CN113265354A - Streptomyces rochei capable of simultaneously degrading aflatoxin and vomitoxin and preparation thereof - Google Patents

Abstract

The invention discloses Streptomyces rochei and a preparation thereof for simultaneously degrading aflatoxin and vomitoxin, wherein the preservation number of the Streptomyces rochei is CGMCC NO.20894, and the preparation contains an extract of a culture of the Streptomyces rochei; the invention has the advantages that: the aflatoxin and the vomitoxin are simultaneously degraded, the degradation rate of the aflatoxin can reach more than 90 percent after 24 hours of reaction, and the degradation rate of the vomitoxin is more than 80 percent; the degradation efficiency is high, the action condition is mild, the concentrated product does not damage the nutrient components in the feed, and the extract can be used for biodegradation detoxification of the feed.

Description

Streptomyces rochei capable of simultaneously degrading aflatoxin and vomitoxin and preparation thereof

Technical Field

The invention relates to Streptomyces rochei N8729 for simultaneously degrading aflatoxin and vomitoxin, belonging to the field of agricultural biology.

Background

Mycotoxins are toxic secondary metabolites produced by fungi of the genera aspergillus, fusarium and penicillium during growth. Over 300 mycotoxins that have toxic effects on humans and animals have been reported, and common mycotoxins include Aflatoxin (AF), Zearalenone (ZEA), vomitoxin (DON), Fumonisin (FB), Ochratoxin (OT), T-2 toxin. In actual production, the situation that several mycotoxins are polluted simultaneously often occurs, and the prior art and products adopt a single microorganism to degrade one toxin. The search for microorganisms that can degrade two or more mycotoxins simultaneously is one of the methods to effectively address the hazards of mycotoxins.

Disclosure of Invention

The invention aims to provide streptomyces rochei and a preparation thereof, which can improve animal intestinal tracts and degrade aflatoxin and zearalenone, and provide technical support for solving mycotoxin pollution in feed and preparation raw materials thereof.

The technical scheme of the invention is as follows:

a Streptomyces rochei capable of degrading aflatoxin and vomitoxin simultaneously is named as Streptomyces rochei and Streptomyces rochei, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is deposited at the institute of microorganisms of China academy of sciences No. 3 of West Lu No. 1 of Beijing area facing the sun, with the preservation number of CGMCC NO. 20894.

The formula weight fraction of the liquid culture medium of the Streptomyces rochei is as follows: 10 per mill of corn flour, 10 per mill of peptone, 8 per mill of yeast powder and 0.2 per mill of magnesium sulfate monohydrate, and the culture conditions are as follows: maintaining pH at 6.6-6.8 at 32 deg.C, and performing aerobic fermentation to obtain Streptomyces rochei liquid culture; the formula weight fraction of the solid culture medium is as follows: 70% of fine bran, 3% of peptone, 5% of yeast powder and 22% of corn flour, wherein the water content is 35%, and the culture conditions are as follows: ventilating and fermenting at 32 ℃ to obtain the Streptomyces rochei solid culture.

A mycotoxin degrading agent contains an extract of the culture of the aforementioned Streptomyces rochei.

The preparation method of the extract comprises the following steps: culturing solid culture of Streptomyces rochei at 32 deg.C for 5 days, leaching with 30 deg.C warm water for 24 hr, adsorbing with carrier, and air drying at low temperature.

The carrier comprises one or more of wheat bran, corncob powder, yeast cell wall, corn starch, rice bran, glucose, zeolite powder, bentonite and montmorillonite.

The content of the extract in the preparation is 20-60% by weight.

The mass ratio of the Streptomyces rochei fermentation extract to the carrier is as follows: 20-60: 80-40.

The action objects of the preparation comprise feed, raw materials for preparing the feed and the like.

The gene sequence determination of the 16SrDNA of the Streptomyces rochei strain comprises the following steps:

GGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCGACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTGAGATTCGCTCCACCTCGCGGTATCGCAGCTCATTGTACCGGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTCCCGTGAGTCCCCAGCACCACAAGGGCCTGCTGGCAACACGGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGGGACCCTGTCTCCAGGGTTTTCCGGTGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCACTTAATGCGTTAGCTGCGGCACGGACAACGTGGAATGTTGCCCACACCTAGTGCCCACCGTTTACGGCGTGGACTACCAGGGTATGTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCGAACTCTAGCCTGCCCGTATCGACTGCAGACCCGGGGTTAAGCCCCGGGCTTTCACAACCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTTCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTGATAGGCCGCGGGCTCATCCTGCACCGCCGGAGCTTTCGAACCTCGCAGATGCCTGCGAGGATCAGTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGCAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCCCACCGAAGTGGTCATCGTTCGAC。

the invention has the beneficial effects that: the aflatoxin and the vomitoxin are simultaneously degraded, the degradation rate of the aflatoxin can reach more than 90 percent after 24 hours of reaction, and the degradation rate of the vomitoxin is more than 80 percent; the degradation efficiency is high, the action condition is mild, the concentrated product does not damage the nutrient components in the feed, and the extract can be used for biodegradation detoxification of the feed.

