Mycotoxin biodecontaminant, its preparation method and application

Abstract

This invention discloses a mycotoxin biological antidote, its preparation method, and its application. The preparation method of the mycotoxin biological antidote includes: using *Aspergillus niger* (accession number CGMCC3.4523) as the strain, performing solid-state fermentation to obtain a *Aspergillus niger* solid culture; mixing the *Aspergillus niger* solid culture with glycyrrhizic acid at a mass ratio of 1-3:1-3 to obtain the mycotoxin biological antidote. Using broiler feeding experiments and serum metabolomics analysis, it was demonstrated that the mycotoxin antidote of this invention can effectively alleviate the harm of various mycotoxins to young broilers by regulating the metabolic pathways in serum, thus improving the health of broilers.

Description

Technical Field

[0001] This invention relates to the field of biological detoxification technology, specifically to a mycotoxin biological detoxification agent, its preparation method, and its application. Background Technology

[0002] In recent years, mycotoxin contamination has frequently occurred in the livestock and feed industries, seriously endangering human and animal health. Mycotoxins are secondary metabolites synthesized by fungi such as Aspergillus, Penicillium, and Fusarium during their growth. Currently, over 400 types of mycotoxins are known and widely present in food and feed. Mycotoxins possess complex and stable chemical structures, containing specific groups that impart toxic effects. These specific groups cause mycotoxins to exhibit carcinogenicity, teratogenicity, mutagenicity, hepatotoxicity, nephrotoxicity, embryotoxicity, and immunosuppression in animals, potentially leading to death in severe cases. When several mycotoxins coexist, their toxicity is cumulative, increasing the harm. Mycotoxins accumulate in the bodies of humans and animals through the food chain, threatening their health. Currently, mycotoxin contamination has attracted widespread attention, and effective control and safe, efficient degradation of mycotoxin contamination are of great significance for the healthy development of the feed, food, and livestock industries.

[0003] Therefore, effectively addressing the hazards of mycotoxins has become a pressing global challenge. Currently, there are three main methods for eliminating or mitigating the harm of mycotoxins: physical, chemical, and biological methods. However, physical and chemical detoxification methods suffer from drawbacks such as strict requirements, high energy consumption, low efficacy, large investment, difficulty in scaling up, impact on feed nutritional value and palatability, and the generation of numerous byproducts that pollute the environment. Consequently, biological detoxification has become the preferred method.

[0004] Biological detoxification methods are widely recognized for their specificity, high efficiency, and environmental friendliness. The nutrients, sensory characteristics, and flavor of the raw materials are preserved without involving harmful chemicals. Screening and isolating naturally occurring microorganisms capable of degrading mycotoxins has become a popular strategy. Biodegradation technology breaks down the toxic groups in mycotoxin molecules, converting them into non-toxic or less toxic degradation products.

[0005] Existing research shows that *Aspergillus niger* can efficiently degrade AFB1, with a degradation rate as high as 93.28%, and the safety of the metabolites after AFB1 degradation by *Aspergillus niger* has been demonstrated through acute toxicity tests in mice. Ji et al. found that a single strain of *Aspergillus niger* could maintain a stable degradation rate of over 95% for ZEN, which was degraded into two low-toxicity products.

[0006] Licorice belongs to the genus *Glycyrrhiza* in the legume family (Fabaceae). It mainly comprises the roots or stems of *Glycyrrhiza uralensis*, *Glycyrrhiza inflata*, or *Glycyrrhiza glabra*. Licorice has a mild odor and a sweet taste. It is believed to have the effects of tonifying the spleen and lungs, clearing heat and relieving cough, promoting qi and blood circulation, unblocking meridians, and harmonizing other herbs, earning it the reputation of "King of Herbs." Glycyrrhizic acid (GA) is the main active substance in licorice, also known as glycyrrhizin or glycyrrhizin saponin, with the molecular formula C2. 42 H 62 O 16 In its pure form, glycyrrhizic acid is a white crystalline solid, readily soluble in hot water but sparingly soluble in cold water. Hydrolysis of glycyrrhizic acid yields two molecules of glucuronic acid and one molecule of 18β-glycyrrhetic acid. It is used to treat various inflammatory and immune diseases, such as hepatitis, urticaria, cirrhosis, and ulcerative colitis. It also possesses anti-inflammatory, immunomodulatory, antioxidant, antiviral, anticancer, and lipid-lowering effects. Glycyrrhizic acid can alleviate liver and intestinal damage. However, research on its use in improving damage caused by ingested mycotoxins is limited.

[0007] No research has been reported on the use of Aspergillus niger culture combined with glycyrrhizic acid to alleviate the harm of AFB1, ZEN and DON to livestock and poultry. Summary of the Invention

[0008] Therefore, embodiments of the present invention provide a mycotoxin biological detoxifier, its preparation method, and its application. Based on the in vitro degradation of AFB1 and ZEN by Aspergillus niger solid culture, this invention selects a strain of Aspergillus niger that efficiently degrades AFB1 and ZEN. The resulting biological agent, obtained by combining its culture with glycyrrhizic acid, has been verified in broiler chicken feeding trials to effectively alleviate the harm caused by AFB1, ZEN, and DON.

[0009] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0010] According to a first aspect of the present invention, the present invention provides a method for preparing a mycotoxin biological antidote, the method comprising: using Aspergillus niger with preservation number CGMCC3.4523 as a strain, performing solid-state fermentation culture to obtain an Aspergillus niger solid culture; mixing the Aspergillus niger solid culture and glycyrrhizic acid at a mass ratio of 1-3:1-3 to obtain the mycotoxin biological antidote.

