CN113621541A

CN113621541A - Marine sediment bacillus and application thereof in degrading ochratoxin A

Info

Publication number: CN113621541A
Application number: CN202110940057.XA
Authority: CN
Inventors: 张健; 杨焱; 李林霖; 张津和; 张颖
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-11-09
Anticipated expiration: 2041-08-09
Also published as: CN113621541B

Abstract

The invention discloses a marine sediment bacillus, which is classified and named asCytobacillus oceanisediminisThe preservation number is CGMCC NO. 22874. The degradation rate of the marine sediment bacillus CO29 and the intracellular lysate thereof on ochratoxin A (OTA) can reach 100 percent, the degradation reaction is irreversible biodegradation, the degradation products are ochratoxin alpha (OT alpha) and beta-phenylalanine, the toxicity is reduced by at least 1000 times, and the immune toxicity is avoided. In addition, the marine sediment bacillus CO29 provided by the invention has the advantages of simple culture condition, fast growth and high OTA degradation efficiency, can be used for preparing OTA biological detoxicant and can be applied to detoxification treatment of OTA pollutants.

Description

Marine sediment bacillus and application thereof in degrading ochratoxin A

Technical Field

The invention belongs to the technical field of microbiology and biodegradation, and particularly relates to a marine sediment bacillus and application thereof in degrading Ochratoxin A (OTA).

Background

Ochratoxin A (OTA) has a chemical name of 7- (L-beta-phenylpropylamino-carbonyl) -carboxy-5-chloro-8-hydroxy-3, 4-dihydro-3R-methylisoxazolene o-ketone, is formed by coupling isocoumarin with beta-phenylalanine through an amide bond, and has a molecular formula of C₂₀H₁₈ClNO₆The molar molecular weight is 403.81 g/mol, and the melting point is 169 ℃.

OTA is a toxic secondary metabolite produced by toxigenic fungi such as Aspergillus and Penicillium, and OTA has been classified as a "class 2B" carcinogen in 1993 by The International Agency for Research on Cancer (IARC). The OTA-producing fungi in nature are of various types, and they mainly infect grains and various agricultural and sideline products, causing OTA pollution in crop raw materials and processed products thereof. This contamination can be transmitted to soil and water, and also to animals and humans through the food chain, with great harm. The major target organs of OTA are liver and kidney, and also teratogenicity, mutagenicity, carcinogenesis and destruction of the immune system.

According to different action mechanisms, the OTA detoxification method can be divided into three methods: physical methods, chemical methods and biological methods, which reduce or eliminate toxicity of OTA by adsorption, modification or decomposition, wherein the biodegradation method has mild reaction conditions, high degradation efficiency and good safety, and does not destroy product quality, thus receiving wide attention. The biodegradation of OTA is generally by hydrolysis of OTA to Ochratoxin α (OT α) and β -phenylalanine, which have very low toxicity. Research on degrading bacteria and degrading enzymes of OTA has been advanced to a certain extent, and currently reported bacteria capable of degrading OTA are Lactobacillus rhamnosus (Lactobacillus rhamnosus: (II))Lactobacillus rhamnosus) Bacillus subtilis preparation (B)Bacillus subtilis) Acinetobacter calcoaceticus (A), (B), (C)Acinetobacter calcoaceticus) Bacillus atrophaeus, Phenylobacterium atrophaeusPhenylobaterium immobile) Bacillus licheniformis (B.licheniformis)Bacillus licheniformis) The fungus is yarrowia lipolytica (A), (B), (C), (D) and (D)Yarrowia lipolytica) Saccharomyces cerevisiae (A)Saccharomyces cerevisiae) Aspergillus fumigatus (Aspergillus fumigatus) And Aspergillus nigerAspergillus niger) And the like. At present, no report about the degradation of OTA by marine sediment bacillus is found.

Disclosure of Invention

The invention aims to provide a bacillus marinus capable of degrading Ochratoxin A (OTA), and a method for degrading OTA by using the bacillus marinus is safe, simple, convenient, green and free of residues.