The invention is further illustrated by the following figures and examples.

Drawings

FIG. 1 is a hyphal pattern of Streptomyces rochei under a 1600X microscope in accordance with an embodiment of the present invention;

FIG. 2 is a spore subgraph of Streptomyces rochei under a 1600X microscope in the embodiment of the invention;

FIG. 3 shows a chromatogram of aflatoxin B1 content of a sample taken at 0 h;

FIG. 4 shows a chromatogram of aflatoxin B1 content in a sample after the strain of the invention is degraded for 24 hours;

FIG. 5 shows a chromatogram of vomitoxin content in 0h samples;

FIG. 6 shows a chromatogram of vomitoxin content in a sample after 24h degradation by the strain of the present invention;

FIG. 7 shows a chromatogram of aflatoxin B1 content of a 0h sample taken;

FIG. 8 shows the content chromatogram of aflatoxin B1 in a sample after the bacterial strain is degraded for 24 hours

FIG. 9 shows a chromatogram of vomitoxin content in 0h samples;

FIG. 10 shows a chromatogram of the content of vomitoxin in a sample after 24 hours of degradation by the strain of the present invention.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Example 1

A Streptomyces rochei capable of degrading aflatoxin and vomitoxin simultaneously comprises the following components in parts by weight in a liquid culture medium: 10 per mill of corn flour, 10 per mill of peptone, 8 per mill of yeast powder and 0.2 per mill of magnesium sulfate monohydrate, and the culture conditions are as follows: maintaining pH at 6.6-6.8 at 32 deg.C, and performing aerobic fermentation to obtain Streptomyces rochei liquid culture; the formula weight fraction of the solid culture medium is as follows: 70% of fine bran, 3% of peptone, 5% of yeast powder and 22% of corn flour, wherein the water content is 35%, and the culture conditions are as follows: ventilating and fermenting at 32 ℃ to obtain the Streptomyces rochei solid culture.

A mycotoxin degrading agent contains an extract of the culture of the aforementioned Streptomyces rochei.

The preparation method of the extract comprises the following steps: culturing solid culture of Streptomyces rochei at 32 deg.C for 5 days, leaching with 30 deg.C warm water for 24 hr, adsorbing with carrier, and air drying at low temperature.

The carrier comprises one or more of wheat bran, corncob powder, yeast cell wall, corn starch, rice bran, glucose, zeolite powder, bentonite and montmorillonite.

The action objects of the preparation comprise feed, raw materials for preparing the feed and the like.

The content of the extract in the biological preparation is 20-60% by weight.

The mass ratio of the Streptomyces rochei fermentation extract to the carrier is as follows: 20: 80 or 30: 70 or 40: 60 or 50: 60 or 60: 40.

the invention relates to Streptomyces rochei which is obtained by screening and separating a large amount of soil in corn fields.

Samples in the soil of the corn field are collected and diluted by 10 times for screening experiments. The diluted solution was spread on a PDA plate and cultured at 30 ℃ for 36 hr. For each single colony on the plate, transfer to a blank PDA plate to obtain pure culture strain. Culturing the purified strain in liquid potato culture medium for 36hr, and testing its ability to degrade aflatoxin and vomitoxin.

The toxin degradation detection method comprises the following steps:

example 2 degradation efficiency of aflatoxin and vomitoxin standards

Firstly, sample treatment:

the strain purified in example 1 was activated in a liquid potato medium for 36hr to obtain a culture solution of the strain of the present invention. Under the premise of aseptic operation, 50mL of the culture solution is accurately measured and placed in a 250mL triangular flask, and if the culture solution is a solid sample, 0.1g of solid material is added into 50mL of sterilized culture medium (the culture medium comprises 10 per mill of corn flour, 10 per mill of peptone, 8 per mill of yeast powder and 0.2 per mill of magnesium sulfate monohydrate). Then 1mL of aflatoxin B1 or 1mL of vomitoxin standard is added to ensure that the concentration of AFB1 in the final reaction system is 50 mug/L and the concentration of DON is 1000 mug/L, the mixture is uniformly mixed, and the mixture is cultured under the conditions of 30 ℃ and 200 rpm. Meanwhile, a blank control without adding any culture solution or solid material is made, and other treatments are the same.

