Fungaltoxin degrading bacterial agent and application thereof in traditional Chinese medicine storage

Abstract

The invention discloses a mycotoxin degrading bacterial agent and application thereof in traditional Chinese medicine storage. According to the microbial agent, bacillus coagulans is used as a core degradation component of ochratoxin, tea residues are used as a carrier, microbial cellulose is generated through kombucha mother fermentation to form a nanometer net-shaped structure, meanwhile, rich pores are formed in the tea residues, an efficient adsorption interface and a diffusion channel are provided for OTA, and an ideal colonization microenvironment is provided for bacillus coagulans. Quercetin can inhibit the synthesis of ochratoxin, and forms a synergistic antibacterial effect with a kombucha fermentation product and bacillus coagulans, so that fungal pollution is doubly controlled from the source and in the process. The microbial agent carrier is stable in structure, can realize sustained release of quercetin and continuous biodegradation of thalli, is natural in raw materials, and is suitable for prevention and control of mycotoxin in a traditional Chinese medicine storage process.

Description

Technical Field

[0001] This invention relates to microbial technology, specifically to a fungal toxin-degrading agent and its application in the storage of traditional Chinese medicine. Background Technology

[0002] Fungal toxins are toxic secondary metabolites produced by fungi such as Aspergillus, Fusarium, and Penicillium. More than 400 types have been discovered and identified, mainly including aflatoxin B1, B2, G1, and G2 (AFB1, AFB2, AFG1, AFG2), and ochratoxin A, B, and C (OTA, OTB, OTC). Ochratoxin A (OTA) is a coumarin-type secondary metabolite produced by fungi of the Aspergillus and Penicillium genera, and is well-known for its nephrotoxicity, hepatotoxicity, immunotoxicity, teratogenicity, carcinogenicity, mutagenicity, and genotoxicity. OTA is stable and difficult to remove through conventional processing. Even at very low concentrations, it can cause serious illness and even death, and is classified as a Group 2B human carcinogen by the International Agency for Research on Cancer (IARC). Due to the potential health hazards of OTA and strict regulatory restrictions, an increasing number of researchers are dedicated to research on controlling OTA pollution. However, due to the diversity of ochratoxin-producing fungi and the complexity of environmental conditions, fungal growth and OTA production in various products are often unavoidable. Therefore, there is an urgent need to develop safe, inexpensive and efficient OTA detoxification methods.

[0003] Currently, the main methods for removing or detoxifying OTA include physical adsorption, oxidation processes (chemical decomposition), and biodegradation. Physical adsorption is based on the complex interaction between mycotoxin binders and OTA. Binders include bentonite, gelatin, chitosan, polyvinylpyrrolidone, oyster mushroom powder, and nano-adsorbents. Oxidation processes can rapidly degrade OTA, including photocatalysis by pulsed light, electron beam irradiation, and decomposition with strong oxidants such as ozone. However, these methods have drawbacks such as product nutrient loss, reagent residues, and secondary pollution to the environment. In contrast, microbial or enzyme-mediated biodegradation has received increasing attention due to its economic and environmental benefits. Various bacterial and fungal isolates have been reported to degrade OTA, including *Narrow-minded Monotrophic Acidophilus* CW117, *Brevibacillus sp. ALJ02*, and *Aspergillus niger*. The reported biodegradation mechanisms of OTA to date include amide bond cleavage, dechlorination, hydroxylation, and opening of the lactone ring, resulting in a series of dihydroisocoumarin derivatives. Among them, hydrolyzing the amide bond of OTA to generate non-toxic ochratoxin α (OTα) and L-β-phenylalanine is the most reliable detoxification route.