Another purpose of the invention is to use the marine sediment bacillus to prepare various products for degrading OTA.

In order to achieve the purpose, the invention discloses the following technical contents:

the invention provides a marine sediment bacillus, classifying marine sediment bacillus (Bacillus:)Cytobacillus oceanisediminis) The bacterial name is CO29, and the classification name isCytobacillus oceanisediminisThe preservation number is CGMCC number 22874. Has been preserved in China general microbiological culture Collection center (CGMCC) (with the address of No. 3 of the institute of microbiology of the national academy of sciences, Japan, No. 1 of the North Chen Xilu, No. 3 of the Korean-Yang district, Beijing) on 13.7 months in 2021, and the preservation number is CGMCC No. 22874. Bacillus marinus with OTA degradation, as in the case of other organisms (ii) ((iii))Cytobacillus oceanisediminis) The mutation is still easy to occur. Thus, mutants of this strain can be obtained using physical and chemical mutagenesis methods known in the art. These mutants are also part of the invention, as long as they retain the characteristic of OTA degradability.

The invention further discloses a marine sediment-containing bacillus (B)Cytobacillus oceanisediminis) Biological detoxicant of CO29 or its intracellular solubles, Bacillus marinus: (A), (B) and (C)Cytobacillus oceanisediminis) The preservation number of the CO29 is CGMCC number 22874, and the biological detoxicant is liquid type or solid type. A method of preparing a biological detoxicant, comprising: activating Bacillus marinus as Bacillus marinus with preservation number of CGMCC No.22874 with CO29, performing multi-stage amplification, and collecting fermentation culture when the thallus is in stationary phase to obtain liquid biological detoxicant; or will send outCentrifuging the fermentation culture, collecting thallus and crushing to obtain cell lysate, and preparing liquid biological detoxicant; or centrifuging the fermentation culture, collecting thallus, and lyophilizing or adding adsorbent to obtain solid biological detoxicant; or centrifuging the fermentation culture, collecting thallus, crushing to obtain cell lysate, and lyophilizing or adsorbing the cell lysate to obtain solid biological detoxicant.

The invention further discloses an application of the marine sediment bacillus CO29 in preparation of an ochratoxin A degrading preparation and an application of the prepared biological detoxicant in preparation of an ochratoxin A degrading preparation. The experimental results show that: the degradation rate of the marine sediment bacillus CO29 and the intracellular lysate thereof on OTA can reach 100%, and the degradation reaction is irreversible biodegradation. The marine sediment bacillus CO29 provided by the invention has a degradation effect on OTA, the degradation products are OT alpha and beta-phenylalanine, the toxicity is reduced by at least 1000 times, and no immune toxicity exists. In addition, the marine sediment bacillus CO29 provided by the invention has the advantages of simple culture condition, fast growth and high OTA degradation efficiency, can be used for preparing OTA biological detoxicant and can be applied to detoxification treatment of OTA pollutants in a large scale.

The invention is described in more detail below:

the invention further discloses a culture method of the marine bacillus sedimentans CO29 and a preparation method of a marine bacillus sedimentans degraded OTA product, wherein the culture method comprises the following steps:

step A, carrying out inverted culture on the marine sediment bacillus CO29 by using NA culture medium at 37 ℃;

and step B, using an NB culture medium during liquid culture, wherein the rotating speed of a shaking table is 180 r/min. The bacillus is pre-cultured for 12h in an NB culture medium, and then inoculated in a shake flask for fermentation culture by 1 percent of inoculum concentration, so as to obtain a fermentation culture of the marine sediment bacillus CO 29;

step C, preferably, the fermentation culture is concentrated by a method which does not influence the activity of the thalli, such as centrifugation, filtration and the like, so as to obtain bacterial suspension with higher concentration; more preferably, the fermentation culture is subjected to centrifugal separation treatment to obtain marine sediment bacillus CO29 wet cells, and the marine sediment bacillus CO29 is subjected to heavy suspension, cell disruption treatment and centrifugal separation treatment to obtain an enzyme preparation; further, the liquid-type biological detoxicant can be prepared into a solid-type biological detoxicant by methods existing in the field, such as freeze-drying or adding an adsorbent.

