A strain of Bacillus amyloliquefaciens and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Bacillus amyloliquefaciens* and its applications. This invention provides a strain of *Bacillus amyloliquefaciens* HW28, with accession number CGMCC No. 32090. This *Bacillus amyloliquefaciens* HW28 was screened and domesticated from cornfield soil and can efficiently degrade zearalenone. Verification has shown that *Bacillus amyloliquefaciens* HW28 can utilize zearalenone as its sole carbon source for growth, achieving effective degradation of zearalenone. When the ZEN concentration is between 100 μg / L and 5000 μg / L, the degradation rate of ZEN by *Bacillus amyloliquefaciens* HW28 reaches over 94%, demonstrating a high degradation rate. Therefore, the *Bacillus amyloliquefaciens* HW28 of this invention has good degradation activity and application prospects for zearalenone.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus amyloliquefaciens and its applications. Background Technology

[0002] Zearalenone (ZEN), also known as F-2 toxin, is an endocrine-disrupting fungal estrogen produced by Fusarium fungi and is widely found in grains such as corn and wheat, as well as their processed products, worldwide. This toxin poses a serious threat to animal and human health due to its potent estrogenic activity. After oral administration, ZEN is rapidly absorbed and metabolized into α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). These metabolites, along with the parent ZEN, can cause morphological and functional disorders of the reproductive systems in livestock and humans, including reproductive disorders in females, abnormal sperm in males, and fetal malformations. Furthermore, ZEN can also cause digestive system dysfunction in animals, leading to significant economic losses in the livestock industry.

[0003] Faced with widespread ZEN pollution, traditional detoxification methods mainly fall into two categories: physical detoxification and chemical detoxification. Physical detoxification methods, such as high-temperature treatment, radiation treatment, and the use of adsorbents, can reduce ZEN levels to some extent, but they suffer from high processing costs, significant nutrient loss, and complex operation. Chemical detoxification methods, such as the use of alkaline solutions and ozone, can effectively destroy ZEN's toxicity, but may introduce new chemical pollutants, posing potential threats to the environment and animal health.

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

[0005] Bacillus, a widely distributed probiotic, possesses strong environmental adaptability and metabolic diversity, making it a potential biomaterial for degrading fungal toxins such as ZEN. Previous studies have shown that certain Bacillus strains can convert ZEN into non-toxic or low-toxic substances by secreting specific enzymes or metabolites. However, the number of commercially available Bacillus strains capable of effectively degrading ZEN remains limited; after 5 days, the ZEN degradation rate is only around 80%, indicating low efficiency. Therefore, screening and optimizing Bacillus strains with high ZEN degradation capabilities is crucial for developing novel ZEN detoxification technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bacillus amyloliquefaciens and its application. The Bacillus amyloliquefaciens HW28 provided by this invention can rapidly and efficiently degrade zearalenone with a high degradation rate.

[0007] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0008] This invention provides a strain of Bacillus amyloliquefaciens HW28, with accession number CGMCCNo.32090.

[0009] The present invention provides a bacterial agent, characterized in that it comprises Bacillus amyloliquefaciens HW28 as described above.

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

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

[0012] The method for preparing the metabolic fluid includes: centrifuging the culture medium, and the resulting supernatant is the metabolic fluid.

[0013] This invention provides the application of Bacillus amyloliquefaciens HW28 or the bacterial agent described in the above technical solution in the preparation of products that degrade zearalenone and / or improve the degradation rate of zearalenone.

[0014] Preferably, when the Bacillus amyloliquefaciens HW28 is used in the form of a culture medium, the inoculum size of the culture medium is 0.5% to 20%, and the effective viable count of Bacillus amyloliquefaciens HW28 in the culture medium is ≥1×10⁻⁶. 9 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume-to-mass ratio of the metabolic liquid to the degraded material containing zearalenone is 1 mL:(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 includes 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 Bacillus amyloliquefaciens HW28 or the bacterial agent described in the above technical solution with the material to be degraded containing zearalenone, and then performing degradation.

[0018] Preferably, when the Bacillus amyloliquefaciens HW28 is used in the form of a culture medium, the effective viable count of the culture medium mixed with the zearalenone-containing material to be degraded is ≥1×10⁻⁶. 7 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume-to-mass ratio of the metabolic liquid to the degraded material containing zearalenone is 1 mL:(5-200) g.

