Speed mixes well in vortex instrument, is placed in 37 DEG C of incubator stationary cultures.In 0h, for 24 hours when take out carry out doubling dilution, Viable count is surveyed with tilt-pour process, equivalent spore solution, which is placed in sterile saline, to be compared.Bacterial strain WXBs-05 simulates cholate Tolerability results are shown in Table 4, and as shown in Table 4, WXBs-05 brood gemma is stronger to simulation Pig cholate tolerance, in simulation cholate Reason still has nearly 92% survival rate after for 24 hours, therefore, bacillus subtilis bacterial strain WXBs-05 is resistant to the environment of enteron aisle cholate.See Table 4.

Tolerance of the 4 bacillus subtilis WXBs-05 of table to cholate

(3) the gastric fluid-resistant test of WXBs-05 bacterial strain

It takes the spore suspension of 0.5mLWXBs-05 bacterial strain to be added in simulate the gastric juice, is mixed well in vortex instrument rapidly, It is placed in 37 DEG C of incubator stationary cultures.It is taken out when 0h, 2h, 4h and carries out doubling dilution, survey viable count with tilt-pour process, etc. Amount spore suspension, which is placed in sterile saline, to be compared.Stomach juice-resistant the results are shown in Table 5, as shown in Table 5, WXBs-05 pH value about For in 2.0 simulate the gastric juice, bacterium activity has no significant change after processing 2,4 hours, and 2h survival rate still has when being up to 93%, 4h Nearly 87% survival rate.Bacillus subtilis bacterial strain WXBs-05 can pass through the acidic environment of gastrointestinal tract and give birth in the gastrointestinal tract It deposits.

5 bacillus subtilis WXBs-05 of table tests the tolerance of gastric acid

(4) the acid and alkali resistance test of WXBs-05 bacterial strain

The PBS buffer solution that pH is 2,4,7,9, filtration sterilization are modulated in advance.Respectively by the bud of 0.5mLWXBs-05 bacterial strain Born of the same parents' suspension is put into the 4.5mL PBS buffer solution that ph is 2,4,7,9, and is sufficiently mixed in vortex instrument rapidly, is subsequently placed in 37 DEG C incubator stationary culture handles 0h, 2h respectively, and taking that treated, bacterium solution carries out doubling dilution, surveys viable count with tilt-pour process, 3 repetitions of every group of test.The acid and alkali resistance of WXBs-05 bacterial strain the results are shown in Table 6.As shown in Table 6, WXBs-05 bacterial strain is in different pH Survival rate variation after handling 2 hours in value differs, and survival rate still has 50% under strong acid, strong alkali environment.The bacterial strain is in pH Survival rate is up to 77% under conditions of 7, and overall survival is good.It is shown in Table 6.

Tolerance of the 6 bacillus subtilis WXBs-05 of table to different pH value

Embodiment 4: bacillus subtilis WXBs-05 detoxification attribute testing

One, the best detoxification condition measurement of bacillus subtilis WXBs-05

(1) influence of the pH value to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose WXBs-05 bacterial strain, 37 DEG C, 200r/min culture for 24 hours, seed liquor is made, by 1% volume ratio switching Into 5mL LB liquid medium, fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours, the pH of fermentation liquid is adjusted to 2 respectively, 4, 6,9 and respectively take 2mL be added 2 μ L T-2 Toxin stock liquid, equivalent T-2 Toxin stock liquid is added with the sterile LB liquid medium of equivalent For control, is cultivated for 24 hours in 37 DEG C, 200r/min, referring to the same method of secondary screening, measure the virus elimination rate under different pH. WXBs- The fermentation liquid of 05 bacterial strain is shown in Table 7 to the virus elimination rate variation of T-2 toxin under different pH environment.It as shown in Table 7, is 2-9 item in pH Under part, virus elimination rate is incremented by with the rising of pH after the fermentation liquid of WXBs-05 reacts for 24 hours with T-2 toxin, is 9 i.e. meta-alkalescence in pH Virus elimination rate highest under environment.And pH is that virus elimination rate is minimum under 2 i.e. strong acid environment, is almost close to zero.It is shown in Table 7.