The biological detoxicant can be packaged by conventional packaging technology in the field, and stored according to specific environmental conditions.

The formula of the fermentation medium is as follows:

NA medium: 10g of tryptone, 3g of beef extract, 5g of NaCl5g and 2% of agar, adding distilled water to 1L, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 20 min.

NB medium: 10g of tryptone, 3g of beef extract, 5g of NaCl5g, adding distilled water to 1L, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 20 min.

Morphological characteristics and physiological and biochemical characteristics of the marine sediment bacillus CO 29. (Table 1)

TABLE 1 morphological characteristics and physio-biochemical characteristics of Bacillus marinus CO29

Drawings

FIG. 1 is a relation between fermentation time and detoxification rate of marine sediment bacillus CO 29;

FIG. 2 Effect of inactivation on the detoxification ability of Bacillus marinus CO 29;

FIG. 3 impact of disruption on the detoxification ability of Bacillus marinus CO 29;

FIG. 4 HPLC-FLD profile of Bacillus marinus CO29 intracellular lysate degrading OTA. Note: A-PBS buffer solution; B-OTA standard substance; C-OTA samples treated with intracellular lysate for 24 h;

FIG. 5 HPLC-MS/MS profile of OTA samples treated with Bacillus marinus CO29 intracellular lysate for 24 h. Note: A-TIC diagram; EIC map of B-OT α; a primary mass spectrum of C-OT α; a secondary mass spectrum of D-OT α;

FIG. 6 is a photograph showing the morphology of Bacillus marinus CO29 observed on the NA medium;

FIG. 7 is a morphological photograph of Bacillus marinus CO29 taken by gram-stained light microscope.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.

Example 1

Separation, purification and identification of marine sediment bacillus CO29

1. Enrichment of

The collection and separation samples of the strains are taken from vineyard soil samples. Removing impurities and grinding after soil sample collection, preparing 5g of sample into suspension in 45 mL of NB culture medium, enriching for 24h at 37 ℃ at 180 r/min, coating the bacterial liquid on an NA flat plate with proper dilution by using normal saline according to a dilution coating method, performing inverted culture for 24h at 37 ℃, picking single bacterial colonies with good separation degree and different bacterial colony morphologies, further performing streak purification on the NA flat plate, and repeating for three times.

2. Preliminary screening

Because the OTA standard substance is expensive and has high toxicity, the coumarin which is the OTA structural analogue with low price and low toxicity is adopted to replace the OTA as the only carbon source in the primary screening culture medium, so the strain which can grow on the primary screening culture medium has the potential of degrading the OTA.

The isolated strain was inoculated in a primary screening solid medium and cultured in an inverted state at 37 ℃ for 48 hours. In order to ensure that the growth of the strain is not influenced by the components of the original culture medium and that coumarin is the only carbon source, the strain with better growth state is inoculated on the primary screening solid culture medium by the same method, and the step is repeated for 2 times. The strains that still grew well were rescreened.

Primary screening of solid culture medium: 0.25g KH₂PO₄，0.25g MgSO₄·H₂O，0.5g KNO₃，0.5g (NH₄)₂SO₄，0.005g CaCl₂，0.003g FeCl₃·6H₂Adding distilled water to 1L, sterilizing at 121 deg.C for 20min at pH 7.0-7.2. Adding the coumarin solution to 1g/L after filter sterilization.

3. OTA pure product detoxification rescreening

And inoculating the primary screened strain into an OTA-NB culture medium with the OTA concentration of 1 mug/mL for a detoxification test, taking an aseptic fermentation culture medium as a blank control, culturing for 72 hours at 37 ℃ at 180 r/min in the dark condition, detecting the OTA content, and repeatedly screening to finally obtain a strain capable of degrading the OTA, wherein the number of the strain is CO 29.