[0019] The beneficial effects of this invention: This invention provides a strain of *Bacillus amyloliquefaciens* HW28, with accession number CGMCC No. 32090. This *Bacillus amyloliquefaciens* HW28 was screened and domesticated from cornfield soil and can efficiently degrade zearalenone. Verification has shown that *Bacillus amyloliquefaciens* HW28 can utilize zearalenone as its sole carbon source for growth, achieving highly efficient degradation of zearalenone. When the ZEN concentration is between 100 μg / L and 5000 μg / L, the degradation rate of ZEN by *Bacillus amyloliquefaciens* HW28 reaches over 94%, with a maximum degradation rate of 99%. Therefore, the *Bacillus amyloliquefaciens* HW28 of this invention can efficiently degrade and metabolize zearalenone and has good application prospects. Attached Figure Description

[0020] Figure 1 To analyze the colony morphology of Bacillus amyloliquefaciens HW28;

[0021] Figure 2 Microscopic observation of Bacillus amyloliquefaciens HW28 after Gram staining;

[0022] Figure 3 This is a diagram showing the positive control results of the zearalenone detection card in Example 1;

[0023] Figure 4 Example 1: Negative control results of the zearalenone detection card;

[0024] Figure 5 HW28 phylogenetic tree;

[0025] Figure 6 The elution time of ZEN toxin was determined by LC-MS.

[0026] Figure 7The ZEN degradation rate under different conditions in Example 3 is shown. The vertical axis “Degradability” represents the degradation rate, and the horizontal axis “Method” represents three different degradation methods: CFE (intracellular), ADS (adsorption), and CFS (extracellular). The concentration of ZEA represents the toxin concentration, the inoculation rate represents the inoculation rate, the temperature represents the temperature, the time represents the time, and the pH represents the acidity or alkalinity.

[0027] Figure 8 The response surface methodology for the degradation of ZEN toxin in Example 3 is shown below.

[0028] Figure 9 The graph shows the effect of adding strain HW28 on the detoxification of ZEN in feed.

[0029] Figure 10 The graph shows the detection of zearalenone in a blank feed that has not been treated with zearalenone toxin.

[0030] Biological Preservation Instructions

[0031] Bacillus amyloliquefaciens HW28 was deposited on September 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32090. Detailed Implementation

[0032] This invention provides a strain of Bacillus amyloliquefaciens HW28, with accession number CGMCC No. 32090. The 16S rRNA gene sequence of Bacillus amyloliquefaciens HW28 described in this invention is shown in SEQ ID No. 1.

[0033] SEQ ID No. 1:

[0034]

[0035] This invention uses cornfield soil samples as raw materials and employs a selective culture medium containing ZEN as the sole carbon source for preliminary screening. The culture medium is then tested using a rapid ZEN detection kit to assess the ZEN degradation ability of the strains, resulting in the isolation of a *Bacillus amyloliquefaciens* strain, HW28. *Bacillus amyloliquefaciens* HW28 can achieve rapid and efficient degradation of zearalenone, and as a non-pathogenic bacillus, it can be used to prepare detoxifying agents for zearalenone. This invention has certain application value in solving ZEN contamination problems and provides a new solution for food safety and livestock development.

[0036] This invention utilizes the 16S rRNA sequence of Bacillus amyloliquefaciens HW28 to perform a BLAST homology sequence search in NCBI, and constructs a phylogenetic tree from the highly homologous sequences obtained, thus identifying strain HW28 as Bacillus amyloliquefaciens.

[0037] This invention provides a microbial agent comprising Bacillus amyloliquefaciens HW28 as described in the above technical solution.

[0038] As an optional implementation, the application of Bacillus amyloliquefaciens HW28 described in this invention includes culture medium or metabolic solution.

[0039] As an optional embodiment, the preparation method of the culture medium of the present invention includes the following steps: inoculating the *Bacillus amyloliquefaciens* HW28 into a culture medium for cultivation to obtain a culture medium. As an optional embodiment, the present invention does not have a specific limitation on the inoculation method, and conventional methods can be used. As an optional embodiment, the cultivation temperature of the present invention is 20℃~37℃, or 28~35℃; in specific embodiments of the present invention, the cultivation temperature is 20, 25, 28, 30, 33, 34, 35, 36, or 37℃. The cultivation time is 1~2 days. In specific embodiments of the present invention, the cultivation time is 1, 1.5, or 2 days. The culture medium used for the cultivation of the present invention includes LB liquid medium. As an optional embodiment, the effective viable count of *Bacillus amyloliquefaciens* HW28 in the culture medium is ≥1×10⁻⁶. 9 CFU / mL.

