Application of bile acid composition in preparation of feed additive for preventing and controlling aspergillus flavus in animal breeding

Abstract

Hyodeoxycholic acid chenodeoxycholic acid

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of bile acid compositions in the preparation of feed additives for the prevention and control of aflatoxin in animal husbandry. Technical Background

[0002] Around the time of harvesting crops such as corn, soybeans, and peanuts, damp environments often breed mold. The mycotoxins produced by these molds can enter the body through food and adversely affect the health of humans and animals. Among these, aflatoxin (Aspergillus flavus) is a particularly harmful toxin. Aspergillus flavus Aflatoxin B1, produced by Aspergillus flavus, is the most toxic form of aflatoxin, classified as a Group 1 carcinogen by the World Health Organization (WHO). Studies show that livestock fed feed containing excessive levels of aflatoxin B1 suffer liver damage and even acute poisoning, and aflatoxin B1 can also threaten the health of young animals through breast milk. Furthermore, aflatoxins have been shown to damage DNA and cause genotoxic effects such as cancer in animals. Therefore, developing technologies to inhibit Aspergillus flavus growth and aflatoxin B1 production is of great significance for the healthy development of the feed and livestock industries and for protecting food safety, and is gradually becoming a hot topic in the industry.

[0003] Bile acid (BA) is a naturally occurring substance secreted by vertebrates with antibacterial properties. It is also a feed additive with liver-protective effects. Numerous studies have shown that bile acids can alleviate liver damage caused by aflatoxin B1. Bile acids are synthesized by the liver, stored in the gallbladder, and enter the intestines with food. Besides promoting lipid digestion, the main function of bile acids in the intestines is to inhibit exogenous microorganisms and maintain the balance of the intestinal microecology. Faced with the rampant spread of drug-resistant pathogens, traditional drugs, represented by antibiotics, are becoming increasingly ineffective, and natural antibacterial substances have gradually become a research hotspot. As natural antibacterial substances, even after hundreds of millions of years of evolution, many pathogens remain sensitive to bile acids. The antibacterial mechanism of bile acids mainly includes two aspects: first, bile acids can disrupt cell membranes, causing intracellular substances to leak out and affecting microbial metabolism; second, bile acids have an amphiphilic structure, allowing them to cross the cell membrane of microorganisms, enter the cell, and induce intracellular oxidative damage. However, the inhibitory effect of bile acids on aflatoxin has not yet been reported.

[0004] There are many types of bile acids, with thousands of species discovered. Studies have shown that bile acids with different structures can have antibacterial effects that differ by as much as 10 times. Summary of the Invention

[0005] Based on the above needs, the application aims to provide the application of the bile acid composition in the preparation of a feed additive for preventing and controlling aspergillus flavus in animal breeding, which can effectively inhibit the growth of aspergillus flavus and the synthesis of aflatoxin B1, and lay a foundation for the application of bile acids in preventing feed mildewing and aflatoxin B1 exceeding the standard.

[0006] To achieve the above application purposes, the application is implemented by the following technical solutions: The application provides the application of the bile acid composition in the preparation of a feed additive for preventing and controlling aspergillus flavus in animal breeding.

[0007] Further, the components of the bile acid composition include, in terms of mass percentage, 78% to 83% of hyodeoxycholic acid, 17% to 22% of chenodeoxycholic acid, Further, the structural formula of the hyodeoxycholic acid is , The structural formula of the chenodeoxycholic acid is .

[0008] Further, the bile acid composition is extracted from pig gall paste by the processes of high-temperature and high-pressure saponification, oxidation and decolorization, acidification and crystallization, water washing and impurity removal, and drying.