[0011] Furthermore, the mass ratio of the Aspergillus niger solid culture to glycyrrhizic acid is 1:1.

[0012] Furthermore, the method for preparing the Aspergillus niger solid culture includes:

[0013] Aspergillus niger was spread onto PDA solid medium plates and incubated statically at 30°C for 3–5 days, yielding 1 × 10⁻⁶ spores. 7 Seed culture per mL;

[0014] Wheat bran, soybean meal, and corn were mixed evenly in a mass ratio of 7:2:1. Water was added at a solid-liquid ratio of 5:3, and the mixture was heated at 121℃ with a temperature of 1.035×10⁻⁶. 5 Autoclaving at Pa for 20 min yields solid culture medium;

[0015] The seed culture was inoculated into the solid culture medium at an inoculation rate of 10% (v / w), cultured in a constant temperature incubator at 30°C for 5-7 days, dried, and pulverized to obtain the Aspergillus niger solid culture.

[0016] Furthermore, the preparation method of the PDA solid culture medium is as follows:

[0017] Prepare a PDA liquid culture medium, which has the following components and contents: glucose 20 g / L, soluble starch 6 g / L, yeast extract 2 g / L, magnesium sulfate 0.3 g / L, potassium dihydrogen phosphate 1 g / L, and peptone 5 g / L.

[0018] Agar was added to the PDA liquid medium at 2% (w / v) to obtain the PDA solid medium.

[0019] According to a second aspect of the present invention, the present invention provides a mycotoxin biological antidote, which is made by the method described in any of the preceding claims.

[0020] According to a third aspect of the present invention, the present invention provides the application of the mycotoxin biological detoxifier as described above as a feed additive in mitigating the harmful effects of three mycotoxins on broiler growth.

[0021] Furthermore, the three mycotoxins are aflatoxin B1 (AFB1), zearalenone (ZEN), and vomitoxin (DON).

[0022] Furthermore, the broiler chickens mentioned are young broiler chickens.

[0023] Furthermore, the amount of the mycotoxin biological detoxifier added is 0.4–0.8 kg / ton of feed.

[0024] The embodiments of the present invention have the following advantages:

[0025] 1. In this study, a strain of Aspergillus niger capable of simultaneously degrading AFB1 and ZEN was screened. Its culture was combined with glycyrrhizic acid to prepare a biological agent with good degradation and detoxification effects on AFB1, ZEN and DON.

[0026] 2. Using broiler feeding experiments and serum metabolomics analysis, it was demonstrated that mycotoxin detoxifiers can effectively alleviate the harm of various mycotoxins to young broilers by regulating the metabolic pathways in serum. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0028] Figure 1 Principal component and clustering heatmap analysis of serum metabolites provided by this invention;

[0029] Figure 2 Analysis of the various OPLS-DA models provided in this invention;

[0030] Figure 3 Volcano plot of differential metabolites between groups provided by this invention;

[0031] Figure 4 Venn diagrams and heatmaps of differential metabolites provided for this invention;

[0032] Figure 5 The KEGG pathway is enriched for the differential metabolites provided by this invention. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] 1. Test materials

[0035] AFB1 and ZEN were purchased from Shanghai Maclean's Biochemical Technology Co., Ltd.; the AFB1 and ZEN detection kits were purchased from Jiangsu Suwei Microbiology Research Co., Ltd. The *Aspergillus niger* used in the experiments was purchased from the China General Microbiological Culture Collection Center (CGMCC 3.4523), and the *Aspergillus oryzae* used in the experiments was selected by our laboratory and is preserved at the China General Microbiological Culture Collection Center (CGMCC 5817). Glycyrrhizic acid was kindly provided by Henan Delin Biological Products Co., Ltd.

[0036] 2. Preparation of reagents and culture media

[0037] Dilution of AFB1 and ZEN standards: Dissolve the toxin standards completely in methanol to prepare a 1 mg / mL standard solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store in a brown bottle at -20°C. Before use, dilute the 1 mg / mL standard solution with 60% methanol to obtain solutions of different concentrations and store at 4°C for later use.

[0038] PDA medium: glucose 20 g / L, soluble starch 6 g / L, yeast extract 2 g / L, magnesium sulfate 0.3 g / L, potassium dihydrogen phosphate 1 g / L, peptone 5 g / L. When preparing the solid medium, simply add 2% (w / v) agar to the above liquid medium.

[0039] Solid culture medium for Aspergillus niger and Aspergillus oryzae fermentation: The mass ratio of wheat bran:soybean meal:corn was 7:2:1, and the solid-liquid ratio was 5:3. Autoclaving conditions for the culture medium: 121℃, 1.035 × 10⁻⁶ °C. 5 Pa, 20 min.

[0040] Example 1

[0041] Screening of biological antidotes for mycotoxins

[0042] 1. Activation and seed culture preparation of Aspergillus niger and Aspergillus oryzae

[0043] The laboratory-preserved *Aspergillus niger* strain was spread onto PDA solid medium plates and incubated statically at 30°C for 3–5 days until a large number of black spores appeared on the plates. 5 mL of physiological saline was added to the mature *Aspergillus niger* plate, and the *Aspergillus niger* spores on the surface of the PDA solid medium were gently scraped off using an autoclaved spreader to prepare a spore suspension. This suspension was then diluted with physiological saline to a spore count of 1 × 10⁻⁶. 7 Seed liquid of 10 ...

