CN105255759A - Aflatoxin and ochratoxin difunctional degradation bacterium and application thereof - Google Patents
Aflatoxin and ochratoxin difunctional degradation bacterium and application thereof Download PDFInfo
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- CN105255759A CN105255759A CN201510681175.8A CN201510681175A CN105255759A CN 105255759 A CN105255759 A CN 105255759A CN 201510681175 A CN201510681175 A CN 201510681175A CN 105255759 A CN105255759 A CN 105255759A
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- aflatoxin
- ochratoxin
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- 239000005409 aflatoxin Substances 0.000 title description 7
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Abstract
The invention provides a difunctional efficient degradation bacterium (Stenotrophomonas sp.) CW117 and application thereof to degradation of low-pollution-concentration aflatoxin B1 and ochratoxin A. Compared with a conventional aspertoxin degradation bacterium, the strain CW117 can achieve an extremely good degradation effect under the low-concentration toxin pollution. When cultivated for 72 h in a fermentation culture solution with the final concentration of aflatoxin B1 and ochratoxin A of 20 [mu]g/L, the strain CW117 has the ochratoxin A degradation ratio of 99.4 percent, and the aflatoxin B1 degradation ratio of 94.3 percent; when used for processing a feed (final concentration is 20 [mu]g/kg) polluted by the combination of aflatoxin B1 and ochratoxin A for 72 h, the strain CW117 has the ochratoxin A degradation ratio of 71.0 percent and the aflatoxin B1 degradation ratio of 54.5 percent. The strain CW117 has a concrete application value and significance in the food and feed biological detoxification aspect.
Description
Technical field
The present invention relates to microbiology and biological degradation field, specifically, relate to a strain aflatoxin B1 and the difunctional degradation bacteria of ochratoxin A and application thereof.
Background technology
The secondary metabolite that aspergillus class mycotoxins produces primarily of aspergillus tubigensis such as flavus, Aspergillus parasiticus and Aspergillus ochraceus, isolation identification 20 various structures analogues, wherein with aflatoxin B1 (AFB1) and ochratoxin A (OTA) toxicity maximum, pollute the most extensive in agriculture production and foodstuffs industry.It is I class carcinogens that aflatoxin in 1993 delimited by the World Health Organization (WHO) Agency for Research on Cancer, because it has strong carcinogenic, teratogenesis and mutagenicity, becomes the focus that publilc health and scientific research field are paid close attention to gradually.Aflatoxin and ochratoxin Polluted grains and oil crops become global problem, in livestock industry, two class aspertoxins are by polluting feed harm animal health, cause Animal husbandry production rate low, cause serious financial loss, and by direct or indirect (animal product propagation) approach contaminated food products, affect the healthy of the mankind.Animal takes in AFB1 or AFB2 from feed, in body, be converted into AFM1 or AFM2, is present in milk, meat and birds, beasts and eggs, pollutes human foods chain.In the end of the year 2011, the aflatoxin event of exceeding standard that China's Dairy Industry occurs is the typical case that AFB1 pollution feed causes milk preparation AFM1 to exceed standard, and has caused the great attention of the Chinese government and food safety department.Along with people are to the progressively intensification of mycotoxins recognizing dangers, detect, the research work of pollution prevention and detoxification technology aspect also deepening continuously for aspergillus class mycotoxins, itself and people's quality of the life, healthy and economic interests are of close concern to each other.
In recent years, found that multiple-microorganism has significant degraded or adsorption to aflatoxin.Such as, Butyrivibrio (Butyrivibriosp.), lactobacillus (Lactobacillussp.), suis (Streptococcussp.), bifidus bacillus (Bifidobacteriumsp.) and acinetobacter (Acinetobactersp.) etc.There is the common problem of following two aspects in most virus-free strains of current report: one in actual applications, the detoxification mechanism of part virus-free strain belongs to physical adsorption, not substantial biological degradation, be adsorbed on the toxin of somatic cells wall, in animal or human's body, may desorption be there is under different physicochemical environment, there is no substantive detoxification meaning; They are two years old, the virus elimination rate majority of report bacterial classification contratoxin measures, for the rarely seen report of actual detoxification capability study of lower concentration aspergillus toxoid in the complex matrices such as food raw material and feed (lower than 20 μ g/kg) under the above higher concentration condition of 100 μ g/kg.But, low dosage, combined pollution and high pollution rate are again the universals that aspergillus class mycotoxins pollutes, and 20 the lower concentration aspergillus class mycotoxins of μ g/kg can damage people and animals' internal organs, especially by the agricultural-food of multiple toxin combined pollution, its toxicity has more significant synergism.Therefore, excavate under having degraded low consistency conditions, the virus-free strain of multiple aspergillus class mycotoxins combined pollution, has great practice significance and using value in agriculture production and field of food safety.
