CN110172425A - The screening and application of zearalenone detoxification type probiotics - Google Patents
The screening and application of zearalenone detoxification type probiotics Download PDFInfo
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Abstract
The invention belongs to agriculture technical field of microbe application, and in particular to the screening and application of zearalenone detoxification type probiotics.Zearalenone (ZEN) is mainly generated by Fusarium, is pollution one of feed and the main mycotoxin of raw material.The present invention screens to obtain the probiotics of one plant of efficient removal zearalenone, by being verified in biological characteristics, detoxification characteristic, detoxification product and detoxification efficiency body, the probiotics is screened, the bacterial strain of one plant of efficient removal zearalenone is filtered out after primary dcreening operation, secondary screening, identification is named as bacillus subtilis ((Bacillus subtilis)) WXBs-36.The bacterial strain has the speed of growth fast, and anti-adversity is strong, and the stronger feature of biocidal property may be used as microorganism fodder additive.Bacterial strain detoxification efficiency of the invention is good, and has good prevention and treatment zearalenone (ZEN) to the effect of the damage of body.
Description
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 zearalenone has the sieve of bacillus subtilis (Bacillus subtilis) bacterial strain of high-efficiency detoxicating effect
Choosing, identification, the identification and detoxification efficiency verifying application of bacteriostasis property, anti-adversity, detoxification characteristic and detoxification substance.
Background technique
Zearalenone (Zearalenone, abbreviation ZEN) is also known as F-2 toxin, mainly by Fusarium graminearum (F.
Graminearum it) generates.Furthermore it is micro- that pearl sickle-like bacteria (F.moniliforme), fusarium tricinctum (F.tricinctum) etc. are altered
Biology also can produce ZEN, and ZEN pollutes cereal all over the world extensively, is one of the widest toxin of global pollution (Mally A
et al 2016)。
Mycotoxin contamination is serious in current agricultural production, and pollution range is wide, main Polluted grains and feed, raw material.It is dynamic
Serious body injury is caused after object feeding pollution feed, leads to heavy economic losses, therefore the whole world is to Mycotoxins in Feed
Removing is lasting to be paid high attention to.Common poison-removing method is mainly physics detoxicity method, chemical detoxication method and biological detoxication method, due to
Biological detoxication method in mycotoxin subtractive process have detoxification is high-efficient, has no toxic side effect, free of contamination unique advantage, by
It has arrived the whole world widely to pay close attention to, therefore has found and the strain excellent of ZEN and can finally develop efficient in efficient removal feed
Detoxicating microbes agent is of great significance, and the removing for mycotoxin in fermented feed provides technical support, be animal husbandry green,
Sound development provides help.
Summary of the invention
It is an object of the invention to overcome defect of the existing technology, filtering out has good removing zearalenone
(ZEN) 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
Zearalenone has the bacillus subtilis (Bacillus subtilis) of high-efficiency detoxicating effect, and the present invention provides withered
Screening, identification, bacteriostasis property, anti-adversity, detoxification characteristic, the identification of detoxification substance and the detoxification efficiency of careless bacillus are tested
The application of card.
The present invention is first using ZEN analogue phenyl ethylene oxide as sole carbon source and nitrogen source, to laboratory bacterium library
In probiotics carry out primary dcreening operation, using ZEN toxin sterling carry out 2 primary dcreening operations, the two result combine filter out 6 plants in ZEN toxin
The strain excellent survived under environment.Secondary screening is carried out to this 6 plants of bacterial strains, using ZEN as substrate, is detected with competitive ELISA method, sieve
Select virus elimination rate highest and most stable of purpose bacterial strain WXBs-36.Bacterial strain identification is carried out to WXBs-36, is seen by morphology
It examines, biochemical indicator identification, 16S rRNA identification, is accredited as bacillus subtilis (Bacillus subtilis).
Growth characteristics, bacteriostasis and the resistance of bacterial strain WXBs-36 studies have shown that the bacterial strain fertility is very strong,
Without lag phase, enter logarithmic phase (0h~6h) quickly, stationary phase is entered after 4h, to E.coli K88, O157, golden yellow grape
Coccus, salmonella have certain bacteriostasis, and high temperature resistant, acid and alkali-resistance, bile tolerance, stomach juice-resistant ability are stronger.
The detoxification condition for studying bacterial strain WXBs-36 obtains, and virus elimination rate is related with reaction time when detoxification, temperature, pH, with
The inoculum concentration of WXBs-36 bacterial strain without obvious relation, and for 24 hours, 7,37 DEG C of pH when virus elimination rate highest, most stable.
