Strain for degrading zearalenone and application thereof

Abstract

The invention relates to a strain for degrading zearalenone and application of the strain, and belongs to the technical field of microbial remediation. The invention provides bacillus subtilis W001 for degrading zearalenone, the Latin name of the bacillus subtilis W001 is bacillus subtilis W001, the bacillus subtilis W001 is preserved in China Center for Type Culture Collection, the preservation date is August 14, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241782. The bacillus subtilis W001 disclosed by the invention can be used for remarkably relieving mouse organ index change caused by the ZEA and improving liver injury, immune function decline and reproductive organ injury caused by the ZEA, and the degradation rate of the ZEA reaches 97.25%.

Description

Technical Field

[0001] The invention relates to the technical field of microbial remediation, and in particular to a strain for degrading zearalenone and application thereof. Background Art

[0002] Zearalenone (ZEA), also known as F-2 toxin, is a hormone-like fungal toxin produced by Fusarium oxysporum. It was first discovered in moldy corn. Most of the zearalenone pollutants are agricultural crops, especially corn and wheat, which are the most seriously polluted. Mold is most common in warm and humid places. Every year, a large number of crops are contaminated by mold around the world, especially in southern my country, where the warm and humid climate is suitable for mold growth, resulting in a large amount of economic losses every year due to mold contamination.

[0003] Mycotoxins are secondary metabolites of molds that can cause acute or chronic poisoning to humans and animals, causing damage to tissues such as the liver, kidneys, testicles, and uterus. Among them, zearalenone is one of the mycotoxins with a consistently high detection rate in crops and their by-products. Zearalenone has an estrogen-like effect, promoting the body's estrogen secretion, affecting the reproductive function of animals, causing redness and swelling of the labia, miscarriage, and degeneration and atrophy of ovarian tissue in female animals, and testicular atrophy, reduced sperm motility, and increased deformity rates in male animals. As a mycotoxin, zearalenone inevitably has toxic effects on important organs such as the liver and kidneys. Studies have shown that zearalenone can significantly increase the weight of the liver and kidneys, causing fatty and vacuolar degeneration in the liver, and tubular degeneration and glomerular atrophy in the kidneys.

[0004] Mycotoxins are widely contaminated and difficult to control. In order to reduce the impact of mycotoxins on agricultural production and life, researchers have been constantly exploring and looking for the most effective and suitable detoxification technology in recent years. Although physical adsorption and chemical methods can effectively remove most toxins from crops and their by-products, the adsorbent method will reduce the nutritional value of the feed, and the chemical method will have residual chemicals, neither of which is the best choice for feed detoxification.

[0005] Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a strain for degrading zearalenone, so as to solve the problem that the degradation efficiency of zearalenone in the prior art feed is low and affects the feed quality.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a Bacillus subtilis W001 capable of degrading zearalenone. The Latin name of the Bacillus subtilis W001 is Bacillus subtilis W001, which is deposited in China Center for Type Culture Collection on August 14, 2024, at Wuhan University, Wuhan, China, and has a deposit number of CCTCC NO: M 20241782.

[0009] The present invention provides the use of the Bacillus subtilis W001 in preparing a product for degrading mycotoxins.

[0010] The invention provides the use of the Bacillus subtilis W001 in preparing a product for degrading zearalenone.

[0011] The present invention provides a zearalenone biodegradation agent, comprising the Bacillus subtilis W001;

[0012] The number of viable bacteria of Bacillus subtilis W001 in the zearalenone biodegradation agent is 0.5 to 1.5×10 9 CFU / mL.

[0013] The present invention provides a method for preparing the zearalenone biodegradation agent, comprising the following steps:

[0014] (1) inoculating the Bacillus subtilis W001 into LB solid culture medium and culturing at 35-39° C. for 10-14 hours to obtain an activated strain;

[0015] (2) inoculating the activated strain obtained in step (1) into LB liquid culture medium and culturing at 35-39° C. for 10-14 h to obtain a seed solution;

[0016] (3) The seed solution obtained in step (2) is inoculated into LB liquid culture medium, and cultured at 35-39° C. for 10-14 h to obtain a zeaxanthin biodegradation agent.

[0017] Preferably, the rotation speed of the culture in step (1) and step (2) is independently 160 to 200 rpm.

[0018] Preferably, the seed liquid inoculation amount in step (3) is 4-6% of the volume of the LB liquid culture medium.

[0019] The present invention also provides the use of the zearalenone biodegradation agent or the zearalenone biodegradation agent prepared by the preparation method in degrading mycotoxins.