Summary of the invention
The object of this invention is to provide a strain aflatoxin B1 and ochratoxin A Double function high efficient degradation bacteria and application thereof.
In order to realize the object of the invention, the present invention isolates a strain from the soil (refinery, Auto repair shop near) and being subject to by polycyclic aromatic hydrocarbons severe contamination the mildew and rot food samples of mycotoxins severe contamination can the bacterial strain CW117 of efficient degradation aflatoxin B1 and ochratoxin A.The Main Morphology of this bacterium is characterized as: Gram-negative, without gemma, shaft-like to curved rod like features, has single polar flagella, can move (Fig. 1).Bacterial strain CW117 is strictly aerobic, and optimum growth temperature is 35 DEG C, and the most suitable growth pH is 7.0-7.5.
Based on morphological specificity and the 16SrDNA sequence (SEQIDNo.1) of bacterial strain CW117, be Stenotrophomonas (Stenotrophomonassp.) by this identification of strains.This bacterial strain is preserved in China typical culture collection center (being called for short CCTCC, address: Wuhan, China, Wuhan University, postcode 430072) on June 14th, 2015, and deposit number is CCTCCNO:M2015371.
In vitro tests shows, bacterial strain CW117 has the degradation effect of significant ochratoxin (OTA) and aflatoxin B1 (AFB1) in the short period of time.Be in the liquid nutrient medium of 20 μ g/L at OTA final concentration, bacterial strain CW117 process 24h, the degradation rate degradation rate of OTA being reached to this bacterial strain of 79.9%, 72h is 99.4%; Be in the liquid nutrient medium of 20 μ g/L at AFB1 final concentration, bacterial strain CW117 process 24h is that 59.0%, 72h degradation rate reaches 94.3% to the degradation rate of AFB1.With the feed (final concentration 20 μ g/kg) of bacterial strain CW117 process endotoxin contamination, the OTA degradation rate of 72h is 71.0%, AFB1 degradation rate is 54.5%.
The zymetology feature detection result display of degradation bacteria strains, the mechanism of degradation of bacterial strain CW117 to OTA is mainly extracellular enzyme and urgees Degradation, the outer degradation rate of crude enzyme liquid 72h to OTA (final concentration 20 μ g/L) of born of the same parents is 83.1%, the mechanism of degradation of bacterial strain CW117 to AFB1 is mainly intracellular enzyme and urgees Degradation, and the outer degradation rate of crude enzyme liquid 72h to AFB1 (final concentration 20 μ g/L) of born of the same parents is only 29.7%.
The present invention also provides microbial inoculum containing Stenotrophomonas CW117 and complex micro organism fungicide thereof.
The present invention also provides the aflatoxin B1 and the difunctional biodegradation agent of ochratoxin A prepared by Stenotrophomonas CW117 or described microbial inoculum.
Preferably, in described biodegradation agent, activeconstituents is the crude enzyme liquid prepared by described microbial inoculum.
More preferably, in described biodegradation agent, activeconstituents is the fermented liquid of bacterial strain CW117, the centrifugal gained supernatant liquor of bacterial strain CW117 fermentation liquor or cellular lysate liquid.
Wherein, the substratum for the preparation of bacterial strain CW117 fermented liquid is: Tryptones 17.0g/L, soya peptone 3.0g/L, glucose 2.5g/L, NaCl5.0g/L, K
2hPO
42.5g/L, pH value 7.0-7.5; Fermentation condition is: 35 DEG C, 180r/min shaking culture 48h.