Different component detoxification is made in WXBs-36 bacterial strain, and as a result viable bacteria suspension detoxification ability is most strong, and supernatant takes second place, tentatively
It is speculated as degradation, absorption two ways detoxification.The detoxification substance of Primary Study WXBs-36, SDS, Proteinase K processing thallus with
ZEN reaction, verifying degradation material are ectoenzyme.Assessment thallus and toxin adsorb the complex stabilities to be formed, and detoxification liquid is multiple
Elution, extraction, it was demonstrated that the compound for adsorbing formation is highly stable.
Bacillus subtilis WXBs-36 of the invention can significantly remove zearalenone (ZEN), avoid corn red
Mould ketenes causes to damage to animal body, and has the function of a degree of prevention and treatment ZEN harm.
The present invention has separately verified the purpose Strains B. subtilis WXBs-36 filtered out by internal, in vitro test
To the excellent detoxification efficiency of ZEN, and studies and measure bacillus subtilis bacterium of the invention there is good fungistatic effect and degeneration-resistant
Property, it can be used as feed addictive use.
Isolated Strain Designation is bacillus subtilis WXBs-36, Bacillus subtilis WXBs- by applicant
36, the China typical culture collection center preservation of the Chinese Wuhan Wuhan University, deposit number are delivered on March 20th, 2019
For CCTCC NO:M 2019178.
The bacteria characteristic of bacillus subtilis WXBs-36:
The fertility of bacillus subtilis WXBs-36 bacterial strain is very strong, no lag phase, enters logarithmic phase after inoculation quickly
(0h~6h) enters stationary phase after 4h, there is certain suppression to E.coli K88, O157, staphylococcus aureus, salmonella
Bacterium ability, while there is stronger high temperature resistant, acid and alkali-resistance, bile tolerance and stomach juice-resistant ability.
The detoxification substance of WXBs-36 bacterial strain, SDS, Proteinase K processing thallus are reacted with ZEN, and verifying degradation material is born of the same parents
Exoenzyme.Assessment thallus and toxin adsorb the complex stabilities to be formed, and detoxification liquid is repeatedly eluted, extracted, and shows thallus toxin
Compound closely, is not easy to elute.
Animal test results show that WXBs-36 bacterial strain can also prevention and treatment ZEN while playing excellent detoxification
Damage to animal body.
The probiotics that the present invention screens has the advantages that
(1) bacterial strain of the invention screen from the bacterial strain of a large amount of candidate probiotics and is obtained, the speed of growth fastly, to common enteron aisle
(staphylococcus aureus, salmonella have apparent fungistatic effect to pathogenic bacteria.
(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 gemma, very strong to the tolerance of high temperature, can tolerate 90 DEG C, 10min, therefore the bacterial strain can
It is subjected to the environment such as high temperature process and granulation.
(4) bacterial strain is absorption, degradation two ways detoxification, high-efficient, substantially irreversible, toxin and thallus compound
It is extremely closely not easy to elute.
(5) in vivo studies proves, bacterial strain WXBs-36 detoxification efficiency is obvious, and also has prevention and treatment ZEN to dynamic
The effect of object body injury, with more environmental-friendly, the advantages of to safety of human and livestock.
More detailed technical solution is shown in the content of " specific embodiment ".
Detailed description of the invention
Fig. 1: being growth conditions of the purpose bacterial strain of the invention screened on LB culture medium.
Fig. 2: being the purpose bacterial strain Gram's staining microscope form that the present invention screens.
Fig. 3: being that the purpose bacterial strain that the present invention screens dyes microscope form in peacock green.
Fig. 4: the purpose bacterial strain-bacillus subtilis WXBs- screened using the 16S rRNA gene PCR augmentation detection present invention
36 result.Description of symbols: M:DL 2000DNA molecular weight standard；Swimming lane 1: bacillus subtilis WXBs-36；Swimming lane
2: negative control
Fig. 5: the growth curve chart of bacterial strain of the present invention.
Fig. 6: the mouse heart pathological section of verification test in bacterial strain detoxification efficiency body of the present invention.Description of symbols:
A figure in Fig. 6 attacks malicious group；B figure in Fig. 6；Detoxification group；C figure in Fig. 6: prevention group；D figure in Fig. 6: prevention
Control group；E figure in Fig. 6: treatment group；F figure in Fig. 6: treatment control group.
Fig. 7 is the mouse liver pathological section 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 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；D figure in Fig. 8: pre-
Anti- control group；E figure in Fig. 8: treatment group；F figure in Fig. 8: treatment control group.