Virus elimination rate of the 7 bacillus subtilis WXBs-05 of table at different pH

(2) influence of the temperature to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose bacterial strain WXBs-05 is cultivated for 24 hours in 37 DEG C, 200r/min, seed liquor is made, turns by 1% volume ratio It is connected in 5mL LB liquid medium, fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours, takes fermentation liquid 2mL that 2 μ L T-2 are added Toxin stock liquid, the sterile LB liquid medium of equivalent add equivalent Toxin stock liquid for control, are respectively placed in 27 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 200r/min is cultivated for 24 hours, using secondary screening same method, measures the virus elimination rate under different temperatures.Bacterial strain WXBs-05 Fermentation liquid 8 are shown in Table to the variation of the virus elimination rate of T-2 toxin under different temperatures environment.It as shown in Table 8, is 27 DEG C, 30 in temperature DEG C, 37 DEG C, under conditions of 40 DEG C, virus elimination rate is passed with the rising of temperature after the fermentation liquid of WXBs-05 reacts for 24 hours with T-2 toxin Increase.

The virus elimination rate of 8 bacillus subtilis WXBs-05 of table at different temperatures

(3) influence of the time to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose bacterial strain WXBs-05 cultivates for 24 hours in 37 DEG C, 200r/min, seed liquor is made, in the ratio of 1% volume It is transferred in 5mL LB liquid medium, fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours, takes 2mL that 2 μ are added fermentation liquid L T-2 Toxin stock liquid, the sterile LB liquid medium of equivalent add equivalent Toxin stock liquid for control, are placed in 37 DEG C, 200r/min Respectively cultivate 12h, for 24 hours, 48h, 72h.Using secondary screening same method, the virus elimination rate under different time is measured.

Virus elimination rate of the 9 bacillus subtilis WXBs-05 of table under different time

Fermentation liquid is shown in Table 9 with the virus elimination rate that T-2 toxin reacts different time.As shown in Table 9, respectively react 12h, for 24 hours, Under 48h, 72h, virus elimination rate and time do not have specific relationship after the fermentation liquid of WXBs-05 reacts for 24 hours with T-2 toxin.

(4) influence of the inoculum concentration to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose bacterial strain WXBs-05,37 DEG C, 200r/min culture for 24 hours, seed liquor is made, respectively by 1%, 3%, 6%, 10% ratio is transferred in 5mL LB culture medium, and fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours, and fermentation liquid is taken 2 μ L T-2 Toxin stock liquid are added in 2mL, and the sterile LB liquid medium of equivalent adds equivalent Toxin stock liquid for control, is placed in 37 DEG C, 200r/min is cultivated for 24 hours, using secondary screening method of the same race, measures the virus elimination rate under different vaccination amount.Different vaccination amount condition Under virus elimination rate be shown in Table 10, there is no specific rule by Biao Ke get, between inoculum concentration and virus elimination rate.

Virus elimination rate of the 10 bacillus subtilis WXBs-05 of table under different vaccination amount

Two, influence of the different component to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose bacterial strain, 37 DEG C, 200r/min culture for 24 hours, seed liquor is made, respectively by 1%, 3%, 6%, 10% ratio Example is transferred in 5mL LB culture medium, and fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours.

Prepare different component detoxification liquid:

1) somatic cells solution: 4 DEG C of fermentation liquid, 8000r/min are centrifuged 10min, liquid are discarded supernatant, with PBS buffer solution weight It is outstanding, it is centrifuged again, PBS buffer solution is resuspended, as somatic cells solution, and equivalent sterile PBS buffer is control；

2) inactivate somatic cells solution: 4 DEG C of fermentation liquid, 8000r/min refrigerated centrifuge 10min discard supernatant liquid, use PBS Buffer is resuspended, and is centrifuged, discards supernatant again, 121 DEG C of sediment, 30min high pressure sterilization, and PBS buffer solution is resuspended after cooling, Somatic cells solution is as inactivated, equivalent sterile PBS buffer is control；

3) extracting solution intracellular: somatic cells solution carries out cell supersonic wave in ice bath and is crushed (100 times, 5s/ times), then will Broken cell is centrifuged 10min under the conditions of 4 DEG C, 8000r/min, takes 0.45 μm of filter filtering of supernatant, the sterile PBS of equivalent Buffer is control；

4) supernatant: 4 DEG C of fermentation liquid, 8000r/min are centrifuged 20min, take supernatant.Equivalent LB culture medium is control.4 Kind component detoxification liquid takes the T-2 Toxin stock liquid of 2 μ L of 2mL addition respectively, and the sterile LB liquid medium of equivalent adds equivalent toxin to store up Standby liquid is control, is placed in 37 DEG C, 200r/min is cultivated for 24 hours.Using secondary screening method method of the same race, the detoxification of different component is measured Rate.