[0044] Similarly, the preserved Aspergillus oryzae culture was spread onto PDA solid medium plates and incubated statically at 30°C for 3–5 days until a large number of yellow spores were produced on the plates. The preparation method for Aspergillus oryzae seed culture is the same as that for Aspergillus niger seed culture.

[0045] 2. Preparation of solid cultures of Aspergillus niger and Aspergillus oryzae

[0046] Mix wheat bran, soybean meal, and corn in a mass ratio of 7:2:1. Add water at a solid-liquid ratio of 5:3 and mix thoroughly. Place 10g of the mixture in a 250mL Erlenmeyer flask and autoclave. Add 1mL of either Aspergillus niger or Aspergillus oryzae seed solution to the sterilized Erlenmeyer flask, mix well, and incubate at 30℃ for 5–7 days. Once the Erlenmeyer flask is confluent with spores, remove the culture and dry it in a 65℃ drying oven. After drying, pulverize the dried culture for later use.

[0047] 3. Single-factor and orthogonal experimental designs for solid cultures of Aspergillus niger and Aspergillus oryzae.

[0048] 3.1 Single-factor mycotoxin degradation experimental design of Aspergillus niger and Aspergillus oryzae solid cultures

[0049] The enzyme digestion system for determining the degradation of AFB1 and ZEN by solid cultures of Aspergillus niger and Aspergillus oryzae was 10 mL of PDA liquid medium, with AFB1 content of 30 μg / L and ZEN content of 100 μg / L. The blank control group was supplemented with only toxins and without solid cultures of Aspergillus niger and Aspergillus oryzae. The experimental groups are shown in Table 1 below. Each treatment was repeated in triplicate and shaken at 100 r / min for 12 h and 24 h at 37 °C.

[0050] Determination methods for AFB1 and ZEN degradation and calculation of degradation rate: Take the solution after reaction and centrifuge at 4000 r / min for 10 min. Take the supernatant after centrifugation and process it according to the instructions of the AFB1 and ZEN detection kit of Jiangsu Suwei Microbial Research Co., Ltd. and detect its residual toxin content.

[0051] AFB1 degradation rate (%) = (AFB1 content in control group - AFB1 content in experimental group) / AFB1 content in control group × 100. The calculation method for ZEN degradation rate is the same as that for AFB1.

[0052] Statistical analysis of data: Experimental data are presented in the format of "mean ± standard deviation". Preliminary calculations were performed using Excel, followed by one-way ANOVA using SPSS 26.0. Significant differences were indicated by P < 0.05.

[0053] Table 1. Single-factor experimental design grouping (%) of Aspergillus niger and Aspergillus oryzae solid cultures

[0054]

[0055] Table 2 shows the degradation results of AFB1 and ZEN by solid cultures of Aspergillus niger and Aspergillus oryzae with different addition amounts after 12 h of enzymatic hydrolysis. The addition amount of solid cultures of Aspergillus niger and Aspergillus oryzae is based on the mass of the enzymatic hydrolysis system (the same applies below).

[0056] Table 2. Degradation of AFB1 and ZEN by solid cultures of Aspergillus niger and Aspergillus oryzae with different addition amounts after 12 h of enzymatic hydrolysis.

[0057]

[0058]

[0059] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), while the same lowercase letter indicates no significant differences (P>0.05). The same applies below.

[0060] Table 2 shows that after 12 hours of enzymatic hydrolysis, the degradation capacity of AFB1 increased with the increase of the amount of Aspergillus niger and Aspergillus oryzae solid cultures added, and the degradation effect of the Aspergillus niger group was significantly higher than that of the Aspergillus oryzae group (P<0.05), with the highest degradation rate reaching 54.36%. For the degradation of ZEN, the Aspergillus oryzae groups with added amounts of 0.06% and 0.09% showed better degradation effects, at 94.18% and 97.99% respectively, and the degradation effects of these two groups were significantly higher than those of other experimental groups (P<0.05), followed by the Aspergillus niger group with added amount of 0.09%.

[0061] Table 3 shows the degradation results of AFB1 and ZEN by enzymatic hydrolysis of solid cultures of Aspergillus niger and Aspergillus oryzae with different addition amounts for 24 h.

[0062] Table 3. Degradation of AFB1 and ZEN by solid cultures of Aspergillus niger and Aspergillus oryzae with different additions after 24 h of enzymatic hydrolysis.

[0063]

[0064] Table 3 shows that after 24 hours of enzymatic hydrolysis, groups 2 and 3 exhibited the best degradation effects on AFB1, with degradation rates of 71.54% and 69.06%, respectively, significantly higher than other groups (P<0.05). The degradation effects on AFB1 were consistent with those after 12 hours of enzymatic hydrolysis, with all Aspergillus niger groups showing significantly higher degradation rates than Aspergillus oryzae groups (P<0.05). All experimental groups showed good degradation effects on ZEN, with the Aspergillus oryzae groups and experimental groups 2-3 achieving degradation rates exceeding 98%, significantly higher than experimental group 1 (P<0.05).