[0009] Further, the extraction method of the hyodeoxycholic acid and the chenodeoxycholic acid in the bile acid composition from pig gall paste includes the following steps: (1) Ultra-high-temperature and ultra-high-pressure saponification: the crushed pig gall paste is added with 10% to 15% of sodium hydroxide, and diluted into a liquid with water, and then treated in a reaction kettle at 105 to 135 DEG C and 1.05 to 1.10 Mpa for 10 to 15 h. After the reaction is completed, the liquid is cooled and separated into layers, and the supernatant is removed to obtain pig gall paste saponification product; (2) Oxidation and decolorization: the pig gall paste saponification product is mixed with water to completely dissolve the pig gall paste saponification product, and then H2O2 is added for decolorization at 25 to 30 DEG C for 24 h; (3) Acidification and crystallization: under the action of stirring, the pH value of the solution is adjusted to 3.0 to 3.5 by using hydrochloric acid, and the stirring is continued until the bile acid crystals are continuously precipitated; (4) Water washing, impurity removal and drying: the supernatant is removed by centrifugation, and the precipitated bile acid is washed and rinsed 2 to 3 times with water, and then filtered by a plate and frame pressure filter to obtain the bile acid, and then dried by using a spray drying device, with the inlet air temperature controlled at 130 to 180 DEG C and the outlet air temperature controlled at 80 to 100 DEG C, to obtain the bile acid composition.

[0010] Further, the total bile acid content and the contents of the hyodeoxycholic acid and the chenodeoxycholic acid in the bile acid composition are determined by high performance liquid chromatography.

[0011] Further, the high performance liquid chromatography conditions are as follows: the chromatography conditions are determined as follows: C18 chromatography column, length 150 mm, inner diameter 4.6 mm, particle size 5 μm; mobile phase: A liquid: 0.05% trifluoroacetic acid solution, B liquid: acetonitrile; flow rate: 1.0 mL / min; detection wavelength: fluorescence excitation wavelength 330 nm, emission wavelength 410 nm; column temperature: room temperature; injection volume: 10 μL.

[0012] Further, the elution gradient is as follows: 0~2 min 40% A liquid + 60% B liquid, 3~18 min 20% A liquid + 80% B liquid, 19~26 min 40% A liquid + 60% B liquid.

[0013] Further, the retention time of poractant alfa is 9.2 min, and the retention time of chenodeoxycholic acid is 14.8 min.

[0014] Further, the Aspergillus flavus is A. flavus a strain (market common sales strain).

[0015] Further, the Aspergillus flavus is A. flavus a strain with a toxin content of 650-730 ng / L.

[0016] Further, the Aspergillus flavus is A. flavus a strain with high yield of aflatoxin B1.

[0017] Further, the MIC value of poractant alfa to Aspergillus flavus is 0.4 mg / mL, and the MIC value of chenodeoxycholic acid to Aspergillus flavus is 0.2 mg / mL.

[0018] Further, the bile acid composition can inhibit the growth of Aspergillus flavus.

[0019] Further, the chenodeoxycholic acid in the bile acid composition has a greater ability to inhibit the growth of Aspergillus flavus than poractant alfa.

[0020] Further, the bile acid composition can inhibit the synthesis of aflatoxin B1 by Aspergillus flavus.

[0021] Further, the ability of poractant alfa in the bile acid composition to inhibit the synthesis of aflatoxin B1 by Aspergillus flavus is greater than the ability of chenodeoxycholic acid to inhibit the synthesis of aflatoxin B1 by Aspergillus flavus.

[0022] Further, the bile acid composition can form water-insoluble precipitates with aflatoxin B1.

[0023] Further, the optimized components and contents of the bile acid composition are as follows: poractant alfa 78%, chenodeoxycholic acid 17%, and the balance being water.

[0024] Further, based on the optimized bile acid composition, the adding amount of the bile acid composition in the feed additive is 3-10 mu g / g, and the mass concentration of the bile acid composition is 0.1-1 mg / mL.

[0025] Further, the bile acid composition plays a bacteriostatic role by destroying the cell membrane of Aspergillus flavus and causing intracellular potassium ions to flow out.

[0026] Compared with the prior art, the present application has the following beneficial effects: 1. The present application separates a strain of Aspergillus flavus with high yield of aflatoxin B1 from corn, and conducts a bacteriostatic experiment on the strain of Aspergillus flavus by using the allowed use of chenodeoxycholic acid and hyodeoxycholic acid in feed additives. The present application explores the inhibitory effect of the bile acid composition on the growth of Aspergillus flavus and the mechanism of interfering with the synthesis of aflatoxin B1, so as to provide a basis for the prevention and control of mycotoxins in crops.