The results show that influence of the fermentation liquid different component to the detoxicated rate of T-2 is shown in Table 11.As seen from table, WXBs-05 Viable bacteria suspension detoxification ability is most strong, and supernatant also has certain detoxification ability.Tentatively judgement hair supernatant mainly passes through after inspection information Extracellular enzyme effect detoxification, viable bacteria and inactivation bacteria suspension demonstrate biological metabolism detoxification to the result difference of the detoxicated rate of T-2 May, while the possibility for also demonstrating absorption detoxification and degradation-detoxification and depositing, show that WXBs-05 thallus is dropped by biological metabolism There is also suction-operated detoxifications while solution detoxification.

The virus elimination rate of 11 bacillus subtilis WXBs-05 different component of table

Three, the Primary Study of detoxification substance

(1) influence of SDS, Proteinase K processing to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose bacterial strain, 37 DEG C, 200r/min culture for 24 hours, seed liquor is made, respectively by 1%, 3%, 6%, 10% ratio Example is transferred in 5mL LB culture medium, and fermentation liquid, fermentation liquid centrifugation, control group 1mL is made in 200r/min, 37 DEG C of cultures for 24 hours PBS buffer solution, untreated fish group are that 100 μ L PBS add 900 μ L bacteria suspensions, and SDS processing group is 900 μ L bacteria suspensions, 50 μ L PBS, 50 μ L SDS liquid, Proteinase K processing group are 900 μ L bacteria suspensions, 50 μ L PBS, 50 μ L Proteinase Ks, equal 37 DEG C of water bath processing 6h 37 DEG C water bath processing 6h, is added equivalent T-2 toxin, and detoxification liquid is made in culture.Using secondary screening method of the same race, SDS, Proteinase K are measured The virus elimination rate that treated.After handling WXBs-05 fermentation liquid respectively using SDS and Proteinase K, virus elimination rate variation such as table 12, As seen from table, virus elimination rate bacterium hangs untreated fish group liquid > SDS processing group > Proteinase K processing group, can be with initial guess WXBs-05 The main active substances for removing toxin are extracellular enzyme material.

The influence of 12 SDS of table, Proteinase K processing bacterial strain WXBs-05 to virus elimination rate

(2) it elutes, influence of the extraction to bacillus subtilis WXBs-05 virus elimination rate

Recovery purpose WXBs-05 bacterial strain, 37 DEG C, 200r/min culture for 24 hours, seed liquor is made, respectively by 1%, 3%, 6%, 10% volume ratio is transferred in 5mL LB culture medium, and fermentation liquid, fermentation liquid are made for 24 hours in 200r/min, 37 DEG C of cultures Equivalent T-2 toxin is added, the sterile LB liquid medium of equivalent adds equivalent Toxin stock liquid for control, is placed in 37 DEG C, 200r/min After culture for 24 hours, 4 DEG C of detoxification liquid, 8000r/min are centrifuged 10min, are resuspended using PBS buffer solution, measurement content of toxins, and continuous 3 Secondary 4 DEG C, 8000r/min centrifugation 10min after PBS buffer solution be resuspended after measure content of toxins again, according to result calculate elute Rate, thus it is speculated that the fastness of its detoxification compound.Equivalent toxin is added in fermentation liquid simultaneously, and the sterile LB liquid medium of equivalent adds equivalent Toxin stock liquid is control, is placed in 37 DEG C, after 200r/min culture for 24 hours, 4 DEG C of detoxification liquid, 8000r/min refrigerated centrifuge 10min, It is resuspended using PBS buffer solution, measures content of toxins, be extracted with dichloromethane 3 times, removing dichloromethane layer, nitrogen is slow at room temperature With volatilize, measure content of toxins again, according to result calculate extraction yield, with elution processing result combine analysis its degradation and inhale Attached combination.