Fig. 9 is the mouse testis pathological section of verification test in bacterial strain detoxification efficiency body of the present invention
A figure in Fig. 9: malicious group is attacked；B figure in Fig. 9: detoxification group；C figure in Fig. 9: prevention group；D figure in Fig. 9: pre-
Anti- control group；E figure in Fig. 9: treatment group；F figure in Fig. 9: treatment control group.
Figure 10: the weight gain result of each processing group mouse.
Figure 11: the cytokines measurement result of each processing group mouse.Description of symbols: the A figure in Figure 11: intestinal inflammatory
Cell factor: TNF-α (Alpha)；B figure in Figure 11: intestinal inflammatory cell factor IL-6；C figure in Figure 11: enteritis
Disease cell factor IL-1；D figure in Figure 11: intestinal inflammatory cell factor IL-12；In Figure 11 E figure: intestinal inflammatory cell because
Sub- IL-13；F figure in Figure 11: intestinal inflammatory cell factor IL-10；
Specific embodiment
To the explanation of sequence table
Sequence table SEQ ID NO:1 is the partial order of bacillus subtilis WXBs-36 16S rDNA gene of the invention
Column.
Embodiment 1: the screening and identification of bacterial strain
One, the primary dcreening operation of bacterial strain: the Hormisch culture first with the analogue primary dcreening operation of ZEN toxin, after configuration improvement
Base (2.0g (NH4)2SO4、0.5g KH2PO4、0.2g MgSO4·H2O、0.1g CaCl2, 15g agar) press 1% volume ratio
The phenyl ethylene oxide solution that concentration is 10% is added, as sole carbon source, by bacillus subtilis WXBs-36 strain inoculated
37 DEG C of incubator stationary culture 72h are placed on, to avoid carbon source interference of the bacterium in former environment, the list that picking grows out
Bacterium colony, continuous 3 pure cultures, finally saves those bacterial strains that can be grown on culture medium, records and analyzes the growth shape of bacterial strain
Condition.Again with ZEN toxin sterling primary dcreening operation, the ZEN toxin sterling that concentration after dilution is 500ng/mL is pressed to the ratio of 0.1% volume
Example is added in the Hormisch culture medium of improvement, and the WXBs-36 bacterial strain of inoculation is placed in 37 DEG C of incubators, carries out continuous 3 again
Secondary pure culture is recorded in the bacterial strain grown above, and is compared with toxin analog primary dcreening operation result, screening obtain 6 plants it is excellent
Gesture bacterial strain, strain number are respectively WXBs-37, WXBs-18, WXBs-33, WXBs-20, WXBs-08, WXBs-36.
Two, the secondary screening that substrate carries out bacterial strain detoxification ability bacterial strain secondary screening: is done with ZEN.Picking isolated strains are in the LB liquid of 5mL
In body culture medium, cultivated at 37 DEG C, under the conditions of 200r/min for 24 hours afterwards as seed liquor.By seed liquor with the inoculation of 1% volume
Amount is inoculated into the LB liquid medium of 10mL, is cultivated in 37 DEG C, under the conditions of 200r/min for 24 hours as fermentation liquid.It takes respectively
The fermentation liquid 2ml of isolated strains, each ZEN that 4 μ l are added stores working solution (500 μ g/ml), at 37 DEG C, under the conditions of 200r/min
Reaction adds the ZEN of equivalent volumes to store working solution as control for 24 hours, with the LB liquid medium of equivalent volumes.Liquid to be checked is mixed
Uniformly, 5min is centrifuged with 10000r/min, the supernatant after taking centrifugation is used for ZEN assay after being filtered with 0.22 μm of filter.
Using the method for competitive ELISA, using ZEN toxin enzyme-linked immune quantitative detection reagent box, (clever biotechnology is limited fastly in Shanghai
Company) ZEN residue content in liquid to be checked is measured, when detection, each sample did multiple holes, and selection has higher detoxification to ZEN
The bacterial strain of rate carries out next step research.As a result WXBs-36 bacterial strain detoxification ability is most strong, is up to the virus elimination rate of ZEN sterling
73.6%.
Three, bacterial strain is identified: observation of the bacterium colony of pure culture through colonial morphology, Gram's staining carry out Preliminary Identification.This hair
Growth characteristics of the bright bacterial strain on LB culture medium are shown in Fig. 1, and Gram's staining characteristic is shown in that Fig. 2, peacock green dyeing property are shown in
Fig. 3.WXBs-36 bacterial strain colonial morphology after LB cultured on solid medium 12h is circle, and white light yellow complexion, edge is irregular,
Bacterium colony is wet, and obvious gauffer is formed among bacterium colony.WXBs-36 can be found for leather by Gram's staining and peacock green coloration result
Lan Shi positive bacteria, is elongated rod shape, and no pod membrane is middle life or nearly middle raw type gemma.