[0020] The present invention also provides the use of the zearalenone biodegradation agent or the zearalenone biodegradation agent prepared by the preparation method in degrading zearalenone and / or alleviating the toxicity of zearalenone to animal organisms.

[0021] The present invention also provides a method for degrading zearalenone in moldy feed, comprising mixing the moldy feed with a zearalenone biodegradation agent, and reacting the mixture at a temperature of 35 to 39° C. and a pH value of 6.9 to 7.1.

[0022] The zearalenone biodegradation agent is the zearalenone biodegradation agent described above or the zearalenone biodegradation agent prepared by the preparation method described above.

[0023] The present invention has the following technical effects and advantages:

[0024] The invention provides a Bacillus subtilis W001 capable of degrading zearalenone. The Bacillus subtilis W001 can significantly alleviate the changes in organ indexes of mice caused by ZEA, improve liver damage, decreased immune function and reproductive organ damage caused by ZEA, and the degradation rate of ZEA reaches 97.25%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the degradation rate of ZEA by 13 bacterial strains;

[0026] Figure 2 The colony and staining diagram of the strain labeled 2. Figure 2 A1 is the colony picture. Figure 2 A2 is the coloring picture;

[0027] Figure 3 These are the testicular sections of mice in different treatment groups. Figure A is for group I, Figure B is for group II, Figure C is for group III, and Figure D is for group VI.

[0028] Figure 4 is the curve of ZEA concentration changing with degradation time;

[0029] Figure 5 is the ZEA concentration in the reaction mixture at different degradation times;

[0030] Figure 6 is the concentration of β-ZOL in the reaction mixture at different degradation times;

[0031] Figure 7 is the concentration of α-ZOL in the reaction mixture at different degradation times;

[0032] Figure 8 is the ZAN concentration in the reaction mixture at different degradation times;

[0033] Fig. 9 is the concentration of β-ZAL in the reaction mixture at different degradation times;

[0034] Fig.10 is the concentration of α-ZAL in the reaction mixture at different degradation times.

[0035] Collection Instructions

[0036] The Latin name of Bacillus subtilis W001 is Bacillus subtilis W001, which is deposited in China Center for Type Culture Collection on August 14, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: M 20241782. DETAILED DESCRIPTION

[0037] The invention provides a Bacillus subtilis W001 capable of degrading zearalenone. The Latin name of the Bacillus subtilis W001 is Bacillus subtilis W001, which is deposited in China Center for Type Culture Collection on August 14, 2024, at Wuhan University, Wuhan, China, and has a deposit number of CCTCC NO: M 20241782.

[0038] The present invention provides the use of the Bacillus subtilis W001 in preparing a product for degrading mycotoxins.

[0039] The invention provides the use of the Bacillus subtilis W001 in preparing a product for degrading zearalenone.

[0040] The present invention provides a zearalenone biodegradation agent, comprising the Bacillus subtilis W001;

[0041] The number of viable bacteria of Bacillus subtilis W001 in the zearalenone biodegradation agent is 0.5 to 1.5×10 9 CFU / mL, preferably 1×10 9 CFU / mL.

[0042] The present invention provides a method for preparing the zearalenone biodegradation agent, comprising the following steps:

[0043] (1) inoculating the Bacillus subtilis W001 into LB solid culture medium and culturing at 35-39° C. for 10-14 h to obtain an activated strain, wherein the culturing temperature is preferably 37° C. and the culturing time is preferably 12 h;

[0044] (2) inoculating the activated strain obtained in step (1) into LB liquid culture medium, and culturing at 35-39° C. for 10-14 h to obtain a seed solution, wherein the culturing temperature is preferably 37° C., and the culturing time is preferably 12 h;

[0045] (3) The seed solution obtained in step (2) is inoculated into LB liquid culture medium and cultured at 35-39° C. for 10-14 h to obtain a zeaxenone biodegradation agent. The culture temperature is preferably 37° C. and the culture time is preferably 12 h.

[0046] In the present invention, the rotation speed of the culture in step (1) and step (2) is independently 160 to 200 rpm, preferably 180 rpm.

[0047] In the present invention, the seed liquid inoculation amount in step (3) is 4-6% of the volume of the LB liquid culture medium, preferably 5%.

[0048] In the present invention, the formula of the LB solid culture medium is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0049] In the present invention, the formula of the LB liquid culture medium is 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0050] The present invention also provides the use of the zearalenone biodegradation agent or the zearalenone biodegradation agent prepared by the preparation method in degrading mycotoxins.

