Preparation and application of skeleton type vomitoxin antidote

Abstract

The invention discloses preparation and application of a skeleton type vomitoxin antidote, and relates to the technical field of feed processing, and the skeleton type vomitoxin antidote is characterized in that the antidote is prepared from 60-70% of Na2S2O5 and 30-40% of a composite carrier through a hot melt extrusion instrument under the conditions that the sleeve outlet temperature is 60-80 DEG C and the screw rotating speed is 30-50 r / min. When the antidote is used for removing vomitoxin in mildewed corn, and the adding level of the effective component skeleton type SMBS is 0.1%, the using effect of the skeleton type vomitoxin antidote is optimal; when the antidote is used for removing DON (deoxynivalenol) in corn alcohol lees polluted by mycotoxin and the addition level of the effective component skeleton-type SMBS is 0.4%, the use effect of the skeleton-type vomitoxin antidote is optimal. A theoretical basis is provided for further development of the corn and the corn alcohol lees in the field of feed processing.

Description

Technical Field

[0001] This invention relates to the field of feed processing technology, and more specifically, to the preparation and application of a skeletal type vomitoxin antidote. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a toxic and harmful secondary metabolite produced by fungi, primarily originating from moldy feed ingredients such as corn and its processing byproducts, and wheat and its processing byproducts. When mammals ingest feed contaminated with DON, they often exhibit symptoms such as reduced feed intake, anorexia, and even vomiting, hence the name "vomiting toxin." Even low doses of DON (1 mg / kg) can still produce toxicity due to mycotoxin interactions, affecting livestock health and production. Among mammals, pigs are considered the most sensitive to DON. Previous studies have thoroughly explored the effects of DON on the physiological health of pigs, clarifying that DON exerts its effects through mechanisms such as inhibiting cellular protein and nucleic acid synthesis and inducing cell death. Studies by Zhang Lei et al. have found that ingestion of certain concentrations of DON in pigs can induce intestinal inflammation and cell necrosis. DON damages the intestinal mucosal epithelial cell barrier, affecting the intestine's ability to absorb nutrients, thereby impacting the overall nutritional status and growth performance of pigs. Khaled Ghareeb et al. found that DON can induce changes in the intestinal structure of pigs, leading to impaired nutrient uptake. Therefore, when pigs ingest feed contaminated with high concentrations of DON, it can severely damage their digestive system, or even cause death, significantly impacting the economic benefits for farmers. However, in actual livestock production, factors such as temperature, humidity, and culture medium can easily cause DON contamination. For example, corn stored at room temperature often becomes moldy, leading to the proliferation of fungi and the production of DON, ultimately affecting corn quality. Therefore, given the severe shortage of feed resources in my country, maximizing the conservation of feed grain resources and eliminating or reducing the toxicity of DON in feed ingredients is crucial for reducing pig farming costs and improving efficiency.

[0003] Corn is the main raw material for the alcohol fermentation industry. China is currently the third largest alcohol producer, with an annual output exceeding 9 million tons. The byproducts left over from corn fermentation for ethanol production, after drying, are called "Distillers Dried Grains with Solubles (DDGS)". The nutritional components of DDGS feed include crude protein, crude fat, neutral detergent fiber, acid detergent fiber, and minerals such as calcium and phosphorus. It is also rich in B vitamins and vitamin E, and incorporates the nutrients and active factors from saccharification koji and yeast. Specifically, the crude protein content ranges from 26.7% to 32.9%, the crude fat content from 8.8% to 12.4%, and the neutral detergent fiber content from 33% to 40%. Furthermore, DDGS has a very low lignin content and is characterized by high protein, high available phosphorus, rich vitamins, and low starch content, making it a nutritionally rich protein feed ingredient. In practical applications, DDGS is widely used as a major component of high-protein feeds in livestock, poultry, and aquatic compound feeds, and can replace traditional protein sources such as soybean meal and fishmeal. It is particularly popular in ruminant feed because ruminants can directly digest this feed. DDGS production is considerable, with approximately 0.85 to 1 ton of DDGS produced for every ton of fuel ethanol produced. However, according to the "2023 Survey Report on Mycotoxin Contamination in Chinese Feed and Raw Materials," the exceedance rate of DON in DDGS was 88.2%, with an average toxin content of 1.36 mg / kg, a minimum of 0.85 mg / kg, and a maximum of 1.72 mg / kg, classifying it as moderate contamination. Furthermore, the lack of relatively effective and economical treatment methods hinders the practical application of DDGS in production. Therefore, how to reduce the DON contamination exceedance rate while utilizing DDGS is of significant research importance.

