CN108102971B

CN108102971B - Pseudomonas monteilii capable of resisting heat and degrading aflatoxin

Info

Publication number: CN108102971B
Application number: CN201810075511.8A
Authority: CN
Inventors: 王明清; 孙杰; 于丽娜; 毕洁; 张初署; 杨伟强; 石程仁; 江晨; 彭娅萍; 杨珍; 张玉凤; 焦坤; 许婷婷; 谢宏峰
Original assignee: Shandong Peanut Research Institute (peanut Engineering Technology Research Center Of Shandong Academy Of Agricultural Sciences)
Current assignee: Shandong Peanut Research Institute (peanut Engineering Technology Research Center Of Shandong Academy Of Agricultural Sciences)
Priority date: 2018-01-26
Filing date: 2018-01-26
Publication date: 2021-04-27
Anticipated expiration: 2038-01-26
Also published as: CN113016984A; CN108102971A; CN113016984B

Abstract

The invention provides a Pseudomonas monteilii (Pseudomonas monteilii) capable of resisting heat and efficiently degrading aflatoxin and application thereof in degrading aflatoxin and preparing products for degrading aflatoxin. The Pseudomonas mendocina (Pseudomonas monteilii) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 14485. The Pseudomonas menhadiensis (Pseudomonas monteilii) can be used for heat-resisting and efficiently degrading the aflatoxin. The strain serving as a biological material for efficient aflatoxin degradation has a good application prospect in the aspect of developing a new aflatoxin biodegradation microbial inoculum or a biodegradation sterile preparation, and particularly has a good application prospect in the feed or food processing industry needing high-temperature treatment.

Description

Pseudomonas monteilii capable of resisting heat and degrading aflatoxin

Technical Field

The invention relates to Pseudomonas monteilii (Pseudomonas monteilii) capable of resisting heat and degrading aflatoxin, belonging to the field of microorganisms and application thereof.

Background

Aflatoxin (Aflatoxin) is a toxic secondary metabolite produced by Aspergillus flavus (Aspergillus flavus), Aspergillus parasiticus (a. parasiticus) and Aspergillus wasabi (a. nomious), has strong toxicity and carcinogenicity, is much higher than cyanide, arsenide and pesticide, and is one of the main pathogens causing human liver cancer and nasopharyngeal cancer. In food and feed, the aflatoxin pollution phenomenon is very common, which causes serious economic loss to food industry and animal husbandry and poses great threat to human and animal health. Brazil peanut meal containing aflatoxin caused more than 10 million turkeys dead in the uk in 1960; the toxin causes up to 11 billion economic losses to the asian swine industry each year.

The basic structure of aflatoxin is difuran ring which is basic toxic structure and oxanellone which has toxicity enhancing and teratogenicity inducing effects. About 20 aflatoxins have been discovered, mainly classified into group B (aflatoxin B)₁And B₂) Group G (aflatoxins G)₁And G₂). Wherein the aflatoxin B₁Has the advantages of widest distribution, highest content and strongest toxicity.

The aflatoxin has stable property, and the toxicity of the aflatoxin cannot be removed by a common processing method. The method for removing aflatoxin at home and abroad mainly comprises physical methods such as ultraviolet irradiation, heat treatment, adsorbent detoxification and the like, and chemical methods such as strong acid, strong base, antioxidant treatment and the like, but the physical and chemical methods have the defects of high cost, large loss of nutrient components, low efficiency, generation of toxic substances in some methods, difficulty in large-scale treatment and the like, and the practical application requirements of high efficiency, safety and low cost are difficult to meet at the same time. The biological method is a method for degrading aflatoxin polluted in feed and food by using microorganisms such as bacteria, fungi and the like and metabolites thereof. The biological detoxification method has no pollution to raw materials, has high specificity, and avoids the regeneration of toxin, thereby being green and safeAnd a high-efficiency detoxification method become a research hotspot in recent years. Zhaoyueju et al, entitled "Pseudomonas aeruginosa strain and application thereof in degrading aflatoxin" (authorization notice No. CN103710292B), reported that a strain of aflatoxin B capable of degrading 82.84% at 37 ℃₁And 46.77% degraded aflatoxin B₂Pseudomonas aeruginosa. It can be seen that although the strain can degrade aflatoxin, the degradation temperature is strict, the strain can degrade aflatoxin only at 37 ℃, and no degradation effect is reported at the temperature exceeding 40-80 ℃. There are processing problems of elevated temperatures in practical food and feed processing applications. We isolated and found a strain which is obviously different from Pseudomonas aeruginosa, and found that the strain is Pseudomonas monteilii through phenotypic characteristics and 16S rRNA gene analysis. The pseudomonas menenginea capable of resisting heat and degrading aflatoxin can degrade 91.5 percent of aflatoxin B at 37 DEG C₁(ii) a Degrading 79.2 percent of aflatoxin B at the temperature of 30 DEG C₂(ii) a Can degrade 83.6 percent of aflatoxin B at the temperature of 80 DEG C₁Degrading 61.1% of aflatoxin B₂(ii) a After being treated at 100 ℃ for 20min, the temperature is reduced to 37 ℃, and 78.9 percent of aflatoxin B can be degraded₁Degrading 50.5% aflatoxin B₂. Compared with pseudomonas aeruginosa, pseudomonas montmorillonii has more efficient capability of degrading aflatoxin, and particularly can still efficiently degrade various aflatoxins at higher temperature. The pseudomonas mendii suspension and/or the fermentation liquor and/or the fermentation supernatant and/or the metabolite can endure high-temperature treatment, and are more suitable for degrading and removing aflatoxin polluted in agricultural product raw materials, feeds, foods, environmental samples and/or products needing high-temperature treatment.

