Application of Armillaria in degrading zearalenone

Abstract

The application discloses application of Armillaria mellea in degradation of zearalenone, adopts Armillaria mellea Am-07-22, and the preservation number is CCTCC NO: M 20221044; the degradation effect on zearalenone is good, the degradation rate can reach 79.37% when the concentration of zearalenone is 5 μg / mL; the optimal condition for degrading zearalenone is that the temperature is 27 DEG C, the culture time is 8d, the initial pH is 7.0, and the inoculation amount is 10%; and Cu 2+ The fermentation supernatant has the best promoting effect on zearalenone degradation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to application of Armillaria mellea in degradation of zearalenone. BACKGROUND

[0002] Fungal toxins exist in contaminated food and feed, and accumulate in the human and animal bodies from the food chain, causing great harm to health. The trichothecene family of mycotoxins is the most common type of contaminating mycotoxins in food, including zearalenone, fumonisin, vomitoxin and the like. The main component of vomitoxin is deoxynivalenol (DON), the chemical name of which is 3α, 7α, 15-trihydroxy-12, 13-epoxy trichothecene-9-anthracene-8-ketone, belonging to the trichothecene family of compounds. DON was first discovered in moldy corn and wheat in Japan in 1970, and was named in 1973 because it could cause sows to refuse to eat and produce emetic reactions. It is colorless needle-like crystals at room temperature, soluble in water and also soluble in methanol, ethyl acetate, acetone and other solvents, and has strong heat resistance, high pressure resistance and acid and alkali resistance. Zearalenone (ZEN) is a non-steroidal estrogen mycotoxin, also known as F-2 toxin, which is a secondary metabolite mainly produced by Fusarium fungi, and mainly exists in contaminated corn, wheat, rice, soybeans and other grains. ZEN can contaminate grains, food and feed through various pathways, seriously threatening human health and life safety. Its chemical structure has similar properties to many estrogens, can activate estrogen receptors, and cause reproductive disorders such as abortion, stillbirth and malformation in farm animals. In addition, ZEN can also enter the human body through the food chain, causing immune damage, liver damage, genetic toxicity, and inducing cancer and the like.

[0003] The results of the survey data in 2020 showed that the over-standard situations of DON, ZEN and AFB1 in corn and its processing by-products were relatively serious, and the toxin pollution of corn by-products was the most serious, and the over-standard rate of DON in corn by-products was 53.57%. At present, the degradation detoxification technologies of DON or zearalenone are divided into physical method, chemical method and biological method. The physical method mainly includes heat treatment, ultraviolet radiation or gamma ray irradiation, adsorption of adsorbent, etc. The chemical method includes ammoniation method, alkali method, ozone treatment, oxygen water treatment, etc. However, the chemical method has the disadvantages of low degradation efficiency, destruction of nutritional components, introduction of other chemical substances, etc. The biological method has the advantages of mild process and reaction conditions, high efficiency, less damage to the flavor and nutritional value of raw materials, etc., and part of the microbial fermentation products can improve the flavor and increase the nutritional value. At present, the action mechanism of biological detoxification is as follows: first, the biological adsorption of microorganisms reduces the content of toxins, such as lactic acid bacteria, yeast and the like, which can adsorb toxins through the β-glucan on the cell wall; second, the biological degradation effect, including microbial growth, secretion of metabolites and enzyme preparation to degrade toxins.

[0004] Studies have shown that the biological degradation of zearalenone mainly comes from the following two types: microbial degradation and enzyme degradation. So far, a large number of microorganisms capable of removing zearalenone have been widely reported. Among them, the bacteria and fungi that degrade ZEN are mainly concentrated in lactic acid bacteria, bacillus, aspergillus and yeast, etc. Zhao et al. isolated plant lactobacillus Lp22, Lp39 and Lp4 from traditional fermented foods, which degraded 47.80%, 38.06% and 39.50% of ZEN in the solution, respectively. Xu et al. found that bacillus amyloliquefaciens ZDS-1 could effectively degrade ZEN in the concentration range of 1mg / L to 100mg / L, and found that ZDS-1 could not only degrade ZEN in the culture medium, but also could degrade ZEN in wheat. Pereyra et al. tested 11 kinds of bacillus strains that had been proved to degrade AFB1, and found that the tested strains could degrade ZEN with a concentration of 400ng / mL after 72 hours, and the degradation rate was 96.9%. In addition, Sun et al. isolated food-grade aspergillus niger FS10 from fermented soybeans, which could effectively remove ZEN in PDB medium, and the mycelium and culture filtrate could also reduce 43.10% and 68.16% of ZEN. SUMMARY

[0005] The purpose of the present application is to provide the application of Armillaria mellea in degrading zearalenone to solve the above problems.

[0006] The application of Armillaria mellea in degrading zearalenone.

[0007] The Armillaria mellea has a preservation number of CCTCC NO: M 20221044.

[0008] The application provides application of Armillaria mellea in degradation of zearalenone. The Armillaria mellea Am-07-22 has a preservation number of CCTCC NO: M 20221044, and has a good degradation effect on zearalenone, and the degradation rate of zearalenone with a concentration of 5 μg / mL can reach 79.37%. The optimal condition for degrading zearalenone is that the temperature is 27 ℃, the culture time is 8 days, the initial pH is 7.0, and the inoculation amount is 10%, and Cu2+ has the best promoting effect on degradation of zearalenone in the fermentation supernatant. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Effect of different edible fungi on degradation of DON (different letters represent significant difference p <0.05);

[0010] Figure 2 Response surface graph of influence of interaction of various factors on DON degradation rate;

[0011] Figure 3 Effect of different DON concentrations on degradation effect of Armillaria mellea (different letters represent significant difference p <0.05);

[0012] Figure 4 Effect of different components of Armillaria mellea on DON degradation rate (different letters represent significant difference p <0.05)

[0013] Figure 5 Effect of different treatment methods on DON degradation rate (different letters represent significant difference p <0.05)

