Bacillus amyloliquefaciens and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens and application thereof. The invention provides bacillus amyloliquefaciens HW28, the preservation number of the bacillus amyloliquefaciens HW28 is CGMCC (China General Microbiological Culture Collection Center) No.32090. The bacillus amyloliquefaciens HW28 provided by the invention is obtained by screening and domesticating from soil of a corn field, and can be used for efficiently degrading zearalenone. Through verification, the bacillus amyloliquefaciens HW28 can grow by using the zearalenone as the unique carbon source, so that the zearalenone can be effectively degraded. When the concentration of the ZEN is 100 mu g / L-5000 mu g / L, the degradation rate of the bacillus amyloliquefaciens HW28 on the ZEN reaches 94% or above, and the degradation rate is high. Therefore, the bacillus amyloliquefaciens HW28 disclosed by the invention has a good degradation effect on zearalenone and a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus amyloliquefaciens and application thereof. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is an endocrine-disrupting fungal estrogen produced by Fusarium fungi and widely found in corn, wheat and other grains and their processed products around the world. This toxin poses a serious threat to animal and human health due to its strong estrogenic activity. After oral administration, ZEN is rapidly absorbed and metabolized into α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). These metabolites and their parent ZEN can cause morphological and functional disorders of the reproductive system of livestock and humans, including reproductive disorders in female animals, sperm abnormalities in male animals, and fetal malformations. In addition, ZEN may also cause dysfunction of the animal digestive system, causing significant economic losses to the animal husbandry industry.

[0003] Faced with the widespread contamination of ZEN, traditional detoxification methods mainly include physical detoxification and chemical detoxification. Physical detoxification methods such as high temperature treatment, radiation treatment, and the use of adsorbents can reduce the content of ZEN to a certain extent, but there are problems such as high treatment costs, large nutrient loss, and complex operations. Chemical detoxification methods such as the use of chemical reagents such as alkali solution and ozone can effectively destroy the toxicity of ZEN, but may introduce new chemical pollutants, posing a potential threat to the environment and animal health.

[0004] With the rapid development of biotechnology, biological detoxification has gradually attracted attention due to its advantages such as high efficiency, no toxic byproducts, and environmental friendliness. Among them, the degradation of ZEN by the biotransformation of microorganisms has become a research hotspot. Microorganisms can transform ZEN into low-toxic or non-toxic products through metabolism, thereby achieving the degradation of ZEN. Compared with traditional methods, biological detoxification has the advantages of simple operation, low cost, and no destruction of nutrients.

[0005] As a widely existing probiotic, Bacillus has strong environmental adaptability and metabolic diversity, and is a potential biomaterial for degrading fungal toxins such as ZEN. Studies have shown that some Bacillus strains can convert ZEN into non-toxic or low-toxic substances by secreting specific enzymes or metabolites. However, there are still limited Bacillus strains on the market that can effectively degrade ZEN. The degradation rate of ZEN by Bacillus is only about 80% when the degradation days are 5 days, and the degradation efficiency is low. Therefore, screening and optimizing Bacillus strains with the ability to efficiently degrade ZEN is of great significance for the development of new ZEN detoxification technology. Summary of the invention

[0006] The purpose of the present invention is to provide a Bacillus amyloliquefaciens strain and an application thereof. The Bacillus amyloliquefaciens HW28 provided by the present invention can quickly and efficiently degrade zearalenone with a high degradation rate.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The invention provides a strain of Bacillus amyloliquefaciens HW28, whose deposit number is CGMCC No.32090.

[0009] The present invention provides a bacterial agent, characterized in that it comprises the Bacillus amyloliquefaciens HW28 of the above technical solution.

[0010] Preferably, the application form of the Bacillus amyloliquefaciens HW28 includes culture solution or metabolic solution.

[0011] Preferably, the method for preparing the culture solution comprises the following steps: inoculating the Bacillus amyloliquefaciens HW28 into a culture medium for culturing to obtain a culture solution;

[0012] The preparation method of the metabolic solution comprises: centrifuging the culture solution to obtain the supernatant, which is the metabolic solution.

[0013] The present invention provides the use of the Bacillus amyloliquefaciens HW28 described in the above technical solution or the bacterial agent described in the above technical solution in preparing a product that degrades zearalenone and / or improves the degradation rate of zearalenone.

[0014] Preferably, when the application form of the Bacillus amyloliquefaciens HW28 is a culture solution, the inoculation amount of the culture solution is 0.5% to 20%, and the effective viable count of the Bacillus amyloliquefaciens HW28 in the culture solution is ≥1×10 9 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume mass ratio of the metabolic liquid to the product to be degraded containing zearalenone is 1mL:(5-200)g.

[0015] Preferably, the degradation temperature is 20-37° C.; the degradation time is 4-15 days; and the degradation pH is 7.0-8.0.

[0016] Preferably, the product comprises one or more of feed additives, food additives and detoxifying agents.

[0017] The present invention provides a method for degrading zearalenone, comprising the following steps: mixing the Bacillus amyloliquefaciens HW28 described in the above technical solution or the bacterial agent described in the above technical solution with a substance to be degraded containing zearalenone for degradation.