[0004] Vomitoxin can be detoxified using various methods, including physical, chemical, and biological approaches. Na₂S₂O₅ (Sodium Metabisulfite, SMBS) can react with the C-9 and C-10 double bonds in the vomitoxin structure, altering its chemical structure and eliminating its toxicity without significantly affecting the growth performance and apparent nutrient digestibility of weaned piglets. However, SMBS also has potential drawbacks in practical applications. It decomposes under the highly acidic conditions of the pig's stomach (pH approximately 2.6), affecting its detoxification efficiency. Furthermore, the SO₂ produced during decomposition damages the gastric mucosa, impacting pig growth. Therefore, developing novel SMBS formulations that are slightly soluble or insoluble in the stomach to avoid damaging the gastric mucosa and affecting growth performance, while rapidly dissolving and releasing in the small intestine to eliminate the toxicity of vomitoxin and prevent its impact on pig growth, has significant theoretical and practical value. Summary of the Invention

[0005] The purpose of this invention is to provide a skeleton-type antidote for vomitoxin and its application. The skeleton-type antidote for vomitoxin comprises 60%–70% SMBS, 30%–40% a composite carrier (composed of 2% tributyl 2-hydroxy-1,2,3-propanetricarboxylate and 98% Eudragit L100), and is produced by hot-melt extrusion at a sleeve outlet temperature of 60°C–80°C and a screw speed of 30–50 r / min using an L9 (3) extruder. 4 An orthogonal experimental screening method was used to obtain a matrix-type vomitoxin antidote (gastric environment dissolution rate ≤20%, intestinal environment dissolution rate ≥70%). The matrix-type vomitoxin antidote is prepared based on the HME process, and its active ingredient is matrix-type SMBS. When used to remove DON from moldy corn, the antidote shows the best effect when the addition level of its active ingredient, matrix-type SMBS, is 0.1%; when used to remove DON from corn distillers grains contaminated with mycotoxins, the antidote shows the best effect when the addition level of its active ingredient, matrix-type SMBS, is 0.4%.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a skeleton-type vomitoxin antidote, wherein the active ingredient in the skeleton-type vomitoxin antidote is skeleton-type SMBS, and the content of SMBS in the skeleton-type vomitoxin antidote is 60% to 70%.

[0007] This invention further provides a method for preparing a skeleton-type vomitoxin antidote:

[0008] The skeleton-type vomiting toxin antidote consists of 60%–70% SMBS, 30%–40% composite carrier (composed of 2% tributyl 2-hydroxy-1,2,3-propanetricarboxylate and 98% Eudragit L100), extruded through a hot melt extruder at a sleeve outlet temperature of 60°C–80°C and a screw speed of 30–50 r / min via L9 (3 4 An antidote for vomitoxin with a skeleton-type structure was obtained through orthogonal screening (dissolution rate in the gastric environment ≤20%, dissolution rate in the intestinal environment ≥70%). The active ingredient in the antidote for vomitoxin with a skeleton-type structure is SMBS. The antidote for vomitoxin with a skeleton-type structure was prepared based on the HME process.

[0009] This invention further provides applications of the above-mentioned skeleton-type vomitoxin antidote, specifically including the following two application methods:

[0010] Application 1: Applying skeletal vomitoxin antidotes in corn-related feed processing.

[0011] When the skeleton-type vomitoxin antidote is used to remove DON from moldy corn, the effect of the skeleton-type vomitoxin antidote is optimal when the addition level of the active ingredient skeleton-type SBMS is 0.1%.

[0012] Application 2: Applying skeletal vomitoxin antidotes in corn alcohol residue feed processing.

[0013] When the skeleton-type vomitoxin antidote is used to remove DON from corn ethanol residue contaminated with mycotoxins, the optimal level of the skeleton-type vomitoxin antidote is achieved when the active ingredient skeleton SMBS is added at 0.4%.

