CN115944046A - Application of armillaria mellea in degrading vomitoxin - Google Patents

Abstract

The invention discloses an application of armillaria mellea in degrading vomitoxin; the invention also discloses armillaria mellea Am-07-22 with a preservation number of CCTCC NO: m20221044; the mycotoxin is vomitoxin or zearalenone. The strain Am-07-22 has the best degradation effect when the concentration of vomitoxin is 2 mu g/mL, and the degradation rate is 64.05 percent; the armillaria mellea has good degradation effect on vomitoxin in two different fermentation raw materials when the corn bran is subjected to a solid fermentation mode, and the corn gluten is subjected to a liquid fermentation mode.

Description

Application of armillaria mellea in degrading vomitoxin

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to application of armillaria mellea in degrading vomitoxin.

Background

Mycotoxins are present in contaminated food and feed, invade into human and livestock from food chains and accumulate, thus causing accumulation, and posing great threat to health. The trichothecene toxins are the most common polluting mycotoxins in food, and comprise zearalenone, fumonisins, vomitoxin and the like. The main component of vomitoxin is Deoxynivalenol (DON), the chemical name is 3 alpha, 7 alpha, 15-trihydroxy-12,13-epoxy trichothecene-9-anthracene-8-ketone, which belongs to trichothecene compounds. DON was first discovered in 1970 in Japanese moldy corn and wheat and was named in 1973 because it causes sow food refusal and emetic reaction. Colorless needle-shaped crystal at normal temperature, can be dissolved in water and solvents such as methanol, ethyl acetate, acetone and the like, and has strong heat resistance, high pressure resistance and acid and alkali resistance. Zearalenone (ZEN) is a nonsteroidal estrogen mycotoxin, also known as F-2 toxin, a secondary metabolite produced mainly by fusarium fungi, and is mainly present in contaminated corn, wheat, rice, soybean and other grains. ZEN can pollute grains, food and feed through various ways, seriously threatens human health and life safety, has similar characteristics with a plurality of estrogen types in chemical structure, can activate estrogen receptors, and causes reproductive disorders such as abortion, stillbirth, abnormal fetus and the like of farm animals. In addition, ZEN can enter human body through food chain to cause harm such as immune injury, liver injury, genetic toxicity, cancer induction and the like.

The results of data survey in 2020 showed DON, ZEN and AFB in corn and its processing by-products ₁ The overproof of three mycotoxins is serious, the pollution of the toxin of the corn by-product is the most serious, and the DON overproof rate is 53.57 percent in the corn by-product. At present, the DON or zearalenone degradation and detoxification technology is divided into a physical method, a chemical method and a biological method. The physical methods are mainly by heat treatment, ultraviolet irradiation or gamma ray irradiation, adsorbent adsorption, and the like. Chemical methods include ammoniation; an alkaline process; carrying out ozone treatment; oxygen water treatment, etc. But the physicochemical method has the limitations of low degradation efficiency, damage to nutrient components, introduction of other chemical substances and other adverse factors, and the like. The biological method has the advantages of mild process and reaction conditions, high efficiency, less damage to the flavor and the nutritive value of the raw materials and the like, and partial microbial fermentation products can improve the flavor and increase the nutritive value. At present, the action mechanism of biological detoxification is that the content of toxin is reduced by utilizing the biological adsorption of microorganisms, for example, lactobacillus, saccharomycetes and the like are utilized to adsorb mycotoxin through beta-glucan on cell walls; secondly, biodegradation comprises the growth and secretion of metabolites by microorganisms and the degradation of toxins by enzyme preparations.

Studies have shown that zearalenone biodegradation is mainly from the following two categories: microbial degradation and enzymatic degradation. To date, numerous microorganisms capable of depriving zearalenone have been widely reported. The ZEN degrading bacteria and fungi are mostly concentrated in lactic acid bacteria, bacillus, aspergillus, yeast and the like. The isolation of Lactobacillus plantarum Lp22, lp39, lp4 from traditional fermented food by Zhao et al degraded ZEN in the solution by 47.80%, 38.06% and 39.50%, respectively. Xu et al found that Bacillus amyloliquefaciens ZDS-1 was effective in degrading ZEN in the concentration range of 1mg/L to 100mg/L, and that ZDS-1 was able to degrade ZEN not only in the medium but also in wheat. Pereyra et al tested 11 Bacillus species that have demonstrated the ability to degrade ZEN by AFB 1-degrading bacteria, and found that the tested strains were able to degrade ZEN at a concentration of 400ng/mL after 72 hours, with a degradation rate of 96.9%. In addition, food grade Aspergillus niger FS10 isolated from fermented soybeans by Sun et al was effective in removing ZEN in PDB medium, and mycelium and culture filtrate were also reduced by 43.10% and 68.16% ZEN.

Disclosure of Invention

The invention aims to solve the problems and provides the application of armillaria mellea in degrading vomitoxin.

Application of Armillaria mellea in degrading vomitoxin is provided.

Armillaria mellea Am-07-22 with the preservation number of CCTCC NO: m20221044.

A method for degrading vomitoxin comprises inoculating Armillaria mellea into testa Maydis or corn yellow powder, and fermenting;

the corn bran is subjected to solid state fermentation;

the maize yellow powder is fermented in a liquid state;

the invention provides an application of armillaria mellea in degrading vomitoxin; the invention also provides armillaria mellea Am-07-22 with a preservation number of CCTCC NO: m20221044; the mycotoxin is vomitoxin or zearalenone. The strain Am-07-22 has the best degradation effect when the DON concentration is 2 mug/mL, and the degradation rate is 64.05%; the armillaria mellea has good degradation effect on vomitoxin in two different fermentation raw materials when the corn bran is subjected to a solid fermentation mode, and the corn gluten is subjected to a liquid fermentation mode.