1mL of the culture solution was sampled at 0h and 24h, and 4mL of chromatographic grade methanol was added and mixed well. Shaking and extracting in shaking table for 30 min. After extraction, centrifuging at 4000rpm for 5min, filtering with glass fiber filter paper, collecting filtrate, purifying with immunoaffinity column, and detecting with high performance liquid chromatography.

Secondly, detection:

1. and (3) a purification process:

5mL of filtrate passing through the glass fiber filter paper is taken, 20mL of PBS buffer solution is added, and the mixture is uniformly mixed and is to be detected.

A10 mL glass syringe was attached to the immunoaffinity column. And (3) removing the plug below the immunoaffinity column, removing the protective solution in the column, and accurately transferring 20mL of the diluted solution to be detected into an injector (which can be transferred for 2 times, and each time is 10 mL). An air pressure pump was connected to the syringe and the pressure was adjusted to allow the solution to pass through the immunoaffinity column at a flow rate of about 1mL/min until air entered the column. Adding 10mL double distilled water to wash for 2 times, adjusting the flow rate to be about 3mL/min until air enters the affinity column, and discarding all effluent liquid.

Finally, accurately adding 0.5 mL (total volume is 1 mL) of chromatographically pure methanol in 2 times, incubating for 2min each time,

the flow rate was around 1mL/min and the eluate was collected in a brown vial for HPLC analysis.

Wherein, DON eluent is collected in a test tube, dried by nitrogen at 40 ℃, and redissolved by 1mL of mobile phase for HPLC analysis.

2. Drawing a standard curve:

according to the use requirement, accurately sucking a certain amount of aflatoxin standard stock solution (B1100 ng/mL), diluting with chromatographic grade methanol, and respectively preparing standard working solutions with a series of concentrations: the concentration of AFB1 was 0. mu.g/L, 1. mu.g/L, 5. mu.g/L, 25. mu.g/L, and 50. mu.g/L, respectively. And (3) drawing a standard working curve by taking the concentration of the standard working solution of the aflatoxin as a horizontal coordinate and the peak area as a vertical coordinate, and quantifying the sample by using the standard curve.

3. And (3) detection:

detecting conditions of aflatoxin:

a chromatographic column: c18 column, 4.6mm × 150mm, 5 μm;

mobile phase: methanol: water =45: 55;

flow rate: 1 mL/min;

fluorescence detector wavelength: excitation wavelength λ ex 360 nm, emission wavelength λ em 440 nm (post-open-column derivatization pool);

sample introduction amount: 20 mu L of the solution;

aflatoxin B1 peak off time: 17-19 min.

Vomitoxin detection conditions:

a chromatographic column: c18 column, 4.6mm × 150mm, 5 μm;

mobile phase: acetonitrile: water = 1: 9;

flow rate: 1 mL/min;

column temperature: 35 ℃;

detection wavelength: λ =218 nm;

sample introduction amount: 20 μ L.

Peak time of vomitoxin: 11-12 min.

4. As a result:

the experimental results show that: according to high performance liquid chromatography detection data, the content of AFB1 in a 0h sample is 43.88 mu g/L, the content of AFB1 in a 24h sample is 0.97 mu g/L, so that the degradation rate of the strain disclosed by the invention to an AFB1 standard product is 97.79%, the content of DON in the 0h sample is 1116.87 mu g/L, and the content of DON in the 24h sample is 153.69 mu g/L, so that the degradation rate of the strain disclosed by the invention to the DON standard product is 86.24%.

Example 3 efficiency of degradation of feed Material and of aflatoxins and vomitoxin in feed

Firstly, sample treatment:

weighing 50g of crushed feed or feed raw materials in a 500mL triangular flask, adding 250mL of methanol, shaking and extracting for 2 hours by a shaking table, filtering by qualitative filter paper, and uniformly mixing the filtrate. The toxin content was determined by immunoaffinity column purification-high performance liquid chromatography.