Identification reference Bu Ruide (Breed) by above-mentioned isolated bacterial strain etc., " primary Jie Shi Bacteria Identification handbook ", the (the 9th
Version) English edition, the correlation technique on 2004, carry out anaerobism, gelatin liquefaction, M.R, V.P, ornithine, lysine, arginine,
Isolated bacterial strain can be identified kind according to test result by the test of salt tolerant growth.
The biochemical identification result of 1 bacillus subtilis WXBs-36 of table
On the basis of above-mentioned identification, 16S rRNA gene order further has been carried out to bacterial strain WXBs-36 of the invention
It is detected, confirmation identification is carried out to the classification of kind belonging to bacterial strain.
(1) purpose strain gene group rapidly extracting
Purpose bacterial strain is inoculated in 5mL LB culture medium with the inoculum concentration of 1% volume, 37 DEG C of 200r/min are cultivated for 24 hours,
2mL bacterium solution is taken, 12000r/min is centrifuged 5min, discards supernatant liquid, is resuspended with the DEPC water of 50 μ L, 100 DEG C of distilled water pretreatments
10min, immediately ice bath 10min, wink is from taking 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 product commission one Hui Yuan biotechnology company of Wuhan Tian that amplification obtains is sequenced.The sequence measured is existed
Blast is carried out on ncbi database compares analysis
16S rRNA PCR amplification result is shown in Fig. 4.Sequencing result is subjected to Blast comparison in ncbi database.Retrieval
It was found that the 16S rRNA for the WXBs-36 bacterial strain that the present invention screens and the sequence homology highest of bacillus subtilis, similitude are
99%.The part 16S rRNA gene order of amplification is shown in sequence attachment 1.
Embodiment 2: growth curve and the bacteriostasis property measurement of isolated strains WXBs-36
Picking purpose bacterial strain WXBs-36, in 37 DEG C, is cultivated for 24 hours in the LB liquid medium of 5mL 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/min
Under the conditions of cultivate for 24 hours as fermentation liquid.Fermentation liquid is inoculated into the LB liquid medium of 50mL with the inoculum concentration of 1% volume,
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.Growth curve result is shown in Fig. 5, can by Fig. 5
Know that WXBs-36 enters logarithmic phase in 2-4h or so, stationary phase is entered after 8h, the decline phase is entered after 20h.
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 in 1% ratio, and 37 DEG C, cultivate 6h under the conditions of 200r/min, than turbid
Method will indicate that bacteria culture fluid is diluted to after suitable concentration and is uniformly coated on LB solid medium using cotton swab；Using identical
Method prepares purpose bacterial strain and positive control (the pig source enterococcus faecium HDRsEf1 that laboratory separates) fermentation liquid, by purpose bacterial strain
It is centrifuged with positive control (enterococcus faecium HDRsEf1) fermentation liquid, takes supernatant filtration sterilization, aseptic filtration is added in Oxford cup
Purpose bacterial strain, positive control (enterococcus faecium HDRsEf1) supernatant afterwards are put into 37 DEG C of constant temperature trainings after 4 DEG C of refrigerator diffusions overnight
It supports and observes inhibition zone after cultivating 12h in case；The inhibition zone of purpose bacterial strain and positive control is measured, to judge purpose bacterial strain to finger
Show the bacteriostasis of bacterium, continuous 3 repetitions.Test result is shown in Table 2.
2 bacillus subtilis WXBs-36 fermented supernatant fluid extracorporeal bacteria inhibitor test of table
Embodiment 3: the adverse-resistant characteristic test of bacillus subtilis WXBs-36
(1) high temperature resistance is tested
The spore solution kept of going bail for is injected into centrifuge tube, is respectively placed in 60,70,80,90 DEG C of water-baths and is heat-treated
5min, the bacterium solution after taking heat treatment carry out doubling dilution step by step, remaining viable count are surveyed with tilt-pour process, with the spore solution of equivalent
Room temperature is placed in as control, 3 repetitions of every group of test.WXBs-036 bacterial strain high temperature resistant the results are shown in Table 3.WXBs-36 as shown in Table 3
Bacterial strain still has 65.58%, 54.55% bioactivity after 80 DEG C, 90 DEG C of processing 5min, it is known that the brood-gemma of the bacterium is to temperature
Degree has higher tolerance.Show bacillus subtilis WXBs-036 resistant against high temperatures, high temperature process and system can be subjected to
The environment such as grain.