[0014] Figure 6 Effect of different culture times of Armillaria mellea on degradation effect of DON;

[0015] Figure 7 Test result of manganese peroxidase color reaction;

[0016] Figure 8 Change rule of DON degradation rate and Mnp enzyme activity at different culture times;

[0017] Figure 9 Liquid chromatogram of strain Am-07-22 degrading ZEN;

[0018] Figure 10The effect of initial ZEN concentration on ZEN degradation by Am-07-22; A. Degradation rate; B. Degradation amount;

[0019] Figure 11 Effects of different conditions on the degradation rate of ZEN by Am-07-22; A. Time; B. Temperature; C. pH value; D. Inoculum size;

[0020] Figure 12 Degradation rates of ZEN by different components of strain Am-07-22;

[0021] Figure 13 The degradation rate of ZEN by fermentation supernatant of strain Am-07-22 after different treatments;

[0022] Figure 14 Effects of different conditions on the degradation rate of ZEN in fermentation supernatant of Am-07-22: A. Different fermentation times; B. Different pH values; C. Different metal ions;

[0023] Figure 15 Effects of Armillaria mellea fermentation of corn husks and corn yellow powder on DON degradation rate (different uppercase letters represent significant differences within groups, different lowercase letters represent significant differences between groups p<0.05).

[0024] Figure 16 Effect of different DON concentrations in corn husks on DON degradation rate during solid-state fermentation by Armillaria mellea (different letters represent significant differences, p<0.05).

[0025] Figure 17 Effect of different DON concentrations of corn yellow powder fermented by Armillaria mellea on DON degradation rate (different letters represent significant differences p<0.05). Detailed Implementation

[0026] Armillaria mellea Am-07-22 ( Armillaria mellea ), preservation number is CCTCC NO:M 20221044; 2) based on the research team's previous experimental results, strains with better (cellulose biodegradation) indicators were screened, and morel mushroom Me-01 was selected respectively. Morchella esculenta Hericium erinaceus He-02-06 ( Hericium erinaceus ) and Armillaria mellea Am-07-22 ( Armillaria mellea), provided by the National Engineering Center for Deep Processing of Wheat and Corn; the three strains were isolated from fruiting bodies of the strains collected in Changbai Mountain, Morchella Me-01 was isolated from Morchella fruiting body, Hericium He-02-06 was obtained by mutation from Hericium fruiting body, and Armillaria Am-07-22 was isolated from Armillaria fruiting body; Morchella Me-01 was disclosed in the research of Morchella solid-state fermentation of corn gluten meal reported by TONG Weina et al.; Hericium He-02-06 was preserved in China Center for Type Culture Collection on July 6, 2022, and the strain preservation number was CCTCC NO: M20221043; Armillaria Am-07-22 was preserved in China Center for Type Culture Collection on July 6, 2022, and the strain preservation number was CCTCC NO: M20221044.

[0027] Example 1 Screening and identification of Armillaria Am-07-22

[0028] I. Mutation method of strain

[0029] The laboratory-preserved Armillaria Am-07 ( Armillaria mellea ) was used as the starting strain, and the protoplast suspension of the strain to be mutated was placed on a plate, the rotor was placed in the plate for stirring, and was placed under a 15W ultraviolet lamp at a distance of 30 cm, and ultraviolet mutation was performed at an irradiation dose of 20-90 s. The bacterial suspension after mutation treatment was coated on the regeneration medium and cultured in a 27℃ constant temperature incubator in the dark for 10 d, the colonies grown were counted, a lethal rate curve was drawn, and well-grown strains were selected as mutant strains. The mutant strains were cultured under appropriate conditions, and the mutant strain with soluble dietary fiber content higher than the starting strain by more than 15% was selected as a positive mutant strain, and was continuously subcultured for 10 times, and fermentation test was performed every generation. The mutant strain with stable production performance was selected, sequenced, identified, and stored in a refrigerator at 4℃.

[0030] II. Comparison of mutation effects

[0031] The Armillaria Am-07-22 strain obtained after ultraviolet mutation treatment was used for solid-state fermentation, and its effect on the soluble dietary fiber content of ginseng residue was as shown in Table 1; after solid-state fermentation of ginseng residue using the mutant strain, the soluble dietary fiber content of ginseng residue was improved, and the hydration properties such as water holding capacity, oil holding capacity and water swelling capacity of insoluble dietary fiber in ginseng residue were also improved, as shown in Table 2. Figure 1 Figure 2 Table 2 Effect of Am-07-22 on the hydration properties of insoluble dietary fiber in ginseng residue

[0032] III. Sequencing results of Am-07-22 strain

[0033] ​The purified bacterial liquid was sent to Jilin Kume Biotechnology Co., Ltd. for detection, the DNA genome of the bacterial liquid was extracted by the company, then the ribosome DNA and ITS sequence were amplified, the PCR product was obtained as shown in the following table, and finally sequencing was performed.

[0034]

[0035] The DNA of the bacteria liquid sent for testing was amplified using ITS4 and ITS5 universal primers for fungi, and successfully spliced into a 623 bp connection fragment, and the splicing result is shown in SEQ ID NO. 1 of the sequence table; the measured sequence is used to draw the phylogenetic tree of the strain using MEGA7.1 software, as shown in 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, named Armillaria Am-07-22. Armillaria Am-07-22 was preserved in China Center for Type Culture Collection on July 6, 2022, and the strain preservation number is CCTCC NO: M20221044.

[0036] Example 2: Strain culture

[0037] I. Strain activation

[0038] The Me-01, He-02-06 and Am-07-22 strains were respectively transferred to the slant medium, and the culture conditions were 27℃, and the culture was carried out for 12d.