[0065] 3.2 Orthogonal experimental design of solid cultures of Aspergillus niger and Aspergillus oryzae

[0066] Solid cultures of Aspergillus niger and Aspergillus oryzae were combined using a two-factor, three-level orthogonal experimental design based on different addition amounts, and the groupings are shown in Table 4. The AFB1 content in the reaction system was 50 μg / L, the ZEN content was 100 μg / L, and all other experimental conditions were the same as in 3.1.

[0067] Table 4. Orthogonal experimental design groupings (%) for Aspergillus niger and Aspergillus oryzae

[0068]

[0069] The degradation results of AFB1 and ZEN by different addition amounts of Aspergillus niger and Aspergillus oryzae solid culture combinations for 24 h are shown in Table 5.

[0070] Table 5. Degradation of AFB1 and ZEN by different combinations of Aspergillus niger and Aspergillus oryzae solid cultures after 24 h.

[0071]

[0072]

[0073] Table 5 shows that after 24 hours of enzymatic hydrolysis, the degradation ability of AFB1 by Aspergillus niger solid culture alone was significantly higher than that by Aspergillus oryzae solid culture (P<0.05), especially in the Aspergillus niger solid culture groups (groups 2 and 3), which showed better degradation effects on AFB1 (P<0.05). There was no significant difference compared to groups 11, 13, and 15 in the orthogonal experiment (P>0.05). Regarding the degradation effect on ZEN, the Aspergillus niger solid culture group was also significantly higher than that of the Aspergillus oryzae solid culture group in the single-factor experiment (P<0.05). However, the orthogonal experiment revealed that the addition of Aspergillus oryzae solid culture reduced the efficacy of the Aspergillus niger solid culture. Through comprehensive analysis, the addition of Aspergillus niger solid culture at levels of 0.04% and 0.06% showed the best simultaneous degradation ability of AFB1 and ZEN, and there was no significant difference between the two groups (P>0.05). Therefore, 0.04% Aspergillus niger solid culture will be used as the reference value for the next step of the experiment.

[0074] 3.3 Orthogonal experimental design of Aspergillus niger solid culture and glycyrrhizic acid

[0075] Two-factor, three-level orthogonal experiments were conducted on Aspergillus niger solid culture and glycyrrhizic acid at addition levels of 0.02%, 0.04%, and 0.06%, respectively. The experimental groups are shown in Table 6 below. The enzymatic hydrolysis system was still 10 mL of PDA liquid culture medium with AFB1 added. The AFB1 content in the reaction system was 50 μg / L and the ZEN content was 100 μg / L. Then, it was shaken in a shaker at 37℃ for 24 h.

[0076] Table 6. Grouping (%) of Aspergillus niger solid culture and glycyrrhizic acid orthogonal experiment

[0077]

[0078] The degradation rates of AFB1 and ZEN by different amounts of Aspergillus niger and glycyrrhizic acid after 24 hours of reaction are shown in Table 7.

[0079] Table 7. Degradation rates of AFB1 and ZEN after 24 hours of reaction with different amounts of Aspergillus niger and glycyrrhizic acid.

[0080]

[0081] As shown in Table 7, after 24 hours of enzymatic hydrolysis, the degradation ability of AFB1 by adding 0.02% and 0.04% *Aspergillus niger* solid culture initially increased and then decreased with increasing glycyrrhizic acid content. However, the degradation ability of the experimental group with 0.06% *Aspergillus niger* solid culture increased with increasing glycyrrhizic acid content. The combination with the best degradation effect on AFB1 and ZEN was group 9, followed by group 5, and the difference in degradation effect between the two groups was not significant (P>0.05). Therefore, from an economic perspective, group 5 (with 0.04% *Aspergillus niger* solid culture and 0.04% glycyrrhizic acid) will be used as a reference value for later experiments.

[0082] Example 2

[0083] Biological detoxifiers alleviate the harmful effects of mycotoxins on the growth performance of broilers.

[0084] 1. Materials and Methods

[0085] 1.1 Test Materials

[0086] The method for preparing Aspergillus niger solid culture is the same as in Example 1. Glycyrrhizic acid was kindly provided by Henan Delin Biological Products Co., Ltd., and the mycotoxin biological detoxifier was prepared by mixing Aspergillus niger solid culture and glycyrrhizic acid at a mass ratio of 1:1. AA broiler chickens were purchased from Henan Longhua Animal Husbandry Co., Ltd.

[0087] 1.2 Animal Experiment Design and Grouping

[0088] Animal experiments were conducted at the Xuchang Experimental Base of Henan Agricultural University. Seven hundred healthy male broiler chickens aged one day with uniform weight were selected and divided into 14 treatment groups, with five replicates per treatment group and ten chickens per replicate. The rearing period was 21 days. A multi-tiered cage rearing system was used, with free access to feed and water, and 24-hour lighting. The immunization program was as follows: Newcastle disease live vaccine on day 1, and Newcastle disease and infectious bronchitis bivalent live vaccine on day 7, administered via drinking water. The experimental groups are as follows:

[0089] Group A: Basal diet (containing AFB1 (0.002 g / t) + ZEN (0.041 g / t) + DON (0.946 g / t)

[0090] Group B: Moldy diet 1 (containing AFB1 (0.03 g / t) + ZEN (0.15 g / t) + DON (1.50 g / t))

[0091] Group C: Moldy diet 2 (containing AFB1 (0.07 g / t) + ZEN (0.50 g / t) + DON (3.00 g / t))

[0092] Group D: Basal diet + Aspergillus niger (0.4 kg / t)