[0040] As an optional implementation, the preparation method of the metabolic fluid includes: centrifuging the culture medium obtained by the preparation method described above, and the resulting supernatant is the metabolic fluid. The centrifugation speed in this invention is 800-1200 rpm, or 1000 rpm; the centrifugation time is 1-2 minutes. The metabolic fluid contains substances produced by Bacillus amyloliquefaciens HW28 that degrade zearalenone.

[0041] This invention provides the application of Bacillus amyloliquefaciens HW28 or the bacterial agent described in the above technical solution in the preparation of products that degrade zearalenone and / or improve the degradation rate of zearalenone.

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

[0043] As an optional implementation, the product of this invention includes one or more of feed additives, food additives, and detoxifying agents. This invention does not impose any particular limitation on the preparation method of the product; conventional methods are acceptable.

[0044] As an optional implementation, the effective viable count of Bacillus amyloliquefaciens HW28 in the zearalenone-containing degradation product of the present invention is ≥1×10⁻⁶. 7 CFU / mL. As an optional implementation, the degradation temperature is 20–37°C, or 25–35°C. The degradation pH is 7–8; the degradation time is 4–15 days, or 5–10 days.

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

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

[0047] As an optional implementation, the zearalenone-containing material to be degraded includes feed and / or food containing zearalenone. In a specific embodiment of the present invention, corn was used as a feed model to verify the effect of Bacillus amyloliquefaciens HW28 on the degradation of zearalenone.

[0048] As an optional implementation, the mass concentration of zearalenone in the zearalenone-containing material to be degraded is 1–5000 μg / L, or even 100–5000 μg / L. In specific embodiments of the present invention, the effectiveness was verified using zearalenone concentrations of 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L, and 5000 μg / L in the culture medium. Degradation is still possible even when the zearalenone concentration is greater than 5000 μg / L, although the degradation efficiency may decrease and the degradation time may be prolonged, because Bacillus amyloliquefaciens HW28 can utilize zearalenone as its sole carbon source for growth.

[0049] As an optional implementation, when the Bacillus amyloliquefaciens HW28 is used in the form of a culture medium, the effective viable count of the culture medium mixed with the zearalenone-containing material to be degraded is ≥1×10⁻⁶. 7 CFU / mL; when the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume-to-mass ratio of the metabolic liquid to the degraded material containing zearalenone is 1 mL:(5-200) g.

[0050] As an optional implementation, the degradation temperature is 20–37°C, more specifically 25–35°C, and even more specifically 30°C. The degradation time is 4–15 days, more specifically 7–13 days; the degradation pH is 7–8.

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

[0052] As an optional implementation, the mixing method is not particularly limited and conventional methods can be used. In this invention, the product to be degraded containing zearalenone includes feed and / or food containing zearalenone, or it can be feed containing zearalenone. In an embodiment of this invention, the degradation effect of feed containing zearalenone on Bacillus amyloliquefaciens HW28 is verified.

[0053] As an optional implementation, the mass concentration of zearalenone in the zearalenone-containing material to be degraded is 1–5000 μg / L, or it can be 100–5000 μg / L. In a specific embodiment of the present invention, the effect was verified by using zearalenone concentrations of 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L, and 5000 μg / L in the culture medium. Degradation is also possible when the zearalenone concentration is greater than 5000 μg / L, although the degradation efficiency may be reduced and the degradation time prolonged, because the Bacillus amyloliquefaciens strain HW28 can utilize zearalenone as the sole carbon source for growth.

[0054] As an optional implementation, when the Bacillus amyloliquefaciens HW28 is used in the form of a culture medium, the effective viable count of the culture medium mixed with the zearalenone-containing material to be degraded is ≥1×10⁻⁶. 7 CFU / mL; When the application form of the Bacillus amyloliquefaciens HW28 is a metabolic liquid, the volume-to-mass ratio of the metabolic liquid to the degraded material containing zearalenone is 1mL:(5-200)g, or it 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 is 7–8.

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

[0057] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

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

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

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

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

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

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

[0064] The composition of MSM medium is: 2 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, 0.001 g / L FeSO4·7H2O and 1.5 g / L Na2HPO4·12H2O.