Drawings

FIG. 1 degradation effect of different edible fungi on DON (different letters represent significant difference)p<0.05）；

FIG. 2 is a graph of response surface of interaction of factors on DON degradation rate;

FIG. 3 is a graph showing the effect of different DON concentrations on the degradation effect of Armillaria mellea (different letters represent significant differences)p<0.05）；

FIG. 4 Effect of different fractions of Armillaria mellea on DON degradation Rate (different letters represent significant differences)p<0.05）

FIG. 5 Effect of different treatments on DON degradation Rate (different)Significant differences in letter representationp<0.05）

FIG. 6 is the effect of different culturing times of Armillaria mellea on the DON degradation effect;

FIG. 7 shows the results of a manganese peroxidase coloration reaction test;

FIG. 8 shows the change rule of DON degradation rate and Mnp enzyme activity at different cultivation times;

FIG. 9 is a liquid chromatogram of ZEN degradation by the strain Am-07-22;

FIG. 10 Effect of initial concentration of ZEN on degradation of ZEN by Am-07-22; A. the degradation rate; B. the amount of degradation;

FIG. 11 is graph showing the effect of different conditions on the degradation rate of Am-07-22 for degrading ZEN; A. time; B. (ii) temperature; C. the pH value; D. inoculating quantity;

FIG. 12 degradation rate of ZEN by different components of strain Am-07-22;

FIG. 13 shows the ZEN degradation rate of fermentation supernatant of the strain Am-07-22 after different treatments;

FIG. 14 is the effect of different conditions on the degradation rate of ZEN by Am-07-22 fermentation supernatant; A. different fermentation times; B. different pH values; C. different metal ions;

FIG. 15 is a graph showing the effect of Armillaria mellea fermentation corn bran and corn gluten meal on DON degradation rate (significant differences exist between different capital letters and between different lower case letters, p is less than 0.05);

FIG. 16 influence of Armillaria mellea solid state fermentation corn husks with different DON concentrations on DON degradation rate (different letters indicate significant difference p < 0.05);

FIG. 17 influence of liquid fermentation of Ecklonia mellea with different DON concentrations of zeaxanthin on the DON degradation rate (different letters indicate significant difference p < 0.05).

Detailed Description

Armillaria mellea Am-07-22 (C)Armillaria mellea) The preservation number is CCTCC NO of M20221044; 2) The strains are screened out with better indexes (cellulose biodegradation) according to the early-stage test results of research teams, and morchella Me-01 (Morchella esculenta) Hericium erinaceus He-02-06: (Hericiumerinaceus) And Armillaria mellea Am-07-22 (Armillaria mellea) Prepared from wheat and jadeThe rice deep processing is provided by the national engineering center; the three kinds of bacteria are separated from the fruiting body of the strain collected in Changbai mountain, morchella Me-01 is separated from the fruiting body of Morchella esculenta, hericium erinaceus He-02-06 is obtained by separating and mutagenizing the fruiting body of Hericium erinaceus, and Armillaria mellea Am-07-22 is separated from the fruiting body of Armillaria mellea; the morchella Me-01 is disclosed in research on morchella solid-state fermented corn protein powder reported by Tong Weina and the like; hericium erinaceus He-02-06 is preserved in China center for type culture Collection at 7 months and 6 days in 2022, and the preservation number of the strain is CCTCC NO: M20221043; armillaria mellea Am-07-22 is preserved in China center for type culture Collection at 7/6/2022, with the preservation number of CCTCC NO: M20221044.

Example 1 screening and identification of Armillaria mellea Am-07-22

1. Mutagenesis method of strain

Armillaria mellea Am-07 (a laboratory-preserved)Armillaria mellea) Respectively placing protoplast suspension of strain to be mutagenized on a flat plate, placing a rotor in the flat plate, stirring, placing under a 15W ultraviolet lamp for 30cm, and performing ultraviolet mutagenesis with irradiation dosage of 20-90 s. Coating the strain suspension subjected to mutagenesis treatment on a regeneration culture medium, culturing for 10 days in a constant-temperature incubator at 27 ℃ in a dark place, counting grown colonies, drawing a lethality curve, selecting a strain with good growth as a mutant strain, culturing under a proper condition, selecting a mutagenized strain with the soluble dietary fiber content of more than 15% of that of a starting strain as a positive mutant strain, carrying out continuous passage for 10 times, carrying out fermentation test at alternate generations, selecting the mutant strain with stable production performance of the mutagenized strain, carrying out sequencing, identifying the strain, and storing in a refrigerator at 4 ℃.

2. Comparison of mutagenesis Effect

The Armillaria mellea Am-07-22 strain obtained after ultraviolet mutagenesis treatment is subjected to solid state fermentation, and the influence on the content of soluble dietary fiber in ginseng residue is shown in figure 1; after the ginseng residue is subjected to solid state fermentation by using the mutant strain, the content of soluble dietary fiber in the ginseng residue is improved, and the hydration characteristics such as water holding capacity, oil holding capacity, water expansion capacity and the like of insoluble dietary fiber in the ginseng residue are also improved, as shown in figure 2.

3. Am-07-22 strain sequencing result

And (3) conveying the separated and purified bacterial liquid to Jilin province U.S. Biotechnology limited company for detection, extracting DNA genome of the bacterial liquid by the company, amplifying ribosomal DNA and ITS sequences as shown in the following table to obtain a PCR product, and finally sequencing.

The DNA of the bacteria liquid sent to be detected is amplified by using ITS4 and ITS5 fungus universal primers, and is successfully spliced into a 623bp connecting fragment, and the splicing result is shown in a sequence table SEQ ID NO.1; drawing a tree building map of the strain by using MEGA7.1 software according to the detected sequence, and showing the tree building map in a figure 3; the figure shows that the Am-07-22 strain has high sequence similarity with Armillaria sp, and the strain is identified as Armillaria mellea and named as Armillaria mellea Am-07-22. Armillaria mellea Am-07-22 is preserved in China Center for Type Culture Collection (CCTCC) at 2022, 7 months and 6 days, and the preservation number of the strain is M20221044.

EXAMPLE 2 Strain culture

1. Strain activation

Transferring the Me-01, he-02-06 and Am-07-22 strains to a slant culture medium respectively, and culturing at 27 deg.C for 12d.

Preparing a strain liquid activation culture medium:

1) Am-07 liquid seed medium: potato 100 g, silkworm chrysalis powder 2.5 g, glucose 5 g, sucrose 5 g, yeast extract powder 10 g, potassium dihydrogen phosphate 0.75 g, magnesium sulfate heptahydrate 0.375 g, vitamin B ₁ 0.005 g, sterilizing at 121 ℃ for 20min, wherein the pH value is natural;

2) Me-01 liquid seed Medium: glucose 10 g, yeast extract powder 5 g, monopotassium phosphate 0.6 g, magnesium sulfate heptahydrate 0.375 g, iron sulfate heptahydrate 0.005 g, natural pH value, sterilization at 121 ℃ for 20 min;

3) He-02 liquid seed Medium: 10 g of glucose, 5 g of yeast extract powder, 10 g of soluble starch, 1.5 g of monopotassium phosphate and 0.3 g of magnesium sulfate heptahydrate, wherein the pH value is natural, and the sterilization is carried out for 20min at 121 ℃. All calculated as 500 mL.

