CN108102963B - Bacillus strain for degrading aflatoxin B1 and screening and application thereof - Google Patents

Abstract

The invention discloses a bacillus strain for degrading aflatoxin B1 and screening and application thereof. The strain is classified and named as Bacillus aryabhattai DT and is preserved in China general microbiological culture collection center with the preservation number of CGMCC 14949. The treatment method comprises strain screening and separation and 16S rDNA identification of a culture medium with coumarin as a unique carbon source; meanwhile, the degradation effect of the strain fermentation culture solution on aflatoxin B1 is explored. A single colony of Bacillus aryabhattai (Bacillus aryabhattai) on a solid culture medium is selected and inoculated in 50mL of growth culture medium, shaking is carried out on a shaking table for 12-24h at the temperature of 30-40 ℃, and a fermentation culture solution and aflatoxin B1 with the concentration of 2 mu g/mL are reacted for 3-4d in the dark, so that the degradation rate of 82.98 percent can be achieved. The screened Bacillus aryabhattai has obvious degradation effect on the aflatoxin B1, has the characteristics of high efficiency, energy conservation, environmental friendliness and the like, and can be applied to preparation of related aflatoxin detoxifiers.

Description

Bacillus strains degrading aflatoxin B1 and their screening and application

technical field

The invention belongs to the field of new strain screening technology and microorganism technology, and particularly relates to a Bacillus strain capable of degrading aflatoxin B1 and its application.

Background technique

Aflatoxins are the most toxic mycotoxins discovered so far, and their basic structures are a difuran ring and a coumarin. It is mainly produced by Aspergillus flavus, Aspergillus parasiticus and other secondary heterocyclic metabolites. Aflatoxin is a highly toxic substance with strong carcinogenicity. Long-term ingestion of toxins in the liver can cause liver cancer and cause cancer in other organs such as kidneys, lungs, and gastrointestinal tract. Among all aflatoxins, aflatoxins are B1 (AFB1) is the most toxic and has been classified as a class I human carcinogen by the International Agency for Research on Cancer (IARC). Aflatoxins are mainly found in moldy peanuts, grains, nuts and rice. According to the United Nations Food and Agriculture Organization (FAO) statistics, 25% of the world's annual cereal production is polluted by mycotoxins to varying degrees, especially aflatoxin pollution. The most serious AFB1 pollution is corn, followed by rice and wheat. The World Health Organization (WHO) stipulates that the maximum allowable concentration of aflatoxin in food is 15μg/kg. In my country, the limit of mycotoxins in food in GB 2761-2017, aflatoxin B1 is found in peanuts, corn, rice, vegetable oil, and beans, respectively. , fermented foods and the limits in dairy products are clearly defined.

Methods to reduce or remove aflatoxins from food and feed have focused on physical and chemical means. Physical methods mainly include light, ultrasonic waves and ultra-high temperature, but these processes may be affected by factors such as temperature and time, resulting in the destruction and loss of nutrients in food. The chemical method is to add some substances to degrade or destroy aflatoxins, such as adding oxidants, ammonium solution method and sodium hydroxide method, etc., but these chemical substances may remain in food and are difficult to remove, so conventional physical and chemical methods remove Aflatoxins in food have shortcomings such as unstable effect, large loss of nutrients, poor feed palatability, and difficulty in large-scale production, which are difficult to be widely used in production practice. Therefore, it is necessary to establish a safe, efficient and environmentally friendly method for controlling aflatoxin contamination to make up for the many application defects of physical and chemical detoxification methods.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of strain and application method that can be used for degrading and removing aflatoxin B1, to overcome and replace the shortcomings and deficiencies of the existing detoxification technology, the present invention provides a novel simple and easy to remove aflatoxin It is an efficient, energy-efficient, and specific microbial treatment method with broad application prospects.

The present invention screened the aflatoxin B1-degrading strain by the culture method with coumarin as the sole carbon source.

Coumarin as the sole carbon source culture method: Weigh soil samples and add them to a triangular flask containing sterile physiological saline, stir and shake evenly to make a suspension, transfer the suspension to a centrifuge tube, and put it under 3000-4000g. Centrifuge, take the supernatant and dilute it with sterile physiological saline, draw an appropriate amount of the diluent and spread it on the coumarin solid medium, and cultivate it at 30-37 °C for 6-7 days. The colonies are separated and streaked repeatedly on the coumarin solid medium, and cultured for three consecutive generations to obtain a genetically stable strain, which is the Bacillus strain that degrades aflatoxin B1.