[0093] Group E: Basal diet + glycyrrhizic acid (0.4 kg / t)

[0094] Group F: Basal diet + Mycotoxin biological detoxifier (0.2 kg / t)

[0095] Group G: Basal diet + Mycotoxin biological detoxifier (0.4 kg / t)

[0096] Group H: Basal diet + mycotoxin biological detoxifier (0.6 kg / t)

[0097] Group I: Moldy diet 1 + Aspergillus niger (0.4 kg / t)

[0098] Group J: Moldy diet 1 + glycyrrhizic acid (0.4 kg / t)

[0099] Group K: Moldy diet 1 + Mycotoxin biological detoxifier (0.2 kg / t)

[0100] Group L: Moldy diet 1 + Mycotoxin biological detoxifier (0.4 kg / t)

[0101] Group M: Moldy diet 1 + Mycotoxin biological detoxifier (0.6 kg / t)

[0102] Group N: Moldy diet 2 + Mycotoxin biological detoxifier (0.4 kg / t)

[0103] 1.3 Dietary composition and nutrient levels in animal experiments

[0104] The experimental basal diet was formulated based on the NRC (1994) broiler feeding standards. Different proportions of naturally moldy corn were used to replace the normal corn in the basal diet to increase AFB1 and ZEN concentrations, and corn slurry powder was used to increase DON content. The diet formulation and nutritional composition are shown in Table 8.

[0105] Table 8 Broiler Chicken Diet Formulation and Nutritional Levels (%, Air-dried Basis)

[0106]

[0107]

[0108] Note: Premixed feed includes (per kg of complete feed): VA 12000 IU; VD3 3000 IU; VE 20 IU; VK3 1.0 mg; VB1 2.0 mg; Riboflavin (VB2) 6 mg; Niacin (nicotinic acid) 35 mg; Choline 1.3 g; Calcium pantothenate 10 mg; VB6 3.5 mg; VB12 0.01 mg; Biotin 0.15 mg; Folic acid 1.25 mg; Copper (copper sulfate) 8 mg; Iron (ferrous sulfate) 100 mg; Manganese (manganese sulfate) 80 mg; Zinc (zinc oxide) 60 mg; Iodine I (calcium iodate) 0.45 mg; Selenium (sodium selenite) 0.35 mg. Crude protein, calcium, and total phosphorus in the nutrient levels are measured values; the rest are estimated from the feed composition and nutritional value table.

[0109] 1.4 Determination of broiler growth performance

[0110] Daily feed intake of broilers was recorded, and their health status and mortality were observed and recorded. On day 21, broilers were weighed after a 12-hour fast, and the average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (F / G), and mortality rate were calculated for each replicate. The effects of the mycotoxin biological antidote on the growth performance of broilers are shown in Table 9.

[0111] Table 9. Effects of mycotoxin biological antidotes on broiler growth performance.

[0112]

[0113]

[0114] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), while the same lowercase letter indicates no significant differences (P>0.05). The same applies below.

[0115] As shown in Table 9, the low-toxin group (Group B) and the high-toxin group (Group C) increased the mortality rate of broilers, and the mortality rate increased with increasing toxin levels (2% / 6%). However, the addition of the mycotoxin biological antidote reduced the mortality rate of all groups to zero, fully demonstrating the effectiveness of the mycotoxin biological antidote. Compared with Group A, high mycotoxin content significantly reduced final weight and average daily feed intake (P<0.05), increasing broiler mortality. Group N, which added the mycotoxin biological antidote, significantly reduced broiler mortality compared with Group C. Compared with Group A, Group G significantly increased final weight and average daily weight gain (P<0.05), and the feed conversion ratio of all experimental groups with added mycotoxin biological antidote showed a decreasing trend.

[0116] 1.5 Determination of conventional nutrient components and the contents of AFB1, ZEN and DON in feed and broiler excrement

[0117] Excrement from 17-19 day old broilers was collected using a total manure collection method. Nitrogen fixation was performed using a 10% sulfuric acid solution, and the excrement was stored at -20℃. At the end of the experiment, excrement collected over three consecutive days was mixed thoroughly and dried at 65℃. The crude protein, crude fat, calcium, and phosphorus contents in the feed and excrement were then determined according to national standards GB / T 6432-2018 (crude protein determination), GB / T 6433-2006 (crude fat determination), GB / T 6436-2002 (calcium determination), and GB / T 6437-2018 (phosphorus determination). The methods for determining AFB1, ZEN, and DON contents in the excrement followed the methods of the Suwei toxin detection kit. The effects of the mycotoxin biological antidote on the nutrient metabolism rate of broilers are shown in Table 10, and the effects of the mycotoxin biological antidote on the toxin content in broiler excrement are shown in Table 11.

[0118] Table 10. Effects of mycotoxin biological antidotes on nutrient metabolism rate in broilers (%)

[0119]

[0120]

[0121] Table 10 shows that group G had the highest crude protein metabolism rate, groups L and N had the highest crude fat metabolism rate, group J had the highest calcium metabolism rate, and group G had the highest phosphorus metabolism rate. Compared with group A, group C significantly decreased crude protein and phosphorus metabolism rates (P<0.05), but groups B and C increased fat metabolism rates (P<0.05). Compared with group A, group G showed an increasing trend in crude protein and phosphorus metabolism rates; compared with group B, group L showed a significant increase in crude fat metabolism, and an increasing trend in crude protein, calcium, and phosphorus metabolism rates; compared with group C, group N showed an increasing trend in crude protein, crude fat, and phosphorus metabolism rates. This indicates that the addition of mycotoxin biological detoxifiers improved the metabolism rate of nutrients in broilers to some extent.