[0051] The present invention also provides the use of the zearalenone biodegradation agent or the zearalenone biodegradation agent prepared by the preparation method in degrading zearalenone and / or alleviating the toxicity of zearalenone to animal organisms.

[0052] The present invention also provides a method for degrading zearalenone in moldy feed, comprising mixing the moldy feed with a zearalenone biodegradation agent, and reacting at a temperature of 35 to 39° C. and a pH value of 6.9 to 7.1, wherein the temperature is preferably 37° C. and the pH value is preferably 7;

[0053] The zearalenone biodegradation agent is the zearalenone biodegradation agent described above or the zearalenone biodegradation agent prepared by the preparation method described above.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1: Screening of strains

[0056] (1) Materials

[0057] Source of bacteria: The bacteria are preserved in the Clinical Veterinary Laboratory of Anhui Agricultural University. The 13 strains were isolated from the intestinal contents of Dabie Mountain yellow cattle. After preliminary identification, they were numbered: 9 strains of Bacillus were numbered 1-9, and 4 strains of lactic acid bacteria were numbered 10-13.

[0058] Medium preparation

[0059] LB solid medium: add 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride to 950 ml of deionized water, adjust the pH to 7.0 with 1 M sodium hydroxide solution, make up to 1 L, then add 15 g of agar powder, sterilize under high pressure at 121°C for 20 min, cool to 50-60°C, invert the plate, and store at 4°C for later use.

[0060] LB liquid culture medium: add 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride to 950 ml of deionized water, adjust the pH to 7.0 with 1 M sodium hydroxide solution, make up to 1 L, sterilize at 121°C for 20 min, and store at 4°C for later use.

[0061] MRS solid culture medium: add 10 g of peptone, 10 g of beef powder, 10 g of yeast powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of diammonium hydrogen citrate, 2 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate and 15 g of agar to 1 L of deionized water, resuspend and dissolve, adjust the pH to 5.7, add 1 mL of Tween 80, sterilize at 121°C for 15 min, and store at 4°C for later use.

[0062] MRS liquid culture medium: take 10g of peptone, 10g of beef powder, 10g of yeast powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 2g of sodium acetate, 0.2g of magnesium sulfate and 0.04g of manganese sulfate, add to 1L of deionized water, resuspend and dissolve, adjust the pH to 5.7, add 1mL of Tween 80, sterilize at 121℃ for 15min, and store at 4℃ for later use.

[0063] (2) Screening of strains

[0064] Preparation of bacterial solution

[0065] Take the strains numbered 1 to 9, streak them on LB solid culture medium plates with an inoculation loop, and place the 9 culture mediums in a 37°C constant temperature incubator for overnight culture. Pick a single colony on the LB solid culture medium and inoculate it in LB liquid culture medium, and culture it in a constant temperature shaker incubator at 37°C, 180r / min for 12h to obtain seed solutions No. 1 to 9, inoculate the seed solutions No. 1 to 9 in the LB liquid culture medium, and the inoculation amount of the seed solution inoculated in the LB culture medium is 5% of the volume of the LB culture medium. After inoculation, culture it in a constant temperature shaker incubator at 37°C, 180r / min for 12h to obtain bacterial solutions No. 1 to 9.

[0066] Take the strains numbered 10 to 13, streak them on MRS solid culture medium plates with an inoculation loop, pick single colonies on the MRS solid culture medium and inoculate them into MRS liquid culture medium, culture them in a constant temperature shaking incubator at 37° C., 180 r / min for 12 h to obtain seed solutions No. 10 to 13, inoculate the seed solutions No. 10 to 13 into MRS liquid culture medium, the inoculation amount of the seed solution inoculated into the MRS liquid culture medium is 5% of the volume of the culture medium, and after inoculation, culture them in a constant temperature shaking incubator at 37° C., 180 r / min for 12 h to obtain bacterial solutions No. 10 to 13.

[0067] Preparation of working solution

[0068] Preparation of ZEA stock solution: Dissolve 25 mg of ZEA standard (Sigma, USA) in 5 mL of dimethyl sulfoxide in a brown glass bottle to prepare a ZEA stock solution with a concentration of 5 mg / mL.

[0069] Preparation of ZEA working solution: Take 10 μL of ZEA stock solution and add 990 μL of normal saline to prepare 50 μg / mL ZEA working solution.