Tolerance of the 3 bacillus subtilis WXBs-036 of table to high temperature
(2) test to Bile salt resistance of bacillus subtilis WXBs-36
0.5mLWXBs-036 bacterial strain spore solution 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.It is resistance to that WXBs-36 bacterial strain simulates cholate
It the results are shown in Table 4 by property, as shown in Table 4, WXBs36 bacterial strain brood-gemma is stronger to simulation Pig cholate tolerance, in simulation cholate
Still there is nearly 91% survival rate after processing for 24 hours, shows that bacillus subtilis bacterial strain WXBs-36 is resistant to the environment of enteron aisle cholate.
Tolerance of the 4 bacillus subtilis WXBs-36 of table to cholate
(3) test to gastric fluid-resistant of bacillus subtilis WXBs-36
It takes the spore solution of 0.5mL WXBs-36 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 solution, which is placed in sterile saline, to be compared.Stomach juice-resistant the results are shown in Table 5, and as shown in Table 5, WXBs-36 bacterial strain is in pH
In the simulate the gastric juice of value about 2.0, bacterium activity has no significant change after processing 2,4 hours, and 2h survival rate is up to 82.91%, 4h
When still have nearly 70% survival rate.Show bacillus subtilis bacterial strain WXBs-36 can pass through the acidic environment of gastrointestinal tract and
It survives in gastrointestinal tract.
Tolerance of the 5 bacillus subtilis WXBs-36 of table to gastric acid
(4) the acidproof and alkali resistance test of bacillus subtilis WXBs-36 bacterial strain
The PBS buffer solution that pH is 2,4,7,9, filtration sterilization are modulated in advance.Respectively by 0.5mLWXBs-36 bacterial strain gemma
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, and tilt-pour process surveys viable count, every group of examination
Test 3 repetitions.Acidproof, alkaline-resisting to the results are shown in Table 6, as shown in Table 6, WXBs-36 bacterial strain is deposited after handling 2 hours in different pH value
Motility rate variation differs, 60% or so.Its survival rate highest under conditions of pH is 4, the survival rate that pH is 2 is minimum, but its
Overall survival is good.
Tolerance of the 6 bacillus subtilis WXBs-36 of table to different pH value
Embodiment 4: the detoxification attribute testing of bacillus subtilis WXBs-36
One, the best detoxification condition measurement of bacillus subtilis WXBs-36
(1) influence of the pH value to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, at 37 DEG C, 200r/min is cultivated for 24 hours, and seed liquor, the switching of 1% volume ratio is made
Into 5mL LB liquid medium, in 200r/min, fermentation liquid is made in 37 DEG C of cultures for 24 hours, fermentation liquid pH is adjusted to 2 respectively, 4,
6,9 and 2mL is respectively taken to be added to the ZEN Toxin stock liquid of 2 μ L, the sterile LB liquid medium of equivalent is added into equivalent Toxin stock liquid
For control, 37 DEG C, 200r/min is cultivated for 24 hours, referring to the same method of secondary screening, measures the virus elimination rate of bacterial strain under different pH.
The fermentation liquid of WXBs-36 bacterial strain is shown in Table 7 to the virus elimination rate variation of ZEN under different pH environment.It as shown in Table 7, is 2-9 in pH
Under the conditions of, virus elimination rate is incremented by with the rising of pH after the fermentation liquid of WXBs-36 reacts for 24 hours with ZEN, is 9 i.e. meta-alkalescence ring in pH
Bacterial strain virus elimination rate highest of the invention under border.It is that virus elimination rate is minimum under 2 under conditions ofs, that is, strong acid environment in pH, is almost close to
Zero.
Virus elimination rate of the 7 bacillus subtilis WXBs-36 of table at different pH
(2) influence of the temperature to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, in 37 DEG C, 200r/min is cultivated for 24 hours, and seed liquor, 1% volume unit ratio is made
It is transferred in 5mL LB liquid medium, in 200r/min, fermentation liquid is made in 37 DEG C of cultures for 24 hours, takes 2mL to be added 2 fermentation liquid
μ L ZEN 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 of the bacterial strain under different temperatures.
The fermentation liquid of WXBs-36 bacterial strain is shown in Table 8 to the virus elimination rate variation of ZEN toxin under different temperatures environment.As shown in Table 8, in temperature
Under conditions of degree is 27 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, virus elimination rate after the fermentation liquid of WXBs-36 bacterial strain reacts for 24 hours with ZEN toxin
Slightly it is incremented by with the rising of temperature, but the virus elimination rate of other temperature is roughly the same in addition to 27 DEG C, without very big difference.