2. Preparation of fermented seed liquid

Preparing a first-stage fermentation seed: in a sterile environment, taking 8 thalli from the slant culture medium of the three activated thalli, inoculating the thalli into a 100 mL triangular flask filled with 30 mL liquid activated culture medium, carrying out shaking table shaking culture at the constant temperature of 27 ℃, and culturing 6d at the constant temperature of 160 r/min to obtain a first-stage fermentation seed;

preparing a secondary fermentation seed solution: breaking the primary fermentation seeds, inoculating the strains into a 500mL triangular flask filled with 200 mL liquid activation culture medium by 8 percent of addition amount, performing shaking table shaking culture at the constant temperature of 27 ℃, and culturing 6d at the constant temperature of 160 r/min to obtain secondary fermentation seed liquid.

Example 3 experiment of degrading emetic toxin by bacterial species

1. Screening of fungi for degrading vomitoxin

1. Degradation of DON by bacterial strain

And transferring the liquid fermentation seed liquid which is full of the uniform bacteria balls and has good growth condition into a basic screening culture medium, and respectively adding DON standard working solution into the basic screening culture medium to ensure that the DON content in the basic screening culture medium is 2 mu g/mL, wherein a control group is the basic screening culture medium which is not inoculated with the bacteria. Each set of three replicates was incubated at 27 ℃ for 7 days at 160rpm in the dark.

The basic screening culture medium: 100 g of potato, 2.5 g of silkworm chrysalis powder, 5 g of glucose, 5 g of cane sugar, 10 g of yeast extract powder, 0.75 g of potassium dihydrogen phosphate, 0.375 g of magnesium sulfate heptahydrate and 0.005 g of vitamin B, wherein the pH value is natural in 500mL, and the potato is sterilized at 121 ℃ for 20min

2. And (4) extracting and detecting DON.

Taking the cultured fermentation liquor, centrifuging at 12000 rpm for 10min, and keeping the temperature at 4 ℃. After 100-fold dilution, the supernatant was filtered through a disposable sterile filtration membrane and placed in a 1.5 mL EP tube and stored at 4 ℃ for testing. The sample solution to be detected is operated according to the method provided by the vomitoxin detection kit, and the OD value of the test hole is measured at the position of the dual-wavelength 450 nm by using an enzyme-labeling instrument.

3. DON degradation Rate calculation

DON residual content in the fermentation broth was calculated by Ridasoft. Win software (4-Parameter). The degradation effect of the strain on DON is expressed by degradation rate. The DON degradation rate is calculated according to the following formula:

4. detection steps of vomitoxin ELISA detection kit

Taking out the required reagents and the required number of micro-porous plates from the environment of 4 ℃, placing the micro-porous plates at room temperature (20-25 ℃) for balancing for more than 30 min, and paying attention to the fact that each liquid reagent needs to be shaken up for use.

Washing solution: the concentrated washing solution (10X) was diluted with ultrapure water at a volume ratio of 1:9.

Adding a sample: adding 50 mu L/hole of vomitoxin enzyme label into the sample, adding 50 mu L/hole of vomitoxin anti-reagent, lightly shaking, mixing, covering with a cover film, keeping out of the sun, and reacting at 25 deg.C for 30 min.

Washing the plate: carefully uncovering the cover plate film, spin-drying the liquid in the holes, fully washing the holes for 4-5 times at intervals of 10 s by 250 mu L of washing liquid, and patting the holes dry by using absorbent paper.

Color development: adding 50 mu L/hole of substrate solution A and 50 mu L/hole of substrate solution B, gently shaking and mixing, and reacting at 25 ℃ in a dark environment for 15 min.

And (3) determination: adding 50 mu L of stop solution into each hole, gently shaking and uniformly mixing, and setting an enzyme-labeling instrument to measure the OD value of each hole at the position of 450 nm.

5. As a result, the

FIG. 1 shows the DON degradation results of three edible fungi in the basic screening culture medium. It can be seen from the figure that the three edible fungi of Me-01, he-02 and Am-07-22 have degradation effects on DON added into the culture medium after fermentation culture, and the degradation rates of the three edible fungi on DON degradation are respectively 11.22%, 32.57% and 51.76%, wherein the degradation capability of the strain Am-07-22 on DON is obviously higher than that of Me-01 and He-02, so that the strain Am-07-22 is selected as the degrading bacteria of the test for subsequent research.

2. Optimization of degradation process conditions

The optimal degrading strain Am-07-22 obtained by early screening is subjected to single factor test to determine the optimal process conditions for degrading vomitoxin by the fungus strain, and the process comprises the following steps: inoculation amount, initial pH value of a culture medium, culture temperature and culture time. After screening by each single-factor test, a preliminary test basis is provided for a response surface optimization test.

On the basis of a single-factor test, three factors of the inoculation amount, the initial pH value of a culture medium and the culture temperature are finally selected as independent variables, the DON degradation rate is used as a response value, a three-factor three-level response surface test is designed, the test design and results are shown in table 1, and the regression model variance analysis is shown in table 2.

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Regression analysis is carried out on the data through Design-Expert software, three factors of A inoculation amount, B initial pH value and C culture temperature are used as independent variables, and the degradation rate (Y) of the strain Am-07 for degrading the vomitoxin is used as a response value, so that a quadratic polynomial regression equation of a quadratic regression model is obtained as follows: y =64.65+5.98A +11.33B +3.04C-3.42AB +5.22AC +0.45BC-8.12A ² -17.25B ² -6.05C ² 。

Through analysis of variance, the p value of the model obtained through analysis is obvious when the p value is less than 0.05, the p values of the inconvertible terms are all not obvious when the p values are more than 0.05, the experimental modeling is proved to be established, and R is ² The value is 0.9522, which shows that the model fitting degree is good; from the significance analysis of the regression model, the following factors were suggested as factors affecting the degradation rate of emetic toxin: initial pH value of B>A inoculum size>C, culture temperature.