[0065] The composition of MM medium is: 2 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.001 g / L FeSO4, 0.002 g / L MnCl2, 4.5 g / L K2HPO4, 2 g / L mannitol and 2 g / L glycerol.

[0066] Example 1: Screening of strains that degrade zearalenone toxin in zearalenone

[0067] 1. Culture media were prepared using zearalenone as the sole carbon source, i.e., different concentrations of zearalenone were directly added to commercially available MSM culture media. The resulting zearalenone-MSM culture media were designated as media 1 to 6, with the following concentrations: 5 μg / L in media 1, 10 μg / L in media 2, 20 μg / L in media 3, 40 μg / L in media 4, 80 μg / L in media 5, and 100 μg / L in media 6.

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

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

[0070] Inoculate culture solution 1 into culture medium 2 at an inoculation rate of 10% of the culture medium volume, and then culture for 7 days to obtain culture solution 2.

[0071] Inoculate culture solution 2 into culture medium 3 at a volume of 10% of the culture medium volume, and culture for another 7 days to obtain culture solution 3. Culture media 1 to 6 are used sequentially until culture solution 5 is inoculated into culture medium 6 at a volume of 10% of the culture medium volume, and cultured for another 7 days to obtain strain screening solution.

[0072] The strain selection solution was serially diluted 10-fold to a final concentration of 10. -1 ...10 -5 、…、10 -8 Each dilution was plated onto LB agar plates and incubated. Single colonies were picked to obtain a bacterial strain, designated as strain HW28. The colony morphology of strain HW28 is shown in [reference needed]. Figure 1 See the microscopic image of HW28 Gram staining. Figure 2 It can be seen that the colony morphology of strain HW28 is white, round with raised bumps, and the colonies are connected with wrinkles. It is a Gram-positive bacterium with rod-shaped and spore-shaped forms.

[0073] 2. Prepare the culture medium using zearalenone (ZEN) as the sole carbon source, i.e., add ZEN to MM medium, with a ZEN concentration of 100 μg / L in MM medium.

[0074] Strain HW28 was inoculated into MM medium and cultured for 4–5 days to obtain a culture medium. Finally, the concentration of ZEN toxin in the culture medium was determined using a zearalenone detection card. 。 The test card showed a negative result, with a ZEN concentration of less than 60 μg / L, indicating that the ZEN toxin concentration was reduced and that strain HW28 was able to effectively degrade the toxin.

[0075] 3. Screening was performed using a zearalenone detection card. ZEN was prepared into a ZEN solution, which was then added to LB liquid medium. Single colonies of strain HW28 were then inoculated into the LB medium. After growing in the medium for a period of time, the culture medium was obtained. The ZEN in the culture medium was then detected using a zearalenone detection card.

[0076] The testing procedure is as follows: Tear open the aluminum foil packaging of the test card, take out the test card, and place it on a flat, clean surface. Use the provided pipette to draw up the prepared sample liquid to be tested, and slowly, drop by drop (avoiding foaming), add 3 drops (approximately 60 μL) into the sample well (S). Let it stand at room temperature for 8–10 minutes to determine the results.

[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 degraded to below the detection standard (60 μg / L), and the result is negative; if only C (control line) on the test card appears as a red line, it indicates that the toxin concentration is still higher than the detection standard (60 μg / L), and the result is positive; if there is no red line at C (control line), the test is invalid.

[0078] LB liquid 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 detection card are shown below. Figure 3 LB liquid medium containing 10 μg / L zearalenone toxin was used as a negative control. The negative control results of the zearalenone detection card are shown below. Figure 4 .according to Figure 3 It is known that the toxin concentration is still higher than 60 μg / L. Figure 4 The toxin concentration is below 60 μg / L, indicating that the zearalenone test card provides accurate results.

[0079] 4. The genome of strain HW28 was extracted using a DNA extraction kit. The extracted sample was the culture medium obtained from steps 2 and 3 of culturing strain HW28. The 16S rRNA target gene of this strain was amplified and sequenced using 16S universal primers. The sequencing results were analyzed using BLAST on NCBI to identify the species of the strain. The phylogenetic tree of strain HW28 is shown below. Figure 5 ,according to Figure 5 It can be known that strain HW28 is Bacillus amyloliquefaciens.

[0080] Example 2: Study on the physiological and biochemical characteristics of strains that degrade zearalenone toxin in zearalenone.