[0027] 2. The bile acid composition plays a bacteriostatic role by destroying the cell membrane of Aspergillus flavus and causing intracellular potassium ions to flow out. The inhibitory effect of chenodeoxycholic acid in the bile acid composition on the growth of Aspergillus flavus is stronger than that of hyodeoxycholic acid, and the inhibitory effect of hyodeoxycholic acid on the synthesis of aflatoxin B1 by Aspergillus flavus is stronger than that of chenodeoxycholic acid.

[0028] 3. The bile acid composition is extracted from pig gall paste by high-temperature and high-pressure saponification, oxidation and decolorization, acidification and crystallization, and water washing and impurity removal drying process. The bile acid composition containing 78% of hyodeoxycholic acid and 17% of chenodeoxycholic acid has the most significant effect on inhibiting the growth of Aspergillus flavus and the synthesis of aflatoxin B1 in feed. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art according to these drawings without exceeding the scope of the present application.

[0030] Figure 1 The left graph is the colony morphology characteristics, and the right graph is the cotton dyeing characteristics of the Aspergillus flavus HL2 strain with high yield of aflatoxin B1.

[0031] Figure 2 The phylogenetic tree of Aspergillus flavus HL2 is shown.

[0032] Figure 3 The ability evaluation of Aspergillus flavus HL2 to synthesize aflatoxin B1 is shown.

[0033] Figure 4 Inhibition of Aspergillus HL2 by hyodeoxycholic acid.

[0034] Figure 5 Inhibition of Aspergillus HL2 by hyodeoxycholic acid.

[0035] Figure 6 Antibacterial mechanism of hyodeoxycholic acid on Aspergillus HL2.

[0036] Figure 7 Inhibition of aflatoxin B1 synthesis by Aspergillus HL2 by different bile acids.

[0037] Figure 8 Effect of different bile acids on aflatoxin B1 related genes of Aspergillus HL2.

[0038] Figure 9 Effect of hyodeoxycholic acid on the transcriptome of Aspergillus HL2 (left panel: up-regulated genes, right panel: down-regulated genes).

[0039] Figure 10 Effect of hyodeoxycholic acid on the transcriptome of Aspergillus HL2 (left panel: up-regulated genes, right panel: down-regulated genes).

[0040] Figure 11 Ability of different bile acids to form co-precipitates with aflatoxin B1.

[0041] Figure 12 Inhibition of Aspergillus HL2 growth in feed by different bile acids. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0043] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application or the field to which the term applies. Although any materials or methods similar or equivalent to those described herein can be used in the practice of the present application, the preferred materials and methods are described herein.

[0044] Example 1, Isolation, identification of Aspergillus producing high levels of aflatoxin B1 and determination of aflatoxin B1 production by the Aspergillus The sample with the highest content of aflatoxin B1 (185 ppb) was selected from 45 corn samples for the isolation of Aspergillus flavus. The sample was sequentially soaked with 0.05% sodium hypochlorite solution for 3 min, washed with sterile water for 30 s, soaked with 75% ethanol for 1 min, and finally washed with sterile water for 4-6 times. The corn particles were evenly spotted on high-salt Sabouraud agar medium, and incubated at 28°C until single colonies were formed. Single colonies were inoculated on Sabouraud medium and purified by continuous streaking until no mixed bacteria appeared. The colony morphology was observed and stained with cotton blue, and the bacterial morphology was observed under a microscope. The isolated strain was named HL2. The ITS sequence of HL2 was amplified by PCR and sent to Shanghai Sangon for sequencing. The measured ITS gene sequence was uploaded to the National Microbiology Data Center (NMDC), and the sequence of the model strain with high similarity to the test strain was downloaded after CLUSTAL_W (Larkin et al. 2007) comparison with the sequence of the related strain in the GenBank database. MEGA 7 software was used for multiple sequence alignment, and then a phylogenetic tree was constructed according to the maximum likelihood model.