And classifying and identifying the degrading strains by using classification methods such as morphology, physiological and biochemical methods, 16S rDNA sequence analysis and the like.

The morphological characteristics and physiological and biochemical characteristics of CO29 are shown in table 1, fig. 6, and fig. 7.

16S rDNA sequence analysis: carrying out PCR amplification by using primers 27f (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3'), carrying out BLAST comparison on a 16S rDNA sequence (16S rDNA is shown in a sequence table 2) with the length of 1409 bp and an existing sequence in a Genbank database, wherein the homology of the sequence and the 16S rDNA of the marine sediment bacillus reaches 100.00%;

TABLE 2 16S rDNA sequence of CO29 Strain

TGGCTCCAATGGTTACCCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAATGGTTTTATGGGATTCGCTTAACCTCGCGGTCTCGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATCCTGTCCCCCGAAGGGGAACGCCCTATCTCTAGGGTTGTCAGGAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTTGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGGAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGGCAGTTACTCCGGTACTTGTTCTTCCCTAACAACAGAGTTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGCCCATCTGTAAGTGATAGCCGAAACCATCTTTCAGCTTTCCCTCATGTGAGGGAAAGAATTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCTGACTTCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTG

Example 2

Detection method of OTA and OT alpha

HPLC-FLD detection method:

the method comprises the following specific steps: a200. mu.L sample was aspirated, centrifuged at 12000r/min for 5min, and the supernatant was filtered through a 0.22 μm filter and analyzed by HPLC-FLD or the sample was stored at 4 ℃. HPLC-FLD detection conditions were performed using a Kromstar HPLC reversed phase C18 column (250X 4.6 mm, 5 μm); excitation wavelength: 333 nm, emission wavelength: 460 nm; mobile phase acetonitrile: water: acetic acid =99:99:2 (V/V); flow rate: 1.0 mL/min, and a sample size of 20. mu.L.

HPLC-MS/MS detection method:

the hydrolysates were all filtered through a filter of 0.22. mu.L pore size and detected by HPLC-MS/MS.

Chromatographic conditions are as follows: phenomenex Gemini 3u C18110A column (150 mM × 2.00 mM, 3 μm), mobile phase a acetonitrile, B5 mM ammonium acetate in water, elution gradient: 0-5 min, 95% B; linearly reducing to 5% B within 5-35 min; 35-45 min, 5% B; increasing the linear rate to 95% B within 45-50 min; 50-60 min, 95% B; sample introduction amount: 2 mu L of the solution; flow rate: 0.3 mL/min; column temperature: at 40 ℃.

Mass spectrum conditions: atmospheric pressure electrospray ion source (API-ESI), negative ion scan mode; capillary voltage 4 kV; the drying gas temperature was 300 deg.C, the atomizer pressure was 60 psi, and the drying gas flow rate was 10L/min.

Degradation rate (detoxification rate) = (supernatant OTA content in blank group-supernatant OTA content in control group)/supernatant OTA content in blank group ] × 100%

Example 3

Use of marine sediment bacillus CO29 for degrading OTA

Culturing marine sediment bacillus CO 29: a single colony of marine sediment bacillus CO29 (preservation number: CGMCC number 22874) is selected and inoculated in 5mL of NB medium, the culture temperature is 37 ℃, the rotation speed is 180 r/min, and the culture time is 12 h. After the fermentation is finished, the fermentation culture is reserved for standby.

Inoculating 500 μ L of prepared Bacillus marinus CO29 fermentation culture into 50mL of OTA-NB culture medium, culturing at 37 deg.C for 24h, 36h, 48h, and 72h, respectively, sampling, determining OTA content with non-inoculated OTA-NB culture medium as blank control group, and calculating detoxification rate. The test was repeated at least 3 times.

The preparation method of the OTA-NB culture medium comprises the following steps: transferring a certain volume of OTA mother liquor (1mg/mL) into a sterile triangular flask, adding a certain volume of NB culture medium, fully dissolving, preparing OTA-NB culture medium with OTA final concentration of 1 mug/mL, and subpackaging according to needs.