Disclosure of Invention

The invention aims to provide a Pseudomonas menhadiensis (Pseudomonas monteilii) which can resist heat and degrade aflatoxin so as to overcome the defects that the existing strain for degrading aflatoxin has low degradation capability and the degradation temperature is limited to the temperature condition of less than 40 ℃. The pseudomonas menhadiensis (Ps eudomonas monteilii) disclosed by the invention has the advantages that the bacterial suspension and/or the fermentation liquid and/or the fermentation supernatant and/or the metabolite thereof can endure high-temperature treatment, the degradation efficiency is high, and the pseudomonas menhadiensis is more suitable for degrading and removing aflatoxin polluted in agricultural product raw materials, feeds, foods, environmental samples and/or products which need to be treated at high temperature.

The Pseudomonas monteilii provided by the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 14485.

A strain of Pseudomonas monteilii (Pseudomonas monteilii) phenotypically gram-negative, rod-like, polar flagella; the biochemical characteristics of the compound are that the compound can utilize Tween 40, Tween 80, L-arabinose, alpha-D-glucose, D-mannose, methyl pyruvate, aconitic acid, citric acid, D-gluconic acid, beta-hydroxybutyric acid, alpha-ketoglutaric acid, D, L-lactic acid, propionic acid, quinic acid, D-glucaric acid, succinic acid, D-alanine, L-alanylglycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-hydroxyproline, L-proline, L-pyroglutamic acid, L-serine, L-carnitine, gamma-aminobutyric acid, inosine, aminoethylaniline, putrescine and glycerol; the 16S rRNA gene sequence is characterized by SEQ ID No. 1.

The method for degrading the aflatoxin by using the Pseudomonas mendii comprises the steps of degrading the aflatoxin by using the Pseudomonas mendii at normal temperature and still having stronger degradation processing capacity by using the Pseudomonas mendii at higher temperature.

Preferably, the method for degrading the aflatoxin by using the Pseudomonas menhadiensis (Pseudomonas monteilii) is to mix a bacterial suspension and/or a fermentation liquid and/or a fermentation supernatant and/or a metabolite of the Pseudomonas menhadiensis with a sample and/or a product containing the aflatoxin at 30-80 ℃. Further, the aflatoxin degradation can be carried out at 37-80 ℃, and the mixing time is 40-72 h.

Preferably, the method for degrading the aflatoxin by using the Pseudomonas menhadenii is implemented, and the sample and/or product containing the aflatoxin is agricultural product raw materials, feed, food, environmental samples and/or products such as peanuts, corns and the like polluted by the aflatoxin.

Preferably, the method for degrading the aflatoxin by using the Pseudomonas mongolicus (Pseudomonas monteilii), wherein the aflatoxin is aflatoxin B in B-family aflatoxin₁Or aflatoxin B₂And/or aflatoxin G of the group G aflatoxins₁Or aflatoxin G₂。

Preferably, the bacterial suspension and/or fermentation liquor and/or fermentation supernatant and/or metabolite of the Pseudomonas menhadenii can be applied to preparing products for degrading aflatoxin.