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

[0082] (1) Amylase production test: strain HW28 was spot-inoculated onto the surface of the solid medium for amylase production screening and incubated upside down at 37°C for 24 hours. After colonies grew, Luvé's iodine solution was added to cover the entire plate. The appearance of clear zones was observed. The size of the colonies and clear zones was measured with calipers to assess the amylase production activity. The experiment was repeated three times independently.

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

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

[0085] After the artificial gastric fluid and artificial intestinal fluid were cultured, 100 μL of each liquid was serially diluted and then evenly spread on LB solid medium. The medium was inverted and incubated at 37°C for 24 h. The viable number of each bacteria was counted using the plate colony counting method, and the survival rate was calculated.

[0086] The survival rate (%) of the strain is calculated as lgN1 / lgN2 × 100, where N1 is the number of viable bacteria after culturing in artificial intestinal fluid or artificial gastric fluid, and N2 is the number of viable bacteria without artificial intestinal fluid or artificial gastric fluid treatment. The unit of viable bacteria count is CFU / mL. The experiment was independently repeated three times.

[0087] (3) Acid tolerance test: First, the pH of LB medium was adjusted to 2.0, 3.0, and 4.0. Then, HW28 bacterial suspension was added to LB medium at different pH values ​​and incubated at 37℃ for 5 h. 100 μL of the treated HW28 bacterial suspension was spread onto LB solid medium and incubated at 37℃ for 24 h. Untreated HW28 bacterial suspension was used as a control. Colonies with a count of 30–300 were selected for counting. The experiment was independently repeated three times.

[0088] (4) Hemolytic activity test: HW28 bacterial suspension was streaked onto the surface of Columbia blood agar medium and incubated overnight at 37°C. The presence of a transparent hemolytic zone around the colony was observed. The experiment was repeated three times independently.

[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: The growth of the strain in Example 1 was observed and compared using a carbon dioxide incubator, a regular incubator and an oxygen-filled incubator.

[0091] (7) Antimicrobial susceptibility test: Mix 100 μL of HW28 bacterial suspension with 5 mL of semi-solid LB agar medium, spread the mixture evenly on the surface of solid medium, and wait for the medium to solidify before use. Use sterilized tweezers to pick up antimicrobial susceptibility test discs, spread them on the surface of the medium, and incubate at 37℃ for 24 h. Observe whether there is an inhibition zone around the antimicrobial susceptibility test discs, and measure the diameter (d) of the inhibition zone with calipers to assess the antimicrobial resistance of the strain.

[0092] (8) Gelatin liquefaction test: The bacterial strain of Example 1 was activated, and 100 μL of bacterial solution was spread on a gelatin culture medium and inverted in a refrigerator at 4°C for 7 days. If the gelatin can be removed in one piece after 7 days, it proves that the bacteria do not have the ability to liquefy gelatin; if the gelatin is brittle or softens significantly after 7 days, it proves that the bacteria have the ability to liquefy gelatin.

[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, has a higher number of viable bacteria under hypoxic conditions than under normal conditions, exhibits γ-hemolysis (meaning it is insoluble in blood), and is sensitive to most antibiotics.

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

[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 cells)]]> + hemolytic gamma hemolysis Gentamicin (120μg / tablet) ++ Ciprofloxacin (5μg / tablet) ++ Ampicillin (10 μg / tablet) + Chloramphenicol (30μg / tablet) ++ Compound Sulfamethoxazole (25μg / tablet) + Lincomycin (2μg / tablet) - Erythromycin (15μg / tablet) ++ Tetracycline (30μg / tablet) - Ceftriaxone (30μg / tablet) ++ Penicillin (10μg / tablet) +

[0096] Acid resistance and artificial gastric juice: "+" indicates a bacterial survival rate higher than 90%; "-" indicates a bacterial survival rate lower than 90%.

[0097] Enzyme activity "+" indicates the presence of that enzyme activity; "-" indicates the absence of that enzyme activity.

[0098] Antibiotics are indicated by "++" for exceptionally sensitive (inhibition zone >= 28 mm); and "+" for sensitive (28 > inhibition zone >= 14 mm).

[0099] "-" indicates insensitivity (14mm > inhibition zone).

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

[0101] 1. Add ZEN after it has been prepared into a 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 suspension. The effective viable count of HW28 bacterial suspension was ≥1×10⁻⁶. 9 CFU / mL; used for the following experiments.