[0018] Preferably, when the application form of the Bacillus amyloliquefaciens HW28 is a culture solution, the effective viable cell count after the culture solution is mixed with the material to be degraded containing zearalenone is ≥1×10 7 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume mass ratio of the metabolic liquid to the product to be degraded containing zearalenone is 1mL:(5-200)g.

[0019] Beneficial effects of the present invention: The present invention provides a strain of Bacillus amyloliquefaciens HW28, with a deposit number of CGMCC No. 32090. The Bacillus amyloliquefaciens HW28 provided by the present invention is screened and domesticated from corn field soil, and can efficiently degrade zearalenone. It has been verified that Bacillus amyloliquefaciens HW28 can grow using zearalenone as the sole carbon source to achieve efficient degradation of zearalenone. When the concentration of ZEN is 100 μg / L to 5000 μg / L, the degradation rate of ZEN by Bacillus amyloliquefaciens HW28 reaches more than 94%, and the degradation rate is as high as 99%. It can be seen that the Bacillus amyloliquefaciens HW28 of the present invention can efficiently degrade and metabolize zearalenone, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony morphology of Bacillus amyloliquefaciens HW28;

[0021] Figure 2 This is a Gram staining microscope observation of Bacillus amyloliquefaciens HW28;

[0022] Figure 3 This is a positive control result diagram of the zearalenone test card in Example 1;

[0023] Figure 4 Negative control result diagram of the zearalenone test card of Example 1;

[0024] Figure 5 HW28 phylogenetic tree;

[0025] Figure 6 The peak time of ZEN toxin detected by LC-MS;

[0026] Figure 7It is the degradation rate of ZEN under different conditions of Example 3, wherein the ordinate "Degradability" represents the degradation rate, the abscissa "Method" represents three different degradation methods: CFE (intracellular), ADS (adsorption), CFS (extracellular), Concentration of ZEA represents the toxin concentration, INoculation Rate represents the inoculation rate, Temperature represents the temperature, Time represents the time, and PH represents the acidity;

[0027] Figure 8 is the response surface curve of ZEN toxin degradation in Example 3;

[0028] Fig. 9 This is a graph showing the detoxification effect of ZEN in feed after adding strain HW28;

[0029] Fig.10 This is the detection chart of zearalenone in blank feed without added zearalenone toxin.

[0030] Biological Deposit Description

[0031] Bacillus amyloliquefaciens HW28 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on September 27, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32090. DETAILED DESCRIPTION

[0032] The present invention provides a strain of Bacillus amyloliquefaciens HW28, with a deposit number of CGMCC No. 32090. The 16s rRNA gene sequence of the Bacillus amyloliquefaciens HW28 of the present invention is shown in SEQ ID No.1.

[0033] SEQ ID No.1:

[0034]

[0035] The present invention uses cornfield soil samples as raw materials, uses a selective culture medium containing ZEN as the sole carbon source for preliminary screening, and then uses a ZEN rapid detection kit to detect the culture medium, evaluates the strain's ability to degrade ZEN, and isolates a strain of Bacillus amyloliquefaciens HW28. Bacillus amyloliquefaciens HW28 can achieve rapid and efficient degradation of zearalenone, and Bacillus amyloliquefaciens HW28 is a non-pathogenic Bacillus, which can be used to prepare a detoxifying agent for zearalenone, has certain application value in solving the problem of ZEN pollution, and provides a new solution for food safety and animal husbandry development.

[0036] The present invention uses the 16s rRNA sequence of Bacillus amyloliquefaciens HW28 to perform BLAST homologous sequence search in NCBI, constructs a phylogenetic tree with sequences with high homology, and determines that the strain HW28 is Bacillus amyloliquefaciens.

[0037] The present invention provides a bacterial agent, comprising the Bacillus amyloliquefaciens HW28 described in the above technical solution.

[0038] As an optional embodiment, the application form of the Bacillus amyloliquefaciens HW28 of the present invention includes culture solution or metabolic solution.

[0039] As an optional embodiment, the method for preparing the culture solution of the present invention comprises the following steps: inoculating the Bacillus amyloliquefaciens HW28 into a culture medium for culturing to obtain a culture solution. As an optional embodiment, the present invention does not specifically limit the method of inoculation, and a conventional method may be used. As an optional embodiment, the culture temperature of the present invention is 20°C to 37°C, or 28 to 35°C; in a specific embodiment of the present invention, the culture temperature is 20, 25, 28, 30, 33, 34, 35, 36 or 37°C. The culture time is 1 to 2 days. In a specific embodiment of the present invention, the culture time is 1, 1.5 or 2 days. The culture medium used for the culture of the present invention includes LB liquid culture medium. As an optional embodiment, the effective viable count of Bacillus amyloliquefaciens HW28 in the culture solution is ≥1×10 9 CFU / mL.

[0040] As an optional embodiment, the preparation method of the metabolic solution comprises: centrifuging the culture solution obtained by the preparation method described in the above technical solution, and the obtained supernatant is the metabolic solution. The centrifugal speed of the present invention is 800-1200rpm, or 1000rpm; the centrifugal time is 1-2min. The metabolic solution contains a substance produced by Bacillus amyloliquefaciens HW28 for degrading zearalenone.