[0004] Furthermore, in biodegradation technology, the choice of carrier material directly affects the colonization stability, functional efficiency, and application effect of microorganisms. Tea residue, a byproduct of tea processing, is abundant, widely available, and inexpensive. It also possesses a natural porous structure, a large specific surface area, and good mechanical stability, providing ample space for microbial colonization. In addition, tea residue contains natural active ingredients such as tea polyphenols, cellulose, and hemicellulose, which not only have certain adsorption properties but also provide a small amount of nutrients for microbial growth, enhancing cell survival. Compared to traditional carriers (such as activated carbon, silica gel, and resin), tea residue as a carrier material not only realizes the resource utilization of agricultural waste and reduces the cost of formulation production but also has significant advantages such as environmental friendliness and good biocompatibility, showing broad application prospects in the field of microbial formulation carriers. However, the adsorption performance and microbial loading stability of single tea residue carriers still need improvement, and it lacks the ability to inhibit OTA synthesis at its source, making it difficult to meet the needs of efficient control of OTA pollution in complex environments. Summary of the Invention

[0005] Technical problem to be solved: To address the issues of poor stability and insufficient degradation efficacy of biodegradable agents, this invention combines the adsorption characteristics of tea residue, the adsorption capacity of kombucha cells, the degradation function of Bacillus coagulans, and the synergistic effect of quercetin to develop a microbial agent with synergistic effects of adsorption, degradation, synergistic effect, and sustained antibacterial activity, thereby achieving efficient removal of OTA.

[0006] Technical solution: This invention provides a method for preparing a fungal toxin-degrading agent, the steps of which are as follows: S1. Crush and filter the tea residue without impurities, add water, stir and mix, sterilize, cool to room temperature, add sucrose to dissolve, let stand, inoculate kombucha mother and let it ferment, stir and ferment again, and after the fermentation is completed, inactivate to obtain fermented tea residue mixture. S2. Quercetin is dissolved in ethanol, and ultrasonic treatment is performed to obtain a quercetin-ethanol solution. This solution is added to the fermented tea residue mixture, sheared and homogenized, then ultrasonically treated, homogenized again, and finally spray-dried to obtain the tea residue carrier. S3. Take a strain of Bacillus coagulans, pick a single colony and inoculate it into a liquid culture medium, culture overnight, subculture twice, and prepare Bacillus coagulans seed culture; S4. Slowly add Bacillus coagulans seed liquid to the tea residue carrier while stirring, ferment and culture, and freeze-dry after intermittent stirring to obtain the toxin-degrading agent.

[0007] Furthermore, in step S1, the tea residue is pulverized to 40-60 mesh, and the mass ratio of tea residue to water is 1:(5-8); the cooling time is 2-4 hours; the amount of sucrose added is 3-5 wt.%; the standing time is 10-20 minutes; the amount of kombucha starter culture inoculated is 8-12 wt.%; the standing fermentation time is 2-4 days; the stirring fermentation speed is 50-80 rpm, and the time is 36-48 hours.

[0008] Furthermore, in step S2, the ultrasonic treatment power is 200-300W, and the time is 10-20min; the quercetin content in the quercetin-ethanol solution is 5-8wt.%; the mass ratio of the quercetin-ethanol solution to the fermented tea residue mixture is 1:(12-18); the shear homogenization speed is 3000-5000rpm, and the time is 15-30min; the rehomogenization speed is 1000-2000rpm, and the time is 10-20min.

[0009] Furthermore, the Bacillus coagulans strain in step S3 is BC361, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 13, 2014, with accession number CGMCCNo.9951, and classified as Bacillus coagulans.

[0010] Furthermore, in step S3, the culture rotation speed is 180 rpm and the temperature is 35-37℃; the concentration of the Bacillus coagulans seed solution is 1×10⁻⁶. 9 - 1×10 10 CFU / mL.

[0011] Furthermore, in step S4, the mass ratio of tea residue carrier to Bacillus coagulans seed liquid is 1:(2-3); the fermentation temperature is 35-37℃, and the time is 18-36h; the interval between intermittent stirring is 6-9h.

[0012] The fungal toxin degrading agent prepared by the above preparation method.

[0013] The above-described application of a fungal toxin-degrading agent in the storage of traditional Chinese medicine.

[0014] Furthermore, the fungal toxin degrading agent can be prepared as a powder or tablet and mixed into traditional Chinese medicine. Beneficial effects

[0015] This invention utilizes kombucha mother fermentation to produce microbial cellulose, which forms a nano-network structure with an extremely high specific surface area. This provides an attachment interface and diffusion channel for OTA (over-the-counter acid bacteria), enabling rapid capture of OTA molecules. During the stirring fermentation process, free microorganisms such as yeast, lactic acid bacteria, and acetic acid bacteria from the mother fermentation enter the tea residue, forming a rich porous structure that enhances its adsorption capacity for OAT. Furthermore, the porous structure and abundant nutrients provide an ideal colonization space and microenvironment for the selected Bacillus coagulans, preventing loss and allowing it to continuously perform its biodegradation function.