[0122] Table 11 Effects of mycotoxin biological antidotes on toxin content in broiler excrement (μg / kg)

[0123]

[0124] Table 11 shows that, regarding the toxin content in broiler excrement, compared with group A, the levels of the three mycotoxins detected in the excrement of group B increased significantly after the addition of mycotoxins (P<0.05). After the addition of the mycotoxin biological antidote in group G, compared with group A, the content of AFB1 decreased significantly (P<0.05), while the contents of ZEN and DON showed a decreasing trend. Compared with group B, after the addition of the mycotoxin biological antidote, the content of ZEN in group L decreased significantly (P<0.05), and the contents of AFB1 and DON showed a decreasing trend, but the difference from group B was not significant (P>0.05).

[0125] 1.6 Determination of serum biochemical indicators

[0126] At 21 days, based on the production performance indicators of each broiler group, groups A, B, G, and L were selected as the key research subjects. In each replicate of each group, one chicken with a weight close to the average was selected, and 2 mL of blood was collected from the wing vein. The blood was placed in a refrigerator at 4℃ until some serum separated from the collection tube. The tube was centrifuged at 4000 rpm for 10 min, and the supernatant serum was transferred to a sterilized cryovial and stored at -20℃ for later use. After the experiment, the contents of glucose, urea, triglycerides, high-density lipoprotein, total cholesterol, low-density lipoprotein, aspartate transferase, lactate dehydrogenase, alkaline phosphatase, total protein, albumin, and globulin were measured using a fully automated blood biochemistry analyzer. The results of the effect of the mycotoxin biological antidote on the serum biochemical indicators of broilers are shown in Table 12.

[0127] Table 12 Effects of mycotoxin biological antidotes on serum biochemical parameters of broilers

[0128]

[0129] Table 12 shows that compared with control group A, group B had significantly lower levels of ALP, total protein, albumin, glucose, total cholesterol, and triglycerides (P<0.05), while AST levels were significantly higher (P<0.05), and ALT levels showed an upward trend. This indicates that low-dose mycotoxins had adverse effects on metabolism and organs. Group L had significantly lower ALT, AST, and ALP activities and triglyceride levels than group B (P<0.05), while albumin and total cholesterol levels were significantly higher (P<0.05). Adding 0.4 kg / t of mycotoxin biological antidote alleviated the damage of mycotoxins to organs and tissues, which is beneficial to improving the health of broilers.

[0130] Example 3

[0131] Effects of various mycotoxin biological antidotes on broiler serum metabolomics

[0132] 1. Materials and Methods

[0133] 1.1 Test Materials

[0134] Based on the growth performance of broilers in each group during the feeding trial period (1-21 days), serum metabolomics analysis was performed on broilers in groups A, B, G, and L. Acetonitrile, formic acid, ammonium formate, and 2-chloro-L-phenylalanine (internal standard) were the main reagents used in the analysis.

[0135] 1.2 Instruments and Equipment

[0136] Filter membranes, refrigerated centrifuges (H1850-R, Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China), mixers (BE-2600, Haimen Qilin Bell Instrument Manufacturing Co., Ltd., Nantong, China), vacuum concentrators (5305, Eppendorf, Hamburg, Germany), liquid chromatographs (Vanquish, Thermo, Massachusetts, USA), and mass spectrometers (Orbitrap Exploris 120, Thermo, Massachusetts, USA).

[0137] 1.3 Extraction of serum metabolites

[0138] 1) Thaw the test sample at 4℃, and vortex the sample for 1 minute after thawing to mix it evenly;

[0139] 2) Accurately transfer an appropriate amount of sample into a 2mL centrifuge tube;

[0140] 3) Add 400 μL of methanol solution (stored at -20℃) and vortex for 1 min;

[0141] 4) Place in a centrifuge and centrifuge at 12000 rpm and 4℃ for 10 min. Transfer all the supernatant from the centrifuge tube to another new 2 mL centrifuge tube, concentrate and dry.

[0142] 5) Add 150 μL of 2-chloro-L-phenylalanine (4 ppm) solution prepared with 80% methanol (store at 4℃) to reconstitute the sample. Take the supernatant and filter it through a 0.22 μm filter membrane. Add the filtrate to the UPLC-MS sample detection bottle for UPLC-MS detection.

[0143] 1.4 UPLC-MS Analysis

[0144] (1) Chromatographic conditions: see Tables 13 and 14.

[0145] Thermo Vanquish ultra-high performance liquid chromatography system, using ACQUITY HSS T3 (2.1×100mm, 1.8μm) column, flow rate 0.3mL / min, column temperature 40℃, injection volume 2μL. Positive ion mode, mobile phase: 0.1% formic acid in water (A1) and 0.1% formic acid in acetonitrile (B1); negative ion mode, mobile phase: 5mM ammonium formate in water (A2) and acetonitrile (B2).