[0041] The present invention provides the use of the Bacillus amyloliquefaciens HW28 described in the above technical solution or the bacterial agent described in the above technical solution in preparing a product that degrades zearalenone and / or improves the degradation rate of zearalenone.

[0042] As an optional implementation, the application includes: applying the Bacillus amyloliquefaciens HW28 described in the above technical solution or the bacterial agent described in the above technical solution to a material to be degraded containing zearalenone for degradation.

[0043] As an optional embodiment, the product of the present invention comprises one or more of feed additives, food additives and detoxifying agents. The present invention has no special limitation on the preparation method of the product, and conventional methods can be used.

[0044] As an optional embodiment, the effective viable count of Bacillus amyloliquefaciens HW28 in the material to be degraded containing zearalenone of the present invention is ≥1×10 7 CFU / mL. As an optional embodiment, the degradation temperature is 20-37°C, or 25-35°C. The degradation pH is 7-8; the degradation time is 4-15d, or 5-10d.

[0045] The present invention provides a method for degrading zearalenone, comprising the following steps: mixing the Bacillus amyloliquefaciens HW28 described in the above technical solution or the bacterial agent described in the above technical solution with a substance to be degraded containing zearalenone for degradation.

[0046] As an optional implementation, the mixing method is not particularly limited and a conventional method may be used.

[0047] As an optional embodiment, the object to be degraded containing zearalenone includes feed and / or food containing zearalenone. In a specific embodiment of the present invention, corn is used as a feed model to verify the effect of Bacillus amyloliquefaciens HW28 in degrading zearalenone.

[0048] As an optional embodiment, the mass concentration of zearalenone in the degradable material containing zearalenone is 1 to 5000 μg / L, or 100 to 5000 μg / L. In a specific embodiment of the present invention, the concentration of zearalenone in the culture medium is 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L and 5000 μg / L for effect verification. When the concentration of zearalenone is greater than 5000 μg / L, it can also be degraded, but the degradation efficiency may be reduced and the degradation time is prolonged, because Bacillus amyloliquefaciens HW28 can use zearalenone as the sole carbon source for growth.

[0049] As an optional embodiment, when the application form of the Bacillus amyloliquefaciens HW28 is a culture solution, the effective viable cell count after the culture solution is mixed with the material to be degraded containing zearalenone is ≥1×10 7 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume mass ratio of the metabolic liquid to the product to be degraded containing zearalenone is 1mL:(5-200)g.

[0050] As an optional embodiment, the degradation temperature is 20-37° C., further 25-35° C., and more preferably 30° C. The degradation time is 4-15 days, further 7-13 days; and the degradation pH value is 7-8.

[0051] The present invention provides a method for degrading zearalenone, comprising the following steps: mixing the Bacillus amyloliquefaciens HW28 described in the above technical solution or the bacterial agent described in the above technical solution with a substance to be degraded containing zearalenone for degradation.

[0052] As an optional embodiment, the mixing method is not particularly limited, and a conventional method can be used. In the present invention, the material to be degraded containing zearalenone includes feed and / or food containing zearalenone, and can also be feed containing zearalenone. In an embodiment of the present invention, the degradation effect of Bacillus amyloliquefaciens HW28 is verified using feed containing zearalenone.

[0053] As an optional embodiment, the mass concentration of zearalenone in the degradable material containing zearalenone is 1 to 5000 μg / L, or 100 to 5000 μg / L. In a specific embodiment of the present invention, the concentration of zearalenone in the culture medium is 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L and 5000 μg / L for effect verification. When the concentration of zearalenone is greater than 5000 μg / L, it can also be degraded, but the degradation efficiency may be reduced and the degradation time is prolonged, because the strain Bacillus amyloliquefaciens HW28 can use zearalenone as the sole carbon source for growth.

[0054] As an optional embodiment, when the application form of the Bacillus amyloliquefaciens HW28 is a culture solution, the effective viable cell count after the culture solution is mixed with the material to be degraded containing zearalenone is ≥1×10 7 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume mass ratio of the metabolic liquid to the product to be degraded containing zearalenone is 1mL:(5-200)g, or can be 1mL:(15-100)g.

[0055] As an optional implementation, the degradation temperature is 20-37° C., or 25-35° C.; the degradation time is 4-15 days, or 7-13 days; and the degradation pH value is 7-8.

[0056] The pH value, temperature, culture time, and mass volume ratio of Bacillus amyloliquefaciens HW28 to zearalenone are set in the present invention to improve the degradation rate of zearalenone by Bacillus amyloliquefaciens HW28.

[0057] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0058] The composition of the amylase screening medium is: 10g / L starch, 3g / L beef extract, 10g / L peptone, 5g / L NaCl and 15g / L agar.

[0059] The composition of artificial gastric juice is: 16.4 mL of dilute hydrochloric acid and 10 g / L of pepsin; the preparation method of dilute hydrochloric acid is to dilute 1 mol / L HCl with water to pH=1.5.

[0060] The composition of artificial intestinal fluid is: 6.8g / L potassium dihydrogen phosphate and 10g / L pancreatic enzyme.

[0061] The composition of LB solid medium is: 10g / L NaCl, 10g / L peptone, 5g / L yeast powder and 15g / L agar. The agar in LB semi-solid medium is 7.5g / L, and the concentrations of other components remain unchanged.