The present invention also provides the application in aflatoxin B1 and ochratoxin A biological degradation of Stenotrophomonas CW117, described microbial inoculum and described biodegradation agent.
The present invention also provides Stenotrophomonas CW117, described microbial inoculum and described biodegradation agent in the application in food raw material or feed detoxification treatment of going mouldy.
Wherein, described in the content of aflatoxin B1 and ochratoxin A in food raw material or feed that goes mouldy be respectively 20 μ g/kg and following, but be not limited to 20 μ g/kg and following.
The present invention further provides Stenotrophomonas CW117 or the application of described microbial inoculum in preparation aflatoxin B1 and ochratoxin A Double function high efficient biodegradation agent.
Compared with existing aspertoxin degradation bacteria, Stenotrophomonas CW117 of the present invention can be issued to splendid degradation effect in lower concentration aflatoxin B1 and ochratoxin A pollutional condition.According to state food and forage health standard regulation, cereals agricultural-food aflatoxin content must lower than 20 μ g/kg, ochratoxin content must not more than 5 μ g/kg, ochratoxin A in feed also must not more than 100 μ g/kg (GB2761-2011, GB13078.2-2006).In fact, animal toxicology experiment shows, feeds the feed containing 20 μ g/kg toxin continuously, just can cause serious injury (Weietal., 2014DOI10.1002/jsfa.6649) the internal organs of rat.Therefore, bacterial strain CW117 has efficient degradation-detoxification ability to lower concentration AFB1 and OTA, has substantial using value and important meaning in foods/feeds biological detoxication application aspect.
Accompanying drawing explanation
Fig. 1 is the electron micrograph of bacterial strain CW117 of the present invention.
Fig. 2 be in the embodiment of the present invention 2 and 3 bacterial strain CW117 to the dynamic degradation curve of aspertoxin.
Fig. 3 be in the embodiment of the present invention 2 bacterial strain CW117 crude enzyme liquid to the dynamic degradation curve of aspertoxin.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The screening of embodiment 1 aflatoxin B1 and ochratoxin A degradation bacteria, qualification and cultivation
1, the screening of bacterial strain
Sample collecting is from the soil (near refinery, Auto repair shop) by polycyclic aromatic hydrocarbons severe contamination and the mildew and rot food by mycotoxins severe contamination.Get collected specimens 0.5g and add (OTA and AFB1 content is respectively 20 μ about g/L) in 150mL enrichment medium, enrichment culture 7 days in 30 DEG C of constant incubators.After enrichment culture terminates for the first time, get 5mL enrichment culture liquid and be inoculated in fresh OTA and the AFB1 enrichment medium of 150mL, the concentration of OTA and AFB1 is brought up to 30 μ g/L, continue enrichment culture 7 days under the same conditions.Complete third time enrichment culture experiment with identical enriching method, improve concentration to the 50 μ g/L of OTA and AFB1.After enrichment culture terminates, with sterilized water, enrichment culture liquid is made gradient dilution, gradient dilution liquid is coated OTA or AFB1 solid separation culture medium respectively, cultivate 3 days under 30 DEG C of conditions.After cultivation terminates, picking culture dish list colony inoculation carries out degraded test cultures 48h in the enrichment medium of 20 μ g/L each containing OTA and AFB1, arranges the negative control not connecing bacterium simultaneously.After degraded cultivation terminates, nutrient solution is through dichloromethane extraction, according to national standard High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD, carry out with reference to National Standard of the People's Republic of China GB/T25220-2010 and GB/T18979-2003) detect two toxoid residual concentrations in nutrient solution, calculate degradation rate compared with the control.Finally, be separated acquisition one strain (OTA and AFB1 content≤20 μ g/L) under lower concentration OTA and AFB1 condition and still keep the bacterial strain CW117 of efficient degradation rate.
Wherein, enrichment medium is: glucose 5.0g/L, peptone 5.0g/L, yeast powder 2.5g/L, ammonium citrate 1.0g/L, NaAc3H
2o2.5g/L, MnSO
44H
2o0.05g/L, K
2hPO
41.0g/L, MgSO
47H
2o0.2g/L, tween-80 0.5mL, OTA add concentration 20-50 μ g/L, and AFB1 adds concentration 20-50 μ g/L, adjusts pH to 6.2-6.5.