[0014] In summary, the present invention has the following beneficial effects:

[0015] When applying the skeleton-type vomitoxin antidote to remove DON from moldy corn according to Application 1, the coating effect of the skeleton-type vomitoxin antidote was best when the addition level of the active ingredient skeleton-type SMBS was 0.1%. The DON content in the sample decreased from 18045.53 ppb to 16323.55 ppb after in vitro simulated digestion (P>0.05), providing a basis for the application of corn in feed processing.

[0016] When applying the skeleton-type vomitoxin antidote to remove DON from corn distillers grains contaminated with mycotoxins according to Application 2, the coating effect of the enteric-coated DON antidote was best when the active ingredient skeleton-type SMBS was added at a level of 0.2%. The DON content in the sample decreased from 3721.70 ppb to 2934.12 ppb after simulated digestion in vitro. However, compared with the 0.4% skeleton-type vomitoxin antidote addition level, the protective effect in the gastric digestive tract was not significantly different, and the removal effect on DON in the intestinal environment was poor, with a removal rate of only 7.93%. Therefore, the 0.4% skeleton-type vomitoxin antidote addition level had better protective and removal effects, which was more in line with the experimental expectations and provided a basis for the application of corn distillers grains in feed processing. Attached Figure Description

[0017] Figure 1 This is the DON content standard curve in Example 1 of the present invention;

[0018] Figure 2 This is the DON content standard curve in Example 2 of the present invention. Detailed Implementation

[0019] The following is in conjunction with the appendix Figure 1-2 The present invention will be described in further detail below.

[0020] Example 1: A method for preparing a skeleton-type vomitoxin antidote based on HME process

[0021] 1. The materials and instruments used in Example 1 are as follows:

[0022] 1.1 Test Materials:

[0023] DON antidote, Eudragit L100, tributyl citrate, sodium thiosulfate, citric acid, disodium hydrogen phosphate, iodine, potassium iodide, potassium dichromate, hydrochloric acid (1+3), distilled water, starch.

[0024] 1.2 Preparation of test reagents:

[0025] (1) Citric acid-disodium hydrogen phosphate with pH=2.6: Accurately weigh 1.000g of disodium hydrogen phosphate and 9.000g of citric acid, dissolve them in distilled water, and determine the pH value.

[0026] (2) Citric acid-disodium hydrogen phosphate with pH=6.2: Accurately weigh 3.000g of disodium hydrogen phosphate and 0.700g of citric acid, dissolve them in distilled water, and measure the pH value.

[0027] (3) 0.05 mol / L iodine titrant: Take 13.0 g of iodine, add 36 g of potassium iodide and 50 mL of water to dissolve, add 3 drops of hydrochloric acid and an appropriate amount of water to make 1000 mL, shake well, filter through a sintered glass filter and standardize.

[0028] (4) 0.1 mol / L sodium thiosulfate titrant: Take 26 g of sodium thiosulfate and 0.20 g of anhydrous sodium carbonate, add an appropriate amount of freshly boiled and cooled water to dissolve to 1000 mL, shake well, let stand for 1 month and then filter, and then standardize with potassium dichromate solution.

[0029] (5) Starch indicator solution: Take 0.5g of soluble starch, add 5mL of water and stir well. Slowly pour the solution into 100mL of boiling water while stirring. Continue to boil for 2 minutes, let cool, and pour off the supernatant. This solution should be freshly prepared before use.

[0030] (6) Potassium dichromate solution: Weigh approximately 0.15g of potassium dichromate that has been dried to constant weight at 120℃, place it in an iodine bottle, add 50mL of water to dissolve it, add 2.0g of potassium iodide, shake gently to dissolve it, add 40mL of dilute sulfuric acid, shake well, and seal tightly; after placing it in the dark for 10 minutes, dilute it with 250mL of water.

[0031] 1.3 Experimental Instruments and Equipment:

[0032] Hot melt extruder: JH-16 single screw extruder manufactured by Shandong Tengfei Plastic Machinery Factory

[0033] Dissolution tester: RC-3 type dissolution tester manufactured by Tianjin Xintianguang Analytical Instrument Technology Co., Ltd.