Further, the bacterial suspension and/or fermentation liquor and/or fermentation supernatant and/or metabolite of a Pseudomonas menhadenii (Pseudomonas monteilii) can be applied to the preparation of a heat-resistant product for degrading aflatoxin.

Preferably, a bacterial suspension and/or fermentation broth and/or fermentation supernatant and/or metabolite of a strain of Pseudomonas monteilii (Pseudomonas monteilii) can be used for degrading aflatoxin.

Furthermore, a bacterial suspension and/or fermentation supernatant and/or metabolite of a strain of Pseudomonas monteilii (Pseudomonas monteilii) can be applied to the heat-resistant degradation of aflatoxin.

Preferably, the bacterial suspension and/or fermentation liquor and/or fermentation supernatant and/or metabolite of a strain of Pseudomonas menhadenii (Pseudomonas monteilii) are applied to the preparation of a product for degrading aflatoxin, a product for degrading heat-resistant aflatoxin, degrading aflatoxin and degrading aflatoxin in B-group aflatoxin₁Or aflatoxin B₂And/or aflatoxin G of the group G aflatoxins₁Or aflatoxin G₂。

The beneficial effects of the invention include:

1. the invention reports that Pseudomonas mendocina (Pseudomonas monteilii) can degrade various aflatoxins for the first time, including aflatoxin B in B-family aflatoxins₁Or aflatoxin B₂And/or yellow in group G aflatoxinsAspergillus toxin G₁Or aflatoxin G₂Before, no study about that the pseudomonas mendocina can degrade various aflatoxins exists, and the degradation efficiency of the pseudomonas mendocina for degrading the aflatoxins is obviously higher than that of other reported pseudomonas;

2. the Pseudomonas menhadiensis (Pseudomonas monteilii) can degrade various aflatoxins in a heat-resistant way, including aflatoxin B in B-family aflatoxins₁Or aflatoxin B₂And/or aflatoxin G of the group G aflatoxins₁Or aflatoxin G₂For AFB at 80 deg.C₁The degradation efficiency was 83.6%.

3. The Pseudomonas monteilii (Pseudomonas monteilii) has good application prospect in the aspects of developing new biodegradable bactericides and biodegradable sterile preparations, particularly heat-resistant biodegradable bactericides and biodegradable sterile preparations.

Drawings

FIG. 1: aflatoxin B at 37 deg.C₁Liquid chromatogram of degradation effect.

FIG. 2: aflatoxin B at 80 deg.C₁Liquid chromatogram of degradation effect.

FIG. 3: treating with 100 deg.C boiling water for 20min, and cooling to 37 deg.C₁Liquid chromatogram of degradation effect.

FIG. 4: aflatoxin B at 30 deg.C₂Liquid chromatogram of degradation effect.

FIG. 5: aflatoxin B at 80 deg.C₂Liquid chromatogram of degradation effect.

FIG. 6: treating with 100 deg.C boiling water for 20min, and cooling to 37 deg.C₂Liquid chromatogram of degradation effect.

FIG. 7: aflatoxin G at 37 deg.C₁Liquid chromatogram of degradation effect.

FIG. 8: aflatoxin G at 80 deg.C₁Liquid chromatogram of degradation effect.

FIG. 9: treating with 100 deg.C boiling water for 20min, cooling to 37 deg.C to yellowAspergillus toxin G₁Liquid chromatogram of degradation effect.

FIG. 10: aflatoxin G at 37 deg.C₂Liquid chromatogram of degradation effect.

FIG. 11: aflatoxin G at 80 deg.C₂Liquid chromatogram of degradation effect.

FIG. 12: treating with 100 deg.C boiling water for 20min, and cooling to 37 deg.C₂Liquid chromatogram of degradation effect.

Detailed Description

The experimental procedures used in the following examples are conventional unless otherwise specified.

Materials such as reagents used in the following examples are commercially available unless otherwise specified.

The quantification experiments in the following examples were repeated three times and the results were averaged.

PNG liquid medium: the solvent is water, and the solute is peptone, beef extract, glucose, sodium chloride and potassium dihydrogen phosphate; there were 10g peptone, 5g beef extract, 3g glucose, 10g sodium chloride and 1g potassium dihydrogen phosphate per liter PNG medium.

PNG solid medium: the PNG solid medium was prepared by adding 2% agar to the PNG liquid medium, i.e., 20g agar per liter of the PNG medium.