Isolation medium is: glucose 10.0g/L, peptone 10.0g/L, yeast powder 5.0g/L, ammonium citrate 2.0g/L, MgSO
47H
2o0.58g/L, NaAc3H
2o5.0g/L, MnSO
44H
2o0.28g/L, tween-80 1.0mL, adjust pH to 6.2-6.5.
Above-mentioned substratum is autoclaving 15min under 120 DEG C of conditions, adds aseptic OTA and AFB1 toxin standardized solution to normality before using, and solid medium adds the agar powder of 2.0% by above formula.
2, the qualification of bacterial strain CW117
The Main Morphology of bacterial strain CW117 is characterized as: Gram-negative, without gemma, shaft-like to curved rod like features, has single polar flagella, can move (Fig. 1).This bacterium is strictly aerobic, and optimum growth temperature is 35 DEG C, and the most suitable growth pH is 7.0-7.5.
16SrDNA identifies: a pair universal primer being designed for amplification bacterial strain CW11716SrDNA sequence: 27F5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R5'-GGTTACCTTGTTACGACTT-3'.
The genomic dna extracting bacterial strain CW117, as template, carries out pcr amplification, and checks order to amplified production.The 16SrDNA sequence of bacterial strain CW117 is as shown in SEQIDNo.1.
3, the cultivation of bacterial strain
The suitableeest solid medium is: Tryptones 17.0g/L, soya peptone 3.0g/L, glucose 2.5g/L, NaCl5.0g/L, K
2hPO
42.5g/L, agar 20.0g/L, pH value 7.0-75.The suitableeest liquid nutrient medium is not containing agar in above-mentioned formula.
Optimal culture conditions is: 35 DEG C, 180r/min shaking culture 48h.
Embodiment 2 high performance liquid chromatography detects the degradation characteristic of bacterial strain CW117
Degradation bacteria strains degradation dynamic detects: degradation bacteria strains CW117 was activated for 2 generations continuously on the suitableeest solid medium, is inoculated in 4mL liquid nutrient medium incubated overnight, obtains fresh bacterium liquid.The fresh bacterium liquid of 50 μ L is inoculated in respectively 4mLOTA test media (containing 20.0 μ g/LOTA) and AFB1 test media (containing 20.0 μ g/LAFB1), by postvaccinal test tube shaking culture 0,24,48 and 72h, each degradation experiment arranges 3 repetitions.With e. coli k12 (E.coliK12) for negative control bacterial strain.Cultivation terminates rear mixing, after the centrifugal 10min of 8000r/min, collects supernatant liquor and bacterial sediment respectively.
Degraded supernatant liquor is respectively through OTA immune affinity column and AFB1 immune affinity column, methanol-eluted fractions, collect elutriant in glass sample injection bottle, carry out the high performance liquid chromatography fluoroscopic examination (HPLC-FLD) of OTA and AFB1 residual quantity according to national standard detection method (GB/T25220-2010 and GB/T18979-2003).Bacterial sediment after centrifugal, adopts chromatographically pure methyl alcohol to carry out resuspended mixing, and concuss 10min extracts the residual toxin of cell adsorption, abandons precipitation after centrifugal, is carried out the residual toxin detection of cell adsorption after 0.22 μm of filter membrane crossed by supernatant liquor by HPLC-FLD.
Degradation bacteria strains zymetology feature detection: degradation bacteria strains CW117 was activated for 2 generations continuously on the suitableeest solid medium, by the inoculation incubated overnight in 50mL liquid nutrient medium after activation, obtains OD
600the fresh bacterium liquid of=0.8.Mixed by bacterium liquid, be sub-packed in 50mL centrifuge tube, the centrifugal 15min of 10000r/min, abandons precipitation.Accurately measure culture supernatant 40mL in aseptic clean triangular flask, add ochratoxin A/aflatoxin B1 respectively, make its final concentration reach 20 μ g/L.By the triangular flask 30 DEG C of lucifuge quiescent culture prepared, detect the residual concentration of OTA/AFB1 in 0,24,48 and 72h sampling.Each degradation experiment arranges 3 repetitions, and is negative control bacterial strain with E.coliK12.The sample of taking-up is sub-packed in 2mL centrifuge tube, centrifugal, abandon precipitation.Supernatant liquor is respectively through OTA immune affinity column and AFB1 immune affinity column, and methanol-eluted fractions, collects elutriant in glass sample injection bottle, carry out OTA and AFB1 residues detection respectively.