[0146] Table 13 Elution gradient program in positive ion mode

[0147]

[0148] Table 14 Elution gradient program in negative ion mode

[0149]

[0150] (2) Mass spectrometry conditions:

[0151] A Thermo Orbitrap Exploris 120 mass spectrometer with an electrospray ionization (ESI) source was used to acquire data in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, and the negative ion spray voltage was -2.50 kV. The sheath gas concentration was 40 alb, and the auxiliary gas concentration was 10 alb. The capillary temperature was 325 °C. A first-stage full scan was performed at a resolution of 60,000 m / z, with a first-stage ion scan range of 100–1000 m / z. Second-stage fragmentation was performed using high-energy induced fragmentation (HCD) with a collision energy of 30% and a second-stage resolution of 15,000 m / z. The first four ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion.

[0152] 1.5 Differential Metabolite Analysis

[0153] Multivariate statistical methods were used to perform dimensionality reduction and classification analysis on omics data, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), to reveal differential metabolites among different groups. Differential analysis was performed using predefined statistical tests, and significant differences were screened using t-tests and OPLS-DA first principal component variable importance value projection (VIP). Subsequently, KEGG pathway enrichment analysis was performed on differential metabolites to assess whether they play an important role in biological responses.

[0154] 1.6 Data Analysis

[0155] The XCMS package in R was used to perform peak detection, peak filtering, and peak alignment on the raw mass spectrometry files converted by Proteowizard. Substance identification was performed using spectral databases such as HMDB, massbank, LipidMaps, mzcloud, KEGG, and Nomi Metabolism's self-built metabolite standard database. After obtaining metabolite information, differentially expressed metabolites were screened using the P<0.05 and VIP>1 criteria. Spearman correlation analysis was then performed on the differentially expressed metabolites and related indicators such as production performance, serum biochemistry, and residues of three toxins in excrement using Nomi Metabolism's self-built platform.

[0156] 2 Results and Analysis

[0157] 2.1 Principal Component and Cluster Heatmap Analysis of Serum Metabolites

[0158] Depend on Figure 1 Principal component analysis (PCA) of serum metabolites revealed that each point represents a sample, with points of the same color indicating samples from the same experimental group. Significant differences were observed between samples, and three sample points from the same experimental group showed a certain clustering trend, indicating significant differences in metabolites among the four experimental groups. The data stability and repeatability of each experimental group were good, meeting the experimental requirements. Figure 1 The total differential metabolite clustering heatmap of B shows that two or more samples from different experimental groups showed the same result for the same metabolite, indicating that the results of this experiment have good consistency among samples in the same group and small differences within the group.

[0159] 2.2 OPLS-DA method for analyzing serum metabolites

[0160] Serum metabolites from the four experimental groups were analyzed using the OPLS-DA method. Figure 2 . Figure 2In diagram A, the horizontal axis PC1 represents the score of the first principal component, and the vertical axis OC2 represents the score of the first orthogonal component. Each point represents a test sample, and points of the same color indicate that they come from the same test group. The horizontal axis represents the differences between groups, and the vertical axis represents the differences within groups. Samples from different test groups are relatively dispersed, while the three sample points from the same test group have a higher degree of clustering. The more clustered the samples within a group and the more dispersed the samples between groups, the more reliable the results are. Figure 2 The x-axis of B represents the similarity between the true sample group and 100 random groups, and the y-axis represents the model evaluation parameters. The upper right corner points Q2 and R2 represent the model evaluation parameters for the true sample group. If Q2 is always below R2, the results are considered reliable. Figure 2 The results showed that the addition of mycotoxins and mycotoxin biological detoxifiers had a significant impact on broiler serum metabolites.

[0161] 2.3 Identification and screening of serum differential metabolites

[0162] A total of 302 differentially expressed metabolites were detected in broiler serum in this experiment. The number of differentially expressed metabolites in each group is shown in Table 15. In the comparisons of A vs. B, B vs. L, A vs. L, and A vs. G, a total of 26, 16, 31, and 18 differentially expressed metabolites were screened, respectively. Compared with group A, group B significantly downregulated 9 differentially expressed metabolites and upregulated 17 differentially expressed metabolites (P<0.05); group L significantly downregulated 17 differentially expressed metabolites and upregulated 14 differentially expressed metabolites (P<0.05); and group G significantly downregulated 6 differentially expressed metabolites and upregulated 12 differentially expressed metabolites (P<0.05). Compared with group B, group L significantly downregulated 11 differentially expressed metabolites and upregulated 5 differentially expressed metabolites (P<0.05). Subsequently, volcano plots were used to analyze the distribution and changes of differentially expressed metabolites in the two experimental groups. The results are as follows: Figure 3 As shown. In each pair of differentially expressed metabolites, the volcano plot displays the top five most significantly different metabolites. In A vs. B, these are nervonic acid ((15Z)-Tetracosenoic acid), 2,3-butanediol, N-acetylaspartylglutamate, phthalic acid, and 9-cis-retinol (…). Figure 3 A). In B vs. L, the five most significantly different metabolites were hydroquinone, oxalureate, 2,3-butanediol, linoleic acid, and gycitein. Figure 3B). In A vs. L, the five most significantly different metabolites were nervonic acid ((15Z)-Tetracosenoic acid), 2,3-butanediol, nicotinic acid, phthalic acid, and 3-hydroxy-DL-kynurenine. Figure 3 C). In A vs. G, the five most significantly different metabolites were aminomalonic acid, resveratrol, nervonic acid ((15Z)-tetracosenoic acid), 3-hydroxy-DL-kynurenine, and cyromazine. Figure 3 D).