[0039] Prepare the strain liquid activation medium:

[0040] 1) Am-07 liquid seed culture 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 B1 0.005 g, pH value is natural, 121℃ sterilization for 20 min;

[0041] 2) Me-01 liquid seed culture medium: glucose 10 g, yeast extract powder 5 g, potassium dihydrogen phosphate 0.6 g, magnesium sulfate heptahydrate 0.375 g, iron sulfate heptahydrate 0.005 g, pH value is natural, 121℃ sterilization for 20 min;

[0042] 3) He-02 liquid seed culture medium: glucose 10 g, yeast extract powder 5 g, soluble starch 10 g, potassium dihydrogen phosphate 1.5 g, magnesium sulfate heptahydrate 0.3 g, pH value is natural, 121℃ sterilization for 20 min. The above are calculated at 500 mL.

[0043] II. Preparation of fermentation seed liquid

[0044] Preparation of primary fermentation seed: In a sterile environment, 8 pieces of bacteria from the activated slant medium of the three bacteria were inoculated into a 100 mL triangular flask containing 30 mL of liquid activated culture medium, and the constant temperature was maintained at 27°C. The culture was incubated at 160 r / min for 6 days to obtain the primary fermentation seed;

[0045] Preparation of secondary fermentation seed liquid: The primary fermentation seed was broken up, and the strain was inoculated into a 500 mL triangular flask containing 200 mL of liquid activated culture medium at an addition amount of 8%. The constant temperature was maintained at 27°C. The culture was incubated at 160 r / min for 6 days to obtain the secondary fermentation seed liquid.

[0046] Example 3: Experiment of strain degradation of vomitoxin

[0047] I. Screening of fungi for degrading vomitoxin

[0048] 1. Degradation of DON by the strain

[0049] The liquid fermentation seed liquid with uniform bacterial balls and good growth conditions was inoculated into the basic screening medium, and DON standard working solution was added to the basic screening medium to make the DON content 2 μg / mL. The control group was the basic screening medium without inoculation of the strain. Each group had three repeats, and the culture was incubated at 27°C and 160 rpm for 7 days in the dark.

[0050] The basic screening medium is as follows: 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 B1 0.005 g, 500 mL, pH value is natural, 121°C sterilization for 20 min.

[0051] 2. Extraction and detection of DON

[0052] The fermented liquid after culture was centrifuged at 12000 rpm for 10 min, and the temperature was kept at 4°C. After 100-fold dilution, the supernatant was filtered with a disposable sterile filter membrane and placed in a 1.5 mL EP tube, which was stored at 4°C for detection. The sample was detected according to the method provided by the vomitoxin detection kit, and the OD value of the test hole was measured by an enzyme-labeled instrument at a double wavelength of 450 nm.

[0053] 3. Calculation of DON degradation rate

[0054] The residual content of DON in the fermentation liquid was calculated by Ridasoft.win software (4-Parameter). The degradation effect of the strain on DON was represented by the degradation rate. The DON degradation rate was calculated according to the following formula:

[0055]

[0056] 4. Vomitoxin ELISA test kit detection steps

[0057] The required reagents and the required number of microplates are taken out of the 4°C environment and placed at room temperature (20-25°C) for 30 min or more, and each liquid reagent must be shaken well before use.

[0058] Washing solution: dilute the concentrated washing solution (10x) with ultrapure water at a volume ratio of 1:9.

[0059] Add sample: 50 μL of sample per well, add 50 μL of vomitoxin enzyme label per well, and then add 50 μL of vomitoxin anti-reagent per well, gently shake to mix, cover with film to avoid light, and react at 25°C for 30 min.

[0060] Wash the plate: carefully remove the cover film, shake off the liquid in the wells, add 250 μL of washing solution per well, wash thoroughly 4-5 times with 10 s intervals, and dry with a water-absorbing paper.

[0061] Color development: add 50 μL of substrate solution A per well and 50 μL of substrate solution B per well, gently shake to mix, and react at 25°C in the dark for 15 min.

[0062] Determination: add 50 μL of termination solution per well, gently shake to mix, and set the enzyme label at 450 nm to determine the OD value of each well.

[0063] 5. Results

[0064] Figure 1 The results of the degradation of DON by three edible fungi in the basic screening medium. As can be seen from the figure, Me-01, He-02, and Am-07-22 three edible fungi after fermentation have degradation effect on the added DON in the medium, and their degradation rates for degrading DON are 11.22%, 32.57%, and 51.76% respectively, among which the degradation ability of strain Am-07-22 for DON is obviously higher than that of Me-01 and He-02, so strain Am-07-22 is selected as the degradation strain for subsequent study.

[0065] II. Optimization of degradation process conditions

[0066] The optimal degradation strain Am-07-22 obtained in the early stage of screening was subjected to single factor test to determine the optimal process conditions for the degradation of vomitoxin by the fungal strain, including inoculum size, initial pH of the culture medium, culture temperature, and culture time. After screening by each single factor test, the early test basis is provided for the response surface optimization test.

[0067] On the basis of single factor experiment, inoculum, initial pH value of medium and culture temperature were selected as independent variables, and DON degradation rate as response value, three-factor and three-level response surface experiment was designed, and the experiment design and results were shown in Table 1, and the regression model variance analysis was shown in Table 2.

[0068]

[0069]

[0070] The data were analyzed by Design-Expert software, and A inoculum, B initial pH value and C culture temperature were selected as independent variables, and the degradation rate (Y) of strain Am-07 to vomit toxin was selected as response value, and the quadratic polynomial regression equation of quadratic regression model was 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 .

[0071] According to variance analysis, the p value of model was less than 0.05, and the loss of inverse item p value was greater than 0.05, which was not significant, indicating that the modeling experiment was established, and the R 2 value was 0.9522, indicating that the model fitting degree was good; according to the significance analysis of regression model, the degree of the factors affecting the degradation rate of vomit toxin was as follows: B initial pH value > A inoculum > C culture temperature.

[0072] After response surface experiment optimization, the optimal degradation process conditions of vomit toxin were as follows: inoculum 7.455%, initial pH value of medium 5.789, and culture temperature 27.458 ℃, and the degradation rate of vomit toxin was 68.3457%. According to the actual control conditions of experiment, the inoculum was adjusted to 7.5%, the initial pH value of medium was 5.8, and the culture temperature was 27.5 ℃. Under the conditions, three parallel experiments of strain Am-07-22 fermentation degradation of vomit toxin were carried out. The final degradation rate of strain Am-07-22 fermentation degradation of vomit toxin was 66.27%, which was consistent with the response surface prediction value, indicating that the optimization conditions could be applied to the next degradation process.