After repeatedly being eluted using PBS buffer solution to detoxification liquid and being extracted using organic solvent to detoxification liquid, Virus elimination rate variation is such as table 13, and as seen from table, repeatedly elution and extraction does not reduce virus elimination rate significantly, therefore can be with Tentatively judge purpose bacterial strain WXBs-05 it is irreversible removing toxin based on and detoxification formation toxin thallus compound very Securely, it is not easy to elute.

The elution of table 13, influence of the extraction processing bacterial strain WXBs-05 to virus elimination rate

Embodiment 5: verification test in bacillus subtilis WXBs-05 detoxification efficiency body

Malicious concentration (100 μ g/mL) is suitably attacked in determination, randomly selects 48 four week old BALB/c mouses, after adapting to 3d, with Machine is divided into 8 groups, and blank control group, positive control (attacking poison) group, probiotic group, detoxification group, prevention group, prevention is respectively set Control group, treatment group, treatment control group.Every group 6, for male BALB/c mouse, the method for continuous stomach-filling daily, each group is equal 0.2ml is gavaged, specific grouping and testing program are shown in Table 14, and mouse is tested after adapting to 3d.Respectively at experimental period 14d and 28d, mouse fasting 12h, free water, each group randomly selects 3 mouse, eye socket blood sampling, 4 DEG C, 2000r/min centrifugation 15min separates serum, measures Serum markers.Such as intestinal inflammatory cell factor: IL-6, IL-1 β, TNF-α, IL-12, IL-10,IL-13.The measurement of growth performance: timing is weighed daily during raising, is recorded its weight, is calculated after the test small The average weight gain of mouse；Pathological section production: sampling (heart, liver, duodenum, colon, testis) in mouse same area, The tissue sample for taking about 1cm2 size is placed in the fixed liquor of general organization and fixes, and it is molten that testis is placed in gonadal tissue fixer It is fixed in liquid.Slice, the processing such as trimmed, flushing, dehydration, saturating wax, embedding, slice (with a thickness of 4 μm) step is made in the company of being sent to Suddenly, it observed, take figure and take pictures, observe each phase mice organs changes in histopathology of each group, analyze villus length, quantity, Crypt depth, the indexs such as hidden ratio of suede, T-2 toxin and relevant group observe and record the male mouse testicular weight of analysis, Sperm state (sperm Vigor, abnormal rate etc.), sexual glands have it is without exception etc.；It tests after data carry out preliminary treatment with 2007 software of Excel, then uses 18.0 software of SPSS is for statistical analysis.Significance test of difference and Multiple range test, (P < 0.05) are carried out using ANOVA, LSD Statistical value be considered as significant difference.Test result is indicated in the form of mean+SD (Mean ± SD).Animal experiment Grouping situation is shown in Table 14.