The virus elimination rate of 8 bacillus subtilis WXBs-36 of table at different temperatures
(3) influence of the time to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, at 37 DEG C, 200r/min is cultivated for 24 hours, and seed liquor, the switching of 1% volume ratio is made
Into 5mL LB liquid medium, fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours, takes 2mL that 2 μ L ZEN are added fermentation liquid
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 difference
Cultivate 12h, for 24 hours, 48h, 72h.Using secondary screening same method, the virus elimination rate of the bacterial strain of the present invention under different time is measured.Fermentation
The virus elimination rate that liquid reacts different time with ZEN is shown in Table 9.As shown in Table 9, respectively react 12h, for 24 hours, under 48h, 72h, WXBs-
Virus elimination rate increase at any time after 36 fermentation liquid reacts for 24 hours with ZEN toxin and be slightly incremented by.
Virus elimination rate of the 9 bacillus subtilis WXBs-36 of table under different time
(4) influence of the inoculum concentration to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, at 37 DEG C, 200r/min is cultivated for 24 hours, and seed liquor is made, respectively by 1%, 3%,
6%, 10% volume ratio is transferred in 5mL LB culture medium, and fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours, by fermentation liquid
Take 2mL that 2 μ L ZEN Toxin stock liquid are added, 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 same method, measures the virus elimination rate of bacterial strain of the present invention under different vaccination amount.It is different
Virus elimination rate under the conditions of inoculum concentration is shown in Table 10, does not have specific rule by Biao Ke get, between inoculum concentration and virus elimination rate.
Virus elimination rate of the 10 bacillus subtilis WXBs-36 of table under different vaccination amount
Two, influence of the different component to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, at 37 DEG C, 200r/min is cultivated for 24 hours, and seed liquor is made, and presses 1% volume ratio respectively
Example is transferred in 5mL LB culture medium, and fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours.
The preparation of different component detoxification liquid:
1) somatic cells solution: taking the fermentation liquid of bacillus subtilis WXBs-36 prepared by the present invention, in 4 DEG C, 8000r/
Min is centrifuged 10min, discards supernatant liquid, is resuspended with PBS buffer solution, is centrifuged again, then be resuspended with PBS buffer solution, as thallus
Cell solution；It is control with equivalent sterile PBS buffer；
2) it inactivates somatic cells solution: taking manufactured fermentation liquid, in 4 DEG C, 8000r/min refrigerated centrifuge 10min is discarded
Clear liquid is resuspended with PBS buffer solution；It is centrifuged, discards supernatant again, sediment is cooling at 121 DEG C, high pressure steam sterilization 30min
It is resuspended afterwards with PBS buffer solution, as inactivation somatic cells solution；It is control with equivalent sterile PBS buffer；
3) extracting solution intracellular: somatic cells solution in ice bath with cell supersonic wave be crushed instrument be crushed (100 times, 5s/
It is secondary), then broken cell is centrifuged 10min, supernatant is taken to filter in 0.45 μm of filter at 4 DEG C under the conditions of 8000r/min, with
Equivalent sterile PBS buffer is control；
4) supernatant: taking 4 DEG C of fermentation liquid, and 8000r/min is centrifuged 20min, takes supernatant.It is pair with equivalent LB culture medium
According to.4 kinds of component detoxification liquid take 2mL that the ZEN Toxin stock liquid of 2 μ L is added respectively, add equivalent with the sterile LB liquid medium of equivalent
Toxin stock liquid is control, is placed in 37 DEG C, 200r/min is cultivated for 24 hours.Using secondary screening same method, the de- of different component is measured
Malicious rate.
The results show that influence of the different component of fermentation liquid to ZEN virus elimination rate is shown in Table 11.As shown in Table 11, WXBs-36 bacterium
The viable bacteria suspension detoxification ability of strain is most strong, and supernatant also has certain detoxification ability.Tentatively judge that supernatant mainly leads to after inspection information
Extracellular enzyme effect detoxification is crossed, viable bacteria and inactivation bacteria suspension demonstrate life to the result difference of zearalenone (ZEN) virus elimination rate
Object is metabolized the possibility of detoxification, while the possibility for also demonstrating absorption detoxification and degradation-detoxification and depositing, and shows that WXBs-36 thallus is logical
There is also the detoxifications of suction-operated while crossing biological metabolism degradation-detoxification.