[0073] Three, the degradation effect of strain Am-07-22 on different concentrations of DON

[0074] In the basic screening medium, different volumes of DON standard working solution were added to a final concentration of 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, respectively, and the strain Am-07-22 was inoculated with a 7.5% inoculum to seed the fermentation broth, and the control group was not inoculated with the seed broth. Each group was tested in triplicate, and the culture was incubated at 27.5°C and 160 rpm for 7 days.

[0075] The results of the effect of different DON concentrations on the degradation effect of Armillaria mellea are shown in Table 1. Figure 3 As the concentration of the toxin increases, the DON degradation rate decreases. As the concentration of DON toxin increases, the degradation effect of Armillaria mellea on DON decreases. At high concentrations (4, 5 μg / mL), DON significantly inhibits the growth of Armillaria mellea, resulting in a large number of cell death and the inability to normally metabolize active substances that can degrade DON. Therefore, the highest DON degradation rate is 64.05% when the concentration of DON is 2 μg / mL.

[0076] Four, source positioning of the substance degrading vomitoxin and qualitative analysis of active substances

[0077] Preparation of fermentation broth, supernatant, bacterial suspension and intracellular fluid: the strain was inoculated into the liquid medium and cultured at 27.5°C and 160 rpm for 7 days to obtain the fermentation broth; 5 mL of the fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was filtered through a sterile filter membrane to obtain a sterile supernatant, which was stored at 4°C for later use; the lower layer of bacterial cells was washed with buffer solution for 3 times and then resuspended in 5 mL, and the mixture was shaken to obtain the bacterial suspension; an appropriate amount of bacterial cells was completely broken under high-pressure homogenization, and the supernatant after centrifugation was the intracellular fluid.

[0078] Source positioning of active substances: 980 μL of the fermentation broth, supernatant, bacterial suspension and intracellular fluid were obtained, and 20 μL of mycotoxin was added to each to make the final concentration of DON 2 μg / mL. The reaction system was incubated in a 27°C, 160 rpm constant temperature and speed shaking incubator for 48 h, and samples were taken. Each test was set in triplicate.

[0079] Results: To preliminarily analyze the degradation mechanism of DON, the degradation effects of the fermentation broth, supernatant, bacterial suspension and intracellular fluid of Armillaria mellea on DON were compared, as shown in Table 2. Figure 4The degradation rates of DON were 65.52%, 60.46%, 13.69% and 16.67%, respectively. The degradation effect of the supernatant was slightly lower than that of the fermentation broth, which might be due to the fact that the fermentation broth contained part of the mycelium. It can be inferred that the active substance of Armillaria mellea for degrading DON exists in the supernatant, which might be the extracellular active substance produced by Armillaria mellea fermentation, rather than relying on the adsorption of the bacterial body or the intracellular substance produced by fermentation. The degradation of each component of the degradation strain to mycotoxins was consistent with the test conclusion of Wang Mingqing, Tang Yu, et al.

[0080] Qualitative analysis of active substance: Referring to the method of Fu Miaomiao et al., the supernatant was treated as follows:

[0081] (1) Heat treatment of supernatant: After the supernatant was treated in a water bath at 95℃ for 10 min and cooled to room temperature, it was filtered with a sterile disposable filter membrane. After the supernatant was treated in a boiling water bath at 100℃ for 10 min and cooled to room temperature, it was filtered with a sterile filter membrane.

[0082] (2) Treatment of supernatant with proteinase K: 100 μL of proteinase K (final concentration 0.1 mg / mL) was added to 2 mL of supernatant, which was treated at 55℃ for 2 h and filtered with a 0.22 μm sterile disposable filter membrane.

[0083] (3) Treatment of supernatant with SDS: 100 μL of SDS (final concentration 0.5 mg / mL) was added to 2 mL of supernatant, which was treated at 55℃ for 2 h and filtered with a 0.22 μm sterile disposable filter membrane.

[0084] (4) Treatment of supernatant with proteinase K and SDS: 100 μL of proteinase K (final concentration 0.1 mg / mL) and 100 μL of SDS (final concentration 0.5 mg / mL) were added to 2 mL of supernatant, which was treated at 55℃ for 2 h and filtered with a 0.22 μm sterile disposable filter membrane.

[0085] 980 μL of the supernatant obtained after the above treatments was taken and 20 μL of mycotoxin was added to each, so that the final concentration of DON was 2 μg / mL. After the reaction system was cultured in a 27℃, 160 rpm constant temperature shaking incubator for 48 h, samples were taken. Three replicates were set for each test.

[0086] Results: In order to further analyze the properties of the extracellular active substance of Armillaria mellea for degrading DON, the supernatant of Armillaria mellea was treated in different ways. The degradation rates of the supernatant treated by heat, proteinase K, SDS and other six methods were as follows: Figure 5The values ​​are 61.92%, 19.35%, 24.97%, 52.35%, 8.67%, and 39.16%, respectively. Heat treatment can usually destroy the spatial structure of most proteins (enzymes). The degradation effect of DON on the supernatant treated with 95 ℃ water bath and boiling water bath decreased significantly, indicating that the proteins or enzymes involved in degradation are heat-labile, and short-term heat treatment has a significant impact on them, easily destroying their structure. SDS, as a denaturant, destroys the spatial structure of proteins. The degradation effect of the supernatant after treatment was significantly reduced. Therefore, it is further inferred that Armillaria mellea secretes a protein-like active substance.