Preferably, the components of the coumarin solid medium are: coumarin 1 g/L, KH ₂ PO ₄ 0.25 g/L, CaCl ₂ ·2H ₂ O 0.005 g/L, FeCl ₃ ·6H ₂ O 0.003 g /L, KNO ₃ 0.5g/L, MgSO ₄ ·7H ₂ O 0.25g/L, (NH ₄ ) ₂ SO ₄ 0.5g/L, agar 15-20g/L, and the balance is distilled water.

The invention also discloses a fermentation culture liquid of a Bacillus strain that degrades aflatoxin B1. The preparation method is as follows: inoculating the strain screened by the invention into LB liquid medium or nutrient broth medium, and culturing After 18-24h, the fermentation broth was obtained.

Preferably, the components of the LB liquid medium are: 8-10g/L peptone, 5-8g/L yeast powder, 10-15g/L sodium chloride, pH 6.8-7.0.

Preferably, the components of the nutrient broth medium are 8-10g/L peptone, 3-5g/L beef extract, 5-8g/L sodium chloride, pH 7.0-7.2.

The inoculum of the strain was 1-2% (V/V).

The invention also discloses a fermentation supernatant of a Bacillus strain that degrades aflatoxin B1. The preparation method includes centrifuging the fermentation culture solution at 9000 g for 5-10 minutes to obtain the fermentation supernatant.

The invention also discloses a bacterial dry powder of a Bacillus strain that degrades aflatoxin B1. The preparation method is as follows: centrifuging the fermentation culture liquid, and 12-24 h freeze-drying to obtain the bacterial dry powder.

The invention also discloses an application of the strain for degrading aflatoxin B1 in food or feed, which is characterized in that the strain is inoculated into LB liquid medium or nutrient broth medium, and cultured for 18-24 hours Then, obtain the fermentation medium, or centrifuge the fermentation medium to obtain the fermentation supernatant; or dry the sediment after the centrifugation of the fermentation medium to obtain the dry powder; One or more are added to food, feed or agricultural products containing aflatoxin, 1 mL of fermentation broth is diluted 10-30 times for 1-3 kg of agricultural products, mixed evenly, and continuously reacted in the dark at 30-40 °C for 3 -4d.

Through the above method, the present invention screened out a microbial strain, which turned purple by Gram staining, and was a Gram-positive bacteria. The genomic DNA of this strain was extracted and amplified, and the strain was identified by sequencing 16S rDNA. The detected sequences of this species were compared and analyzed by BLAST in the GenBank database, and the results with the highest matching degree were found to have 100% homology with the 16S rDNA sequence of Bacillus aryabhattai. . The strain can be determined to be Bacillus Ayerbodot, named BacillusaryabhattaiDT. The biological preservation information of this strain is: China General Microorganism Culture Collection and Management Center (Address: No. 1, Beichen West Road, Chaoyang District, Beijing), strain collection number CGMCC 14949.

The strain of the present invention can use coumarin as the sole carbon source. The strain DT that degrades aflatoxin B1 screened and isolated by the invention can make the degradation rate of 2 μg/mL of aflatoxin B1 reach 82.98% within 3 days, and the degradation ability of aflatoxin is better than the existing commercial physical and chemical The method has the characteristics and advantages of high efficiency, strong specificity, and no pollution to feed agricultural and sideline products and the environment.

Description of drawings

Fig. 1 plate culture character of bacterial strain DT of the present invention;

The Gram staining result of Fig. 2 strain DT of the present invention;

Fig. 3 degrading effect of strain DT of the present invention on aflatoxin B1 in different reaction times, wherein the ordinate represents the degradation rate of aflatoxin B1, and the abscissa represents the action time;

Fig. 4 Neighbor-Joining phylogenetic tree of the 16S rDNA sequence of the strain DT of the present invention.

Detailed ways

The embodiments of the present invention are described in detail below, but the following embodiments are not intended to limit the protection scope of the present invention.