The HPLC-FLD detection method of OTA is shown in example 2.

And (3) detection results: when the bacillus marinus is cultured at 37 ℃, the bacillus marinus CO29 acinetobacter cutaneus AP19 reacts for 48 hours, and the degradation rate exceeds 90 percent; the reaction is carried out for 72 hours, and the degradation rate is 100 percent. (FIG. 1).

Example 4

Preliminary study on degradation mechanism of marine sediment bacillus CO29

Culturing marine sediment bacillus CO 29: 500 mu L of the fermentation liquid of the marine bacillus sedimentans CO29 prepared in the example 3 is inoculated in 50mLNB liquid culture medium, the culture temperature is 37 ℃, the rotation speed is 180 r/min, and the culture time is 48 h. After the fermentation is finished, the fermentation culture is reserved for standby.

1. And (3) degrading the thalli OTA:

taking 10mL of fermentation liquid, centrifuging to remove supernatant, resuspending the thallus by using PBS buffer solution after ice bath, and washing the thallus for 2 times. Finally, 1mLOTA final concentration of 1 u g/mL OTA-PBS heavy suspension bacteria. Measuring the content of OTA at 4h and 24h, and calculating the detoxification rate.

2. And (3) inactivating bacteria to degrade OTA:

taking 10mL of fermentation liquor, centrifuging to remove supernatant, resuspending the thalli by using PBS buffer solution after ice bath, washing the thalli for 2 times, and inactivating the thalli. Finally, 1mLOTA final concentration of 1 u g/mL OTA-PBS heavy suspension bacteria. Measuring the content of OTA at 4h and 24h, and calculating the detoxification rate.

3. Degrading OTA by fermentation supernatant:

taking 1mL of fermentation supernatant, adding a certain volume of OTA mother liquor (1mg/mL), mixing with the fermentation supernatant (OTA final concentration is 1 μ g/mL), measuring OTA content at 12h and 24h, and calculating detoxification rate.

4. Intracellular lysate degradation OTA:

taking 10mL of fermentation liquor, centrifuging to remove supernatant, resuspending the thallus by using PBS buffer solution after ice bath, and carrying out ultrasonic disruption. Taking 1mL of intracellular lysate, adding a certain volume of OTA mother solution (1mg/mL), fully mixing with the intracellular lysate (the final concentration of OTA is 1 mug/mL), measuring the content of OTA in 12h and 24h, and calculating the detoxification rate.

PBS phosphate buffered saline (pH 7.4): NaCl 8.5 g, Na₂HPO₄·12H₂O 2.9 g，KH₂PO₄0.24 g, pH7.2-7.4, and distilled water to a constant volume of 1L.

Wherein the detection method of HPLC-FLD is shown in example 2.

And (3) detection results: the reduction rate of OTA in 24h of inactivated thallus treatment is 47.62%; the degradation rate of untreated bacteria for 24h is 100%, which indicates that bacteria treated OTA has adsorption effect but mainly has degradation effect. (FIG. 2).

The reduction rate of OTA after 24h of fermentation supernatant treatment is 10.27%; the degradation rate of intracellular lysate for 24h is 100%, which indicates that the marine sediment bacillus CO29 mainly has degradation effect on OTA and active substances mainly exist in bacteria. (FIG. 3).

Example 5

OTA degradation product analysis

Degradation products and blanks were prepared as described in example 3 and analyzed after 24h of reaction by HPLC-FLD and HPLC-MS/MS detection.

And (3) detection results: detecting OTA by HPLC-FLD, wherein the retention time of OTA is 10.567min, reacting for 24h, and the degradation rate of OTA is 100%; a new peak, probably a degradation product of OTA, appeared at 3.952 min (FIG. 4), and was analyzed by HPLC-MS/MS at 255.2465 for OT α hydrogen-reducing ion. This can be determined as the ionic peak of OT α. Therefore, the degradation product of the marine sedimentary bacillus CO29 did contain OT α (fig. 5).