[0016] In this invention, quercetin can inhibit the synthesis of OTA, thereby inhibiting the generation of ochratoxin from the source. In addition, the antibacterial effect of quercetin can produce a superimposed or synergistic effect with the antibacterial properties of tea residue, kombucha fermentation products, and the biological competition with Bacillus coagulans, which can more effectively inhibit the colonization of toxin-producing molds in the environment. The porous structure of tea residue is an excellent carrier for loading quercetin, which can achieve controllable loading and slow release of quercetin.

[0017] The fermentation of tea residue in this invention not only significantly increases the number of multi-level pores in the carrier, allowing OTA molecules to quickly enter the pores and be "intercepted" by the porous structure, providing a high-concentration reaction field for subsequent biodegradation, but also produces a series of enzymes such as peptidases and hydroxylases. These enzymes can destroy the original highly toxic OAT molecule structure and transform it into ochratoxin α or other metabolites with significantly reduced toxicity.

[0018] Bacteria can adsorb OTA through some proteins, carbohydrates or functional groups on the surface of their cell walls. In this invention, the bacteria are inactivated after fermentation, which preserves the bacterial structure, enhances the adsorption capacity of the carrier, and the bacterial residue also serves as an anchoring point for Bacillus coagulans.

[0019] The Bacillus coagulans used in this invention can efficiently secrete specific degradation enzymes. These enzymes can act on the toxic functional groups of OTA molecules, gradually decomposing them into non-toxic small molecules, ultimately achieving complete mineralization of OTA and fundamentally eliminating its carcinogenic, teratogenic, and other toxic risks. Attached Figure Description

[0020] Figure 1 The graph shows the degradation rate of ochratoxin in the examples and comparative examples; Figure 2 The graph shows the inhibition rate of ochratoxin synthesis in the examples and comparative examples. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Example 1

[0022] A method for preparing a fungal toxin-degrading agent, comprising the following steps: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Example 2

[0023] A method for preparing a fungal toxin-degrading agent, comprising the following steps: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 400g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 18g of sucrose to dissolve, let stand for 10min, then inoculate with 40g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 45g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Example 3

[0024] A method for preparing a fungal toxin-degrading agent, comprising the following steps: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 4d, then stir at 50rpm to ferment for 36h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Example 4

[0025] A method for preparing a fungal toxin-degrading agent, comprising the following steps: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 35g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Example 5

[0026] A method for preparing a fungal toxin-degrading agent, comprising the following steps: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 100g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Example 6

[0027] A method for preparing a fungal toxin-degrading agent, comprising the following steps: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year.10 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Comparative Example 1

[0028] The difference between this comparative example and Example 1 is that the tea residue is not fermented, as detailed below: S1. Crush 50g of tea dregs without impurities into a 40-mesh filter, add 250g of water, stir and mix, sterilize at 121℃ for 20min, cool and soak for 3h to room temperature to obtain tea dregs mixture; S2.4g of quercetin was dissolved in 46g of ethanol and sonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the tea residue mixture, and the mixture was sheared and homogenized at 5000rpm for 15min, sonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Comparative Example 2

[0029] The difference between this comparative example and Example 1 is that it uses lactic acid bacteria and yeast to ferment tea residue, as detailed below: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of lactic acid bacteria and yeast suspension and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Comparative Example 3

[0030] The difference between this comparative example and Example 1 is that quercetin is not added, as detailed below: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2. After shearing and homogenizing the fermented tea residue mixture at 5000 rpm for 15 min, ultrasonication at 200 W for 15 min, homogenization at 2000 rpm for 10 min, and finally spray drying, the tea residue carrier is obtained. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Comparative Example 4

[0031] The difference between this comparative example and Example 1 is that in S2, after shear homogenization, there is no further ultrasonic homogenization, as detailed below: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonically treated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture and sheared and homogenized at 5000rpm for 15min before spray drying to obtain the tea residue carrier. S3. Take Bacillus coagulans strain BC361, pick a single colony and inoculate it into liquid culture medium, incubate overnight at 37°C with 180 rpm, subculture twice, and prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Comparative Example 5