The grouping of 14 animal experiment of table

It is analyzed according to statistical result, detoxification group mouse is in good condition in entire experimental period body weight increase, with blank group, benefit Raw bacterium group is extremely significant with the mouse difference of attacking malicious T-2 toxin group without significant difference, hence it is demonstrated that WXBs-36 has stronger detoxification Effect.Treatment group gavages purpose bacterial strain WXBs-36 in 14d~28d, hence it is evident that alleviates the shadow that T-2 toxin increases weight to mouse It rings, difference is extremely significant compared with prevention control group does not gavage the weight gain of WXBs-36；Prevention group gavages purpose bacterial strain in 1d~14d After WXBs-36, it is little to its increase heavy influence that 14d~28d experimental period, gavages T-2 toxin, extremely significant with prevention control group difference, The influence for effectively T-2 toxin being prevented to increase weight mouse.It is each according to heart, liver, testis, enteron aisle as shown in Fig. 6,7,8,9 The pathological section interpretation of result of group, the generation without obvious lesion compared with attacking poison group of detoxification group, each organ lesion of prevention group are bright Aobvious to be lighter than prevention control group, compared with treating control group, lesion, which has, largely to be alleviated for treatment group；By immune factor result Significant up-regulation mouse proinflammatory factor IL-1 β (the P < 0.01), TNF-α, IL-6 (P < of poison group is attacked in analysis in 14d experimental period 0.05) expression, at the same it is significant lower the expression (P < 0.05) for pressing down scorching factor IL-10, it is scorching to proinflammatory factor IL-12, suppression because The expression of sub- IL-13 has not significant impact.In 14~28d experimental period, attack poison group still significantly up-regulation TNF-α (P < 0.01), The expression of IL-1 β (P < 0.05), does not make significant difference to the expression of IL-1 β, IL-12, IL-10, IL-13, and detoxification group and sky White group not significant compared to difference；Same analysis can obtain, and purpose bacterial strain WXBs-36 group is equal in 14d and 14~28d experimental period The significant expression for lowering mouse proinflammatory factor TNF-α (P < 0.05), IL-6 (P < 0.01), does not have the expression of other factors It significantly affects；When 14~28d, prevention group compared with preventing control group downward mouse proinflammatory factor IL-1 β of significant difference, The expression (P < 0.05) of TNF-α, IL-6, has down regulation trend to the expression of IL-12 but difference is not significant, factor IL- scorching to suppression 10, the expression of IL-13 has up-regulation trend but equally not significant；Treatment group is compared with treating control group, mouse proinflammatory factor IL-1 The expression of β (P < 0.05), TNF-α (P < 0.05), IL-6 (P < 0.01) are significantly lowered, and the expression for pressing down scorching factor IL-13 is aobvious Up-regulation (P < 0.05) is write, the differential expression of IL-12 and IL-10 is not significant.

Therefore it can be obtained according to animal test results, bacillus subtilis WXBs-36 can significantly remove T-2 toxin, keep away Exempt from T-2 toxin animal body is caused to damage, and has the function of a degree of prevention and treatment T-2 toxin harm.

Bibliography

1. history is competing etc., zearalenone Progress in study of biodegradation China grain and oil journal, and 2013,28:111-114

2. pavilion Sun Zhi etc. decomposes separation, identification and its producing enzyme feature the microbiology notification of zearalenone bacterial strain, 2014,41:909-915

3.Abbes S, Ben Salah-Abbes J, Sharafi H, Oueslati R, Noghabi KA.Lactobacillus paracasei BEJ01 prevents immunotoxic effects during chronic Zearalenone exposure in Balb/c mice.Immunopharmacol Immunotoxicol, 2013,35: 341-348.

4.Abrunhosa L, In ê s A, Rodrigues AI,A, Pereira VL, Parpot P, Endes-Faia A,A.Biodegradation of ochratoxin A by Pediococcus Parvulus isolated from Douro wines.Int J Food Microbiol, 2014,188:45-52

5.Ashraf R, Shah NP.Immune system stimulation by probiotic Microorganisms.Crit Rev Food Sci Nutr, 2014,54:938-56

6.Awad WA, Ghareeb K, Bohm J, Zentek J.Decontamination and detoxification strategies for the Fusarium mycotoxin deoxynivalenol in animal feed and the effectiveness of microbial biodegradation.Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2010,27:510-520

7.Beloglazova NV, Shmelin PS, Speranskaya ES, Lucas B, Helmbrecht C, Knopp D, Niessner R, De Saeger S, Goryacheva IY.Quantumdot loaded liposomes as Fluorescent labelsfor immunoassay.Analytical Chemistry, 2013,85:7197-7204

8.Binder SB, Schwartz-Zimmermann HE, Varga E, Bichl G, Michlmayr H, Adam G, Berthiller F.Metabolism of zearalenone and its major modified forms in Pigs.Toxins (Basel), 2017,9, doi:10.3390/toxins9020056

9.De Baere S, Devreese M, Watteyn A, Wyns H, Plessers E, De Backer P, Croubels S.Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantitative determination of gamithromycin in animal plasma,lung tissue and pulmonary epithelial lining fluid.J Chromatogr A, 2015,1398:73-82

10.Do EJ, Hwang SW, Kim SY, Ryu YM, Cho EA, Chung EJ, Park S, Lee HJ, Byeon JS, Ye BD, Yang DH, Park SH, Yang SK, Kim JH, Myung SJ.Suppression of colitis- associated carcinogenesis through modulation of IL-6/STAT3 pathway by Balsalazide and VSL#3. J Gastroenterol Hepatol, 2016,31:453-461