The virus elimination rate of the bacillus subtilis WXBs-36 different component of the invention of table 11
Three, to the Primary Study of detoxification substance
(1) influence of SDS, Proteinase K processing to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, at 37 DEG C, 200r/min is cultivated for 24 hours, and seed liquor is made, and presses 1% volume ratio respectively
Example is transferred in 5mL LB culture medium, and fermentation liquid is made in 200r/min, 37 DEG C of cultures for 24 hours.Fermentation liquid is centrifuged, control group is
1mL 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, in 37 DEG C of water-baths
6h is handled, 6h is handled in 37 DEG C of water-baths, equivalent ZEN toxin is added, detoxification liquid is made in culture.Use secondary screening same method, measurement
SDS, the Proteinase K virus elimination rate that treated.After handling WXBs-36 fermentation liquid respectively using SDS and Proteinase K, virus elimination rate
Variation such as table 12.As shown in Table 12, virus elimination rate bacterium hangs untreated fish group liquid > SDS processing group > Proteinase K processing group, Ke Yichu
Step speculates that the main active substances of WXBs-36 bacterial strain removing toxin are extracellular enzyme material.
The influence of 12 SDS of table, Proteinase K processing bacterial strain WXBs-36 to virus elimination rate
(2) it elutes, influence of the extraction to bacillus subtilis WXBs-36 virus elimination rate
Recovery purpose bacterial strain WXBs-36, in 37 DEG C, seed liquor is made in 200r/min culture for 24 hours, presses 1% volume ratio respectively
Example is transferred in 5mL LB culture medium, cultivates at 200r/min, 37 DEG C and fermentation liquid is made for 24 hours；Be added again into fermentation liquid etc.
Toxin is measured, the sterile LB liquid medium of equivalent adds the ZEN Toxin stock liquid of equivalent volumes for control, is placed in 37 DEG C, 200r/min
After culture for 24 hours, by detoxification liquid in 4 DEG C, 8000r/min is centrifuged 10min, is resuspended using PBS buffer solution, and measurement ZEN toxin contains
Amount, at 4 DEG C, measures ZEN content of toxins, root after being resuspended after 8000r/min centrifugation 10min with PBS buffer solution continuous 3 times again
Eluting rate is calculated according to result, thus it is speculated that the fastness of its detoxification compound.Equivalent ZEN toxin is added into fermentation liquid simultaneously, with etc.
Measuring sterile LB liquid medium adds equivalent Toxin stock liquid for control, 37 DEG C is placed in, after 200r/min culture for 24 hours, by detoxification liquid
It in 4 DEG C, 8000r/min refrigerated centrifuge 10min, is resuspended using PBS buffer solution, measures ZEN content of toxins, extracted with methylene chloride
It takes 3 times, removing dichloromethane layer, nitrogen mitigation volatilizes at room temperature, measures ZEN content of toxins again, is calculated and extracted according to result
Rate combines its degradation of analysis and absorption combination with elution processing result.
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 the ZEN toxin thallus compound that based on the irreversible removing toxin of purpose bacterial strain WXBs-36 and detoxification is formed
Very securely, it is not easy to elute.
The elution of table 13, influence of the extraction processing bacterial strain WXBs-36 to virus elimination rate
Embodiment 5: verification test in bacillus subtilis WXBs-36 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, separate serum, measure Serum markers, such as intestinal inflammatory cell factor: IL-6, IL-1 β, TNF-α (Alpha),
IL-12,IL-10,IL-13.The measurement of growth performance: timing is weighed daily during raising, records its weight, after the test
Calculate the average weight gain of mouse；Pathological section production: mouse same area sampling (heart, liver, duodenum, colon,
Testis), the tissue sample of about 1cm2 size is taken, is placed in the fixed liquor of general organization and fixes, it is solid that testis is placed in gonadal tissue
Determine fixed in liquor.Slice, trimmed, flushing, dehydration, saturating wax, embedding, slice (with a thickness of 4 μm) etc. is made in the company of being sent to
Processing step is observed, takes figure and take pictures, and each phase mice organs changes in histopathology of each group is observed, and analysis villus is long
Degree, quantity, Crypt depth, the indexs such as hidden ratio of suede, ZEN and relevant group observe and record the male mouse testicular weight of analysis, Sperm state
(sperm motility, abnormal rate etc.), sexual glands have without exception etc.；It tests after data carry out preliminary treatment with 2007 software of Excel,
It is for statistical analysis with 18.0 software of SPSS again.Significance test of difference and Multiple range test are carried out using ANOVA, LSD, (P <
0.05) statistical value is considered as significant difference.Test result is indicated in the form of mean+SD (Mean ± SD).