After the response surface test optimization, the optimal degradation process conditions of the vomitoxin are as follows: the inoculation amount is 7.455%, the initial pH value of the culture medium is 5.789, the culture temperature is 27.458 ℃, and the degradation rate of the vomitoxin is 68.3457% at the moment. According to the actual control conditions of the experiment, the inoculum size is adjusted to 7.5%, the initial pH value of the culture medium is 5.8, and the culture temperature is 27.5 ℃. Under the condition, three parallel tests of degrading vomitoxin by fermenting the strain Am-07-22 are carried out. The degradation rate of the final strain Am-07-22 for fermenting and degrading vomitoxin is 66.27%, and the degradation rate is consistent with the response surface prediction value, which indicates that the optimization condition can be applied to the next degradation process.

3. Research on degradation effect of strain Am-07-22 on DON with different concentrations

Different volumes of DON standard working solution are respectively added into the basic screening culture medium until the final concentration is 2 mug/mL, 3 mug/mL, 4 mug/mL and 5 mug/mL, the strain Am-07-22 fermentation seed solution is inoculated according to 7.5 percent of inoculation amount, three repeated experiments are carried out on each group by taking the non-inoculation seed solution as a control group, and 7d is cultured under the conditions of 27.5 ℃ culture temperature and 160rpm rotation speed.

The results of the effect of different DON concentrations on the degradation effect of Armillaria mellea are shown in FIG. 3, and the DON degradation rates are 64.05%, 42.34%, 17.32% and 8.01%, respectively. The DON degradation rate is continuously reduced with the increase of the toxin concentration. With the increase of the concentration of the DON toxin, the degradation effect of the armillaria mellea on the DON is continuously reduced, and at high concentration (4 and 5 mu g/mL), the DON obviously inhibits the growth of the armillaria mellea, so that a large amount of thalli die, and cannot be metabolized normally to generate active substances capable of degrading the DON. Therefore, the DON degradation rate is determined to be the highest when the DON concentration is 2 mu g/mL, and the highest degradation rate is 64.05%.

4. Source location of substance and active substance characterization for degrading vomitoxin

Preparing fermentation liquor, supernatant, bacterial suspension and intracellular fluid: inoculating the strain into liquid culture medium, culturing at 27.5 deg.C and 160rpm for 7d to obtain fermentation broth; centrifuging fermentation liquor 5mL at 4 deg.C and 12000 rpm for 10min, filtering the supernatant with sterile filter membrane to obtain thallus-free supernatant, and storing at 4 deg.C; washing the lower layer of thallus with buffer solution for 3 times, then resuspending 5mL, and shaking and mixing uniformly to obtain a thallus suspension; taking a proper amount of somatic cells, completely crushing the somatic cells under high-pressure homogenization, and obtaining a clear solution after centrifugation as intracellular fluid.

Active material source localization: 980. Mu.L of each of the obtained fermentation liquid, supernatant, bacterial suspension and intracellular liquid was added with 20. Mu.L of mycotoxin to give a final DON concentration of 2. Mu.g/mL. The reaction system is placed in a full-temperature shaking incubator with 27 ℃ and 160rpm to culture 48 h, then samples are taken, and 3 times of experiments are set.

As a result: in order to preliminarily analyze the degradation mechanisms of the DON, the degradation effects of fermentation liquor, supernatant, bacterial suspension and intracellular fluid of the armillaria mellea on the DON are respectively compared, as shown in FIG. 4, the DON degradation rates are 65.52%, 60.46%, 13.69% and 16.67%, respectively, and the degradation effect of the supernatant on the DON is slightly lower than that of the fermentation liquor, which may be because the fermentation liquor obtained by the test contains part of hypha, so that it can be inferred that active substances for degrading the DON by the armillaria mellea exist in the supernatant, and may be that extracellular active substances produced by fermentation of the armillaria mellea act on the DON instead of depending on the adsorption of thalli or intracellular substances produced by fermentation. Each component of the degrading strain degrades mycotoxins, and this is consistent with the experimental conclusion of Wang Mingqing, tang et al.

Active substance characterization: and (3) respectively processing the supernatant by referring to seedling seedlings and the like:

(1) And (3) heat treatment of supernatant: treating the supernatant in water bath at 95 deg.C for 10min, cooling to room temperature, and filtering with disposable sterile filter membrane; treating the supernatant in 100 deg.C boiling water bath for 10min, cooling to room temperature, and filtering with sterile filtration membrane.

(2) Treating the supernatant with proteinase K: 2 mL supernatant was added 100. Mu.L proteinase K (final concentration 0.1 mg/mL), treated at 55 ℃ for 2h, and filtered through a 0.22 μm disposable sterile filter membrane.

(3) Treatment of the supernatant with SDS: 2 mL supernatant was treated with 100. Mu.L SDS (final concentration of 0.5 mg/mL) at 55 ℃ for 2h and filtered through a 0.22 μm disposable sterile filter membrane.

(4) Protease K, SDS supernatant was treated: the supernatant of 2 mL was added 100. Mu.L proteinase K (final concentration of 0.1 mg/mL) and 100. Mu.L SDS (final concentration of 0.5 mg/mL), treated at 55 ℃ for 2h, and filtered through a 0.22 μm disposable sterile filter membrane.

980. Mu.L of each supernatant obtained by the above treatment was taken, and 20. Mu.L of mycotoxin was added thereto so that the final DON concentration was 2. Mu.g/mL. The reaction system is placed in a full-temperature shaking incubator with 27 ℃ and 160rpm to culture 48 h, then samples are taken, and 3 times of experiments are set.

As a result: in order to further analyze the properties of extracellular active substances for degrading DON by fermentation of Armillaria mellea, different treatments were performed on the supernatant of Armillaria mellea, and the degradation rates of the supernatant after six treatments, such as heat treatment and protease K, SDS, are shown in FIG. 5, which are 61.92%, 19.35%, 24.97%, 52.35%, 8.67% and 39.16%, respectively. The heat treatment can usually destroy the space structure of most of protein (enzyme), the degradation effect of the supernatant treated by 95 ℃ water bath and boiling water bath on DON is greatly reduced, which indicates that the protein or enzyme participating in degradation is unstable to heat, the short-time heat treatment obviously affects the protein or enzyme, and the structure of the protein or enzyme is easy to destroy; SDS as a denaturant destroys the spatial structure of protein, and the degradation effect of the supernatant after treatment is obviously reduced, so that the Armillaria mellea is further presumed to be a protein active substance.