Example 1: Rapid screening of aflatoxin-degrading microbial strains according to the present invention

1. Implementation steps

A number of soil samples were randomly collected from the fruit and vegetable park of the Institute of Landscape Architecture, Zhejiang University, Hangzhou City, Zhejiang Province, in ziplock bags, and the name, location, time and other information of the collection were indicated. The collected soil samples were weighed 5g into 50mL of 0.85% sterile physiological saline, and the soil suspension was uniformly made by magnetic stirring and shaking. Transfer the soil suspension to a sterilized centrifuge tube and centrifuge at 3000-4000g for 3-5min, take 1 mL of the supernatant of the soil suspension, and dilute it step by step by concentration gradient dilution method for 10 ^-1 , 10 ^-2 , 10 ^{- 3} , 10 ^-4 , 10 ^-5 , 10 ^-6 , draw 200 μL and spread evenly on the solid medium with coumarin as the sole carbon source [components: coumarin 1g/L, KH ₂ PO ₄ 0.25g/L , CaCl ₂ ·2H ₂ O 0.005g/L, FeCl ₃ ·6H ₂ O 0.003g/L, KNO ₃ 0.5g/L, MgSO ₄ ·7H ₂ O 0.25g/L, (NH ₄ ) ₂ SO ₄ 0.5g /L, agar 15-20g/L], cultured at 30-37°C for 6-7d. After the colonies grow in the petri dish, pick a single colony on the coumarin solid medium for three times, and then separate the single colony.

2. Implement the result analysis

The strains screened through the medium with coumarin as the only carbon source are round in shape, not shiny, dry and slightly wrinkled on the surface, rough in the edge, white and opaque in color, and not easy to be provoked by the inoculation ring (Figure 1). It was Gram-stained purple and was Gram-positive (Figure 2). After consulting the literature, it is known that aflatoxin B1 is a derivative of dihydrofuran oxa-naphthalene, and the basic structure is a difuran ring and an oxa-naphthalene (coumarin). Therefore, in the above-mentioned example operation, selecting a solid medium with a partial structure of aflatoxin B1 such as coumarin as the sole carbon source for screening, the strains that degrade aflatoxin B1 can be screened in a targeted manner, and it also has high efficiency, Advantages such as low cost, and then use aflatoxin B1 for validation, enabling fast, safe and efficient screening.

Example 2: Determination of the degradation ability of the microbial strains of the present invention to aflatoxin B1

1. Implementation steps

The selected single colony was inoculated into a 250mL conical flask containing sterilized 50mL LB liquid medium (components: 8-10g/L peptone, 5-8g/L yeast powder, 10-15g/L sodium chloride), Placed in a constant temperature shaker, cultured at 37°C and 180rpm for 18-24h, take 900μL of fermentation broth into a sterilized 2mL centrifuge tube, add 100μLAFBl toxin and mix evenly to make the final concentration 2μg/mL , and placed in a 37°C incubator for 3-4d in the dark. The control group was a sterile liquid medium containing the same concentration of AFB1, and the extraction and detection of aflatoxin B1 were carried out at 0h, 6h, 12h, 24h, 48h and 96h respectively after the reaction.

Extraction of aflatoxin B1: After the above reaction mixture was added, 1 mL of dichloromethane was added and extracted 3 times on a vortex shaker for 30 s each time, fully shaken, and allowed to stand for 5 minutes until obvious stratification appeared, then combined The dichloromethane in the lower layer of each time was collected and placed in a 5 mL centrifuge tube, slowly blown dry with nitrogen at room temperature, and the residue was dissolved with 0.5 mL of methanol. Carefully aspirate the supernatant, properly dilute it to within the measurement range, filter it with a 0.22 μm organic microporous membrane, and use it as a sample for HPLC.

Detection of aflatoxin B1: The content of aflatoxin B1 after the reaction was determined by high performance liquid chromatography (HPLC). Aflatoxin B1 standard product treatment: Weigh aflatoxin B1 standard product powder, dissolve it in methanol to make a 1 mg/mL stock solution, take 250 μL, 100 μL, 20 μL, 10 μL, 2 μL, 0.5 μL of the stock solution and add methanol to 1 mL (concentration). 250 μg/mL, 100 μg/mL, 20 μg/mL, 10 μg/mL, 2 μg/mL, 0.5 μg/mL, respectively).