[0087] V. Study on the dynamic degradation of vomitoxin by Armillaria mellea

[0088] Degradation of DON by the strain: Liquid seed culture containing uniformly grown mycelial pellets in good condition was inoculated into the culture medium at an inoculum of 7.5%. DON standard working solution was added to achieve a DON concentration of 2 μg / mL in the medium, and the pH was adjusted to 5.8. The control group consisted of uninoculated screening medium. Each group was in triplicate and cultured at 27.5℃ and 160 rpm in the dark for 7 days. Fermentation broth was sampled every 12 h, with three replicates for each sample.

[0089] Depend on Figure 6 It can be seen that with the extension of fermentation time, the DON degradation rate showed a trend of first significantly increasing and then gradually decreasing. In the period of 0-144 h, the DON degradation rate showed an upward trend, reaching the highest value at 144 h, with a maximum DON degradation rate of 63.83%. After 144 h, the DON degradation rate began to gradually decrease, reaching 62.86% at 156 h and 63.08% at 168 h. Therefore, this study determined that the optimal fermentation time for Armillaria mellea to degrade DON was 144 h.

[0090] Enzyme activity assay of manganese peroxidase produced by fermentation strain:

[0091] (1) Plate color development test

[0092] Refer to the methods of Du Haiping et al.

[0093] Basic culture medium: potato 20%, glucose 2%, agar 2%, KH2PO4 0.3%, MgSO4·7H2O 0.15%, VB1 0.002%.

[0094] RB Brilliant Blue Assay: Add 0.003% RB Brilliant Blue to the basal medium and observe whether an orange-yellow ring appears in the medium.

[0095] Guaiacin medium: Add 0.1 g·L⁻¹ to the basal medium. -1Guaiacol (final concentration) was added to the plates and the appearance of a brownish ring around the hyphae was observed.

[0096] The extracellular manganese peroxidase of edible fungi can make the guaiacol culture medium produce a red-brown ring, and the production of peroxidase can appear an orange-yellow ring in RB bright blue. The orange-yellow ring produced by Armillaria mellea in the RB bright blue culture medium proves that there is production of peroxidase, and the manganese peroxidase (MnP) can oxidize guaiacol into tetra-ortho-methoxy benzene, which is red-brown. The results of the plate color reaction test show that Armillaria mellea can produce extracellular manganese peroxidase during fermentation.

[0097] (2) Determination of manganese peroxidase (MnP) enzyme activity

[0098] Refer to the method of Du Haiping et al. Preparation of crude enzyme solution: 2 mL of fermentation broth in the fermentation medium was centrifuged at 10000 r / min for 10 min to obtain the crude enzyme solution, which was used for the following enzyme activity determination experiments.

[0099] MnP enzyme activity determination: the reaction system was 3.4 mL of acetic acid-sodium acetate buffer solution (concentration of 200 mmol / L, pH value of 4.5), 0.1 mL of 6 mmol / L MnSO4 solution, 0.4 mL of crude enzyme solution and 0.1 mL of 1.6 mmol / L H2O2 solution, which was reacted at 37℃ for 3 min, and the absorbance value at 240 nm was determined.

[0100] One enzyme activity unit (U) oxidizes 1 μmol Mn 2+ per minute to become Mn 3+ . Each sample was operated in triplicate, and then the average value was taken.

[0101] By Figure 8It can be seen that the DON degradation rate in this experiment is basically consistent with the growth trend of MnP enzyme activity. With the increase of MnP enzyme activity produced by Armillaria mellea fermentation, the DON degradation rate also increases continuously. The MnP enzyme activity increases continuously with the extension of fermentation culture time before 144 h, and the highest value appears at 144 h, with the highest enzyme activity of 105.23 U / L. The MnP enzyme activity starts to decline after 144 h. With the extension of fermentation time, the DON degradation rate also gradually increases, and the degradation rate is the highest at 144 h, with the degradation rate of 63.83%. The degradation rate starts to decline steadily after 144 h, and the degradation rates at 156 h and 168 h are 62.86% and 63.08% respectively. At 36-60 h, the DON degradation rate and enzyme activity both increase significantly, and the DON degradation rate increases from 15.23% to 38.24%, and the MnP enzyme activity increases from 43.69 U / L to 72.34 U / L. Through Pearson correlation analysis of the correlation between the DON degradation rate and the MnP enzyme activity, the results show that at the 0.01 significant level, the DON degradation rate is significantly correlated with the MnP enzyme activity, with a correlation of 0.989, indicating that the degradation of Armillaria mellea on DON is related to manganese peroxidase.

[0102] Example 4: Experiment of Armillaria mellea Am-07-22 strain degrading zearalenone

[0103] The zearalenone ZEN solid standard was purchased from Qingdao Pribolab Biological Engineering Co., Ltd. The solid standard was diluted with methanol solution to prepare a ZEN standard stock solution with a concentration of 100 μg / mL, which was stored at -20°C in the dark.

[0104] I. Determination of the effect of Armillaria mellea on ZEN degradation

[0105] The prepared Armillaria mellea Am-07-22 secondary fermentation seed culture solution was broken and inoculated into 10 mL liquid medium containing 5 μg / mL of ZEN at an inoculation amount of 10%. Then the reaction system was oscillated in a constant temperature shaker at 27°C and 160 rpm / min for 7 d in the dark. 1 mL of supernatant was added with 1 mL of methanol solution for ZEN extraction, and then centrifuged at 10000 rpm / min for 10 min at low temperature. After filtration through a 0.22 μm filter membrane, the content of ZEN was detected using a high performance liquid chromatograph, and the degradation rate of ZEN was calculated. The calculation formula of ZEN degradation rate is as follows:

[0106]

[0107] Results: By adding 5 μg / mL of ZEN to the culture medium and using Armillaria mellea Am-07-22 strain for fermentation and degradation, the ZEN degradation rate was 63.83% after 7 d of fermentation, which was significantly higher than that of the control group without ZEN addition (0.00%). Figure 9The high performance liquid chromatogram of Armillaria mellea Am-07-22 shows that the degradation effect of Armillaria mellea Am-07-22 on 5 μg / mL ZEN is good, and the degradation rate can reach 73.83%.