Placing the above extract in a 250mL fat bottle, and spin-drying at 65 deg.C (the amount of extract to be spin-dried is determined based on the measured AFB1 and DON content in the sample, such that the concentration of AFB1 in the solution dissolved in methanol is about 2.5ppm and the concentration of DON is about 50ppm after spin-drying)

The strain purified in example 1 was activated in a liquid potato medium for 36hr to obtain a culture solution of the strain of the present invention. Under the premise of aseptic operation, 50mL of the culture solution is accurately measured and placed in a 250mL triangular flask, and if the culture solution is a solid sample, 0.1g of solid material is added into 50mL of sterilized culture medium (the culture medium comprises 10 per mill of corn flour, 10 per mill of peptone, 8 per mill of yeast powder and 0.2 per mill of magnesium sulfate monohydrate). Then 1mL of concentrated aflatoxin B1 or 1mL of concentrated vomitoxin was added to make the concentration of AFB1 in the final reaction system 50. mu.g/L and the concentration of DON 1000. mu.g/L, mixed well, and cultured at 30 ℃ and 200 rpm. Meanwhile, a blank control without adding any culture solution or solid material is made, and other treatments are the same.

1mL of the culture solution was sampled at 0h and 24h, and 4mL of chromatographic grade methanol was added and mixed well. Shaking and extracting in shaking table for 30 min. After extraction, centrifuging at 4000rpm for 5min, filtering with glass fiber filter paper, collecting filtrate, purifying with immunoaffinity column, and detecting with high performance liquid chromatography.

Secondly, detection:

1. and (3) a purification process:

5mL of filtrate passing through the glass fiber filter paper is taken, 20mL of PBS buffer solution is added, and the mixture is uniformly mixed and is to be detected.

A10 mL glass syringe was attached to the immunoaffinity column. And (3) removing the plug below the immunoaffinity column, removing the protective solution in the column, and accurately transferring 20mL of the diluted solution to be detected into an injector (which can be transferred for 2 times, and each time is 10 mL). An air pressure pump was connected to the syringe and the pressure was adjusted to allow the solution to pass through the immunoaffinity column at a flow rate of about 1mL/min until air entered the column. Adding 10mL double distilled water to wash for 2 times, adjusting the flow rate to be about 3mL/min until air enters the affinity column, and discarding all effluent liquid.

Finally, accurately adding 0.5 mL (total volume is 1 mL) of chromatographically pure methanol in 2 times, incubating for 2min each time,

the flow rate was around 1mL/min and the eluate was collected in a brown vial for HPLC analysis.

Wherein, DON eluent is collected in a test tube, dried by nitrogen at 40 ℃, and redissolved by 1mL of mobile phase for HPLC analysis.

2. Drawing a standard curve:

according to the use requirement, accurately sucking a certain amount of aflatoxin standard stock solution (B1100 ng/mL) and vomitoxin standard stock solution (DON 50 mug/mL), diluting with chromatographic grade methanol, and respectively preparing standard working solutions with a series of concentrations: the concentration of AFB1 is 0. mu.g/L, 1. mu.g/L, 5. mu.g/L, 25. mu.g/L and 50. mu.g/L respectively; the concentrations of DON were: and drawing a standard working curve by taking the concentration of the standard working solution as the abscissa and the peak area as the ordinate at 0. mu.g/L, 100. mu.g/L, 200. mu.g/L, 1000. mu.g/L and 5000. mu.g/L, and quantifying the sample by using the standard curve.

3. And (3) detection:

detecting conditions of aflatoxin:

a chromatographic column: c18 column, 4.6mm × 150mm, 5 μm;

mobile phase: methanol: water =45: 55;

flow rate: 1 mL/min;

fluorescence detector wavelength: excitation wavelength λ ex 360 nm, emission wavelength λ em 440 nm (post-open-column derivatization pool);

sample introduction amount: 20 mu L of the solution;

aflatoxin B1 peak off time: 17-19 min.

Vomitoxin detection conditions:

a chromatographic column: c18 column, 4.6mm × 150mm, 5 μm;

mobile phase: acetonitrile: water = 1: 9;

flow rate: 1 mL/min;

column temperature: 35 ℃;

detection wavelength: λ =218 nm;

sample introduction amount: 20 μ L.

Peak time of vomitoxin: 11-12 min.

4. As a result:

the experimental results show that: according to high-performance liquid chromatography detection data, the content of AFB1 in a 0h sample is 73.95 mu g/L, the content of AFB1 in a 24h sample is 2.27 mu g/L, the degradation rate of the strain to AFB1 in the feed or feed raw materials is 96.93%, the content of DON in the 0h sample is 1061.72, and the content of DON in the 24h sample is 274.42 mu g/L, so the degradation rate of the strain to DON in the feed or feed raw materials is 74.15%.