[0163] Table 15 Statistical Analysis of Differential Metabolites

[0164]

[0165] Venn diagram analysis was performed on the differential metabolites from each group. Figure 4 A) Two differentially expressed metabolites were found in the four comparisons Avs.B, B vs.L, Avs.L, and Avs.G. The comparisons between the Avs.B and B vs.L groups revealed seven common differentially expressed metabolites: dimethylglycine, 2,3-butanediol, selenocysteine, (-)-jasmonic acid, 3-hydroxy-DL-kynurenine, 3-ketosphingosine, and glycitein. Dimethylglycine and jasmonic acid were downregulated in group A vs. B, but upregulated in group B vs. L; 3-ketosphingosine and glycitein were upregulated in group A vs. B, but downregulated in group B vs. L. The specific differentially expressed metabolites between groups are shown in Table 16. To better illustrate the concentration changes of different metabolites in different experimental groups, we performed heatmap cluster analysis on 56 differentially expressed metabolites from 12 samples in 4 experimental groups. Figure 4B) Cluster heatmaps showed good homology among the three samples in the experimental group, and the metabolites could be divided into two major categories and four subcategories. Compared with group B, group L had 26 downregulated metabolites and 30 upregulated metabolites.

[0166] Table 16 Expression levels of differentially expressed metabolites in different samples

[0167]

[0168]

[0169] Note: E represents scientific notation, which is 10 to the power of negative numbers.

[0170] 2.4 KEGG enrichment analysis of serum differential metabolites

[0171] To further elucidate the metabolic pathways involved in all differentially metabolized metabolites in serum, KEGG pathway enrichment was performed on the differentially metabolites in the four groups: Avs.B, B vs. L, Avs.L, and Avs.G. The results are as follows: Figure 5 As shown. Compared with group A, the differential metabolites in group B are mainly concentrated in the following pathways: alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism; arginine and proline metabolism; and steroid hormone biosynthesis. Figure 5 A). Compared with group B, the differential metabolites in group L were mainly concentrated in pathways such as glycine, serine and threonine metabolism, cysteine ​​and methionine metabolism, selenium compound metabolism, and linoleic acid metabolism. Figure 5 B). Compared with group A, the differential metabolites in group L were mainly related to pathways involving selenium compound metabolism, arginine and proline metabolism, ABC transporters, and arginine biosynthesis. Figure 5C). Compared to group A, the differential metabolites in group G were mostly concentrated in the alanine, aspartate, and glutamate metabolism pathways; ABC transporters; linoleic acid metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis pathways. Figure 5 D).

[0172] Further KEEG enrichment analysis of common differential metabolites in groups A vs. B and B vs. L revealed that their metabolites were mainly associated with three pathways: selenium compound metabolism, alpha-linolenic acid metabolism, and glycine, serine, and threonine metabolism. Figure 5 E). The results showed that mycotoxins damage the body by affecting the synthesis and metabolism of amino acids and the biosynthesis of steroid hormones. However, the addition of mycotoxin biological detoxifiers can alleviate the damage caused by mycotoxins by affecting pathways such as the synthesis and metabolism of amino acids, selenium complex metabolism, and α-linolenic acid metabolism.

[0173] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a mycotoxin biological antidote, characterized in that, The method includes: Using Aspergillus niger with accession number CGMCC3.4523 as the strain, solid-state fermentation was carried out to obtain Aspergillus niger solid culture; A solid culture of Aspergillus niger and glycyrrhizic acid were mixed at a mass ratio of 1-3:1-3 to obtain the mycotoxin biological antidote. The method for preparing the Aspergillus niger solid culture includes: Aspergillus niger was spread onto PDA solid medium plates and incubated statically at 30°C for 3–5 days, yielding 1 × 10⁻⁶ spores. 7 Seed culture per mL; Wheat bran, soybean meal, and corn were mixed evenly in a mass ratio of 7:2:

1. Water was added at a solid-liquid ratio of 5:3, and the mixture was heated at 121℃ with a temperature of 1.035×10⁻⁶. 5 Autoclaving at Pa for 20 min yields solid culture medium; The seed culture was inoculated into the solid culture medium at an inoculation rate of 10% v / w, and cultured in a constant temperature incubator at 30℃ for 5-7 days. After drying and pulverizing, the Aspergillus niger solid culture was obtained.

2. The method for preparing the mycotoxin biological antidote according to claim 1, characterized in that, The mass ratio of Aspergillus niger solid culture to glycyrrhizic acid is 1:

1.

3. The method for preparing the mycotoxin biological antidote according to claim 1, characterized in that, The preparation method of the PDA solid culture medium is as follows: Prepare a PDA liquid culture medium, which has the following components and contents: glucose 20 g / L, soluble starch 6 g / L, yeast extract 2 g / L, magnesium sulfate 0.3 g / L, potassium dihydrogen phosphate 1 g / L, and peptone 5 g / L. Agar was added to the PDA liquid medium at a rate of 2% w / v to obtain the PDA solid medium.

4. A biological antidote for mycotoxins, characterized in that, It is made by the method of any one of claims 1-3.

5. The application of the mycotoxin biological detoxifier according to claim 4 as a feed additive in mitigating the harmful effects of three mycotoxins on broiler growth, characterized in that... The three mycotoxins are aflatoxin B1, zearalenone, and vomitoxin.

6. The application according to claim 5, characterized in that, The broiler chickens mentioned are young broiler chickens.

7. The application according to claim 5, characterized in that, The amount of the mycotoxin biological detoxifier added is 0.4~0.8 kg / ton of feed.

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