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 ZEN group without significant difference, hence it is demonstrated that WXBs-36 has stronger detoxification to imitate
Fruit.Treatment group gavages purpose bacterial strain WXBs-36 in 14d-28d, hence it is evident that the influence that ZEN increases weight to mouse is alleviated, with prevention
The weight gain that control group does not gavage WXBs-36 is extremely significant compared to difference；Prevention group after 1d-14d gavages purpose bacterial strain WXBs-36,
It is little to its increase heavy influence that 14d-28d experimental period, gavages ZEN, extremely significant with prevention control group difference, effectively prevents ZEN pairs
The influence of mouse weight gain.As shown in Fig. 6,7,8,9, according to the pathological section result of heart, liver, testis, enteron aisle each group point
Analysis, the generation without obvious lesion compared with attacking poison group of detoxification group, each organ lesion of prevention group are obviously lighter than prevention control group,
Compared with treating control group, lesion, which has, largely to be alleviated for treatment group.By immune factor interpretation of result, in 14d experimental period
The expression of significant up-regulation mouse proinflammatory factor IL-1 β (the P < 0.01), TNF-α, IL-6 (P < 0.05) of poison group is attacked, while significant
The expression (P < 0.05) for pressing down scorching factor IL-10 is lowered, it is unobvious to the expression of proinflammatory factor IL-12, the scorching factor IL-13 of suppression
It influences.In 14-28d experimental period, the still significantly expression of up-regulation TNF-α (P < 0.01), IL-1 β (P < 0.05) of poison group is attacked, it is right
The expression of IL-1 β, IL-12, IL-10, IL-13 do not make significant difference, and difference is not significant compared to the blank group for detoxification group；
Same analysis can obtain, and purpose bacterial strain WXBs-36 group significantly lowers mouse proinflammatory factor in 14d and 14~28d experimental period
The expression of TNF-α (P < 0.05), IL-6 (P < 0.01), has no significant effect the expression of other factors；When 14~28d, in advance
Expression (the P < for lowering mouse proinflammatory factor IL-1 β, TNF-α, IL-6 of the anti-group of significant difference compared with preventing control group
0.05), there is down regulation trend to the expression of IL-12 but difference is not significant, have up-regulation to the expression for pressing down scorching factor IL-10, IL-13
Trend but equally not significant；Treatment group is compared with treating control group, mouse proinflammatory factor IL-1 β (P < 0.05), TNF-α (P <
0.05), the expression of IL-6 (P < 0.01) is significantly lowered, and (P < 0.05), IL-12 are significantly raised in the expression for pressing down scorching factor IL-13
It is not significant with the differential expression of IL-10.
It can be obtained by above-mentioned animal test results, bacillus subtilis WXBs-36 of the invention can be removed significantly
ZEN avoids ZEN from causing to damage to animal body, and has the function of a degree of prevention and treatment ZEN harm.
Probiotics bacterial bacterial strain WXBs-36 of the invention has good removing zearalenone (ZEN) and can be used as
Feed addictive uses.
The present invention has separately verified the purpose bacterial strain filtered out i.e. bacillus subtilis WXBs- by internal, in vitro test
The excellent detoxification efficiency of 36 pairs of zearalenones (ZEN), bacillus subtilis of the invention have good fungistatic effect and
Resistance can be used as feed addictive use.
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Sequence table
<110>Hua Zhong Agriculture University
<120>screening and application of zearalenone detoxification type probiotics
<141> 2019-05-26
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1429
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<220>
<221> gene
<222> (1)..(1429)
<400> 1
ctatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg 60
gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta 120
ataccggatg gttgtctgaa ccgcatggtt cagacataaa aggtggcttc ggctaccact 180
tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcgacga 240
tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc 300
tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg 360
cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtgccgt 420
tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac tacgtgccag 480
cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg 540
caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 600
actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 660
agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg 720
agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg 780
agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc 840
gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc 900
ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct 960
ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt 1020
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc 1080
ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag 1140
gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg 1200
gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt 1260
tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag 1320
catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt 1380
tgtaacaccc gaagtcggtg aggtaacctt tatggagcca gcccgcccc 1429
Claims (2)
1. the Bacillus subtillis (Bacillus subtilis) that a kind of pair of zearalenone has high-efficiency detoxicating effect
WXBs-36, is deposited in China typical culture collection center, and deposit number is CCTCC NO:M2019178.
2. Bacillus subtillis (Bacillus subtilis) WXBs-36 described in claim 1 is in efficient removal Gibberella zeae
Application in ketenes.
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