[0045] HL2 was inoculated on PDA plates and incubated in a 28°C incubator for 5 days. Spores were washed off with PBS solution (pH 7.2), counted with a hemocytometer, and adjusted to a final concentration of 1×10 6 CFU / mL, and stored in a 4°C refrigerator for standby. The prepared spore suspension was inoculated in Sabouraud medium and incubated at 28°C and 180 r / min for 72 h. The supernatant was obtained by centrifugation, filtered through a 0.22 μm filter membrane, and stored in a 4°C refrigerator for detection. The content of aflatoxin B1 was detected according to GB5009.22—2016 "National Food Safety Standard Determination of Aflatoxin B and G in Food", and the content of aflatoxin B1 was determined by HPLC. A. flavus CICC 41668 was used as the control group, and the sample was also detected.

[0046] In the sample library, one strain of mold was isolated from one corn sample with the highest content of aflatoxin B1. The colony morphology was semi-woolly, and the color changed from white to yellow and yellow-green as it grew. Staining and microscopic observation showed that the conidial phialides were rough, the conidial heads were loose and radial, and the top cysts were flask-shaped ( Figure 1 ). The above colony morphology and strain morphology characteristics were basically consistent with the description of Aspergillus in the fifth volume of "Chinese Fungus" ( A. flavus ), so it could be preliminarily determined that the strain was A. flavus By constructing the ITS sequence phylogenetic tree of the strain, it was found that it was in the same evolutionary branch as Aspergillus flavus ( Figure 2 ). Further determination showed that the strain was Aspergillus flavus, and it was namedA. flavus HL2. The ITS gene sequence of HL2 was uploaded to NMDC, and the sequence number was NMDCN007RDL, A. flavus HL2 can also be obtained through market sales channels.

[0047] To explore the ability of synthesizing aflatoxin B1, the model strain A. flavus CICC 41668 was selected as the control, and A. flavus the ability of HL2 to synthesize aflatoxin B1 was evaluated. The results are shown in Figure 3 , A. flavus HL2 showed stronger toxin-producing ability, and 48 h A. flavus HL2 detected 465.68 ng / L of AFB1. At the same time, A. flavus the amount of AFB1 synthesized by HL2 showed a rapid increase trend with the extension of time, and was higher than that of the model strain A. flavus CICC41668 (P<0.05) at different time points. After 72 h of culture, the toxin content of the model strain A. flavus CICC41668 was 564.55 ng / L, while A. flavus the toxin content of HL2 reached 693.89 ng / L, which was 22.91% higher than that of the model strain. The above results show that the isolated A. flavus HL2 is a strain with high yield of aflatoxin B1.

[0048] Example 2, preparation of bile acid composition The present embodiment provides a preparation method of bile acids, and the prepared bile acids include hyodeoxycholic acid and chenodeoxycholic acid, which are obtained from the extraction method of pig gall paste.

[0049] 1. Preparation of bile acid composition (1) Ultra-high temperature and ultra-high pressure saponification: After the pig gall paste is crushed, 10%-15% sodium hydroxide is added, and water is used to dilute it into a liquid. In the reaction kettle, the conditions are 105-135℃ and 1.05-1.10 Mpa for 10-15 h. After the reaction is completed, the liquid is cooled and separated into layers, and the supernatant is removed to obtain pig gall paste saponification product.

[0050] (2) Oxidation and decolorization: Mix the pig gall paste saponification product with water to completely dissolve the pig gall paste saponification product, then add H2O2, and decolorize at 25-30℃ for 24 h.

[0051] (3) Acidification and crystallization: Under the action of stirring, adjust the pH value of the solution to 3-3.5 using hydrochloric acid, and continue to stir until bile acid crystals are continuously precipitated.

[0052] (4) Washing and drying: Centrifuge to remove supernatant, wash the precipitated bile acids with water 2-3 times, filter through plate and frame filter to obtain bile acids, and then use spray drying equipment with the inlet air temperature controlled at 130-180℃ and the outlet air temperature controlled at 80-100℃ to obtain bile acid composition.

[0053] 2. Measurement of the components of the bile acid composition The total bile acid content and the contents of porcine deoxycholic acid and chenodeoxycholic acid in the bile acid composition obtained above were determined by high performance liquid chromatography (HPLC) using the following experiments: (1) Before detection by high performance liquid chromatography, the bile acid composition was derivatized in acetonitrile solution using 4-bromomethyl-7-methoxycoumarin as a derivatizing reagent to obtain bile acid fluorescent derivatives.