Example 1

Separation, purification and identification of bacteria

First, separation of bacteria

First, soil samples were collected from the peanut field of the leice, Qingdao in 2016, and 1g of soil was placed in 10ml of sterile distilled water in a super clean bench to prepare a soil suspension by shaking dilution, followed by dilution with a gradient of 100 times, 1000 times, and 10000 times with sterile distilled water.

And (II) coating the diluted soil suspension with different concentrations on a PNG medium solid plate, culturing for 36 hours at 37 ℃, enabling bacterial colonies to grow over the plate, selecting bacterial strains with different morphological characteristics, colors and sizes on the plate, carrying out plate streaking purification, carrying out 3 times of subculture purification, inoculating the purified bacterial strains to carry out an aflatoxin degradation test, and analyzing to obtain a strain with the highest degradation efficiency, wherein the strain is A3-1.

II, identification

The morphological characteristics and physiological and biochemical characteristics of the strain A3-1 were identified according to the method described in Bergey's Manual of identification of bacteria (eighth edition), and the specific results are as follows:

the shape and physiological and biochemical characteristics of the thallus: gram-negative; is rod-shaped; has polar flagella; aerobic bacteria; the salinity range for growth was 0-5% (w/v) NaCl, round and smooth clones on solid plates.

Biolog GN2 growth experiments showed that strain A3-1 can be prepared from Tween 40, Tween 80, L-arabinose, alpha-D-glucose, D-mannose, methyl pyruvate, aconitic acid, citric acid, D-gluconic acid, beta-hydroxybutyric acid, alpha-ketoglutaric acid, D, l-lactic acid, propionic acid, quinic acid, D-glucaric acid, succinic acid, D-alanine, L-alanylglycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-hydroxyproline, L-proline, L-pyroglutamic acid, L-serine, L-carnitine, gamma-aminobutyric acid, inosine, aminoethylaniline, putrescine and glycerol.

Gram-negative, rod-shaped, root-polar flagella, and the ability to utilize the phenotypic and biochemical properties of Tween 40 and Tween 80, etc., are consistent with the characteristics of Pseudomonas sp.

(II) 16S rRNA Gene analysis

Extracting total DNA of A3-1, performing PCR amplification by using a 16S rRNA gene universal primer to obtain an amplification product with the length of about 1486bp, connecting the amplification product to a pMD19-T vector, transforming a recombinant plasmid to escherichia coli, and sequencing to obtain a sequence shown as SEQ ID No. 1. Based on the comparison of the sequence homology of the standard strains in the database of EzTaxon-e server, the 16S rRNA gene of the strain A3-1 is compared with the NBRC 103158 of the standard strain Pseudomonas monteilii^TThe 16S rRNA gene homology of (A) was 100%, and gene analysis revealed that the bacterium was a bacterium belonging to the genus Pseudomonas monteilii (Pseudomonas monteilii).

Strain a3-1 was identified as Pseudomonas monteilii (Pseudomonas monteilii) based on morphological, physiological and biochemical characteristics and gene sequence characteristics. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 Xilu-Beijing, Chaoyang, national academy of sciences, Japan, and postal code 100101) in 2017, 8.4.8.4.4.A preservation number is CGMCC No. 14485.

Example 2

Culture of Pseudomonas monteilii A3-1

Inoculating Pseudomonas mendocina A3-1(Pseudomonas monteilii) into liquid PNG culture medium, and shake culturing at 37 deg.C and 150rpm for 70h to obtain bacterial liquid for aflatoxin degradation experiment.

Example 3

Degradation of aflatoxin B1 by pseudomonas menbergii A3-1 at 37 DEG C

A, aflatoxin B₁Is arranged in

Mixing 5mg aflatoxin B₁(AFB₁) Dissolving the standard substance in 100ml of chromatographic grade methanol to prepare AFB with the concentration of 50ppm₁The solution was stored. Taking 1ml of 50ppm AFB₁Adding 9ml of chromatographic grade methanol to prepare AFB with the concentration of 5000ppb₁And (4) working mother liquor.

II, Pseudomonas mengyuensis A3-1 to AFB₁Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb of AFB was added₁Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l 5000ppb of AFB were added to 1.96ml of non-inoculated medium₁The working stock solution was used as a control and was noted as a control solution.

Three, A3-1 pair AFB under the condition of 37 DEG C₁Analysis of the ability to degrade

Firstly, adding methanol into an experimental group solution or a control group solution for extraction respectively, then purifying and extracting residual toxins of samples extracted by the experimental group solution and the control group solution by using an immunoaffinity column, and finally detecting the samples obtained by purification and extraction by using HPLC provided with a photochemical derivatization column.