HPLC detects OTA condition: moving phase: acetonitrile: water: acetic acid (volume ratio 96:102:4), fluorimetric detector: excitation wavelength 333nm, emission wavelength 460nm, flow velocity: 1.0mL/min, sample size: 20 μ l, chromatographic column: C
18chromatographic column (4.6 × 250mm, filler diameter 5 μm of Waters); HPLC detects AFB1 condition: moving phase: methyl alcohol: water (volume ratio 45:55), fluorimetric detector: excitation wavelength 360nm, emission wavelength 420nm, flow velocity: 0.8mL/min, sample size: 20 μ l, chromatographic column: C
18chromatographic column (4.6 × 250mm, filler diameter 5 μm of Waters), 0.05% iodine solution post-column derivation method.
To the dynamic degradation curve of aspertoxin as shown in Figure 2, experimental result shows bacterial strain CW117, and CW117 bacterial strain fermentation liquor process 24h, reaches 79.9% to the degradation rate of OTA, and the degradation rate of this bacterial strain of process 72h is 99.4%; Process 24h is 59.0% to the degradation rate of AFB1, and after process 72h, degradation rate is more than 94.3%.In addition, thalline contratoxin is without adsorption.
Bacterial strain CW117 crude enzyme liquid to the dynamic degradation curve of aspertoxin as shown in Figure 3, degradation bacteria strains zymetology feature detection result shows, the mechanism of degradation of bacterial strain CW117 to OTA is mainly extracellular enzyme and urgees Degradation, the outer degradation rate of crude enzyme liquid 72h to OTA (final concentration 20 μ g/L) of born of the same parents is 83.1%, the mechanism of degradation of bacterial strain CW117 to AFB1 is mainly intracellular enzyme and urgees Degradation, and the outer degradation rate of crude enzyme liquid 72h to AFB1 (final concentration 20 μ g/L) of born of the same parents is only 29.7%.
The application of embodiment 3 bacterial strain CW117 in corn bean pulp type feed detoxification treatment
1, experiment material
Strain activation and culture base I: Tryptones 17.0g/L, soya peptone 3.0g/L, glucose 2.5g/L, NaCl5.0g/L, K
2hPO
42.5g/L, agar 20.0g/L.
Strain activation and culture base II: peptone 5.0g/L, extractum carnis 30.0g/L, NaCl5.0g/L, pH7.0-7.2.
Above-mentioned substratum is autoclaving 15min at 120 DEG C all, and experiment feed is corn bean pulp type daily ration.
2, experimental technique
Appropriate OTA and AFB1 standard reserving solution is added in 50mL phosphate buffered saline buffer, be poured in the Feed Sample that 100g crushed after mixing immediately, stir, make the final concentration of OTA and AFB1 reach 40.0 μ g/kg respectively, feed dries stand-by in shady and cool ventilation place.Solid activation medium I activates bacterial classification 2 generation to be measured continuously, the strain inoculation after activation is cultivated 48h in 150mL liquid activation medium II, obtain OD
600the fresh bacterium liquid of=1.0.Get the fresh bacterium liquid of 100mL to mix with the combined pollution feed that 100g prepares, cultivated by the sample prepared under 35 DEG C of conditions, in 0h, 24h, 48h and 72h sampling, each degradation experiment arranges 3 repetitions.Meanwhile, to mix with the pollution toxin that 100g prepares using the fresh blank cultures of 100mL and mix as negative control process.Treatment group and the every sub-sampling 15g of blank group, carry out OTA and AFB1 immediately and extract and analyze.