[0054] (2) High-performance liquid chromatography (HPLC) conditions were as follows: A C18 column, 150 mm long, 4.6 mm inner diameter, and 5 μm particle size. Mobile phase: Solution A: 0.05% trifluoroacetic acid solution; Solution B: acetonitrile. Flow rate: 1.0 mL / min. Detection wavelength: fluorescence excitation wavelength 330 nm, emission wavelength 410 nm. Column temperature: room temperature. Injection volume: 10 μL. Elution gradient: 0–2 min 40% Solution A + 60% Solution B, 3–18 min 20% Solution A + 80% Solution B, 19–26 min 40% Solution A + 60% Solution B. The retention time of porcine deoxycholic acid was 9.2 min, and the retention time of chenodeoxycholic acid was 14.8 min.

[0055] Example 3: Inhibitory effect of bile acids on the growth of Aspergillus flavus To investigate the antibacterial efficacy of the bile acid composition, porcine deoxycholic acid and chenodeoxycholic acid obtained in Example 2 were subjected to a diffusion experiment. The plate diffusion method was used to investigate the effects of different bile acids on... A. flavus For the antibacterial experiment of HL2, different concentrations of porcine deoxycholic acid and chenodeoxycholic acid solutions were prepared using methanol solution, with final concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 mmol / mL, and evenly spread on PDA plates. 10 μL of spore suspension was spotted onto the center of the plate, and after the colonies air-dried naturally, they were incubated upright at 28℃ for 5 days, and the colony diameter was recorded.

[0056] The effects of different bile acids on the body were determined using a gradient dilution method. A. flavus The MIC was determined. In a 96-well plate, 100 μL of spore suspension (10 μL / well) was added to each well. 6(CFU / mL), then methanol solutions of chenodeoxycholic acid were added to final concentrations of 25.60, 12.80, 6.40, 3.20, 1.60, 0.80, 0.40, 0.20, 0.10, and 0.05 mg / mL. After mixing, the mixture was incubated at 28℃ for 24 h, and the effects of chenodeoxycholic acid on... A. flavus The MIC value of HL2. Using the same method, the effect of porcine deoxycholic acid on... A. flavus MIC value of HL2.

[0057] Different bile acids A. flavus The effect of HL2 growth diameter as follows Figure 4 and Figure 5 As shown, untreated with bile acids A. flavus HL2 grew normally in PDA, starting to produce yellow spores on the second day and producing a large number of spores on the fifth day. Treatment with 100 mmol / mL bile acids significantly inhibited [the growth of HL2]. A. flavus The growth of HL2 indicates that porcine deoxycholic acid and chenodeoxycholic acid have antibacterial activity. Based on this concentration, the two bile acids were serially diluted; as the concentration of the bile acids decreased, A. flavus The colony diameter of HL2 cells gradually increases. Figure 4 and Figure 5 It is known that porcine deoxycholic acid and chenodeoxycholic acid have significant antibacterial activity against Aspergillus flavus and can effectively inhibit its growth.

[0058] Cultured by treatment with different concentrations of chenodeoxycholic acid and porcine deoxycholic acid A. flavus After 24 hours of HL2 culture, when chenodeoxycholic acid reached 0.2 mg / mL and porcine deoxycholic acid reached 0.4 mg / mL, no mycelial growth was observed in the 96-well plate. This confirmed the presence of chenodeoxycholic acid and porcine deoxycholic acid. A. flavus The minimum inhibitory concentrations for HL2 were 0.2 and 0.4 mg / mL, respectively.