[0062] Columbia blood agar plates: purchased from Guangdong Huankai Microbiological Technology Co., Ltd., catalog number: CP0160.

[0063] Gelatin medium (g / L): 5g / L NaCl, 10g / L peptone, 3g / L beef extract and 120g / L gelatin.

[0064] The composition of MSM medium is: 2g / L (NH4)2SO4, 0.2g / L MgSO4·7H2O, 0.01g / LCaCl2·2H2O, 0.001g / LFeSO4·7H2O and 1.5g / LNa2HPO4·12H2O.

[0065] The composition of MM medium is: 2g / L (NH4)2SO4, 0.2g / LMgSO4·7H2O, 0.01g / L CaCl2, 0.001g / LFeSO4, 0.002g / LMnCl2, 4.5g / LK2HPO4, 2g / L mannitol and 2g / L glycerol.

[0066] Example 1 Screening of strains capable of degrading zearalenone toxin

[0067] 1. The culture medium was prepared with zearalenone as the sole carbon source, that is, different concentrations of zearalenone were directly added to the commercially available MSM culture medium, and the obtained zearalenone-MSM culture medium was recorded as culture medium 1 to 6, respectively. The zearalenone concentration in culture medium 1 was 5 μg / L, the zearalenone concentration in culture medium 2 was 10 μg / L, the zearalenone concentration in culture medium 3 was 20 μg / L, the zearalenone concentration in culture medium 4 was 40 μg / L, the zearalenone concentration in culture medium 5 was 80 μg / L, and the zearalenone concentration in culture medium 6 was 100 μg / L.

[0068] Soil samples were taken from corn fields in Qujing City, Yunnan Province, and were used to screen strains that can degrade zearalenone toxin (hereinafter referred to as toxin).

[0069] Add 5 g of soil sample to medium 1 and culture for 7 days to obtain culture 1 solution;

[0070] The culture 1 solution was inoculated into the culture medium 2 at an inoculum amount of 10% of the volume of the culture medium, and then cultured for 7 days to obtain the culture 2 solution;

[0071] The culture 2 solution was inoculated into the culture medium 3 at an inoculation amount of 10% of the culture medium volume, and then cultured for 7 days to obtain the culture 3 solution; the culture media 1 to 6 were used in sequence until the culture 5 solution was inoculated into the culture medium 6 at an inoculation amount of 10% of the culture medium volume, and then cultured for 7 days to obtain the strain screening solution;

[0072] The strain screening solution was diluted 10-fold in a gradient manner until the strain screening solution was diluted to 10 -1 ,…10 -5 , …, 10 -8 Each gradient dilution was spread on LB medium plates for culture, and a single clone colony was picked to obtain a strain, which was recorded as strain HW28. The colony morphology of strain HW28 is shown in Figure 1 , HW28 Gram staining microscope observation diagram is shown in Figure 2 It can be seen that the colony morphology of strain HW28 is white, round and convex, and the colonies are connected and wrinkled. It is a Gram-positive bacterium with rod-shaped and spore morphology.

[0073] 2. Prepare the culture medium with zearalenone (ZEN) as the sole carbon source, that is, add ZEN to the MM culture medium, and the ZEN concentration in the MM culture medium is 100 μg / L.

[0074] The strain HW28 was inoculated into MM medium and cultured for 4-5 days to obtain the culture fluid, and finally the ZEN toxin concentration in the culture fluid was determined using the zearalenone test card. The test card was negative and the ZEN concentration was less than 60 μg / L, indicating that the ZEN toxin concentration was reduced and the strain HW28 could effectively degrade the toxin.

[0075] 3. Screening using the zearalenone detection card. Prepare ZEN into a ZEN solution, add the ZEN solution to the LB liquid culture medium, then inoculate the monoclonal colony of strain HW28 into the LB culture medium, wait for it to grow in the culture medium for a period of time to obtain a culture solution, and use the zearalenone detection card to detect ZEN in the culture solution.

[0076] The test process is as follows: tear open the aluminum foil packaging bag of the test card, take out the test card, and place it on a flat, clean surface. Use the matching pipette to absorb the prepared sample liquid to be tested, and slowly, drop by drop (avoiding the generation of foam) add 3 drops (about 60μL) to the sample well (S). Place it at room temperature for 8 to 10 minutes to judge the result.

[0077] If red lines appear at both C (control line) and T (test line) on the test card, it proves that the ZEN toxin has been degraded to below the test standard (60μg / L), and the result is negative; if a red line only appears at C (control line) on the test card, it means that the toxin concentration is still higher than the test standard (60μg / L), and the result is positive; if there is no red line at C (control line), the test is invalid.

[0078] LB liquid culture medium containing 100 μg / L zearalenone toxin was used as a positive control. The positive control results of the culture medium obtained from the zearalenone test card are shown in Figure 3 LB liquid culture medium containing 10 μg / L zearalenone toxin was used as negative control. The negative control results of the zearalenone test card are shown in Figure 4 .according to Figure 3 It can be seen that the toxin concentration is still higher than 60μg / L. Figure 4 It can be seen that the toxin concentration is lower than 60μg / L, which means that the test results of the zearalenone test card are accurate.