5. Study on dynamic degradation of vomitoxin by Armillaria mellea

Degrading DON by the strain: liquid seeds full of the uniform bacteria balls and good in growth condition are transferred into a culture medium according to the inoculation amount of 7.5%, DON standard working solution is added into the culture medium, the DON content in the culture medium is respectively 2 mu g/mL, the pH value of the culture medium is adjusted to 5.8, and a control group is a basic screening culture medium without inoculated bacteria. Each set of three replicates was incubated at 27.5 ℃ for 7 days at 160rpm protected from light. The fermentation broth of the strain cultured by fermentation is sampled once every 12 h, and 3 times of sampling are set for each time.

As can be seen from FIG. 6, as the fermentation culture time is prolonged, the DON degradation rate has a trend of first significant increase and then gradual decrease, the DON degradation rate is in an increasing trend from 0 h to 144h, reaches a maximum value at 144h, has a maximum DON degradation rate of 63.83%, and begins to gradually decrease after 144h, has a DON degradation rate of 62.86 at 156 h, and has a DON degradation rate of 63.08% at 168h, so that the research determines that the optimal fermentation time for the fermentation and degradation of the DON by the armillaria mellea is 144 h.

Measuring enzyme activity of manganese peroxidase produced by strain fermentation:

(1) Plate color development test

See Du Haiping et al.

Basic culture medium: 20% of potato, 2% of glucose, 2% of agar and KH ₂ PO ₄ 0.3%，MgSO ₄ ·7H ₂ O 0.15%,VB ₁ 0.002%。

RB brilliant blue test: 0.003% RB brilliant blue was added to the basal medium and the medium was observed for orange rings.

Guaiacol medium: adding 0.1 g.L into the basic culture medium ^-1 Guaiacol (final concentration) was observed for the presence of dark brown rings around the hyphae.

The extracellular manganese peroxidase of the edible fungi can enable a culture medium of the guaiacol to generate a reddish brown ring, and the generation of the peroxidase can generate an orange yellow ring in RB brilliant blue. Armillaria mellea produces orange-yellow rings in RB brilliant blue medium, which proves the production of peroxidase, while manganese peroxidase (MnP) can oxidize guaiacol into tetrao-methoxycerol, which is reddish brown. The results of the plate color reaction test show that the Armillaria mellea fermentation process can produce extracellular manganese peroxidase.

(2) Determination of manganese peroxidase (MnP) enzyme activity

Refer to Du Haiping, et al. Preparation of crude enzyme solution: the fermentation liquid in the 2 mL fermentation medium is sucked and centrifuged for 10min at 10000 r/min, and the crude enzyme solution is obtained and used for the following enzyme activity determination experiment.

And (3) measuring the MnP enzyme activity: the reaction system is 3.4 mL acetic acid-sodium acetate buffer solution (the concentration is 200 mmol/L, pH value is 4.5), 0.1 mL 6 mmol/L MnSO ₄ Solution, 0.4 mL crude enzyme solution and 0.1 mL of 1.6 mmol/L H ₂ O ₂ The solution was reacted at 37 ℃ for 3 min and the absorbance value of 240 nm was determined.

One enzyme activity unit (U) oxidizes 1. Mu. Mol Mn per minute ²⁺ To Mn ³⁺ The required amount of enzyme. Each sample was run in triplicate and then averaged.

As can be seen from FIG. 8, the DON degradation rate in this experiment is substantially consistent with the growth trend of MnP enzyme activity, and the DON degradation rate is increased with the increase of MnP enzyme activity produced by Armillaria mellea fermentation. The MnP enzyme activity is continuously improved along with the extension of the fermentation culture time before 144h, the highest value is shown at 144h, the highest enzyme activity is 105.23U/L, and the MnP enzyme activity begins to decline after 144 h; along with the extension of the fermentation time, the DON degradation rate is gradually improved, the DON degradation rate is the highest in the 144h, the DON degradation rate is 63.83%, the DON degradation rate starts to stably decline after 144h, and the DON degradation rates of 156 h and 168h are 62.86% and 63.08% respectively; when the degrading rate is 36-60 h, both DON degrading rate and enzyme activity are greatly improved, the DON degrading rate is increased from 15.23% to 38.24%, and the MnP enzyme activity is increased from 43.69U/L to 72.34U/L. The correlation between the DON degradation rate and the MnP enzyme activity is analyzed through Pearson correlation, and the result shows that the DON degradation rate and the MnP enzyme activity have obvious correlation at the significance level of 0.01, the correlation is 0.989, and the degradation effect of the armillaria mellea on the DON is related to the manganese peroxidase.

Example 4 experiment of degrading zearalenone by the species Armillaria mellea Am-07-22

Zearalenone ZEN solid standard substance is purchased from Qingdao Pop (Pribolab) bioengineering limited company, and the solid standard substance is diluted by methanol solution to prepare ZEN standard stock solution with the concentration of 100 mug/mL, and is stored at minus 20 ℃ in a dark place.

1. Determination of ZEN degradation effect of Armillaria mellea

Smashing the prepared armillaria mellea Am-07-22 secondary fermentation seed culture solution, inoculating the smashed armillaria mellea Am-07-22 secondary fermentation seed culture solution into 10mL of liquid culture medium containing 5 mu g/mL of ZEN, wherein the inoculation amount is 10%, and then oscillating the reaction system in a constant-temperature shaking table at the temperature of 27 ℃ and at the speed of 160rpm for reaction in a dark place for 7d. Adding 1mL of methanol solution into 1mL of supernatant to extract the ZEN, centrifuging at 10000rpm/min for 10min, filtering with a 0.22 μm filter membrane, detecting the ZEN content with a high performance liquid chromatograph, and calculating the degradation rate of the ZEN. The ZEN degradation rate calculation formula is as follows:

as a result: by adding 5 mu g/mL of ZEN into the culture medium and utilizing the Armillaria mellea Am-07-22 strain for fermentation degradation, the high performance liquid chromatogram of figure 9 shows that the Armillaria mellea Am-07-22 has good degradation effect on 5 mu g/mL of ZEN, and the degradation rate can reach 73.83%.