[0059] Example 4: Mechanisms of action of different bile acids in inhibiting the growth of Aspergillus flavus 1% vaccination dose, inoculated and stored A. flavusHL2 spore suspension was transferred to Sabouraud dextrose agar and cultured at 28°C and 180 r / min for 48 h. After filtration to obtain mycelial pellets, the pellets were rinsed three times with 0.85% NaCl saline. Three equal-weight mycelial pellets were weighed and added to three 100 mL bottles of saline solution. 1×MIC (0.2 mg / mL) and 2×MIC (0.4 mg / mL) chenodeoxycholic acid were added to each, respectively, with an equal volume of methanol as a control. The mixture was shaken at 180 r / min, and samples were taken at 0, 5, 10, 30, and 60 min. The samples were filtered through a 0.22 μm filter, and the potassium (K) content was determined using atomic absorption spectrometry. + Content. Using the same method, the effect of porcine deoxycholic acid on... A. flavus HL2's K + The impact of release amount.

[0060] To investigate the effect of bile acids on the cell membrane permeability of Aspergillus flavus, the potassium ion content within Aspergillus flavus spores was measured after treatment with different concentrations of chenodeoxycholic acid and sucrase deoxycholic acid, respectively. Figure 6 As shown, with the increase of chenodeoxycholic acid concentration and porcine deoxycholic acid concentration, intracellular K... + Increased release leads to higher K content in the filtrate. + Increased content. When the sampling time was 80 min, 1×MIC chenodeoxycholic acid K + The content is 5.30 μg / mL, 2×MIC chenodeoxycholic acid K + The concentration was 6.57 μg / mL, representing increases of 68.79% and 109.23% compared to the control (CK), respectively. This indicates that bile acids alter the permeability of the Aspergillus flavus cell membrane, affecting cell membrane transport and transmembrane transport. However, no K was observed in Aspergillus flavus treated with the same concentration of porcine deoxycholic acid. + Increased content.

[0061] Example 5: Inhibition of aflatoxin B1 synthesis by different bile acids from Aspergillus flavus 1% vaccination dose, inoculated and stored A. flavus HL2 spore suspensions were transferred to three bottles of Sabouraud dextrose agar, and sucrase deoxycholic acid and chenodeoxycholic acid were added to a final concentration of 0.5 × MIC, respectively. The treatment without bile acid was used as a control group. The culture was carried out at 28℃ and 150 rpm for 5 days, with samples taken every 24 h. After filtering through a 0.22 μm filter, the aflatoxin B1 content in the supernatant was determined using a kit.

[0062] To further study the effects of bile acids on A. flavus The effects of HL2 toxicity were investigated under different bile acid treatments. A. flavus AFB1 production of HL2 in Sabouraud broth. The effects of different bile acids on... A. flavusThe effect of HL2 on the synthesis of aflatoxin B1, such as Figure 7 As shown, after 32 h of culture, the AFB1 content in the control group showed a significant upward trend ( P <0.05), reaching 138.22 μg / mL, while the addition of porcine deoxycholic acid and chenodeoxycholic acid significantly inhibited the production of aflatoxin. At 96 h, the AFB1 content in the chenodeoxycholic acid group was 596.28 μg / mL, and the AFB1 content in the porcine deoxycholic acid group was 495.53 μg / mL, which were reduced by 15.40% and 29.69% respectively compared with the control group, indicating that the addition of different bile acids had an effect on the production of aflatoxin. A. flavus The HL2 group showed an inhibitory effect on the production of aflatoxin B1, with the porcine deoxycholic acid group showing a stronger inhibition of aflatoxin B1 synthesis.

[0063] 1% vaccination dose, inoculated and stored A. flavus HL2 spore suspensions were transferred to three bottles of Sabouraud dextrose agar, and methanol solutions of sucrase deoxycholic acid (SDCA) and chenodeoxycholic acid (CDO) at a final concentration of 0.5 × MIC were added, respectively. An equal volume of methanol solution was added to the control group. The culture was incubated at 30°C and 180 rpm for 48 h. The bacterial suspension was centrifuged at 5000 rpm for 2 min, the supernatant was discarded, and the bacterial cells were washed twice with physiological saline and collected by centrifugation. RNA was extracted from the bacterial cells using an RNA extraction kit. The samples were sent to Shanghai Sangon Biotech, where a cDNA library was constructed using a reverse transcription kit. After quality control, transcriptome sequencing was performed using Illumina Hiseq™.