[0108] II. Effect of ZEN concentration on degradation of Armillaria mellea Am-07-22

[0109] The prepared secondary fermentation seed culture solution of Armillaria mellea Am-07-22 was broken, inoculated into 10 mL liquid medium containing ZEN with concentrations of 2 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL and 15 μg / mL respectively, the inoculation amount was 10%, and each strain had 3 parallel samples, then the reaction system was oscillated in a constant temperature shaker at 27°C and 160 rpm / min for 7 days in the dark. After extracting ZEN, the content of zearalenone was detected by high performance liquid chromatograph, each group had three parallel samples, and the degradation rate of ZEN was calculated.

[0110] As shown in Figure 10 A, the degradation rates of Armillaria mellea Am-07-22 on ZEN with different concentrations are different, when the initial concentration is 2 μg / mL, the degradation rate can reach 97.32%, when the concentration of ZEN is 5 μg / mL, the degradation rate is 73.83%, and when the concentration of ZEN is 10 μg / mL and 15 μg / mL, the degradation rates are 49.47% and 32.55% respectively. It can be seen that the degradation rate decreases with the increase of the concentration of ZEN. But from Figure 10 B, it can be seen that when the initial concentration is 2 μg / mL, the degradation amount of ZEN is 19.46 μg, when the initial concentration is 5 μg / mL, the degradation amount of ZEN is 36.5 μg, and when the initial concentration is 10 μg / mL and 15 μg / mL, the degradation amount of ZEN is more than 48.82 μg, and the difference is not significant, so the degradation amount of ZEN by Armillaria mellea Am-07-22 gradually increases and tends to be stable. At low concentration, the initial content of ZEN is low, although the degradation rate is high, but the final degradation amount is still low, while at high concentration, the initial content of ZEN is high, even if the degradation rate decreases, but the degradation amount increases constantly, and finally gradually stabilizes to the upper limit of degradation.

[0111] III. Effect of culture time, temperature, pH and inoculation amount on degradation of ZEN by Armillaria mellea Am-07-22

[0112] The broken Armillaria Am-07-22 secondary seed liquid was inoculated into 10 mL liquid medium containing ZEN with a concentration of 5 μg / mL, pH was 4, 5, 6, 7, 8, 9, respectively, and the inoculation amount was 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, respectively. Then the reaction system was oscillated in a constant temperature shaker at 18℃, 21℃, 24℃, 27℃, 30℃, 33℃, 160 rpm / min in the dark for 1-12 d. The content of zearalenone was detected by high performance liquid chromatograph, each group had three parallel samples, and the degradation rate of ZEN was calculated.

[0113] From Figure 11 It can be seen from A that the degradation rate of ZEN by Armillaria Am-07-22 gradually increased with the extension of culture time (P<0.05), from the initial 1 d degradation rate of only 21.37% to 7 d of 73.83%. The degradation rate increased to about 80% from 8 d to 12 d, and the difference in degradation rate was not significant at this time, indicating that the degradation rate was the highest and tended to be stable from 8 d to 12 d.

[0114] From Figure 11 B can be known that in the range of 18℃-27℃, the degradation rate of ZEN by Armillaria Am-07-22 increased with the increase of temperature (P<0.05), from 39.92% at 18℃ to 73.83% at 27℃. However, in the condition of 27℃-33℃, the degradation rate decreased with the increase of temperature (P<0.05), and the degradation rate of ZEN at 33℃ was 45.52%. This shows that too high or too low temperature has a greater impact on the degradation of ZEN by Armillaria Am-07-22. In the unsuitable temperature conditions, the growth environment of Armillaria is poor, so the degradation rate of ZEN will also decrease.

[0115] From Figure 11 C can be known that in the range of pH value 4.0-9.0, with the increase of pH value, the degradation rate of ZEN by Armillaria Am-07-22 showed a trend of first increasing and then decreasing (P<0.05), and the degradation rate at pH 7.0 was the highest, reaching 75.22%. The degradation rates at pH 4.0 and 9.0 were only 45.89% and 61.97%, respectively. This shows that in the environment of too acid or too alkali, the degradation effect will be affected. In the strong acid or strong alkali culture conditions, the degradation of ZEN will be affected, and the degradation effect is the best in the environment of pH 7.0-8.0, and the degradation rate is the highest. This is similar to the research results of Tan et al.

[0116] From Figure 11As shown in D, the degradation rate of ZEN gradually increased with the increase of the inoculum amount of Armillaria mellea Am-07-22 (P<0.05). It eventually stabilized at an inoculum amount of 10% to 15%, at which point the degradation rate was highest, exceeding 73%. This indicates that Armillaria mellea Am-07-22 had already reached its maximum growth rate in this environment, and increasing the inoculum amount did not lead to an increase in the ZEN degradation rate.

[0117] IV. Distribution of ZEN-degrading active components by Armillaria mellea Am-07-22

[0118] Armillaria mellea Am-07-22 was re-inoculated into liquid seed culture medium and cultured for 6 days at 27℃ with shaking at 160 rpm / min. The obtained mycelium and fermentation supernatant were collected by filtration. The active components of the ZEN-degrading mycelium seed fermentation broth were located. The experiment was conducted in 3 groups, with a control group containing 5 μg / mL of ZEN. Three replicate samples were taken from each group for the experiment.

[0119] Following Jin Bowen's method, different groups were treated as follows: Group A (fermentation supernatant): The filtered upper fermentation broth was centrifuged at 4℃ and 10000 rpm / min for 10 min, and 1 mL of supernatant was collected. Group B (mycelium group): 1 g of the filtered lower mycelium was weighed and gently washed 3-5 times with PBS buffer. Group C (cell disruption group): 1 g of the filtered lower mycelium was weighed and gently washed 3-5 times with PBS buffer, and the cells were ultrasonically disrupted for 2 h (200W, 5 s on, 5 s off), then centrifuged at 10000 rpm / min for 10 min, and 1 mL of intracellular fluid was collected. The prepared experimental groups were added to diluted ZEN stock solution, with a ZEN concentration of 5 μg / mL. The reaction system was cultured at 27℃ and 160 rpm in the dark with shaking for 7 days.