HPLC detection conditions were mobile phase methanol: water 1:1 (volume ratio); the flow rate is 0.8 ml/min; column C18(150 mm. times.4.6 mm, 0.5 μm); the excitation wavelength is 350nm, and the detection wavelength is 450 nm; the sample volume is 20 mul; the column temperature was 30 ℃.

AFB₁Percent (%) degradation (control AFB)₁content-Experimental group AFB₁Content)/control AFB₁The content is multiplied by 100 percent

The results are shown in FIG. 1 and Table 1. In fig. 1, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFB under the condition of 37 DEG C₁The degradation effect is better, and the degradation rate is 91.5%.

Example 4

Pseudomonas mendii A3-1 for AFB at 80 deg.C₁Degradation of

One, AFB₁Is arranged in

5mg of AFB₁Dissolving the standard substance in 100ml of chromatographic grade methanol to prepare AFB with the concentration of 50ppm₁The solution was stored. Taking 1ml of 50ppm AFB₁Adding 9ml of chromatographic grade methanol to prepare AFB with the concentration of 5000ppb₁And (4) working mother liquor. II, Pseudomonas mengyuensis A3-1 to AFB₁Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb of AFB was added₁Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 50h at 80 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l 5000ppb of AFB were added to 1.96ml of non-inoculated medium₁Working stock was incubated at 80 ℃ as a control and noted as control solution.

Three, A3-1 pairs of AFB under the condition of 80 DEG C₁Analysis of degradability

The results are shown in FIG. 2 and Table 1. In fig. 2, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFB under the condition of 80 DEG C₁The degradation effect is good, and the degradation rate is 83.6%.

Example 5

Treating Pseudomonas mendii A3-1 at 100 deg.C for 20min, and cooling to 37 deg.C for AFB₁Degradation of

One, AFB₁Is arranged in

Taking 1.96ml of the bacterial liquid obtained in the example 2, placing the bacterial liquid in a 10ml sample tube, heating the sample tube in a boiling water bath at the temperature of 100 ℃ for 20min, cooling the sample tube to 37 ℃, and adding 40 mu l of 5000ppb AFB₁Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l 5000ppb of AFB were added to 1.96ml of non-inoculated medium₁The working stock solution was used as a control and was noted as a control solution.

Thirdly, heating in 100 ℃ boiling water bath for 20min, and then cooling to 37 ℃, wherein A3-1 pairs of AFB₁Analysis of the ability to degrade

The results are shown in FIG. 3 and Table 1. In fig. 3, a: a control group; and B, experimental group. The results show that A3-1 pairs of AFB are obtained by heating in a boiling water bath at 100 ℃ for 20min, cooling to 37 DEG C₁The degradation rate was 78.9%.

Example 6

Pseudomonas mendii A3-1 was tested for aflatoxin B at 30 deg.C₂Degradation of

A, aflatoxin B₂Preparation of

1mg of aflatoxin B₂(AFB₂) Dissolving the standard substance in 20ml of chromatographic grade methanol to prepare AFB with the concentration of 50ppm₂The solution was stored. Taking 1ml of 50ppm AFB₂Adding 9ml of chromatographic grade methanol to prepare AFB with the concentration of 5000ppb₂And (4) working mother liquor.

II, Pseudomonas mengyuensis A3-1 to AFB₂Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb of AFB was added₂Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 30 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l 5000ppb of AFB were added to 1.96ml of non-inoculated medium₂The working stock solution was used as a control and was noted as a control solution.

Three, A3-1 pairs of AFB under the condition of 30 DEG C₁Analysis of degradability

AFB₂Percent (%) degradation (control AFB)₂content-Experimental group AFB₂Content)/control AFB₂The content is multiplied by 100 percent

The results are shown in FIG. 4 and Table 1. In fig. 4, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFB under the condition of 30 DEG C₂The degradation rate was 79.2%.