2. Effect of ZEN concentration on Armillaria mellea Am-07-22 degradation

The prepared armillaria mellea Am-07-22 secondary fermentation seed culture solution is smashed, inoculated into 10mL of liquid culture medium containing ZEN with the concentration of 2 mug/mL, 5 mug/mL, 7.5 mug/mL, 10 mug/mL and 15 mug/mL respectively, the inoculum size is 10 percent, 3 strains are paralleled, and then the reaction system is vibrated in a constant temperature shaking table at 27 ℃ and 160rpm/min for light-shielding reaction for 7d. And (3) detecting the content of zearalenone by using a high performance liquid chromatograph after extracting the ZEN, wherein each group comprises three groups in parallel, and calculating the degradation rate of the ZEN.

As shown in FIG. 10A, the degradation rate of Armillaria mellea Am-07-22 on ZEN with different concentrations is different, and can reach 97.32% when the initial concentration is 2 mug/mL, has 73.83% when the ZEN concentration is 5 mug/mL, and is 49.47% and 32.55% when the ZEN concentration is increased to 10 mug/mL and 15 mug/mL respectively. It can be seen that the degradation rate decreases with increasing ZEN concentration. However, as can be seen from FIG. 10B, the degradation amount of ZEN is 19.46 μ g at the initial concentration of 2 μ g/mL and 36.5 μ g at the initial concentration of 5 μ g/mL, while the degradation amount of ZEN exceeds 48.82 μ g at the initial concentrations of 10 μ g/mL and 15 μ g/mL, and the difference is not significant, so the degradation amount of ZEN by Armillaria mellea Am-07-22 gradually increases and becomes stable. At low concentrations, the initial ZEN content itself is low, the final degradation amount is still low despite the high degradation rate, while at high concentrations the initial ZEN content is high, even though the degradation rate is reduced, the degradation amount is continuously increased, and finally gradually stabilizes to the upper degradation limit.

3. Influence of culture time, temperature, pH and inoculum size on ZEN degradation of Armillaria mellea Am-07-22

Inoculating the smashed armillaria mellea Am-07-22 secondary seed solution into 10mL of liquid culture medium with pH values of 4, 5, 6, 7, 8 and 9 and ZEN concentration of 5 mu g/mL respectively, wherein the inoculation amounts are 2.5%, 5%, 7.5%, 10%, 12.5% and 15% respectively, and then carrying out oscillation and light-shielding reaction on the reaction system in a constant-temperature shaking table at 18 ℃, 21 ℃, 24 ℃,27 ℃, 30 ℃, 33 ℃ and 160rpm/min for 1-12 d. And detecting the content of the zearalenone by using a high performance liquid chromatograph, wherein each group comprises three parallels, and calculating the degradation rate of the ZEN.

As shown in FIG. 11A, the degradation rate of ZEN by Armillaria mellea Am-07-22 gradually increased (P < 0.05) with the increase of the culture time, and the degradation rate from the first 1d was only 21.37% to 73.83% of the 7d. And the degradation rate of the 8 th to 12 th days is increased to about 80 percent, and the degradation rate difference is not obvious at the moment, which shows that the degradation rate of the 8 th to 12 th days is the highest and tends to be stable.

As can be seen from FIG. 11B, the degradation rate of ZEN by Armillaria mellea Am-07-22 increases with increasing temperature (P < 0.05) in the range of 18 ℃ to 27 ℃ and increases from 39.92% at 18 ℃ to 73.83% at 27 ℃. And under the condition of 27-33 ℃, the degradation rate is reduced along with the temperature rise (P < 0.05), wherein the degradation rate to ZEN at 33 ℃ is 45.52 percent. This indicates that too high or too low temperature has a large influence on the degradation of ZEN by Armillaria mellea Am-07-22, and the growth environment of Armillaria mellea becomes poor under unfavorable temperature conditions, so that the degradation rate of ZEN is also reduced.

As can be seen from FIG. 11C, the degradation rate of Armillaria mellea Am-07-22 on ZEN shows a tendency of increasing first and then decreasing (P < 0.05) with the increase of pH value in the range of pH value 4.0-9.0, and the degradation rate can reach 75.22% at pH value of 7.0 at most. The degradation rates at pH 4.0 and 9.0 were 45.89% and 61.97%. This indicates that the degradation effect is affected in the peracid or overbase environment. The degradation of ZEN can be influenced under the culture condition of strong acid or strong base, the degradation effect is optimal in the environment with the pH value of 7.0-8.0, and the degradation rate is highest. This is similar to the results of Tan et al.

As shown in FIG. 11D, the degradation rate of ZEN was gradually increased (P < 0.05) with the increase of the inoculum size of Armillaria mellea Am-07-22. Finally, the inoculation amount tends to be constant when the inoculation amount is 10 to 15 percent, and the degradation rate is higher than 73 percent at most. This indicates that the Armillaria mellea Am-07-22 reaches the maximum growth amount in the growth environment at this time, and the increase of the inoculation amount does not show the increase of the ZEN degradation rate.

4. Distribution of degraded ZEN active ingredients of armillaria mellea Am-07-22

Inoculating Armillaria mellea Am-07-22 in liquid seed culture medium, and shake culturing at 160rpm/min in 27 deg.C constant temperature shaker for 6d. The obtained mycelia and fermentation supernatant were collected by filtration, and the active ingredients of the fermentation broth of the cell seeds degrading ZEN were localized for 3 groups in total, and 3 replicate samples were taken for each group to conduct the test, with ZEN contained at a concentration of 5 μ g/mL as a control group.

Reference Jin Bowen the different groups were treated, group a (fermentation supernatant): taking the filtered upper layer fermentation liquor, centrifuging at 4 ℃ and 10000rpm/min for 10min, and collecting 1mL of supernatant. Group B (mycelium group): the filtered lower mycelium was weighed at 1g and gently rinsed 3-5 times with PBS buffer. Group C (cell disruption group): weighing 1g of filtered lower-layer mycelium, gently rinsing with PBS buffer solution for 3-5 times, performing cell ultrasonic wall breaking treatment for 2h (200W, working for 5s, and intermittent 5 s), centrifuging at low temperature of 10000rpm/min for 10min, and collecting 1mL of intracellular fluid. The prepared experimental group is added with diluted ZEN stock solution, the concentration of ZEN in the system is 5 mug/mL, and the reaction system is subjected to shake culture for 7 days at the temperature of 27 ℃ and under the condition of 160rpm in the dark.