[0079] 4. Use a DNA extraction kit to extract the genome of strain HW28. The sample is the culture fluid obtained by culturing strain HW28 in steps 2 and 3. Use 16S universal primers to amplify and sequence the 16S rRNA target gene of the strain. The sequencing results are compared with Blast on NCBI to identify the species of the strain. The phylogenetic tree of strain HW28 is shown in Figure 5 ,according to Figure 5 It was found that strain HW28 was Bacillus amyloliquefaciens.

[0080] Example 2 Study on the physiological and biochemical characteristics of the strain that degrades zearalenone toxin

[0081] The strain HW28 was inoculated into LB liquid medium and cultured at 37°C for 24 h to obtain HW28 bacterial liquid, which was used in the following experiments.

[0082] (1) Amylase production test: The strain HW28 was spotted onto the surface of an amylase screening solid culture medium and incubated at 37°C for 24 h. After colonies grew, Lu's iodine solution was added to cover the entire plate and the appearance of a transparent circle was observed. The size of the colonies and the transparent circle was measured with a caliper to evaluate the amylase activity. The experiment was repeated three times.

[0083] (2) Experiment on resistance to artificial gastrointestinal fluid: The activated bacterial strain HW28 was inoculated into artificial intestinal fluid at a volume ratio of 5% and cultured statically at 37°C for 3 h.

[0084] The activated bacterial strain HW28 was inoculated into artificial gastric fluid at a volume ratio of 5%, and statically cultured at 37°C for 4 hours.

[0085] After the artificial gastric juice and artificial intestinal juice culture was completed, 100 μL of the obtained liquid was taken for gradient dilution. After dilution, the liquids were evenly spread on LB solid culture medium, inverted and cultured at 37°C for 24 h. The number of live bacteria of each bacteria was counted by the plate colony counting method, and the survival rate was calculated.

[0086] Strain survival rate (%) = lgN1 / lgN2×100, wherein N1 is the number of viable bacteria after culture in artificial intestinal fluid or artificial gastric fluid, N2 is the number of viable bacteria without treatment with artificial intestinal fluid or artificial gastric fluid, and the unit of viable bacteria is CFU / mL; the experiment was repeated three times independently.

[0087] (3) Acid tolerance test: First, adjust the pH of the LB medium to 2.0, 3.0, and 4.0. Then, add the HW28 bacterial solution to the LB medium with different pH values, and culture at 37°C for 5 h. Take 100 μL of the treated HW28 bacterial solution and spread it on the LB solid medium, and culture at 37°C for 24 h. Use the untreated strain HW28 bacterial solution as a control, select 30 to 300 colonies for counting, and repeat the experiment three times.

[0088] (4) Hemolytic activity assay: The HW28 bacterial suspension was streaked onto the surface of Columbia blood agar medium and cultured overnight at 37°C to observe whether a transparent hemolytic ring appeared around the colonies. The experiment was repeated three times.

[0089] (5) Catalase activity test: The catalase activity test was performed using a catalase kit purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKAO003-1M.

[0090] (6) Anaerobic experiment: A carbon dioxide incubator, a common incubator and an oxygen incubator were used to observe and compare the growth of the bacteria in Example 1.

[0091] (7) Drug-sensitive paper test: Take 100 μL of HW28 bacterial solution and mix it with 5 mL of semi-solid agar LB medium, spread it evenly on the surface of the solid medium, and wait for the medium to solidify before use. Use sterilized tweezers to pick up drug-sensitive paper strips, spread them on the surface of the medium, and culture them at 37°C for 24 hours. Observe whether there is an inhibition zone around the drug-sensitive paper strips, and use a caliper to measure the diameter (d) of the inhibition zone to evaluate the drug resistance of the strain.

[0092] (8) Gelatin liquefaction test: The bacterial strain of Example 1 was activated, 100 μL of bacterial solution was spread on the gelatin culture medium, and then inverted in a 4°C refrigerator for 7 days. If the gelatin can be removed intact after 7 days, it proves that the bacteria has no gelatin liquefaction ability; if the gelatin is easily broken or obviously softened after 7 days, it proves that the bacteria has gelatin liquefaction ability.

[0093] The results of steps (1) to (8) are shown in Table 1. It can be seen that Bacillus amyloliquefaciens HW28 is not acid-resistant, cannot colonize in artificial gastric juice, has hydrogen peroxide and amylase activity, has no gelatin liquefaction ability, the number of viable bacteria under anaerobic conditions is higher than that under normal conditions, the hemolytic property is γ-hemolytic (insoluble in blood), and is sensitive to most antibiotics.

[0094] Table 1 Study on the strain characteristics of Bacillus amyloliquefaciens

[0095] characteristic result characteristic result Acid resistance (pH 4.0) - Artificial intestinal fluid (pH 6.8) + Acid resistance (pH 3.0) - Amylase + Acid resistance (pH 2.0) - Gelatin liquefaction - Artificial gastric juice (pH 1.5) - Anaerobic + <![CDATA[Catalase activity (U / 10 4 cell)]]> + Hemolytic gamma hemolysis Gentamycin (120 μg / tablet) ++ Ciprofloxacin (5 μg / tablet) ++ Ampicillin (10μμg / tablet) + Chloramphenicol (30 μg / tablet) ++ Co-trimoxazole (25 μg / tablet) + Lincomycin (2 μg / tablet) - Erythromycin (15 μg / tablet) ++ Tetracycline (30 μg / tablet) - Ceftriaxone (30 μg / tablet) ++ Penicillin (10 μg / tablet) +

[0096] Acid resistance and artificial gastric juice "+" means that the survival rate of the bacteria is higher than 90%; "-" means that the survival rate of the bacteria is lower than 90%.