[0070] Test methods

[0071] A blank control group and a test group were set up. In the test group, 120 μL of ZEA working solution was added to 3.88 mL of No. 1 to No. 13 bacterial solution to make the final concentration 1.5 μg / mL, and the solution was shaken to ensure that the bacterial solution and the toxin were fully mixed. In the blank control group, 120 μL of ZEA working solution was added to 3.88 mL of LMRs liquid culture medium and LB liquid culture medium, and the solution was shaken to ensure that the bacterial solution and the toxin were fully mixed. Three parallel groups were set up in each group, and all were cultured at 37°C and 180 r / min for 24 hours. After the culture was completed, the ZEA concentration was determined by fluorescent immunochromatography. The determination method was as follows:

[0072] The experimental group and blank control group of the test sample were restored to room temperature; 1.5mL EP centrifuge tubes were taken out and marked according to the names of the test groups, and divided into two groups: extraction tubes and dilution tubes; the rapid fluorescence quantitative analyzer was taken out, turned on, and long-pressed the high-sensitivity fluorescence power button (the light was on); the mobile phone Weiruiyun detection APP was started; the QR code on the test kit was scanned to obtain the standard curve online; a working directory was established in the software grouping document, and the corresponding standard curve was selected (the instrument has a built-in standard curve, which can save time during operation, speed up the detection speed, and avoid errors caused by time differences); the sample was added, and the sample to be tested was fully shaken in the test tube, 1mL of the sample to be tested was taken into the 1.5mL EP tube, and centrifuged at 3500r / min for 15min to separate the supernatant. 380μL of the dilution solution was taken into the dilution tube, and then 20μL of the supernatant to be tested was fully shaken and mixed to ensure that the toxins in the solution were evenly distributed, and then 80μL of the sample to be tested was taken with a pipette and added to the corresponding numbered reagent card sample well. Incubate the immune system in a dark place for 15 minutes for immune reaction. For testing, insert the reagent card into the high-sensitivity fluorescence analyzer and click the "test" button to instantly display and store the mold test value. The test environment temperature is 18-30℃. Temperature that is too high or too low will affect the result.

[0073] The ZEA degradation rate of the test group was calculated using the following formula:

[0074] Degradation rate (%) = [(toxin content in control group - toxin content in experimental group) / toxin content in experimental group] × 100%

[0075] The ZEA degradation rates of the 13 bacterial strains were calculated according to the formula. The results are as follows: Figure 1 As shown in the figure, the horizontal axis represents the strain number, and the vertical axis represents the ZEA degradation rate (%).

[0076] according to Figure 1 It can be seen that the ZEA degradation rate of the strain labeled 2 is 97.25%, which is significantly higher than that of other strains.

[0077] (3) Identification of bacterial species

[0078] Colony morphology identification

[0079] Streak the strain numbered 2 on LB solid culture medium and culture it in a constant temperature incubator at 37°C for 24 hours to grow a single colony. Select a plate with good growth status from multiple LB solid culture plates and observe its morphological characteristics, such as Figure 2 As shown in A1; the operation was carried out in a clean bench, a single colony was picked out in a sterile environment, diluted with saline, spread, fixed, Gram-stained, and examined under a microscope. The results were as follows Figure 2 As shown in A2. Figure 2A1 shows that the colony of the strain labeled 2 is larger, with a rough and irregular surface, wrinkles, and multiple ridges. The colony color is grayish white or slightly yellowish. Figure 2 A2 shows that after Gram staining, the strain is observed under a microscope to be rod-shaped and stained blue-purple, indicating that it is a Gram-positive bacterium.

[0080] Molecular bioassay

[0081] The strain numbered 2 was inoculated into a liquid culture medium, and grown overnight at 37°C at 180 r / min. 2 mL of the bacterial solution was dispensed into sterile EP tubes and sent to Beijing Qingke Biotechnology Co., Ltd. for 16SrDNA sequencing. The sequencing results were compared using BLAST in NCBI, and the 16SrDNA sequence was compared for homology in the database. The final result was that the homology between the strain numbered 2 and Bacillus subtilis reached 99%, so the strain numbered 2 was named Bacillus subtilis W001 and was deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20241782.

[0082] Example 2: Preparation of zeaxenone biodegradation agent

[0083] (1) The Bacillus subtilis W001 preserved in Example 1 was inoculated into LB solid medium and cultured at 37° C. and 180 rpm for 12 h to obtain an activated strain;

[0084] (2) inoculating the activated strain obtained in step (1) into LB liquid culture medium, and culturing at 37° C. and 180 rpm for 12 h to obtain a seed solution;

[0085] (3) The seed solution obtained in step (2) was inoculated into LB liquid culture medium at an inoculation amount of 5% of the volume of the LB liquid culture medium, and then cultured at 37°C for 12 h to obtain a viable bacterial count of 10 9 CFU / mL of michelinone biodegradation agent for future use.