From FIG. 12, it can be seen that the different components of Armillaria mellea Am-07-22 have obvious degradation effect on ZEN (P < 0.05). Wherein the highest degradation rate of fermentation supernatant to ZEN is 47.42%, the degradation rate of Am-07-22 thallus cells is 37.05%, and the degradation rate of broken thallus cells to ZEN is only 13.08% at least. From this, it is inferred that the fermentation supernatant, which is the main effect of Armillaria mellea Am-07-22 on the degradation of ZEN, is presumed to be an extracellular active substance, and the cells of the strain have a certain degree of adsorption on ZEN.

5. Preliminary analysis of property of armillaria mellea Am-07-22 for degrading ZEN active substance

The active substances are analyzed by referring to a treatment method of Jing Sai source, and the treatment is divided into 3 groups: group 1 (proteinase K group): 5mL of the fermentation supernatant was added with 250. Mu.L of proteinase K (final concentration: 0.1 mg/mL) and treated at 55 ℃ for 2 hours. Group 2 (SDS group): 5mL of the fermentation supernatant was added with 250. Mu.L DSS (final concentration: 0.5 mg/mL) and treated at 55 ℃ for 2 hours. Group 3 (proteinase K + SDS group): 5mL of the fermentation supernatant was added with 250. Mu.L of proteinase K (final concentration of 0.1 mg/mL) and 250. Mu.L of LSDS (final concentration of 0.5 mg/mL), and treated at 55 ℃ for 2 hours. 4 groups (100 ℃ boiling water bath group): 5mL of fermentation supernatant was taken and treated in a boiling water bath at 100 ℃ for 20min.

After 4 groups of fermentation supernatants after different treatments were filtered by a 0.22 μm filter, a ZEN stock solution was added thereto, the concentration of ZEN in the system was 5 μ g/mL, and the reaction system was subjected to shake culture at 27 ℃ and 160rpm for 7 days in the dark. And after the end, detecting the ZEN content by using a high performance liquid chromatograph and calculating the degradation rate. Meanwhile, fermentation supernatant with initial concentration of 5 μ g/mL ZEN was used as a control group.

As can be seen from fig. 13, the fermentation supernatant of armillaria mellea was treated with protease K, SDS, boiling water bath, etc., and it was found that the above treatment methods had different effects on ZEN degradation (P < 0.05). The degradation rates of the protease K and the SDS after treatment are respectively 12.65 percent and 22.89 percent, and the degradation effect of the protease K and the SDS after the joint treatment is not obviously different from the degradation effect of the protease after the single treatment. And the degradation rate of the ZEN after boiling water bath is only 5.55%, and the degradation capability is obviously reduced. Therefore, the active substance which mainly plays a role in degrading ZEN in the fermentation supernatant of the armillaria mellea Am-07-22 can be further deduced to be extracellular enzyme.

6. Influence of fermentation time, pH and metal ions on ZEN degradation of armillaria mellea Am-07-22 fermentation supernatant

Referring to the method of Wei Jinfan and modified, the fermentation supernatant of Armillaria mellea Am-07-22 was collected as described above, and ZEN standard solution was added thereto at a concentration of 5. Mu.g/mL, and the reaction was shaken at 27 ℃ and 160 rpm. The change of ZEN content in the reaction system was measured by sampling at reaction times of 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d, and 10d, respectively, and the degradation rate was calculated. Analyzing the influence of different time on the degradation of ZEN by the fermentation supernatant of the armillaria mellea Am-07-22. Further taking fermentation supernatant, adjusting pH to 3, 4, 5, 6, 7, 8, 9, 10, and adding Na with concentration of 10mmol ⁺ 、K ⁺ 、Mg ²⁺ 、Zn ²⁺ 、Cu ^{2 +} 、Fe ²⁺ 、Fe ³⁺ 、Mn ²⁺ Adding metal ions into ZEN standard solution with a concentration of 5 μ g/mL, oscillating at 27 deg.C and 160rpm, and reacting with fermentation supernatant crude enzyme solution without adding metal ions for 7dEach group was replicated in triplicate. And after the reaction is finished, adding 1mL of reaction solution into an equal volume of methanol solution for extraction of zearalenone, fully shaking uniformly, centrifuging at a low temperature of 10000rpm/min for 10min, filtering by using a 0.22 mu m filter membrane, detecting the content of zearalenone by using a high performance liquid chromatograph, and calculating the degradation rate of ZEN. Analyzing the influence of different pH values and different metal ions on the degradation of ZEN by the fermented supernatant of the armillaria ammdii-07-22.

As can be seen from FIG. 14A, the degradation rate of the fermentation supernatant of the armillaria mellea Am-07-22 on ZEN is gradually increased (P < 0.05), the degradation rate is increased from 21.69% at the 1 st d to 62.50% at the 10 th d, the enzyme activity of the armillaria mellea laccase is also gradually increased along with the extension of the fermentation time, the enzyme activity at the 1 st d is only 169.26U/L, the enzyme activity at the 10 th d is increased to 521.11U/L, and the enzyme activity of the laccase is continuously increased (P < 0.05) in the process, which is consistent with the increase of the degradation rate of ZEN.

As can be seen from FIG. 14B, different pH values greatly affect the degradation rate of ZEN by the fermented supernatant of armillaria mellea Am-07-22. Wherein the degradation rate of ZEN is 46.98% and 51.84% at pH 6-7. The two were not significantly different from the control group of fermentation supernatant without adjusting pH. And under acidic and alkaline conditions, the degradation rate of the product on ZEN is low (P < 0.05), and the degradation rates at pH values of 3 and 10 are only 8.29% and 11.22%. The method shows that the enzyme damage effect on fermentation supernatant is stronger under the condition of peracid or over alkali, so that ZEN cannot be effectively degraded.

As can be seen from FIG. 14C, the effect of different metal ions on the degradation of ZEN in the fermentation supernatant of Armillaria mellea Am-07-22 is different, wherein Na is ⁺ 、K ⁺ And Fe ²⁺ The effect of adding the metal ion on degrading ZEN in the fermentation supernatant is not obviously different from that of adding the metal ion. Zn ²⁺ 、Fe ³⁺ The addition of the (B) has certain inhibition effect on the degradation rate of ZEN (P)<0.05 42.09 percent and 37.76 percent respectively, and Fe ³⁺ The inhibition effect of the compound is reduced by 9.66 percent compared with that of a control. And Cu ²⁺ 、Mg ²⁺ 、Mn ²⁺ The effect of the addition of (B) on the degradation of ZEN in the fermentation supernatant is enhanced (P)<0.05 Adding M)g ²⁺ And Mn ²⁺ The degradation rate of the copper alloy to ZEN is 55.57 percent and 57.80 percent respectively, and Cu is added ²⁺ The degradation effect of the fermentation supernatant on the degradation of ZEN is improved by 63.37% to the maximum extent, and is improved by 15.95% compared with a contrast. From this it is possible to infer Cu ²⁺ The enzyme activity of the laccase can be obviously enhanced.