[0097] Enzyme activity "+" means that the enzyme has this activity; "-" means that the enzyme does not have this activity.

[0098] Antibiotics “++” indicates particularly sensitive (inhibition zone >= 28 mm); “+” indicates sensitive (28>inhibition zone >= 14 mm);

[0099] “-” indicates insensitive (14mm>inhibition zone).

[0100] Example 3 Optimization of the Zearalenone Toxin Degradation System

[0101] 1. Add ZEN after preparing it into ZEN solution.

[0102] 2. Bacillus amyloliquefaciens HW28 was inoculated into LB liquid medium and cultured at 37°C for 1 day to obtain HW28 bacterial solution. The effective viable count of HW28 bacterial solution was ≥1×10 9 CFU / mL; used in the following experiments.

[0103] 3. Liquid chromatography-mass spectrometry (LC-MS) conditions are as follows:

[0104] LC-MS was performed in accordance with the national standard GB 5009.209-2016 “National Food Safety Standard for the Determination of Zearalenone in Foods”.

[0105] LC-MS detection was performed using commercially available ZEN as a standard. The peak time of ZEN is shown in Figure 6 , the ZEN peak time is 2.622min, the ZEN standard curve is y=931.661916×x, R 2 =0.99190923, where y represents the peak area and x represents the ZEN concentration in μg / L.

[0106] 4. Degradation rate (SC, %) = (SO-ST) / SO*100

[0107] Wherein: SO: peak area of ​​ZEN standard solution at standard concentration, ST: peak area of ​​ZEN in the treatment group.

[0108] The specific measurement process is as follows:

[0109] (1) Extracellular substance degradation experiment (abbreviated as extracellular): The HW28 bacterial solution was centrifuged (centrifuged at room temperature for 1 min at 1000 rpm). The supernatant was taken and ZEN solution was added to make the ZEN concentration in the supernatant 100 μg / L. The supernatant was kept in the dark for 5 days and the toxin concentration was determined by LC-MS. The results are shown in Table 2 and Figure 7 .

[0110] (2) Intracellular substance degradation experiment (abbreviated as intracellular): The HW28 bacterial solution was centrifuged (centrifuged at room temperature for 1 min at 1000 rpm). The supernatant was removed, sterile water was added to the bacteria, and the cells were ultrasonically disrupted to obtain a cell disruption solution. The cell disruption solution was filtered using a 0.22 μm filter membrane to obtain a bacterial intracellular substance solution. ZEN solution was added to make the ZEN concentration in the bacterial intracellular substance solution 100 μg / L. The solution was kept away from light for 5 days, and the toxin concentration was determined using LC-MS. The results are shown in Tables 2 and Figure 7 .

[0111] (3) Cell adsorption experiment (abbreviated as adsorption): ZEN solution was added to the HW28 bacterial solution to make the ZEN concentration in the bacterial solution 100 μg / L. The solution was kept in the dark for 5 days and then centrifuged (centrifuged at room temperature at 1000 rpm for 1 min). The supernatant was removed, sterile water was added to the bacteria, and the cells were ultrasonically disrupted and filtered using a 0.22 μm filter membrane to obtain a sterile solution. The toxin concentration was determined using LC-MS. If the toxin was detected, it means that the cells of strain HW28 can adsorb ZEN. The results are shown in Tables 2 and Figure 7 According to Table 2 and Figure 7 It can be seen that the way in which Bacillus amyloliquefaciens HW28 degrades zearalenone toxin is through degradation of extracellular substances.

[0112] Table 2 Degradation rate of extracellular, intracellular and cell-adsorbed ZEN (unit: %)

[0113] Degradation rate Degradation rate Degradation rate intracellular (CFE) 3.886944489 8.03366829 17.17216435 Adsorption (ADS) 64.52272164 57.30792898 56.34760867 Extracellular (CFS) 91.76307458 85.10125268 93.7678024

[0114] (4) Add ZEN solution to LB liquid culture medium to make the ZEN concentration in the LB liquid culture medium be 100 μg / L, which is recorded as culture medium 1.

[0115] The HW28 bacterial solution was added to the medium 1 at a volume ratio of 0.5%, 1%, 2%, 5%, 10%, and 20%. After standing for 1 day in the dark, the toxin concentration was determined by LC-MS. The determination was performed three times and the average value was taken. The results are shown in Tables 3 and Figure 7 .