[0086] Example 3: In vivo experiments in mice

[0087] 1. Materials and Methods

[0088] 1.1 Experimental animal grouping

[0089] Seventy-two healthy Kunming male mice aged 5 weeks were randomly divided into 4 groups, with 3 replicates in each group. The grouping is shown in Table 1.

[0090] Table 1 Test groups

[0091]

[0092] 1.2 Preparation of animal test solution

[0093] ZEA solution preparation

[0094] Dissolve 25 mg of ZEA standard (Sigma, USA) in 1 mL of dimethyl sulfoxide in a brown glass bottle to prepare a 25 mg / mL stock solution, store it at -20°C away from light for later use, return it to room temperature before gavage, and dilute it with normal saline to a concentration of 40 mg / L.

[0095] 1.3 Animal husbandry and management

[0096] Before the experiment, all mouse cages, drinking bottles and other supplies were disinfected with 75% alcohol or Sanisol, sprayed, wiped or soaked for disinfection. The ambient temperature in the mouse room was maintained at 25±1℃ to ensure a clean environment and regular disinfection of the room. During the experiment, mice were free to eat and drink, the bedding was changed every 3 days, and the mice were observed regularly. After one week of pre-feeding, gavage was performed every morning at 9:00 am starting from the second week according to the different requirements of each group for 3 consecutive weeks.

[0097] 1.4 Sample collection

[0098] The experimental period was 21 days, and the mice were weighed and sampled on an empty stomach at 9:00 am on the 22nd day. After the mice were anesthetized, blood was collected from the eyeballs. To prevent hemolysis, the whiskers on both sides of the mice were cut off, and the blood was collected in a 1.5 mL EP tube. After being placed at 4°C for 2 hours, the serum was separated and stored at -20°C for later use.

[0099] Mice were killed by cervical dislocation, dissected, and observed for obvious lesions in organs. The testicles on both sides were separated, excess fat was removed, and the testicles were rinsed with saline and then dried with filter paper. The testicles were weighed and fixed in 4% paraformaldehyde solution. The spleen and pancreas were removed, rinsed with saline, dried, and weighed. The organ index was calculated using the following formula:

[0100] Organ index (%) = (organ mass / body mass) × 100%

[0101] 2. Analysis of in vivo test results

[0102] 2.1 Mouse organ index

[0103] The organ indexes of mice in each group were calculated respectively. The results are shown in Table 2. In the same column of the table, the same or no superscript letters indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0104] Table 2 Organ indexes of liver, spleen and testis of mice treated with different methods

[0105] Group Liver index (%) Spleen index (%) Testicular index (%) Group I 5.29±0.47 <![CDATA[0.23±0.04 b ]]> <![CDATA[0.58±0.07 b ]]> Group II 4.98±0.18 <![CDATA[0.31±0.07 ab ]]> <![CDATA[0.82±0.04 a ]]> Group III 4.88±0.29 <![CDATA[0.28±0.07 ab ]]> <![CDATA[0.84±0.03 a ]]> Group IV 4.52±0.26 <![CDATA[0.27±0.05 ab ]]> <![CDATA[0.81±0.16 a ]]>

[0106] Liver organ index: There was no statistically significant difference in liver index among the groups (P>0.05), but the liver index of group I showed an upward trend compared with groups II, III, and IV.

[0107] Organ index of spleen: The spleen index of group I was significantly lower than that of group IV (P<0.05), while the spleen index of groups II and III was not significantly different from that of group IV (P>0.05).

[0108] Testicular organ index: The testicular index of group IV was significantly higher than that of group I (P<0.05), while there was no significant difference in the testicular index of groups II and III compared with group IV (P>0.05).

[0109] 2.2 Mouse serum biochemical parameters

[0110] The serum collected from the sample was taken out of the -20℃ refrigerator, thawed at room temperature and mixed, and used immediately to avoid repeated freezing and thawing affecting the test results. The detection kit (Changchun Huili Biotechnology Co., Ltd.) was used according to the instructions of the S-220 automatic biochemical analyzer to measure the serum biochemical indicators of mice treated with different methods. The results are shown in Table 3. In the same column of the table, the same or no superscript letters indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different capital letters indicate extremely significant difference (P<0.01).