[0064] Combination Figure 8 and Figure 9 and Figure 10 As shown in the transcriptome analysis of *Aspergillus flavus*, among the 46 genes involved in aflatoxin B1 synthesis, 38 genes were downregulated and 8 were upregulated in the chenodeoxycholic acid (CDO) treatment group; while in the porcine CDO treatment group, 44 genes were downregulated and only 2 were upregulated, indicating that porcine CDO more significantly inhibited the expression of genes related to aflatoxin B1 synthesis. In the CDO treatment group, due to its stronger cell membrane disruption effect, some cell membrane synthesis-related genes were upregulated. Furthermore, cell membrane disruption led to the efflux of some small molecules, resulting in a significant upregulation of some molecular synthesis-related genes. Among the downregulated genes, due to the stronger antibacterial effect of CDO, the number of downregulated nucleic acid-related genes was higher than that of genes downregulated in the aflatoxin B1 synthesis pathway.

[0065] Example 6: Application of bile acids in mycotoxin binders In 100 ng / mL aflatoxin B1 solution, 20 mmol / mL of chenodeoxycholic acid and hyodeoxycholic acid solutions (pH 8.0) were added respectively, and stirred until the sample was completely dissolved, and then continued to stir for 30 min, with a sample without bile acid as a control. Then, the pH of the solution was slowly adjusted to 5.0 using 50% HC1, and continued to stir for 30 min until insoluble substances precipitated, and then centrifuged at 10000 r / min for 5 min to separate the supernatant and the precipitate. After adjusting the pH of the supernatant solution to 8.0 using 2 mol / L NaOH, the aflatoxin B1 content was detected. An equal volume of pH 8.0 phosphate buffer was added to the precipitate, and continued to stir until the precipitate was completely dissolved. The pH of the re-dissolved precipitate solution was adjusted to 8.0, and the aflatoxin B1 content in the solution was detected using an aflatoxin B1 detection kit. The results of the co-precipitation experiment of bile acids and aflatoxin B1 are shown in Table 1. The aflatoxin B1 content in the supernatant at pH 8.0 was 109.216 ng / mL, and after adding bile acids to form precipitates, the aflatoxin B1 content in the supernatant was greatly reduced, with a 55.5% reduction in the chenodeoxycholic acid group and a 29.9% reduction in the hyodeoxycholic acid group, while the aflatoxin B1 content in the control group remained unchanged. The above results show that bile acids have the ability to bind aflatoxin B1 and form co-precipitates.

[0066] Table 1. Aflatoxin B1 content under different conditions

[0067] To further verify whether aflatoxin B1 is contained in the precipitate, the co-precipitate of bile acids and aflatoxin B1 was collected and freeze-dried. It was placed on a solid sample holder and tested for fluorescence spectrum using a fluorescence spectrometer (Hitachi FL-4700) in steady-state mode with an excitation wavelength of 365 nm. As shown in Table 2, the fluorescence spectrum of aflatoxin B1 appeared in the co-precipitate of hyodeoxycholic acid and chenodeoxycholic acid with aflatoxin B1. Figure 11 Figure 11 In the precipitate, aflatoxin B1 appeared, and the fluorescence signal intensity of aflatoxin B1 in the chenodeoxycholic acid precipitate was higher than that in the co-precipitate of hyodeoxycholic acid and aflatoxin B1, indicating that chenodeoxycholic acid has a stronger co-precipitation effect on aflatoxin B1, which is consistent with the results in Table 1.

[0068] Example 7. Application of bile acid composition in preventing mold growth in feed raw materials ​The bile acid combination in this embodiment is a combination of porcine deoxycholic acid and chenodeoxycholic acid in different mass percentages, specifically as follows: Combination 1 (porcine deoxycholic acid 100%, chenodeoxycholic acid 0%), Combination 2 (porcine deoxycholic acid 0%, chenodeoxycholic acid 100%), Combination 3 (porcine deoxycholic acid 20%, chenodeoxycholic acid 80%), Combination 4 (porcine deoxycholic acid 50%, chenodeoxycholic acid 50%), Combination 5 (porcine deoxycholic acid 80%, chenodeoxycholic acid 20%), Combination 6 (porcine deoxycholic acid 78%, chenodeoxycholic acid 17%, with the remainder being water).