Example 7

Pseudomonas mendii A3-1 for AFB at 80 deg.C₂Degradation of

One, AFB₂Is arranged in

1mg of AFB₂Dissolving the standard substance in 20ml of chromatographic grade methanol to prepare AFB with the concentration of 50ppm₂The solution was stored. Taking 1ml of 50ppm AFB₂Adding 9ml of chromatographic grade methanol to prepare AFB with the concentration of 5000ppb₂And (4) working mother liquor. II, Pseudomonas mengyuensis A3-1 to AFB₂Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb of AFB was added₂Working mother liquor is turned over until the final concentration is 100ppb, and is incubated for 60h at 80 ℃ after being evenly mixed, and then is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l 5000ppb of AFB were added to 1.96ml of non-inoculated medium₂Working stock was incubated at 80 ℃ as a control and noted as control solution.

Thirdly, A3-1 is subjected to AFB under the condition of 80 DEG C₂Analysis of degradability

HPLC detection conditions were mobile phase methanol: water 1:1 (volume ratio); the flow rate is 0.8 ml/min; column C18(150 mm. times.4.6 mm, 0.5 μm); the excitation wavelength is 350nm, and the detection wavelength is 450 nm; the column temperature is 30 ℃; the amount of the sample was 20. mu.l.

The results are shown in FIG. 5 and Table 1. In fig. 5, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFB under the condition of 80 DEG C₂The degradation rate of (2) was 61.1%.

Example 8

Treating Pseudomonas mendii A3-1 at 100 deg.C for 20min, and cooling to 37 deg.C for AFB₂Degradation of

One, AFB₂Is arranged in

Taking 1.96ml of the bacterial liquid obtained in the example 2, placing the bacterial liquid in a 10ml sample tube, heating the sample tube in a boiling water bath at the temperature of 100 ℃ for 20min, cooling the sample tube to 37 ℃, and adding 40 mu l of 5000ppb AFB₂Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l 5000ppb of AFB were added to 1.96ml of non-inoculated medium₂The working stock solution was used as a control and was noted as a control solution.

Thirdly, heating in 100 ℃ boiling water bath for 20min, and then cooling to 37 ℃, wherein A3-1 pairs of AFB₂Analysis of the ability to degrade

AFB₂Degradation ofPercent (%) as (control group AFB)₁content-Experimental group AFB₂Content)/control AFB₁The content is multiplied by 100 percent

The results are shown in FIG. 6 and Table 1. In fig. 6, a: a control group; and B, experimental group. The results show that A3-1 pairs of AFB are obtained by heating in a boiling water bath at 100 ℃ for 20min, cooling to 37 DEG C₂The degradation rate was 50.5%.

Example 9

Pseudomonas mendii A3-1 was tested for aflatoxin G at 37 deg.C₁Degradation of

A, aflatoxin G₁Is arranged in

1mg of aflatoxin G₁(AFG₁) Dissolving the standard substance in 20ml of chromatographic grade methanol to prepare AFG with the concentration of 50ppm₁The solution was stored. Taking 1ml of 50ppm AFG₁Adding 9ml of chromatographic grade methanol to prepare AFG with the concentration of 5000ppb₁And (4) working mother liquor.

II, Pseudomonas mengypti A3-1 vs AFG₁Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb AFG was added₁Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l of 5000ppb AFG were added to 1.96ml of non-inoculated medium₁The working stock solution was used as a control and was noted as a control solution.

Three, A3-1 pairs of AFG at 37 DEG C₁Analysis of degradability

AFG₁Percent (%) degradation(control group AFG₁content-Experimental group AFG₁Content)/control AFG₁The content is multiplied by 100 percent

The results are shown in FIG. 7 and Table 1. In fig. 7, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFG at 37 DEG C₁Has better degradation effect, and the degradation rate is 98.4 percent.

Example 10

Pseudomonas mendii A3-1 at 80 ℃ to AFG₁Degradation of

One, AFG₁Is arranged in

A stock solution of aflatoxin B2 at a concentration of 50ppm was prepared by dissolving 1mg of AFG1 standard in 20ml of chromatographic grade methanol. Taking 1ml of 50ppm AFG₁Adding 9ml of chromatographic grade methanol to prepare AFG with the concentration of 5000ppb₁And (4) working mother liquor.

II, Pseudomonas mengypti A3-1 vs AFG₁Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb AFG was added₁Working mother liquor is turned over until the final concentration is 100ppb, and is incubated for 60h at 80 ℃ after being evenly mixed, and then is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l of 5000ppb AFG were added to 1.96ml of non-inoculated medium₁Working stock was incubated at 80 ℃ as a control and noted as control solution.