11.Fan W, Lv Y, Ren S, Shao M, Shen T, Huang K, Zhou J, Yan L, Song S. Zearalenone(ZEA)-induced intestinal inflammation is mediated by the NLRP3 Inflammasome.Chemosphere, 2018,190:272-279

12.Flores-Flores ME, Gonz á lez- E.An LC-MS/MS method for multi- Mycotoxin quantification in cow milk.Food Chem, 2017,218:378-385

13.Genuis SJ, Kyrillos E.The chemical disruption of human Metabolism.Toxicol Mech Methods, 2017,27:477-500

14.Green, DH, Wakeley, PR, Page, A, Barnes, A, Baccigalupi, L, Ricca, E,； Cutting, SM.Characterization of two Bacillus probiotics.Appl Environ Microbiol.Appl Environ Microbiol, 1999,65:4288-4291

15.He JW, Hassan YI, Perilla N, Li XZ, Boland GJ, Zhou T.Bacterial Epimerization as a Route for Deoxynivalenol Detoxification:the Influence of Growth and Environmental Conditions.Front Microbiol, 2016,7:572

16.Henryk H Jele ń, Dziadas M, Magorzata Majcher.Different headspace solid phase microextraction-Gas chromatography/mass spectrometry approaches To haloanisoles analysis in wine [J] .J Chromatogr A, 2013,1313:185-193

17.Hu J, Ma L, Nie Y, Chen J, Zheng W, Wang X, Xie C, Zheng Z, Wang Z, Yang T, Shi M, Chen L, Hou Q, Niu Y, Xu X, Zhu Y, Zhang Y, Wei H, Yan X.A Microbiota-Derived Bacteriocin Targets the Host to Confer Diarrhea Resistance in Early-Weaned Piglets.Cell Host Microbe, 2018,24:817-832

18.Huang C, Zong MH, Wu H, Liu QP.Microbial oil production from rice Straw hydrolysate by Trichosporon fermentans.Bioresour Technol, 2009,100:4535- 4538

19.Hueza IM, Raspantini PC, Raspantini LE, Latorre AO, G ó rniak SL.Zearalenone,an estrogenic mycotoxin,is an immunotoxic compound.Toxins (Basel), 2014,6:1080-1095

20.Karlovsky P, Suman M, Berthiller F, De Meester J, Eisenbrand G, Perrin I, Oswald IP, Speijers G, Chiodini A, Recker T, Dussort P.Impact of food processing and detoxification treatments on mycotoxin contamination.Mycotoxin Res, 2016,32:179-205

Khan FA, Zahoor M.In vivo detoxification of aflatoxinB1 by magnetic Carbon nanostructures prepared from bagasse.BMC Vet Res, 2014,10:255.

Sequence table

<110>Hua Zhong Agriculture University

<120>there is the bacillus subtilis of high-efficiency detoxicating to T-2 toxin

<141> 2019-05-27

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1417

<212> DNA

<213>bacillus subtilis (Bacillus subtilis)

<220>

<221> gene

<222> (1)..(1417)

<400> 1

ctataatgca gttcgagcgg acagatggga gctcgctccc tgaggtagcg gcggacgggt 60

gagtaacacg tgggtaacct gcctgtaaga gtgggagaac tccgggaaac cggggctaat 120

accggagggt tgtttgaacc gcatggttca aacataaaag gtggcttcgg ctaccactta 180

cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg 240

cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300

cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360

tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtaccgttc 420

gatagggcgg taccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag 480

ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540

gcggtttctt aagtctgatg tgaaagcccc cggctcaccc ggggagggtc attggaaact 600

ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga 660

gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720

gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780

gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc 840

tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt 900

ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 960

acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc 1020

gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1080

gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg 1140

gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200

agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg 1260

gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagctt 1320

gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttta 1380

cacccgaagt cggtaggtac ctttaggagc cagccgc 1417

Claims (2)

1. a kind of pair of T-2 toxin has bacillus subtilis (Bacillus subtilis) WXBs-05 of high-efficiency detoxicating, preservation In China typical culture collection center, deposit number is CCTCC NO:M2019177.

2. Bacillus subtillis (Bacillus subtilis) WXBs-05 described in claim 1 is in efficient removal T-2 toxin In application.