[0032] The difference between this comparative example and Example 1 is that Bacillus coagulans strain BC361 was replaced with other Bacillus coagulans strains, as detailed below: S1. Crush 50g of tea residue without impurities into a 40-mesh filter, add 250g of water and stir to mix, then sterilize at 121℃ for 20min, cool and soak for 3h to room temperature, add 9g of sucrose to dissolve, let stand for 10min, then inoculate with 25g of kombucha starter culture and let stand to ferment for 2d, then stir at 50rpm to ferment for 48h, and finally heat at 80℃ for 30min to inactivate and terminate fermentation to obtain fermented tea residue mixture; S2.4g of quercetin was dissolved in 46g of ethanol and ultrasonicated at 200w for 15min to obtain a quercetin-ethanol solution. 27g of the solution was added to the fermented tea residue mixture, sheared and homogenized at 5000rpm for 15min, ultrasonicated at 200w for 15min, homogenized at 2000rpm for 10min, and finally spray-dried to obtain the tea residue carrier. S3. Take a strain of Bacillus coagulans, pick a single colony and inoculate it into liquid culture medium. Incubate overnight at 37°C with 180 rpm. Subculture twice to prepare 1×10⁻⁶ cells / year. 9 CFU / mL Bacillus coagulans seed solution; Slowly add 150g of Bacillus coagulans seed liquid to 4.50g of tea residue carrier while stirring. Ferment at 37℃ for 24 hours, stirring once every 6 hours. After the fermentation is completed, freeze dry to obtain the toxin-degrading agent. Indicator Testing

[0033] 1. Degradation rate determination The bacterial agent was prepared into a mixture, and 995 μL of it was mixed with 5 μL of OTA at a concentration of 500 μg / mL. The mixture was then incubated at 37°C for 48 h. After incubation, the residual amount of OTA in each sample was determined by high performance liquid chromatography, and the degradation rate was calculated.

[0034] The results are as follows Figure 1 As shown, this invention achieves highly efficient degradation of OTA. Under the synergistic effect of the components, the degradation rate in the embodiments remained stable at over 90%; among them, Example 6 had the highest degradation rate of 96%, while Comparative Example 1 had the lowest at 78.6%. Unfermented tea residue lacks a microbial cellulose nano-network structure and abundant pores, which reduces the OTA adsorption capacity (no efficient adhesion interface) and affects the colonization stability of Bacillus coagulans, leading to a decrease in the adsorption and degradation capacity of the composite system. Although Comparative Example 2 incorporated probiotic fermentation, it was difficult to synthesize cellulose to provide structural optimization and toxin adsorption.

[0035] 2. Antibacterial properties The agar perforation diffusion method was used. Bacterial suspension was added to an agar plate in a petri dish, spread evenly with a spreader, and perforated with a perforator. 100 μl of the bacterial solution, a positive control, and a negative control were added to each perforator, and parallel groups were prepared. After incubation at 37°C for at least 12 hours, the diameter of the inhibition zone was visually observed and measured.

[0036] The results are shown in Table 1. The inoculants in the embodiments of the present invention have a significant inhibitory effect on these two bacteria. The largest difference between Comparative Example 1 and Example 1 is the inhibition zone of *Aspergillus* (11.5 mm) and *Penicillium* (9.8 mm), which are significantly lower than the *Aspergillus* inhibition zone of 16.8 mm and *Penicillium* inhibition zone of 13.2 mm in Example 1. This may be because the unfermented tea residue lacks the antibacterial effect of kombucha fermentation products. Comparative Example 4 lacks the ultrasonic and secondary homogenization steps, and the inhibition zones are also smaller, indicating that the multi-step homogenization process can ensure the dispersibility of the tea residue carrier and avoid local enrichment or loss of effective components.

[0037] Table 1. Antibacterial properties of the inoculant.

[0038] 3. Ability to inhibit toxin production 0.5 g of the examples and comparative examples were added to 100 mL of sterilized PDA medium. PDA medium without samples was used as a blank control. 5 μL of Aspergillus charcoal spore suspension was inoculated. The plates were placed in a constant temperature culture at 28℃ for 7 days. OTA was extracted on the 7th day for measurement, and the inhibition rate was calculated.