Three, A3-1 pairs of AFG at 80 DEG C₁Analysis of degradability

AFG₁Percent (%) degradation (control AFG)₁content-Experimental group AFG₁Content)/control AFG₁The content is multiplied by 100 percent

The results are shown in FIG. 8 and Table 1. In fig. 8, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFG under the condition of 80 DEG C₁Has better degradation effect, and the degradation rate is 93.5 percent.

Example 11

Treating Pseudomonas mendii A3-1 at 100 deg.C for 20min, and cooling to 37 deg.C for AFG₁Degradation of

One, AFG₁Is arranged in

1mg of AFG₁The standard substance is dissolved in 20ml of chromatographic grade methanol to prepare a 50ppm aflatoxin B2 storage solution. Taking 1ml of 50ppm AFG₁Adding 9ml of chromatographic grade methanol to prepare AFG with the concentration of 5000ppb₁And (4) working mother liquor.

II, Pseudomonas mengypti A3-1 vs AFG₁Degradation of

Taking 1.96ml of the bacterial liquid obtained in the example 2, placing the bacterial liquid in a 10ml sample tube, heating the sample tube in a boiling water bath at the temperature of 100 ℃ for 20min, cooling the sample tube to 37 ℃, and adding 40 mu l of 5000ppb AFG₁Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l of 5000ppb AFG were added to 1.96ml of non-inoculated medium₁The working stock solution was used as a control and was noted as a control solution.

Thirdly, heating in 100 ℃ boiling water bath for 20min, and then cooling to 37 ℃, wherein A3-1 is used for AFG₁Analysis of the ability to degrade

AFG₁Percent (%) degradation (control AFG)₁content-Experimental group AFB₁Content)/control AFG₁The content is multiplied by 100 percent

The results are shown in FIG. 9 and Table 1. In fig. 9, a: a control group; and B, experimental group. The results show that A3-1 pairs of AFG are obtained by heating in a boiling water bath at 100 ℃ for 20min, cooling to 37 DEG C₁The degradation rate was 73.6%.

Example 12

Pseudomonas mendii A3-1 was tested for aflatoxin G at 37 deg.C₂Degradation of

A, aflatoxin G₂Is arranged in

1mg of aflatoxin G₂(AFG₂) Dissolving the standard substance in 20ml of chromatographic grade methanol to prepare AFG with the concentration of 50ppm₂The solution was stored. Taking 1ml of 50ppm AFG₂Adding 9ml of chromatographic grade methanol to prepare AFG with the concentration of 5000ppb₂And (4) working mother liquor.

II, Pseudomonas mengypti A3-1 vs AFG₂Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb AFG was added₂Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l of 5000ppb AFG were added to 1.96ml of non-inoculated medium₂The working stock solution was used as a control and was noted as a control solution.

Three, A3-1 pair AFG under the condition of 37 DEG C₂Analysis of degradability

AFG₂Percent (%) degradation (control AFG)₂Content-Experimental group AFG₂Content)/control AFG₂The content is multiplied by 100 percent

The results are shown in FIG. 10 and Table 1. In fig. 10, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFG at 37 DEG C₂Has better degradation effect, and the degradation rate is 99.2 percent.

Example 13

Pseudomonas mendii A3-1 for AFG at 80 deg.C₂Degradation of

One, AFG₂Is arranged in

1mg of AFG₂Dissolving the standard substance in 20ml of chromatographic grade methanol to prepare AFG with the concentration of 50ppm₂The solution was stored. Taking 1ml of 50ppm AFG₂Adding 9ml of chromatographic grade methanol to prepare AFG with the concentration of 5000ppb₂And (4) working mother liquor. II, Pseudomonas mengypti A3-1 vs AFG₂Degradation of

1.96ml of the bacterial suspension obtained in example 2 was placed in a 10ml sample tube, and 40. mu.l of 5000ppb AFG was added₂Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 80 ℃, and then centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l of 5000ppb AFG were added to 1.96ml of non-inoculated medium₂Working stock was incubated at 80 ℃ as a control and noted as control solution.

Three, A3-1 pairs of AFG at 80 DEG C₂Analysis of degradability

AFG₂Percent (%) degradation (control AFG)₂content-Experimental group AFG₂Content)/control AFG₂Content 100% results are shown in FIG. 11 and Table 1。

In fig. 11, a: a control group; and B, experimental group. The results show that A3-1 is corresponding to AFG under the condition of 80 DEG C₂Has better degradation effect, and the degradation rate is 93.3 percent.