[0103] 3. The specific conditions for liquid chromatography-mass spectrometry (LC-MS) are as follows:

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

[0105] LC-MS analysis was performed using commercially available ZEN as a standard. The peak time of ZEN is shown in [reference needed]. Figure 6 The ZEN elution time was 2.622 min, and the ZEN standard curve was y = 931.661916 × x, R0. 2 =0.99190923 (see figure), 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] Where: SO: peak area of ​​ZEN standard solution at standard concentration, ST: peak area of ​​ZEN in treatment group.

[0108] The specific measurement process is as follows:

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

[0110] (2) Intracellular degradation experiment (hereinafter referred to as intracellular): HW28 bacterial culture was centrifuged (1000 rpm for 1 min at room temperature). The supernatant was discarded, sterile water was added to the bacterial cells, and the cells were sonicated to disrupt the cell lysis, obtaining a cell lysate. The cell lysate was filtered through 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 incubated 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 .

[0111] (3) Cell Adsorption Assay (hereinafter referred to as adsorption): ZEN solution was added to the HW28 bacterial culture to make the ZEN concentration in the bacterial culture 100 μg / L. After standing in the dark for 5 days, centrifugation was performed (centrifugation at 1000 rpm for 1 min at room temperature). The supernatant was discarded, sterile water was added to the bacterial cells, and the cells were sonicated and filtered through a 0.22 μm filter membrane to obtain a sterile solution. The toxin concentration was determined by LC-MS. If the toxin was detected, it indicates that the cells of strain HW28 can adsorb ZEN. The results are shown in Table 2 and Figure 7 According to Table 2 and Figure 7 It can be seen that Bacillus amyloliquefaciens HW28 degrades zearalenone toxin through extracellular degradation.

[0112] Table 2. Degradation rates of ZEN adsorbed extracellularly, intracellularly, and within cells (in %)

[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 medium to make the ZEN concentration in LB liquid medium 100 μg / L, and denot it as medium 1.

[0115] HW28 bacterial suspension was added to culture medium 1 at inoculum concentrations of 0.5%, 1%, 2%, 5%, 10%, and 20% (v / v). After standing in the dark for one day, the toxin concentration was determined by LC-MS. Three measurements were performed, and the average value was taken. The results are shown in Table 3. Figure 7 .

[0116] Table 3. ZEN degradation rate at different inoculum sizes (unit: %)

[0117] Inoculation rate (%) 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 medium to achieve ZEN concentrations of 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L, and 5000 μg / L, respectively, designated as media 1–5. HW28 bacterial suspension was inoculated into media 1–5 at 1% of the media volume. After standing in the dark for 5 days, the toxin concentration was determined by LC-MS. Three measurements were taken, and the average value was recorded. The results are shown in Table 4 and [Table data missing]. Figure 7 As shown in Table 4, Bacillus amyloliquefaciens HW28 exhibits 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 medium to make the ZEN concentration in LB liquid medium 100 μg / L, and denot it as medium 1.

[0122] Add 1% (v / v) of HW28 bacterial suspension to culture medium 1, and incubate at 20℃, 25℃, 30℃, 35℃, and 40℃ respectively. After standing in the dark for 5 days, the toxin concentration is determined by LC-MS. Three measurements are performed, and the average value is taken. The results are shown in Table 5. Figure 7 As shown in Table 5, the toxin degradation rate can reach over 90% in environments ranging from 20℃ to 40℃.

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

[0124] Temperature (°C) 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 medium to make the ZEN concentration in LB liquid medium 100 μg / L, and denot it as medium 1.

[0126] Add 1% (v / v) of HW28 bacterial suspension to culture medium 1, and adjust the pH of the medium to 5.0, 6.0, 7.0, 8.0, and 9.0 respectively. After standing in the dark for 5 days, the toxin concentration was determined by LC-MS. Three measurements were performed, and the average value was taken. The results are shown in Table 6. Figure 7 As shown in Table 6, the ZEN degradation rate is highest at pH 7–8.

[0127] Table 6. ZEN degradation rate at different culture pH levels (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) Add ZEN solution to LB liquid medium to make the ZEN concentration in the LB liquid medium 100 μg / L, and designate this as medium 1. Add 1% (v / v) of HW28 bacterial suspension to medium 1, and incubate in the dark for 1–10 days. Then, determine the toxin concentration by LC-MS. Perform three measurements and take the average value. The results are shown in Table 7 and [Table 8]. Figure 7 As shown in Table 7, the degradation rate of ZEN reaches 99% after 10 days. The lower ZEN degradation rates in Table 6 and 7 are mainly due to measurement errors. The large sample size means that some samples may deviate from the actual degradation time. Furthermore, the samples need to be filtered before testing, and different filters may also cause variations in toxin concentration. In summary, these factors can lead to data errors. The data in Table 7 are normal and consistent with common understanding.