[0069] Select peanut, soybean, and corn seeds with intact seed coats and uniform size, and sterilize at 121℃ for 20 min. Weigh 20 g of seeds and place them in a petri dish. Take 100 μL of the preserved seeds. A. flavus HL2 spore suspension was inoculated onto the seed surface. 100 μL of 0.4 mg / mL bile acid combination 1–6 samples were added and thoroughly mixed. The treatment without added bile acids served as a control group. The seeds were sealed with sealing film and incubated at 28℃ for 5 days. The mold growth status of the plant seeds was observed, and the growth of Aspergillus flavus and the synthesis of aflatoxin B1 in the maize samples were detected by dilution plating and an aflatoxin B1 kit.

[0070] To investigate the effect of bile acids on the pathogenicity of Aspergillus flavus on peanut, corn, and soybean seeds, this experiment determined the infection status of Aspergillus flavus on peanut, corn, and soybean seeds treated with 0.4 mg / mL bile acid combinations 1 to 6. The results are as follows: Figure 12 As shown, after 5 days of cultivation, peanut, corn, and soybean seeds in the control group showed obvious mold growth, with Aspergillus flavus covering the seed surface. However, no Aspergillus flavus mycelium was observed in the peanut, corn, and soybean seeds of the treatment groups, indicating that bile acids can be used as an additive to prevent mold growth in feed. Table 2 shows the Aspergillus flavus count and aflatoxin B1 content detection results for corn samples treated with bile acid combinations 1 to 6. The mold count in corn samples treated with bile acid combinations 1 to 6 was less than the 10-1 specified in the "Feed Hygiene Standard". 4 The concentrations of aflatoxin B1 were all less than 30 ppb (CFU / g). Based on the prices of chenodeoxycholic acid (500 yuan / kg) and sucrase deoxycholic acid (200 yuan / kg), and considering the quantity of mold and the content of aflatoxin B1 in the raw materials, the optimal combination of sucrase deoxycholic acid and chenodeoxycholic acid in the bile acid products for controlling Aspergillus growth and aflatoxin B1 synthesis in feed was determined to be: 78% sucrase deoxycholic acid and 17% sucrase deoxycholic acid.

[0071] Table 2. Determination of total mold count and aflatoxin B1 content in corn samples.

[0072] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the present application; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features thereof can be replaced by equivalent replacements; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. Application of bile acid compositions in the preparation of feed additives for the prevention and control of aflatoxin in animal husbandry.

2. The application according to claim 1, characterized in that, The components of the bile acid composition include, by mass percentage, 78% to 83% porcine deoxycholic acid and 17% to 22% chenodeoxycholic acid.

3. The application according to claim 2, characterized in that, The bile acid composition is extracted from pig bile paste through a process of high-temperature, high-pressure saponification, oxidation decolorization, acidification crystallization, water washing to remove impurities, and drying.

4. The application according to claim 1, characterized in that, The MIC value of the porcine deoxycholic acid against Aspergillus flavus is 0.4 mg / mL; the MIC value of the chenodeoxycholic acid against Aspergillus flavus is 0.2 mg / mL.

5. The application according to claim 2, characterized in that, The bile acid composition can inhibit the growth of Aspergillus flavus, and the bile acid composition has a greater inhibitory effect on Aspergillus flavus growth than porcine deoxycholic acid.

6. The application according to claim 2, characterized in that, The bile acid composition can inhibit the synthesis of aflatoxin B1 by Aspergillus flavus.

7. The application according to claim 6, characterized in that, The bile acid composition can form a water-insoluble precipitate with aflatoxin B1.

8. The application according to claim 2, characterized in that, The optimized composition and content of the bile acid composition are as follows: by mass percentage, 78% porcine deoxycholic acid, 17% chenodeoxycholic acid, and the balance being water.

9. The application according to claim 1, characterized in that, The bile acid composition exerts its antibacterial effect by disrupting the cell membrane of Aspergillus flavus, leading to the outflow of intracellular potassium ions.

10. The application according to claim 1, characterized in that, The bile acid composition is added to the feed additive at a rate of 3-10 μg / g, and the mass concentration of the bile acid composition is 0.1-1 mg / mL.

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