[0111] Table 3 Serum biochemical parameters of mice treated with different

[0112]

[0113] According to the results in Table 3, after oral administration of toxin, the serum ALT, AST, and ALP activities of group I were significantly higher than those of group VI in the control group (P<0.05), and there were no significant differences in the serum ALT, AST, and ALP activities of groups II and III compared with those of group VI (P>0.05). The serum TP, ALB, and GLB contents of group I were significantly lower than those of group VI in the control group (P<0.05), and there were no significant differences in the serum TP contents of groups II and III compared with those of group VI (P>0.05); the serum ALB content of group II was not significantly different from that of group VI (P>0.05), and the serum ALB content of group III was significantly higher than that of group I (P<0.05); the serum GLB content of groups II, III, and IV was significantly higher than that of group I (P<0.05).

[0114] 2.3 Content of cellular inflammatory factors

[0115] ELISA kits were used to detect the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in serum. The results are shown in Table 4. In the same column of the table, the same or no superscript letters indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0116] Table 4 Serum inflammatory cytokine levels in mice treated with different methods

[0117] Group IL-1β (mg / mL) IL-6 (mg / mL) TNF-α(mg / mL) Group I <![CDATA[173.54±9.2 Aa ]]> <![CDATA[151.10±32.74 Aa ]]> <![CDATA[726.47±52.53 Aa <!-- 7 -->]]> Group II <![CDATA[94.36±5.16 Dd ]]> <![CDATA[105.3±3.8 CDbc ]]> <![CDATA[414.67±14.18 Cc ]]> Group III <![CDATA[120.8±6.72 Cc ]]> <![CDATA[117.01±8.6 BCb ]]> <![CDATA[561.42±45.23 Bb ]]> Group IV <![CDATA[87.34±6.03 Dd ]]> <![CDATA[86.54±5.42 Dc ]]> <![CDATA[417.33±22.59 Cc ]]>

[0118] According to the results in Table 4, the serum IL-1β and IL-6 levels in group I were significantly higher than those in group IV (P<0.01), and there were no significant differences in the serum IL-1β and IL-6 levels in group II compared with group IV (P>0.05). The serum IL-1β level in group III was higher than that in group IV and lower than that in group I (P<0.01), and the serum IL-6 level in group III was significantly lower than that in group I (P<0.01). The serum TNF-α level in group I was significantly higher than that in groups II, III, and IV (P<0.01), and there was no significant difference in the serum TNF-α level in group II compared with group IV (P>0.05).

[0119] 2.4 Immunoglobulin content

[0120] ELISA kits were used and the instructions were followed to detect the levels of immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) in serum. The results are shown in Table 5. In the same column of the table, the same or no superscript letters indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0121] Table 5 Serum immunoglobulin levels in mice treated with different

[0122]

[0123]

[0124] According to Table 5, the IgA content of groups I and II was significantly higher than that of group IV (P<0.01), and there was no significant difference between group III and group IV (P>0.01). The IgG content of group I was not significantly different from that of group IV (P>0.05), and it was significantly higher in group II than that of group IV (P<0.01), and there was no significant difference between group III and group IV (P>0.05). The IgM content of groups I, II, and III was significantly higher than that of group IV (P<0.01).

[0125] 2.5 Reproductive hormone levels

[0126] ELISA kits were used to detect the levels of male hormones testosterone (T) and estradiol (E2) in serum according to the instructions. The results are shown in Table 6. In the same column of the table, the same or no superscript letters indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0127] Table 6 Serum hormone levels of mice treated with different

[0128] Group T(nmol / mL) <![CDATA[E2(nmol / mL)]]> Group I <![CDATA[20.92±1.38 Dd ]]> <![CDATA[73.32±5.16 Aa ]]> Group II <![CDATA[33.12±2.35 Aa ]]> <![CDATA[42.08±2.99 De ]]> Group III <![CDATA[28.87±1.74 Bb ]]> <![CDATA[53.82±3.29 Cc ]]> Group IV <![CDATA[34.95±1.49 Aa ]]> <![CDATA[38.33±2.07 De ]]>

[0129] According to Table 6, the serum testosterone content of group I was significantly lower than that of group IV (P<0.01), the content of group II did not change significantly compared with group IV (P>0.05), and the content of group III was significantly higher than that of group I (P<0.01). The serum estradiol content of group I was significantly higher than that of group IV (P<0.01), the content of group II was not significantly different from that of group IV (P>0.05), and the content of group III was significantly lower than that of group I (P<0.01).