Example 5 application of Armillaria mellea Am-07-22 to degradation of mycotoxins

1. Artificial infection of corn husks and corn gluten meal: referring to the test methods Dou Yong, the test materials (corn bran and corn gluten meal) were mixed with DON solution diluted with sterile water at a concentration of 50. Mu.g/mL, and prepared into corn bran and corn gluten meal with toxin contents of 5. Mu.g/g, 7.5. Mu.g/g, 10. Mu.g/g, and 12.5. Mu.g/g, respectively.

2. Degradation experiments

And (3) degradation test of the solid and liquid fermented corn husks of the armillaria mellea: the feed-liquid ratio of the test is the ratio of the mass of the added solid material (corn bran) to the liquid volume of the culture medium; solid-liquid fermentation was carried out at a feed-liquid ratio of 1. Meanwhile, the corn husks which are not fermented are set as a control group.

Degradation test of the armillaria solid-state and liquid-state fermented maize yellow powder: the feed-liquid ratio is the ratio of the mass of the added solid material (corn gluten meal) to the liquid volume of the culture medium; solid-state and liquid-state fermentations were carried out at 1:1 and 1.5 feed-to-liquid ratios, respectively, with a medium pH of 5.8, and fermentation cultures were carried out at a culture temperature of 27.5 ℃. Meanwhile, maize yellow powder which is not fermented is set as a control group.

As a result: from the results of degrading DON by different fermentation modes, as shown in FIG. 15, in the research on the application of Armillaria mellea in fermenting and degrading DON in corn husks, the DON degradation rates after solid fermentation and liquid fermentation are 89.48% and 76.33%, respectively, and the effect of the solid fermentation mode is better when degrading DON in corn husks; in the research on the application of the armillaria mellea in the fermentation degradation of the DON in the corn gluten meal, the DON degradation rates after solid fermentation and liquid fermentation are 46.26 percent and 74.68 percent respectively, and the effect of degrading the DON in the corn gluten meal by the liquid fermentation mode is better. Therefore, the experiment researches the degradation effect of the armillaria mellea on DON in two different fermentation raw materials, and the degradation effect is good when the corn bran is subjected to a solid fermentation mode and the corn gluten is subjected to a liquid fermentation mode. Corn deep processing byproducts such as corn gluten meal, corn bran and the like are rich in bioactive components such as protein, dietary fiber and the like, and early-stage laboratory researches prove that the edible fungi have high-efficiency biotransformation capability on the active components, thereby greatly widening the application range of the processing byproducts.

And (3) degradation test of corn husks with different DON concentrations by solid state fermentation of armillaria mellea: corn bran was added to the medium at a feed-to-liquid ratio of 1:2.5 so that the DON concentration in the medium was 2, 3, 4, 5. Mu.g/g, the pH of the medium was 5.8, and solid-state fermentation culture was carried out at a culture temperature of 27.5 ℃. Meanwhile, the corn husks which are not fermented are set as a control group.

In the application research of the solid fermentation corn bran of the armillaria mellea, the results of the effect of fermenting the corn bran with different concentrations on the DON degradation rate are shown in FIG. 16, and the DON degradation rate of the armillaria mellea on the corn bran with different toxin concentrations is 89.48%, 85.24%, 87.93% and 73.35%. The degradation rate of the armillaria mellea fermentation degradation vomitoxin tends to be stable when the DON concentration is 2, 3 and 4 mug/g, and the degradation effect is best when the DON concentration is 2 mug/g. However, when the DON concentration is increased to 5 mu g/g, the DON degradation rate is reduced, the degradation effect is reduced, and at higher concentration, DON can inhibit the growth of the strain Am-07-22 and cannot be metabolized normally to generate active substances capable of degrading DON.

And (3) degradation test of the corn gluten meal with different DON concentrations by liquid state fermentation of the armillaria mellea: adding the maize yellow powder into the culture medium according to the feed-liquid ratio of 1:2.5, so that the DON concentration in the culture medium is 2, 3, 4 and 5 mu g/mL, the pH value of the culture medium is 5.8, and the liquid fermentation culture is carried out at the culture temperature of 27.5 ℃ and under the condition of 160 rpm. Meanwhile, maize yellow powder which is not fermented is set as a control group.

As a result: in the application research of the armillaria liquid fermentation of the zeaxanthin powder, the results of the effect of fermenting the zeaxanthin powder with different concentrations on the DON degradation rate are shown in FIG. 17, and the DON degradation rate of the zeaxanthin powder with different toxin concentrations by the armillaria is 74.68%, 75.98%, 57.75% and 56.46%. The DON degradation rate showed a decreasing trend with increasing toxin concentration. The degradation effect difference of the armillaria mellea fermentation for degrading vomitoxin is not obvious when the DON concentration is 2 and 3 mug/g and the DON concentration is 4 and 5 mug/g, and the degradation effect is best when the DON concentration is 3 mug/g. With the increase of the toxin concentration, the DON can inhibit the growth and the generation of the Armillaria mellea, and can not be metabolized normally to generate active substances for degrading the DON, so that the degradation effect is reduced.

Claims (6)

1. Armillaria melleaArmillaria melleaApplication in degrading vomitoxin.

2. Armillaria melleaArmillaria melleaAm-07-22, with the preservation number of CCTCC NO: m20221044.

3. A method for degrading vomitoxin comprises inoculating Armillaria mellea into testa Maydis or corn yellow powder, and fermenting.

4. The method of degrading emetic toxin of claim 3, wherein said Armillaria mellea is the Armillaria mellea of claim 2.

5. A method of degrading emetic toxin according to claim 3 or 4, wherein: the corn bran is subjected to solid state fermentation.

6. A method of degrading emetic toxin according to claim 3 or 4, wherein: the maize yellow powder is fermented in a liquid state.

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