[0120] from Figure 12 The results show that different components of *Armillaria mellea* Am-07-22 exhibit significant differences in their ZEN degradation effects (P<0.05). The fermentation supernatant showed the highest ZEN degradation rate at 47.42%, while the degradation rate of *Armillaria mellea* cells was 37.05%, and the degradation rate of ZEN by lysed cells was the lowest at only 13.08%. Therefore, it can be inferred that the fermentation supernatant plays a major role in ZEN degradation by *Armillaria mellea* Am-07-22, possibly due to some extracellular active substance, while the cells themselves also exhibit some degree of ZEN adsorption.

[0121] V. Preliminary Analysis of the Properties of ZEN-Degrading Active Substances by Armillaria mellea Am-07-22

[0122] Following Jing Siyuan's treatment method, the active substances were analyzed and treated in three groups: Group 1 (Protein K group): 5 mL of fermentation supernatant was mixed with 250 μL of proteinase K (final concentration 0.1 mg / mL) and treated at 55℃ for 2 h. Group 2 (SDS group): 5 mL of fermentation supernatant was mixed with 250 μL of SDS (final concentration 0.5 mg / mL) and treated at 55℃ for 2 h. Group 3 (Protein K + SDS group): 5 mL of fermentation supernatant was mixed with 250 μL of proteinase K (final concentration 0.1 mg / mL) and 250 μL of SDS (final concentration 0.5 mg / mL) and treated at 55℃ for 2 h. Group 4 (100℃ boiling water bath group): 5 mL of fermentation supernatant was treated in a boiling water bath at 100℃ for 20 min.

[0123] Four groups of fermentation supernatants after different treatments were filtered through a 0.22 μm filter membrane, and ZEN stock solution was added to them. The concentration of ZEN in the system was 5 μg / mL. The reaction system was incubated at 27℃ and 160 rpm in the dark with shaking for 7 days. After the reaction, the ZEN content was detected by high performance liquid chromatography and the degradation rate was calculated. Fermentation supernatant with an initial ZEN concentration of 5 μg / mL was used as a control group.

[0124] Depend on Figure 13 It was found that treatments of the *Armillaria mellea* fermentation supernatant with proteinase K, SDS, and boiling water bath had different effects on ZEN degradation (P<0.05). The degradation rates after proteinase K and SDS treatment were 12.65% and 22.89%, respectively, and the degradation effect of the combined proteinase K and SDS treatment was not significantly different from that of proteinase treatment alone. However, the degradation rate of ZEN after boiling water bath was only 5.55%, indicating a significant decrease in degradation capacity. Therefore, it can be further inferred that the main active substance in the *Armillaria mellea* Am-07-22 fermentation supernatant that degrades ZEN is an extracellular enzyme.

[0125] VI. Effects of fermentation time, pH, and metal ions on the degradation of ZEN in the fermentation supernatant of Armillaria mellea Am-07-22

[0126] Following and improving upon Wei Jinfan's method, fermentation supernatant of *Armillaria mellea* Am-07-22 was collected. A ZEN standard solution with a concentration of 5 μg / mL was added, and the reaction was carried out with shaking at 27℃ and 160 rpm. Samples were taken at reaction times of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days to measure the changes in ZEN content and calculate the degradation rate. The effect of different reaction times on ZEN degradation in the fermentation supernatant of *Armillaria mellea* Am-07-22 was analyzed. Additionally, the pH of the fermentation supernatant was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10, and a 10 mmol Na₂SO₄ solution was added.+ , K + , Mg 2+ , Zn 2+ , Cu 2 + , Fe 2+ , Fe 3+ , Mn 2+ Metal ions, to which the concentration of 5 μg / mL ZEN standard solution was added, were oscillated under the conditions of 27℃ and 160 rpm for 7d, and the reaction of the crude enzyme solution of the fermentation supernatant without the addition of metal ions was used as a control, with three repeats in each group. After the reaction, 1 mL of the reaction solution was added with an equal volume of methanol solution for the extraction of zearalenone, and after being fully shaken, low-temperature centrifugation was performed at 10000 rpm / min for 10 min, and then filtration was performed through a 0.22 μm filter membrane, and the content of zearalenone was detected using a high-performance liquid chromatograph, and the degradation rate of ZEN was calculated. The effects of different pH values and different metal ions on the degradation of ZEN by the fermentation supernatant of Armillaria Am-07-22 were analyzed.

[0127] As can be seen from Figure 14 A, with the extension of the fermentation time, the degradation rate of ZEN by the fermentation supernatant of Armillaria Am-07-22 gradually increased (P<0.05), from 21.69% on the 1st day to 62.50% on the 10th day, and the enzyme activity of the laccase produced by Armillaria also gradually increased, from 169.26 U / L on the 1st day to 521.11 U / L on the 10th day, and the enzyme activity of the laccase continuously increased (P<0.05) during this period, which was consistent with the increase of the degradation rate of ZEN.

[0128] As can be seen from Figure 14 B, different pH values had a greater effect on the degradation rate of ZEN by the fermentation supernatant of Armillaria Am-07-22. Among them, when the pH was 6-7, the degradation rate of ZEN was the highest, being 46.98% and 51.84%. There was no significant difference between the two and the fermentation supernatant without pH adjustment. However, under acidic and alkaline conditions, the degradation rate of ZEN was lower (P<0.05), being only 8.29% and 11.22% at pH 3 and 10. It was shown that under the conditions of excessive acid or alkali, the enzyme produced by the fermentation supernatant was more strongly destroyed, resulting in the inability to effectively degrade ZEN.