[0130] 2.6 Oxidation and Antioxidation Indexes of Testicular Tissue

[0131] ELISA kits were used to detect catalase (CAT) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in testicular tissue according to the instructions. The results are shown in Table 7. In the same column of the table, the same or no superscript letters indicate no significant difference (P>0.05), different lowercase letters indicate significant difference (P<0.05), and different uppercase letters indicate extremely significant difference (P<0.01).

[0132] Table 7 Oxidative and antioxidative indexes of testicular tissues in mice treated with different methods

[0133]

[0134]

[0135] According to the results in Table 7, the MDA content in the testicles of group I after gavage with the toxin ZEA was significantly higher than that of the control group VI (P<0.01), the MDA content in the testicles of group II did not change significantly compared with group VI (P>0.05), and the MDA content in the testicles of group III was significantly lower than that of group I (P<0.01).

[0136] The testicular SOD activity of group I was significantly lower than that of group VI (P<0.01), the testicular SOD activity of group II was not significantly changed compared with group VI (P>0.05), and the testicular SOD activity of group III was significantly higher than that of group I (P<0.01).

[0137] The testicular CAT activity of group I was significantly lower than that of group VI (P<0.01), the testicular CAT activity of group II was not significantly different from that of group VI (P>0.05), and the testicular CAT activity of group III was significantly higher than that of group I (P<0.01).

[0138] 2.7 Pathological changes of testicular tissue

[0139] Remove the mouse testis from the 4% paraformaldehyde fixative during sample collection, cut it horizontally, rinse the residual fixative on the tissue with running water in the embedding box, rinse overnight, and then proceed as follows:

[0140] (1) Place the slices in a drying oven at 66°C for 25 min;

[0141] (2) Pass through xylene three times, 5 min each time;

[0142] (2) Pass through ethanol three times (100%-95%-80%), each time for 3 minutes;

[0143] (4) Place the slices in a beaker and rinse slowly with running water to remove the ethanol until the slices are clean and transparent;

[0144] (5) Slice and soak in hematoxylin for 3 min and rinse with running water;

[0145] (6) Dissolve with 1% hydrochloric acid and alcohol for a few seconds and rinse with running water;

[0146] (7) The saturated lithium carbonate solution turns blue for a few seconds and is then rinsed with running water;

[0147] (8) Dehydrate the slices in 95% ethanol for 2 min, shaking up and down 3 times;

[0148] (9) Immerse the sections in eosin stain (alcohol soluble) for 8 seconds;

[0149] (10) Pass through 95% ethanol twice, each time for 8 seconds;

[0150] (11) Dehydration by two passes of 100% ethanol, each pass for 1 min;

[0151] (12) Pass through phenol-xylene, xylene I, and xylene II in sequence, each time for 2 min;

[0152] (13) Seal the slides with neutral gum, air-dry and fix, and observe under a microscope. The results are as follows: Figure 3 shown.

[0153] according to Figure 3 It can be seen that in group I, a large number of spermatogenic cells and supporting cells in the testis were arranged loosely and disorderly, or even fell off, with a small number of cells, a large number of vacuoles between the supporting cells and spermatogonia, and the thickness of the seminogenic epithelium was significantly thinned; in groups IV and II, it can be observed that the seminogenic tubules are intact, and the spermatogenic cells and supporting cells of all levels are arranged neatly in the tubules with clear layers and regular order, and mature sperm can be clearly seen in the tubules; compared with group I, the thickness of the seminogenic epithelium in group III was significantly thickened, and the number of supporting cells and spermatogenic cells increased significantly; the thickness of the seminogenic epithelium in group III was thinner than that in group IV.

[0154] 3. ZEA degradation time and product analysis

[0155] 3.1 ZEA degradation time

[0156] Take 120 μL of the ZEA solution prepared in "1.2 Preparation of Animal Test Solution" and 3.88 mL of the zearalenone biodegradation agent, mix the two, shake well, and culture at 37°C and 180 rpm. Use a fast fluorescence quantifier with a range of 50-2000 ppb (μg / L) to detect the ZEA concentration every 8 hours to obtain a curve of the change of ZEA concentration with degradation time. The results are as follows: Figure 4 shown.