Sequence listing

<110> science and technology development Co., Ltd

<120> Streptomyces rochei capable of simultaneously degrading aflatoxin and vomitoxin and preparation thereof

<141> 2021-05-19

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1370

<212> DNA

<213> Streptomyces rochei (Streptomyces rochei)

<400> 1

gggttgggcc accggcttcg ggtgttaccg actttcgtga cgtgacgggc ggtgtgtaca 60

aggcccggga acgtattcac cgcagcaatg ctgatctgcg attactagcg actccgactt 120

catggggtcg agttgcagac cccaatccga actgagaccg gctttttgag attcgctcca 180

cctcgcggta tcgcagctca ttgtaccggc cattgtagca cgtgtgcagc ccaagacata 240

aggggcatga tgacttgacg tcgtccccac cttcctccga gttgaccccg gcggtctccc 300

gtgagtcccc agcaccacaa gggcctgctg gcaacacggg acaagggttg cgctcgttgc 360

gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcacca cctgtacacc 420

gaccacaagg gggaccctgt ctccagggtt ttccggtgta tgtcaagcct tggtaaggtt 480

cttcgcgttg cgtcgaatta agccacatgc tccgccgctt gtgcgggccc ccgtcaattc 540

ctttgagttt tagccttgcg gccgtactcc ccaggcgggg cacttaatgc gttagctgcg 600

gcacggacaa cgtggaatgt tgcccacacc tagtgcccac cgtttacggc gtggactacc 660

agggtatgta atcctgttcg ctccccacgc tttcgctcct cagcgtcagt atcggcccag 720

agatccgcct tcgccaccgg tgttcctcct gatatctgcg catttcaccg ctacaccagg 780

aattccgatc tcccctaccg aactctagcc tgcccgtatc gactgcagac ccggggttaa 840

gccccgggct ttcacaaccg acgtgacaag ccgcctacga gctctttacg cccaataatt 900

ccggacaacg cttgcgccct acgtattacc gcggctgctg gcacgtagtt agccggcgct 960

tcttctgcag gtaccgtcac tttcgcttct tccctgctga aagaggttta caacccgaag 1020

gccgtcatcc ctcacgcggc gtcgctgcat caggctttcg cccattgtgc aatattcccc 1080

actgctgcct cccgtaggag tctgggccgt gtctcagtcc cagtgtggcc ggtcgccctc 1140

tcaggccggc tacccgtcgt cgccttggtg agccgttacc tcaccaacta gctgataggc 1200

cgcgggctca tcctgcaccg ccggagcttt cgaacctcgc agatgcctgc gaggatcagt 1260

atccggtatt agaccccgtt tccagggctt gtcccagagt gcagggcaga ttgcccacgt 1320

gttactcacc cgttcgccac taatccccac cgaagtggtc atcgttcgac 1370

Claims (7)

1. Streptomyces rochei capable of simultaneously degrading aflatoxin and vomitoxin is characterized in that: the preservation number is CGMCC NO. 20894.

2. The streptomyces rochei for simultaneously degrading aflatoxins and vomitoxin of claim 1, which is characterized in that: the formula weight fraction of the liquid culture medium of the Streptomyces rochei is as follows: 10 per mill of corn flour, 10 per mill of peptone, 8 per mill of yeast powder and 0.2 per mill of magnesium sulfate monohydrate, and the culture conditions are as follows: maintaining pH at 6.6-6.8 at 32 deg.C, and performing aerobic fermentation to obtain Streptomyces rochei liquid culture; the formula weight fraction of the solid culture medium is as follows: 70% of fine bran, 3% of peptone, 5% of yeast powder and 22% of corn flour, wherein the water content is 35%, and the culture conditions are as follows: ventilating and fermenting at 32 ℃ to obtain the Streptomyces rochei solid culture.

3. A mycotoxin degrading formulation characterized by: the preparation contains an extract of a culture of Streptomyces rochei as claimed in claim 1 or 2.

4. A mycotoxin degrading formulation as defined in claim 3, wherein: the preparation method of the extract comprises the following steps: culturing solid culture of Streptomyces rochei at 32 deg.C for 5 days, leaching with 30 deg.C warm water for 24 hr, adsorbing with carrier, and air drying at low temperature.

5. A mycotoxin degrading formulation as defined in claim 4, wherein: the carrier comprises one or more of wheat bran, corncob powder, yeast cell wall, corn starch, rice bran, glucose, zeolite powder, bentonite and montmorillonite.

6. A mycotoxin degrading formulation as defined in claim 3, wherein: the content of the extract in the preparation is 20-60% by weight.

7. A mycotoxin degrading formulation as defined in claim 4, wherein: the mass ratio of the Streptomyces rochei fermentation extract to the carrier is as follows: 20: 80 or 30: 70 or 40: 60 or 50: 60 or 60: 40.

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