It will be apparent to those skilled in the art that various changes and modifications can be made in the above embodiments without departing from the scope and spirit of the invention, and it is intended that all such changes and modifications as fall within the true spirit and scope of the invention be interpreted in accordance with the principles of the invention. And the invention is not limited to the example embodiments set forth in the description.

SEQUENCE LISTING

<110> Tianjin science and technology university

<120> marine sediment bacillus and application thereof in degradation of ochratoxin A

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1409

<212> DNA

<213> Artificial sequence

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tggctccaat ggttacccca ccgacttcgg gtgttacaaa ctctcgtggt gtgacgggcg 60

gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat tactagcgat 120

tccggcttca tgcaggcgag ttgcagcctg caatccgaac tgagaatggt tttatgggat 180

tcgcttaacc tcgcggtctc gcagcccttt gtaccatcca ttgtagcacg tgtgtagccc 240

aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt tgtcaccggc 300

agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg cgctcgttgc 360

gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca cctgtcatcc 420

tgtcccccga aggggaacgc cctatctcta gggttgtcag gagatgtcaa gacctggtaa 480

ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540

attcctttga gtttcagcct tgcggccgta ctccccaggc ggagtgctta atgcgtttgc 600

tgcagcacta aagggcggaa accctctaac acttagcact catcgtttac ggcgtggact 660

accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagcgtc agttacagac 720

caaagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca ccgctacacg 780

tggaattcca ctcttctctt ctgcactcaa gttccccagt ttccaatgac cctccccggt 840

tgagccgggg gctttcacat cagacttaag gaaccgcctg cgcgcgcttt acgcccaata 900

attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 960

gctttctggt taggtaccgt caaggtaccg gcagttactc cggtacttgt tcttccctaa 1020

caacagagtt ttacgatccg aaaaccttca tcactcacgc ggcgttgctc cgtcagactt 1080

tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag 1140

tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgtcgccttg gtgagccgtt 1200

acctcaccaa ctagctaatg cgccgcgggc ccatctgtaa gtgatagccg aaaccatctt 1260

tcagctttcc ctcatgtgag ggaaagaatt atccggtatt agccccggtt tcccggagtt 1320

atcccagtct tacaggcagg ttgcccacgt gttactcacc cgtccgccgc tgacttcagg 1380

gagcaagctc ccatctgtcc gctcgactg 1409

Claims

1. Marine sediment bacillus (Cytobacillus oceanisediminis) The strain is CO29 with the preservation number of CGMCC number 22874.

2. A biological detoxicant contains marine sediment bacillus (Bacillus subtilis)Cytobacillus oceanisediminis) The bacillus marinus has the bacterial name of CO29 or the intracellular lysate thereof, and the preservation number of the bacillus marinus CO29 is CGMCC number 22874.

3. The biodetoxicant of claim 2, wherein the biodetoxicant is a liquid type or a solid type.

4. A method of preparing a biodetoxicant as defined in claim 2, comprising: activating Bacillus marinus as Bacillus marinus with preservation number of CGMCC No.22874 with CO29, performing multi-stage amplification, and collecting fermentation culture when the thallus is in stationary phase to obtain liquid biological detoxicant; or centrifuging the fermentation culture, collecting thallus and crushing to obtain cell lysate, and making into liquid biological detoxicant; or centrifuging the fermentation culture, and collecting thallus to obtain solid biological detoxicant; or centrifuging the fermentation culture, collecting thallus, crushing to obtain cell lysate, and making the cell lysate into solid biological detoxicant.

5. The method of preparing a biodetoxicant as defined in claim 4, further comprising the step of forming the liquid-type biodetoxicant into a solid-type biodetoxicant.

6. Use of the bacillus marinus CO29 according to claim 1 for preparing a preparation for degrading ochratoxin a.

7. Use of a biodetoxicant as claimed in claim 2 or claim 3 in the preparation of a formulation for degrading ochratoxin a.