[0116] Table 3 ZEN degradation rate under different inoculation amounts (unit: %)

[0117] Inoculation amount (%) Degradation rate 1 Degradation rate 2 Degradation rate 3 average value 0.5 29.70505442 37.66577289 26.6147108 31.32851271 1 33.54800627 37.78271535 40.32148038 37.21740067 2 41.75068425 15.84653809 41.98234169 33.19318801 5 53.00277599 48.36851352 59.47025067 53.61384673 10 69.62002055 64.14238022 59.40760293 64.39000123 20 64.52272164 60.27771049 64.1267879 62.97574001

[0118] (5) ZEN solution was added to LB liquid culture medium to make the ZEN concentration in the LB liquid culture medium be 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L, and 5000 μg / L, respectively, denoted as culture medium 1 to 5. HW28 bacterial solution was inoculated in culture medium 1 to 5, respectively, with the inoculation amount being 1% of the culture medium volume. After standing in the dark for 5 days, the toxin concentration was determined by LC-MS. The determination was performed three times and the average value was taken. The results are shown in Tables 4 and Figure 7 As shown in Table 4, Bacillus amyloliquefaciens HW28 has a high ZEN degradation rate.

[0119] Table 4 ZEN degradation rate at different ZEN concentrations (unit: %)

[0120] Toxin concentration (μg / L) Degradation rate 1 Degradation rate 2 Degradation rate 3 average value 100 96.68273233 96.16289527 94.33859296 95.72807352 500 94.85917263 93.6306566 95.63536749 94.70839891 1000 97.30650278 98.06399463 96.92994709 97.4334815 2500 97.00545803 97.35282884 97.17817459 97.17882049 5000 96.39020178 96.07677166 96.09455109 96.18717484

[0121] (6) Add ZEN solution to LB liquid culture medium to make the ZEN concentration in the LB liquid culture medium be 100 μg / L, which is recorded as culture medium 1.

[0122] 1% HW28 bacterial solution was added to the culture medium 1, and the culture medium was placed in an environment of 20°C, 25°C, 30°C, 35°C, and 40°C, and then kept away from light for 5 days. The toxin concentration was determined by LC-MS. The determination was performed three times, and the average value was taken. The results are shown in Table 5 and Figure 7 According to Table 5, the toxin degradation rate can reach more than 90% in an environment of 20℃~40℃.

[0123] Table 5 ZEN degradation rate at different culture temperatures (unit: %)

[0124] Temperature(℃) Degradation rate 1 Degradation rate 2 Degradation rate 3 average value 20 91.76307458 89.64725143 90.70516301 90.70516301 25 95.28415625 94.7854802 97.15189433 95.74051026 30 97.15913362 93.78617907 95.47265635 95.47265635 35 99.27050182 98.08464963 98.67757573 98.67757573 40 99.02895996 98.11277151 99.34790659 98.82987935

[0125] (7) Add ZEN solution to LB liquid culture medium to make the ZEN concentration in the LB liquid culture medium be 100 μg / L, which is recorded as culture medium 1.

[0126] Add 1% HW28 bacterial solution by volume to medium 1, adjust the pH of the medium to 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and place it in the dark for 5 days before using LC-MS to measure the toxin concentration. The results are shown in Tables 6 and Figure 7 According to Table 6, the degradation rate of ZEN is the highest when the pH is 7-8.

[0127] Table 6 ZEN degradation rate at different culture pH (unit: %)

[0128] pH Degradation rate 1 Degradation rate 2 Degradation rate 3 average value 5 15.81368282 16.82217229 20.99451206 17.87678906 6 66.62016812 73.85974145 74.16574087 71.54855015 7 88.79858333 94.35028721 92.81388608 91.98758554 8 83.15527452 93.19116472 82.08385888 86.14343271 9 66.00371432 56.48821805 63.82190221 62.10461152

[0129] (8) ZEN solution was added to LB liquid medium to make the ZEN concentration in the LB liquid medium 100 μg / L, which was recorded as medium 1. 1% by volume of HW28 bacterial solution was added to medium 1, and the toxin concentration was measured by LC-MS after standing in the dark for 1 to 10 days. The measurement was performed three times and the average value was taken. The results are shown in Tables 7 and Figure 7 . According to Table 7, when the degradation time is 10d, the degradation rate of ZEN reaches 99%. The lower degradation rate of ZEN at the 6th and 7th degradation in Table 7 is mainly due to measurement errors. The number of samples tested is large, so some samples will have a certain deviation from the actual time during testing. In addition, the samples need to be filtered with a membrane before being tested on the machine, and different membranes may also cause changes in toxin concentrations. In summary, these problems will lead to data errors. The data in Table 7 are normal and consistent with common knowledge.

[0130] Table 7 ZEN degradation rate at different degradation times (unit: %)

[0131] Degradation days (d) Degradation rate 1 Degradation rate 2 Degradation rate 3 average value 1 33.54800627 37.78271535 40.32203725 37.21758629 2 69.97029105 65.64147114 66.75938533 67.45704917 3 87.60326437 83.03025747 88.04681039 86.22677741 4 89.64725143 93.34820173 90.26648958 91.08731425 5 96.68273233 96.16289527 94.33859296 95.72807352 6 93.78617907 97.56676161 93.00043714 94.78445928 7 92.45303507 94.46667279 93.5971221 93.50560999 8 98.00947234 98.2210825 97.99917027 98.07657503 9 99.24432899 99.41556615 96.91912315 98.52633943 10 99.01239312 98.48754424 99.34790659 98.94928131

[0132] (9) Response surface analysis

[0133] Design Expert 13 software was used to assist in analyzing the effects of culture time, culture temperature and culture pH on the degradation of zearalenone by strain HW28, and a response surface curve was drawn. The results of the response surface analysis are shown in Figure 8 .according to Figure 8 It can be seen that strain HW28 has an excellent degradation effect on zearalenone when the degradation days are 5 days or later, the degradation pH is between 6 and 9, and the temperature is between 20 and 40°C. The single factor curve shows that the degradation days reach the highest around the 7th day, the best effect is between pH 7 and 8, and the best effect is between 20 and 30°C. Figure 8 The first row from top to bottom represents the response surface 3D plot, the second row is the response surface top view, and the third row is the single factor curve; Figure 8 Where A is time, B is pH, and C is temperature.