[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 analyze the effects of culture time, temperature, and pH on the degradation of zearalenone by strain HW28, and response surface methodology (RSM) curves were plotted. The results of the RSM analysis are shown below. Figure 8 .according to Figure 8 It can be seen that strain HW28 has excellent degradation effects on zearalenone when the degradation time is 5 days or more, the degradation pH is between 6 and 9, and the temperature is between 20 and 40℃. The single-factor curve shows that the degradation time reaches the highest level at around 7 days, the pH is between 7 and 8, and the temperature is between 20 and 30℃. Figure 8 The first row from top to bottom represents the 3D response surface plot, the second row is the top-down plan view of the response surface, and the third row is the single-factor curve. Figure 8 In this context, A represents Time, B represents pH, and C represents pH and temperature.

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

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

[0136] Add 600 μL of zearalenone toxin solution to 30 g of corn feed and mix thoroughly. The zearalenone concentration in the zearalenone toxin solution is 5000 μg / L. The resulting corn feed has a zearalenone toxin concentration of 100 μg / kg. Then add 1.5 mL of HW28 bacterial suspension (inoculation amount of 5%) to the feed and mix thoroughly. The effective viable count of HW28 in the feed is ≥1×10⁻⁶. 7 CFU / mL. Virus-free corn feed was obtained after 15 days of incubation at room temperature, pH 7.0, and in the dark, and detected using a test card. Considering the limitations of bacterial growth and reproduction conditions in feed, the incubation period was extended to 15 days.

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

[0138] Tear open the aluminum foil packaging of the test card, remove the test card, and place it on a flat, clean surface. Use the provided pipette to draw up the prepared sample liquid, and slowly, drop by drop (avoiding foaming), add 3 drops (approximately 60 μL) into the sample well (S). Let it stand at room temperature for 8–10 minutes to interpret the results. See the attached image for the results. Figure 9 and Figure 10 . Figure 9 The tested sample contained zearalenone toxin and HW28 bacterial culture, and the test result was negative, indicating that the zearalenone toxin has been degraded to below 60 μg / kg. According to... Figure 10 It can be seen that no HW28 bacterial solution was added. feed The test result was positive, indicating that the concentration of zearalenone toxin in zearalenone was still higher than 60 μg / kg. According to... Figure 9 and Figure 10 It is known that the Bacillus amyloliquefaciens HW28 of the present invention has a good degradation effect on zearalenone and can be used to prepare a detoxifying agent for zearalenone used in feed.

[0139] In summary, this invention provides Bacillus amyloliquefaciens HW28, which exhibits a high degradation rate of zearalenone. Therefore, Bacillus amyloliquefaciens HW28 of this invention shows promising application prospects in the degradation of zearalenone.

[0140] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method of degrading zearalenone, characterized by, Includes the following steps: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens HW28 or an inoculum containing Bacillus amyloliquefaciens HW28 was inoculated into LB medium containing zearalenone at a volume of 1% of the medium and incubated in the dark for 5 days. The preservation number of the Bacillus amyloliquefaciens HW28 is CGMCC No. 32090; The concentration of zearalenone is 100 μg / L, 500 μg / L, 1000 μg / L, 2500 μg / L or 5000 μg / L; The application form of the Bacillus amyloliquefaciens HW28 or the bacterial agent of the Bacillus amyloliquefaciens HW28 includes a HW28 bacterial liquid; the preparation method of the HW28 bacterial liquid includes the following steps: inoculating the Bacillus amyloliquefaciens HW28 into an LB culture medium for 37℃ culture for 1d to obtain the HW28 bacterial liquid; the effective viable count of the HW28 bacterial liquid is ≥1×10 9 CFU / mL.

2. The method of claim 1, wherein, The application of Bacillus amyloliquefaciens HW28 also includes a metabolic liquid, wherein the volume-to-mass ratio of the metabolic liquid to the degraded material containing zearalenone is 1 mL:(5-200) g. The metabolic fluid is prepared by inoculating the Bacillus amyloliquefaciens HW28 into a culture medium and culturing it to obtain a culture medium. The culture medium is then centrifuged, and the supernatant obtained is the metabolic fluid.

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