[0129] As can be seen from Figure 14 C, different metal ions had different effects on the degradation of ZEN by the fermentation supernatant of Armillaria Am-07-22. Among them, Na + , K + and Fe 2+The addition of Zn2+, Fe3+ had no obvious effect on the degradation of ZEN by the fermentation supernatant. The degradation rates of ZEN were 42.09% and 37.76%, respectively, and the inhibitory effect of Fe3+ was the most obvious, which was 9.66% lower than the control. 2+ 3+ The addition of Zn2+, Fe3+ had a certain inhibitory effect on the degradation of ZEN (P<0.05), and the degradation rates of ZEN were 42.09% and 37.76%, respectively. The inhibitory effect of Fe3+ was the most obvious, which was 9.66% lower than the control. 3+ 2+ The addition of Cu2+, Mg2+, and Mn2+ enhanced the effect of the fermentation supernatant on the degradation of ZEN (P<0.05). The degradation rates of ZEN were 55.57% and 57.80% when Mg2+ and Mn2+ were added, respectively. The degradation effect of the fermentation supernatant on ZEN was the highest when Cu2+ was added, which was 63.37%, and it was 15.95% higher than the control. It can be inferred that Cu2+ can significantly enhance the enzyme activity of laccase. 2+ 2+ 2+ 2+ 2+ 2+

[0130] Example 5 Application of Armillaria mellea Am-07-22 Strain in the Degradation of Mycotoxins

[0131] 1. Artificially infected corn husks and corn meal: According to the test method of Dou Yong et al., 50 μg / mL of DON solution diluted with sterile water was added to the test raw materials (corn husks and corn meal), and then mixed uniformly to prepare corn husks and corn meal with DON contents of 5 μg / g, 7.5 μg / g, 10 μg / g, and 12.5 μg / g, respectively.

[0132] 2. Degradation experiment

[0133] Degradation test of Armillaria mellea solid-state and liquid-state fermented corn husks: The ratio of solid-state material (corn husks) to liquid volume of the culture medium was 1:2.5 and 1:5, respectively. The fermentation culture was carried out at a culture temperature of 27.5°C and a pH value of 5.8. Corn husks without fermentation were set as the control group.

[0134] Degradation test of Armillaria mellea solid-state and liquid-state fermented corn meal: The ratio of solid-state material (corn meal) to liquid volume of the culture medium was 1:1 and 1:2.5, respectively. The fermentation culture was carried out at a culture temperature of 27.5°C and a pH value of 5.8. Corn meal without fermentation was set as the control group.

[0135] Results: From the results of DON degradation by different fermentation methods, it can be seen that Figure 15 ​​​​​​​​, the DON degradation rate of solid-state fermentation and liquid fermentation of Armillaria mellea in the application research of degrading DON in corn bran was 89.48% and 76.33%, respectively, and the effect of solid-state fermentation was better; while in the application research of degrading DON in corn gluten meal, the DON degradation rate of solid-state fermentation and liquid fermentation of Armillaria mellea was 46.26% and 74.68%, respectively, and the effect of liquid fermentation was better. Therefore, the degradation effect of Armillaria mellea on DON in two different fermentation raw materials was studied, and the degradation effect was good when corn bran was fermented by solid-state fermentation and corn gluten meal was fermented by liquid fermentation. Corn gluten meal, corn bran and other corn deep processing by-products are rich in bioactive components such as protein and dietary fiber. Previous laboratory studies have confirmed that edible fungi have high biological conversion capacity for these active components, greatly expanding the application range of these processing by-products.

[0136] DON degradation test of Armillaria mellea solid-state fermentation of corn bran with different DON concentrations: corn bran was added to the culture medium at a solid-liquid ratio of 1:2.5, and the DON concentration in the culture medium was 2, 3, 4, and 5 μg / g, the culture medium pH was 5.8, and the solid-state fermentation culture was carried out at a culture temperature of 27.5℃. At the same time, corn bran without fermentation was set as the control group.

[0137] In the application research of Armillaria mellea solid-state fermentation of corn bran, the effect of fermentation of corn bran with different concentrations on the DON degradation rate was as follows Figure 16 , the DON degradation rate of Armillaria mellea on corn bran with different toxin concentrations was 89.48%, 85.24%, 87.93%, and 73.35%. When the DON concentration was 2, 3, and 4 μg / g, the degradation rate of Armillaria mellea on DON tended to be stable, and the degradation effect was best when the DON concentration was 2 μg / g. However, when the DON concentration was increased to 5 μg / g, the DON degradation rate decreased, and the degradation effect decreased. At a higher concentration, DON could inhibit the growth of strain Am-07-22, and could not normally metabolize active substances that could degrade DON.

[0138] DON degradation test of Armillaria mellea liquid-state fermentation of corn gluten meal with different DON concentrations: corn gluten meal was added to the culture medium at a solid-liquid ratio of 1:2.5, and the DON concentration in the culture medium was 2, 3, 4, and 5 μg / mL, the culture medium pH was 5.8, and the liquid-state fermentation culture was carried out at a culture temperature of 27.5℃ and a rotation speed of 160 rpm. At the same time, corn gluten meal without fermentation was set as the control group.

[0139] Results: In the application research of Armillaria mellea liquid-state fermentation of corn gluten meal, the effect of fermentation of corn gluten meal with different concentrations on the DON degradation rate was as follows Figure 17The degradation rates of Armillaria mellea on corn meal with different concentrations of DON were 74.68%, 75.98%, 57.75%, 56.46%. With the increase of toxin concentration, the degradation rate showed a downward trend. There was no significant difference in the degradation effect of Armillaria mellea on DON at the concentration of 2 and 3 μg / g and at the concentration of 4 and 5 μg / g, and the degradation effect was the best at the concentration of 3 μg / g. With the increase of toxin concentration, DON inhibited the growth of Armillaria mellea, and could not normally metabolize to produce active substances to degrade DON, so the degradation effect decreased.

Claims

1. Application of Armillaria in degrading zearalenone; characterized in that: the Armillaria is Armillaria Am-07-22, and the preservation number is CCTCC NO: M 20221044; the zearalenone is degraded by culturing the Armillaria in a culture solution containing zearalenone.

2. Use according to claim 1, characterized in that: Cu was added to the culture medium 2+ .

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