[0134] Example 4 Using corn as a feed model to verify the degradation effect of strain HW28 on zearalenone

[0135] Bacillus amyloliquefaciens HW28 was inoculated into LB liquid culture medium and cultured at 37° C. for 1 day to obtain HW28 bacterial liquid.

[0136] Take 600 μL of zearalenone toxin solution and add it to 30g of corn feed and mix it evenly. The zearalenone concentration of the zearalenone toxin solution is 5000 μg / L. The zearalenone toxin concentration of the corn feed after proportioning is 100 μg / kg. Then add 1.5mL of HW28 bacterial solution (inoculation volume is 5%) to the feed and mix it evenly. The effective live bacteria count of HW28 in the feed is ≥1×10 7 CFU / mL. After culturing for 15 days at room temperature and pH 7.0 in the dark, the detoxified corn feed was obtained and tested using the test card. Considering the limitations of the growth and reproduction conditions of the strain in the feed, the time was extended to 15 days.

[0137] The specific detection is as follows: weigh 5g of detoxified corn feed crushed sample into a 50mL centrifuge tube, add 12mL of 50% ethanol solution by volume for extraction, shake for 5min, and then centrifuge at 4000rpm for 5min at room temperature to obtain the sample liquid.

[0138] Tear open the aluminum foil packaging bag of the test card, take out the test card, and place it on a flat, clean surface. Use the matching pipette to absorb the prepared sample liquid, and slowly, drop by drop (to avoid foaming) add 3 drops (about 60μL) to the sample well (S). Place it at room temperature for 8 to 10 minutes to judge the result. Fig. 9 and Fig.10 . Fig. 9 The test sample contained zearalenone toxin and HW28 bacterial solution, and the test result was negative, indicating that the zearalenone toxin was degraded to below 60μg / kg. Fig.10 It can be seen that no HW28 bacterial solution was added Feed , the test result is positive, indicating that the concentration of zearalenone toxin is still higher than 60μg / kg. Fig. 9 and Fig.10 It can be seen that the Bacillus amyloliquefaciens HW28 of the present invention has a good degradation effect on zearalenone and can be used to prepare a detoxifying bacterial agent for zearalenone used in feed.

[0139] In summary, the present invention provides Bacillus amyloliquefaciens HW28, which has a high degradation rate of zearalenone. It can be seen that the Bacillus amyloliquefaciens HW28 of the present invention has good application prospects in the degradation of zearalenone.

[0140] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens HW28, characterized in that: The deposit number is CGMCC No.32090.

2. A bacterial agent, characterized in that The invention comprises the Bacillus amyloliquefaciens HW28 of claim 1.

3. The bacterial agent according to claim 2, characterized in that: The application form of the Bacillus amyloliquefaciens HW28 includes culture solution or metabolic solution.

4. The bacterial agent according to claim 3, characterized in that: The method for preparing the culture solution comprises the following steps: inoculating the Bacillus amyloliquefaciens HW28 into a culture medium for culturing to obtain a culture solution; The preparation method of the metabolic solution comprises: centrifuging the culture solution to obtain the supernatant, which is the metabolic solution.

5. Use of the Bacillus amyloliquefaciens HW28 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a product that degrades zearalenone and / or improves the degradation rate of zearalenone.

6. The use according to claim 5, characterized in that: When the application form of the Bacillus amyloliquefaciens HW28 is a culture solution, the inoculation amount of the culture solution is 0.5% to 20%, and the effective viable count of the Bacillus amyloliquefaciens HW28 in the culture solution is ≥1×10 9 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume mass ratio of the metabolic liquid to the product to be degraded containing zearalenone is 1mL:(5-200)g.

7. The use according to claim 5, characterized in that: The degradation temperature is 20-37° C.; the degradation time is 4-15 days; and the degradation pH is 7.0-8.

0.

8. The use according to claim 5, characterized in that: The product comprises one or more than two of feed additives, food additives and detoxifying agents.

9. A method for degrading zearalenone, characterized in that: The method comprises the following steps: mixing the Bacillus amyloliquefaciens HW28 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 with a substance to be degraded containing zearalenone, and performing degradation.

10. The method according to claim 9, characterized in that When the application form of the Bacillus amyloliquefaciens HW28 is a culture solution, the effective viable cell count after the culture solution is mixed with the material to be degraded containing zearalenone is ≥1×10 7 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume mass ratio of the metabolic liquid to the product to be degraded containing zearalenone is 1mL:(5-200)g.

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