Example 14

Treating Pseudomonas mendii A3-1 at 100 deg.C for 20min, and cooling to 37 deg.C for AFG₂Degradation of

One, AFG₂Is arranged in

Taking 1.96ml of the bacterial liquid obtained in the example 2, placing the bacterial liquid in a 10ml sample tube, heating the sample tube in a boiling water bath at the temperature of 100 ℃ for 20min, cooling the sample tube to 37 ℃, and adding 40 mu l of 5000ppb AFG₂Working mother liquor is turned over until the final concentration is 100ppb, the mixture is evenly mixed and incubated for 72h at 37 ℃, and then the mixture is centrifuged for 5min at 8000rpm to obtain supernatant which is recorded as an experimental group solution; 40. mu.l of 5000ppb AFG were added to 1.96ml of non-inoculated medium₂The working stock solution was used as a control and was noted as a control solution.

Thirdly, heating in 100 ℃ boiling water bath for 20min, and then cooling to 37 ℃, wherein A3-1 is used for AFG₂Analysis of the ability to degrade

The results are shown in FIG. 12 and Table 1. In fig. 12, a: a control group; and B, experimental group. The results show that A3-1 pairs of AFG are obtained by heating in a boiling water bath at 100 ℃ for 20min, cooling to 37 DEG C₂The degradation rate was 70.7%.

TABLE 1 degradation Effect of Pseudomonas monteilii A3-1 on aflatoxin

The corresponding pseudomonad A3-1 is corresponding to AFB under the condition of 30 DEG C₂The degradation rate of (c).

Sequence listing

<110> institute of peanut of Shandong province (research center of peanut engineering technology, academy of agricultural sciences of Shandong province)

<120> pseudomonas monteilii capable of resisting heat and efficiently degrading aflatoxin

<130> 2018

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1486

<212> DNA

<213> Pseudomonas monteilii (Pseudomonas monteilii)

<400> 1

atcctggctc agattgaacg ctgcggcagg cctaacacat gcaagtcgag cggatgacgg 60

gagcttgctc cttgattcag cggcggacgg gtgagtaatg cctaggaatc tgcctggtag 120

tgggggacaa cgtttcgaaa ggaacgctaa taccgcatac gtcctacggg agaaagcagg 180

ggaccttcgg gccttgcgct atcagatgag cctaggtcgg attagctagt tggtggggta 240

atggctcacc aaggcgacga tccgtaactg gtctgagagg atgatcagtc acactggaac 300

tgagacacgg tccagactcc tacgggaggc agcagtgggg aatattggac aatgggcgaa 360

agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa gcactttaag 420

ttgggaggaa gggcagtaag ttaatacctt gctgttttga cgttaccgac agaataagca 480

ccggctaact ctgtgccagc agccgcggta atacagaggg tgcaagcgtt aatcggaatt 540

actgggcgta aagcgcgcgt aggtggttcg ttaagttgga tgtgaaagcc ccgggctcaa 600

cctgggaact gcatccaaaa ctggcgagct agagtacggt agagggtggt ggaatttcct 660

gtgtagcggt gaaatgcgta gatataggaa ggaacaccag tggcgaaggc gaccacctgg 720

actgatactg acactgaggt gcgaaagcgt ggggagcaaa caggattaga taccctggta 780

gtccacgccg taaacgatgt caactagccg ttggaatcct tgagatttta gtggcgcagc 840

taacgcatta agttgaccgc ctggggagta cggccgcaag gttaaaactc aaatgaattg 900

acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 960

accaggcctt gacatgcaga gaactttcca gagatggatt ggtgccttcg ggaactctga 1020

cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgtaa 1080

cgagcgcaac ccttgtcctt agttaccagc acgtaatggt gggcactcta aggagactgc 1140

cggtgacaaa ccggaggaag gtggggatga cgtcaagtca tcatggccct tacggcctgg 1200

gctacacacg tgctacaatg gtcggtacag agggttgcca agccgcgagg tggagctaat 1260

ctcacaaaac cgatcgtagt ccggatcgca gtctgcaact cgactgcgtg aagtcggaat 1320

cgctagtaat cgcgaatcag aatgtcgcgg tgaatacgtt cccgggcctt gtacacaccg 1380

cccgtcacac catgggagtg ggttgcacca gaagtagcta gtctaacctt cgggaggacg 1440

gttaccacgg tgtgattcat gactggggtg aagtcgtaac aaggta 1486

Claims

1. Pseudomonas monteilii (a)Pseudomonas monteilii) It is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.14485。