[0039]

[0040] Where C0 represents the OTA content in the blank control, C 样 The content of TA in the samples of the examples and comparative examples was added.

[0041] Quercetin can inhibit OTA synthesis, and its loading and the antibacterial properties of the bacterial agent are the core influencing factors on the OTA synthesis inhibition rate. Results are as follows... Figure 2 As shown, Example 4, with the highest quercetin dosage, achieved an inhibition rate of 97.1%, the best among all groups. Comparative Example 3, lacking quercetin, had an inhibition rate of only 55.8%. Comparative Example 1, being unfermented, exhibited weaker antibacterial ability and a lower inhibition rate of OTA synthesis. Meanwhile, although Example 6 used the same quercetin dosage as Example 1, its inhibition rate was higher due to the biological competition from a high concentration of Bacillus coagulans, indicating that biological competition can enhance the inhibitory effect of quercetin and further verifying the synergistic advantage of the composite system. The inhibition rates of the comparative examples were all below 85%, while those of the examples were above 90%, demonstrating the significant effectiveness of the bacterial agent of this invention in inhibiting bacteria and toxin synthesis.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a fungal toxin-degrading agent, characterized in that, The steps are as follows: S1. Crush and filter the tea residue without impurities, add water, stir and mix, sterilize, cool to room temperature, add sucrose to dissolve, let stand, inoculate kombucha mother and let it ferment, stir and ferment again, and after the fermentation is completed, inactivate to obtain fermented tea residue mixture. S2. Quercetin is dissolved in ethanol, and ultrasonic treatment is performed to obtain a quercetin-ethanol solution. This solution is added to the fermented tea residue mixture, sheared and homogenized, then ultrasonically treated, homogenized again, and finally spray-dried to obtain the tea residue carrier. S3. Take a strain of Bacillus coagulans, pick a single colony and inoculate it into a liquid culture medium, culture overnight, subculture twice, and prepare Bacillus coagulans seed culture; S4. Slowly add Bacillus coagulans seed liquid to the tea residue carrier while stirring, ferment and culture, and freeze-dry after intermittent stirring to obtain the toxin-degrading agent.

2. The method for preparing a fungal toxin-degrading agent according to claim 1, characterized in that: In step S1, the tea residue is pulverized to 40-60 mesh, and the mass ratio of tea residue to water is 1:(5-8); the cooling time is 2-4 hours; the amount of sucrose added is 3-5 wt.%; the standing time is 10-20 minutes; the amount of kombucha starter culture inoculated is 8-12 wt.%; the standing fermentation time is 2-4 days; the stirring fermentation speed is 50-80 rpm, and the time is 36-48 hours.

3. The method for preparing a fungal toxin-degrading agent according to claim 1, characterized in that: In step S2, the ultrasonic treatment power is 200-300W, and the time is 10-20min; the quercetin content in the quercetin-ethanol solution is 5-8wt.%; the mass ratio of the quercetin-ethanol solution to the fermented tea residue mixture is 1:(12-18); the shear homogenization speed is 3000-5000rpm, and the time is 15-30min; the re-homogenization speed is 1000-2000rpm, and the time is 10-20min.

4. The method for preparing a fungal toxin-degrading agent according to claim 1, characterized in that: In step S3, the Bacillus coagulans strain BC361 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 13, 2014, with accession number CGMCC No. 9951.

5. The method for preparing a fungal toxin-degrading agent according to claim 1, characterized in that: In step S3, the culture is carried out at a rotation speed of 180 rpm and a temperature of 35-37℃; the concentration of the Bacillus coagulans seed solution is 1×10⁻⁶. 9 - 1×10 10 CFU / mL.

6. The method for preparing a fungal toxin-degrading agent according to claim 1, characterized in that: In step S4, the mass ratio of tea residue carrier to Bacillus coagulans seed liquid is 1:(2-3); the fermentation temperature is 35-37℃ and the time is 18-36h; the interval between intermittent stirring is 6-9h.

7. The fungal toxin degrading agent prepared by the preparation method according to any one of claims 1-6.

8. The application of the fungal toxin degrading agent according to claim 7 in the storage of traditional Chinese medicine.

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