Mobile phase treatment: Each mobile phase (methanol, acetonitrile, ultrapure water) was suction filtered and degassed for 30 min.

Liquid chromatography conditions: 60% ultrapure water, 30% acetonitrile and 10% methanol were used as mobile phases; the flow rate was 1 mL/min; the detection wavelength was 362 nm, and a Promosil C18 chromatographic column 4.6 mm × 250 mm/5 μm was used, and the column temperature was 30℃; the peak time is about 10-12min. The content of aflatoxin B1 was obtained according to the peak area comparison standard curve, and combined with the control group, the degradation rate of aflatoxin B1 was calculated. The calculation formula is as follows:

Degradation rate %=(1-AFBI peak area after treatment/AFB1 peak area in control)×100%

2. Implement the result analysis

Fig. 3 is the degradation effect of the strain of the present invention on aflatoxin B1 in different time periods. Compared with the control group, the content of aflatoxin B1 added in the original culture solution was significantly reduced, and at the same time, the degradation rate of aflatoxin B1 of the strain of the present invention also gradually increased with the progress of the reaction time. high. In the first 12h, the degradation rate of aflatoxin B1 by the strain of the present invention increased linearly, reaching 62.04% in 12h, and in the following 24h and 48h, the degradation rate of aflatoxin B1 gradually increased slowly. Since aflatoxin B1 has a certain inhibitory effect on the strain, coupled with the accumulation of its own metabolic wastes, the degradation of the strain on Aspergillus flavus B1 is slowed down. At 72h and 96h, the degradation of aflatoxin B1 The rate stabilized at 82.98%.

Example 3: Identification of the microbial strains of the present invention

1. Implementation steps

The total DNA of the strain of the present invention was extracted according to the steps of the Takara Bacteria Genomic DNA Extraction Kit, and the 16S rDNA gene of the strain was amplified with the universal primers 27F and 1492R, and the amplified product was recovered and sequenced. , by determining the sequence of 16S rDNA for bacterial species identification. The obtained sequence results were compared by Blast at NCBI, and the recognized standard sequence data homologous to the 16S rDNA of the strain was obtained from the database of GenBank. The MEGA software was used to calculate the sequence similarity and the Neighbor-Joining algorithm was used as the system. Developmental Analysis.

2. Implement the result analysis

The strain of the present invention is identified by 16S rDNA full sequence determination, and its sequence is shown in SEQ ID NO: 1:

SEQ ID NO: 1:

ggccgggggggcgtgctatacatgcagtcgagcgaactgattagaagcttgcttctatgacgttagcggcggacgggtgagtaacacgtgggcaacctgcctgtaagactgggataacttcgggaaaccgaagctaataccggataggatcttctccttcatgggagatgattgaaagatggtttcggctatcacttacagatgggcccgcggtgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcatagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggctttcgggtcgtaaaactctgttgttagggaagaacaagtacgagagtaactgctcgtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggaattattgggcgtaaagcgcgcgcaggcggtttcttaagtctgatgtgaaagcccacggctcaaccgtggagggtcattggaaactggggaacttgagtgcagaagagaaaagcggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcggctttttggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagagggtttccgccctttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaactctagagatagagcgtt ccccttcgggggacagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcatttagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaaagggctgcaagaccgcgaggtcaagccaatcccataaaaccattctcagttcggattgtaggctgcaactcgcctacatgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtggagtaaccgtaaggagctagccgcctaaggtgacaggggg

The 16S rDNA of this strain was compared at NCBI using Blast, and it was found to be Bacillus sp. and was similar to the 16S rDNA sequence of the strain. The 16S rDNA phylogenetic tree was constructed as shown in Figure 4. aryabhattai) has the highest homology, and the strain described in the present invention can be determined to be Bacillus aryabhattai and named Bacillus aryabhattaiDT.

The descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, the present invention can also be appropriately changed and modified, and these changes and modifications also fall within the protection scope of the claims of the present invention.

sequence listing

<110> Zhejiang University

<120> Bacillus strain degrading aflatoxin B1 and its screening and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 1457

<212> DNA

<213> Bacillus aryabhattai

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ggccgggggg gcgtgctata catgcagtcg agcgaactga ttagaagctt gcttctatga 60

cgttagcggc ggacgggtga gtaacacgtg ggcaacctgc ctgtaagact gggataactt 120

cgggaaaccg aagctaatac cggataggat cttctccttc atgggagatg attgaaagat 180

ggtttcggct atcacttaca gatgggcccg cggtgcatta gctagttggt gaggtaacgg 240

ctcaccaagg caacgatgca tagccgacct gagagggtga tcggccacac tgggactgag 300

acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360

tgacggagca acgccgcgtg agtgatgaag gctttcgggt cgtaaaactc tgttgttagg 420

gaagaacaag tacgagagta actgctcgta ccttgacggt acctaaccag aaagccacgg 480

ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggaattattg 540

ggcgtaaagc gcgcgcaggc ggtttcttaa gtctgatgtg aaagcccacg gctcaaccgt 600

ggagggtcat tggaaactgg ggaacttgag tgcagaagag aaaagcggaa ttccacgtgt 660

agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcggct ttttggtctg 720

taactgacgc tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 780

acgccgtaaa cgatgagtgc taagtgttag agggtttccg ccctttagtg ctgcagctaa 840

cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 900

ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960

aggtcttgac atcctctgac aactctagag atagagcgtt ccccttcggg ggacagagtg 1020

acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080

gagcgcaacc cttgatctta gttgccagca tttagttggg cactctaagg tgactgccgg 1140

tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200

acacacgtgc tacaatggat ggtacaaagg gctgcaagac cgcgaggtca agccaatccc 1260

ataaaaccat tctcagttcg gattgtaggc tgcaactcgc ctacatgaag ctggaatcgc 1320

tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380

gtcacaccac gagagtttgt aacacccgaa gtcggtggag taaccgtaag gagctagccg 1440

cctaaggtga caggggg 1457

Claims (8)

1. a Bacillus strain for degrading aflatoxin B1, characterized in that the classification of the bacterial strain is named Bacillus aryabhattai DT, preservation unit: China General Microorganisms Collection Management Center (CGMCC) ), deposit number: CGMCC No.14949, deposit date is November 22, 2017. 2. a fermentation broth of the Bacillus strain of claim 1 degrading aflatoxin B1, is characterized in that its preparation method is as follows: bacterial strain described in claim 1 is inoculated into LB liquid medium or nutrient broth In the medium, after culturing for 18-24 h, a fermentation broth is obtained. 3. fermentation broth as claimed in claim 2, is characterized in that the component of described LB liquid medium is: 8-10 g/L peptone, 5-8 g/L yeast powder, 10-15 g/L Sodium chloride, pH 6.8-7.0. 4. fermentation broth as claimed in claim 2, is characterized in that described nutrient broth medium component is 8-10 g/L peptone, 3-5 g/L beef extract, 5-8 g/L chlorine Sodium chloride, pH 7.0-7.2. 5. fermentation broth as claimed in claim 2 is characterized in that the inoculation amount of described bacterial strain is calculated as 1-2% by volume percentage of medium. 6. A fermentation supernatant of a Bacillus strain that degrades aflatoxin B1, characterized in that the preparation method is as follows: centrifuging the fermentation culture liquid of claim 2 to obtain a fermentation supernatant. 7. A thalline dry powder of a Bacillus strain that degrades aflatoxin B1, characterized in that its preparation method is as follows: centrifuging the fermentation culture solution of claim 2, and precipitation drying to obtain a thalline dry powder. 8. a bacterial strain according to claim 1 is used for degrading the application of aflatoxin B1 in food or feedstuff, it is characterized in that, bacterial strain according to claim 1 is inoculated in LB liquid medium or nutrient broth medium, cultivated After 18-24 hours, the fermentation medium is obtained, or the fermentation medium is centrifuged to obtain the fermentation supernatant; or the precipitate after the centrifugation of the fermentation medium is dried to obtain the dry powder of bacteria; Add one or more of the fermentation broth, fermentation supernatant or dry bacterial powder to aflatoxin-containing food, feed or toxin-containing agricultural products, mix evenly, and continuously react at 30-40°C in the dark for 3- 4d.

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