[0157] according to Figure 4 It can be seen that the initial ZEA concentration was greater than 2000μg / L, which was higher than the maximum range of the rapid fluorescence quantifier; after 16 hours of mixed culture, the ZEA concentration dropped to less than 50μg / L, which was lower than the minimum range of the rapid fluorescence quantifier, and the ZEA concentration was always lower than 50μg / L during the 16-64 hours of reaction with the bacterial solution; it rose again to 96μg / L at 72 hours, and the final ZEA concentration was 959μg / L. The increase in ZEA concentration after 72 hours may be due to the change in culture medium conditions due to the long culture time, resulting in the conditions for the degradation reaction not being met, resulting in degradation failure; or due to the limitations of the fluorescent immunochromatography method, the generated ZEA derivative was mistakenly detected as ZEA.

[0158] 3.2 Analysis of ZEA degradation products

[0159] Take 120 μL of the ZEA solution prepared in "1.2 Preparation of animal test solution" and 3.88 mL of zearalenone biodegradation agent, mix the two, shake well, and incubate at 37°C at 180 rpm. Use ultra-performance liquid phase tandem mass spectrometry (UPLC-MS) to detect the mixed solution at 24 h, 48 h, 72 h, 96 h and 120 h. The concentrations of ZEA and ZEA derivatives are detected at the corresponding time. The results are as follows: Figures 5 to 10 shown.

[0160] according to Figures 5 to 10 It can be seen that with the extension of degradation time, the concentrations of ZEA, β-ZOL, α-ZOL, ZAN, and β-ZAL in the degradation products gradually increased, and the concentration of α-ZAL increased first and then decreased. The concentrations of β-ZOL, α-ZOL, ZAN, and β-ZAL reached the highest level at 120h, which were 1.37978μg / L, 0.74381μg / L, 1.67596μg / L, and 0.91349μg / L, respectively; the concentration of α-ZAL reached the highest level at 48h, which was 0.15059μg / L.

[0161] It can be seen from the above embodiments that the present invention provides a Bacillus subtilis W001 for degrading zearalenone. The Bacillus subtilis W001 in the present invention can significantly alleviate the changes in organ indexes of mice caused by ZEA, improve liver damage, decreased immune function and reproductive organ damage caused by ZEA, and the degradation rate of ZEA reaches 97.25%.

[0162] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Bacillus subtilis W001 for degrading zearalenone, characterized in that: The Latin name of the Bacillus subtilis W001 is Bacillus subtilis W001, which is deposited in the China Center for Type Culture Collection on August 14, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: M 20241782.

2. Use of the Bacillus subtilis W001 according to claim 1 in preparing a product for degrading mycotoxins.

3. Use of the Bacillus subtilis W001 according to claim 1 in preparing a product for degrading zearalenone.

4. A zearalenone biodegradation agent, characterized in that: Comprising the Bacillus subtilis W001 of claim 1; The number of viable bacteria of Bacillus subtilis W001 in the zearalenone biodegradation agent is 0.5 to 1.5×10 9 CFU / mL.

5. The method for preparing the zearalenone biodegradation agent according to claim 4, characterized in that: The steps include: (1) inoculating the Bacillus subtilis W001 described in claim 1 into LB solid culture medium and culturing at 35-39° C. for 10-14 h to obtain an activated strain; (2) inoculating the activated strain obtained in step (1) into LB liquid culture medium and culturing at 35-39° C. for 10-14 h to obtain a seed solution; (3) The seed solution obtained in step (2) is inoculated into LB liquid culture medium, and cultured at 35-39° C. for 10-14 h to obtain a zeaxanthin biodegradation agent.

6. The preparation method according to claim 5, characterized in that: The rotation speed of the culture in step (1) and step (2) is independently 160 to 200 rpm.

7. The preparation method according to claim 5, characterized in that: The seed liquid inoculation amount in step (3) is 4-6% of the volume of the LB liquid culture medium.

8. Use of the zearalenone biodegradation agent according to claim 4 or the zearalenone biodegradation agent prepared by the preparation method according to any one of claims 5 to 7 in degrading mycotoxins.

9. Use of the zearalenone biodegradation agent according to claim 4 or the zearalenone biodegradation agent prepared by the preparation method according to any one of claims 5 to 7 in degrading zearalenone and / or alleviating the toxicity of zearalenone to animal organisms.

10. A method for degrading zearalenone in moldy feed, characterized in that: The moldy feed is mixed with the zeaxenone biodegradation agent, and reacted at a temperature of 35 to 39° C. and a pH value of 6.9 to 7.1; The zearalenone biodegradation agent is the zearalenone biodegradation agent according to claim 4 or the zearalenone biodegradation agent prepared by the preparation method according to any one of claims 5 to 7.

Images