The extraction and purification of toxin.Take the sample after 10g cultivation in 150mL tool plug triangular flask, add 1gNaCl and 20mL methanol-water mixture (volume ratio 4:1), 30min is extracted in normal temperature concussion, take off bottle stopper through Cao Wen filter paper filtering in clean cup (or under 3000r/min the centrifugal 5min of normal temperature), accurately pipette 5.0mL supernatant liquor and add 20mL pure water dilution mixing, filter 1-2 time to filtrate clarification through glass fiber filter paper, carry out immune affinity column purification process.
Accurately pipette 10.0mL (representing 1.0g sample) above-mentioned clear filtrate, in implantation glass syringe, pressure is regulated to make solution slow transit through immune affinity column with the flow velocity of 1-2 drop/sec, until there is partial air to pass through cylinder, with 10mL pure water drip washing pillar (repeating once), discard whole effluent liquid and make partial air pass through cylinder.Accurately add 1.5mL methanol-eluted fractions, flow velocity is 1 drop/sec, collects elutriant in glass sample injection bottle, carries out HPLC-FLD detection.
To the dynamic degradation curve of aspertoxin as shown in Figure 1, experimental result shows bacterial strain CW117, and the feed (final concentration 20 μ g/Kg) of bacterial strain CW117 process toxin combined pollution, 72h is 71.0% to OTA degradation rate, is 54.5% to AFB1 degradation rate.Illustrate that having extracellular enzyme urgees the bacterial strain CW117 of degradation capability to the degradation effect of the aspergillus toxoids A of lower concentration in complex matrices better, takes second place to the degradation effect of aflatoxin B1.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. Stenotrophomonas (Stenotrophomonassp.) CW117, its deposit number is CCTCCNO:M2015371.
2. the microbial inoculum containing Stenotrophomonas CW117 described in claim 1.
3. the aflatoxin B1 prepared by microbial inoculum described in Stenotrophomonas CW117 described in claim 1 or claim 2 and the difunctional biodegradation agent of ochratoxin A.
4. biodegradation agent according to claim 3, is characterized in that, its activeconstituents is the crude enzyme liquid prepared by described microbial inoculum.
5. biodegradation agent according to claim 3, is characterized in that, its activeconstituents is the fermented liquid of bacterial strain CW117, the centrifugal gained supernatant liquor of bacterial strain CW117 fermentation liquor or cellular lysate liquid.
6. biodegradation agent according to claim 5, is characterized in that, the substratum for the preparation of bacterial strain CW117 fermented liquid is: Tryptones 17.0g/L, soya peptone 3.0g/L, glucose 2.5g/L, NaCl5.0g/L, K
2hPO
42.5g/L, pH value 7.0-7.5; Fermentation condition is: 35 DEG C, 180r/min shaking culture 48h.
7. the application of biodegradation agent in aflatoxin B1 and ochratoxin A biological degradation described in microbial inoculum, any one of claim 3-6 described in Stenotrophomonas CW117, claim 2 described in claim 1.
8. biodegradation agent described in microbial inoculum, any one of claim 3-6 described in Stenotrophomonas CW117, claim 2 described in claim 1 is in the application in food raw material or feed detoxification treatment of going mouldy.
9. application according to claim 8, is characterized in that, described in the content of aflatoxin B1 and ochratoxin A in food raw material or feed that goes mouldy be respectively 20 μ g/kg and following, but be not limited to 20 μ g/kg and following.
10. the application of microbial inoculum described in Stenotrophomonas CW117 described in claim 1 or claim 2 in preparation aflatoxin B1 and the difunctional biodegradation agent of ochratoxin A.
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| CN109762761A (en) * | 2019-01-10 | 2019-05-17 | 河南农业大学 | Micro- acidophilus Stenotrophomonas of efficient degradation aflatoxin B1 and its application |
| CN109762761B (en) * | 2019-01-10 | 2022-01-07 | 河南农业大学 | Stenotrophomonas acidiphila capable of efficiently degrading aflatoxin B1 and application thereof |
| CN111607579A (en) * | 2019-02-22 | 2020-09-01 | 安徽农业大学 | Ochratoxin hydrolase, encoding gene, recombinant vector and application thereof |
| CN111607579B (en) * | 2019-02-22 | 2021-10-08 | 安徽农业大学 | Ochratoxin hydrolase, encoding gene, recombinant vector and application thereof |
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