[0034] pHS-3C pH meter: manufactured by Shanghai Leici Company

[0035] 2. Experimental methods and procedures

[0036] 2.1 Design of Experimental Process Parameters

[0037] In this experiment, the main coating process parameters were the ratio of antidote to coating material, the outlet temperature of the hot melt extruder sleeve, and the screw speed of the hot melt extruder. An orthogonal experimental design was used. The factor levels are shown in Table 1.

[0038] Table 1 Factor Level Table

[0039]

[0040] L9 (3) 4 The orthogonal experimental design is shown in Table 2.

[0041] Table 2 L9 (3) 4 Orthogonal experimental design table

[0042]

[0043] The dissolution factor design is shown in Table 3:

[0044] Table 3 Dissolution test conditions

[0045]

[0046] 2.2 Sample Preparation

[0047] The antidote was prepared with two carrier excipients, tributyl citrate and Eudragit L100, in the proportions specified in Table 2.2. The mixture was then thoroughly ground and mixed in a mortar and slowly poured into a preheated hot melt extruder. The mixture was then extruded at the temperature and speed specified in Table 2 to produce the nine samples required for the experiment.

[0048] 2.3 Dissolution of the sample

[0049] Take 5.000g of each of the nine prepared samples and grind them thoroughly. Pour 500mL of pre-prepared buffer solution of the corresponding pH condition into a rotating basket, turn on the dissolution tester, and preheat at the predetermined temperature for 30min. After preheating, pour 5.000g of sample into the buffer solution and dissolve the sample according to the predetermined time and rotation speed.

[0050] 2.4 Sample collection

[0051] At pH 2.6, 50 mL of sample solution was collected using a single-mark pipette at 30 min, 60 min, and 90 min, with 50 mL of buffer added immediately after each sampling. At pH 6.2, 50 mL of sample solution was collected using a single-mark pipette at 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. After each sampling, the sample solution was placed in a 50 mL container, and 50 mL of the corresponding buffer solution was added to the transfer basket immediately.

[0052] 2.5 Sample Titration

[0053] Take 5 mL of the sample solution and place it in a 100 mL Erlenmeyer flask. Dilute with 25 mL of distilled water, accurately add 5 mL of iodine titrant (0.05 mol / L), and then add 1 mL of 1+3 hydrochloric acid. Shake thoroughly. Titrate with sodium thiosulfate titrant (0.1 mol / L). Near the endpoint, add 1 mL of starch indicator solution and continue titrating until the blue color disappears. Record the result accurately. Repeat the titration 3 times for each sample solution.

[0054] 2.6 Standardization of Sodium Thiosulfate Concentration

[0055] Take 0.2000 g of potassium dichromate, dried to constant weight at 120℃, and place it in a 100 mL iodine flask. Dissolve it in 45 mL of water, add 2.000 g of potassium iodide, shake gently, add 5 mL of 1+3 hydrochloric acid, shake well, and stopper tightly. Place in the dark for 10 minutes to allow the potassium dichromate and potassium iodide to react completely. Then, take 5 mL of the solution and dilute it in a volumetric flask with 50 mL of water for the standardization of sodium thiosulfate. Titrate with this solution until near the endpoint, add 3 mL of starch indicator solution, and continue titrating until the blue color disappears and a bright green color appears. Correct the titration result with a blank test. Repeat the standardization of sodium thiosulfate concentration three times.

[0056] 2.7 Test Indicators and Formulas

[0057] This experiment used redox titration to determine the content of the antidote. The formula for calculating the amount of antidote dissolved is as follows:

[0058]

[0059] In the formula:

[0060] —The volume of sodium thiosulfate standard titration solution consumed in the blank test, in mL;

[0061] —The volume of sodium thiosulfate standard titration solution consumed in titrating the sample, in mL;

[0062] c — The actual concentration of the sodium thiosulfate standard titration solution, in mol / L;

[0063] —Mass of the sample, g;

[0064] 0.04752—titrated with 1.00 mL sodium thiosulfate standard solution [[ =1.000 mol / L] is the equivalent mass of the antidote in grams.

[0065] Formula for calculating the dissolution rate of the antidote:

[0066]

[0067] In the formula:

[0068] m—Dissolution amount of the sample under test conditions;

[0069] m0 — Total mass of antidote in the sample.

[0070] Sodium thiosulfate concentration calibration formula:

[0071] c

[0072] In the formula:

[0073] m K2Cr2O7 —The mass of potassium dichromate

[0074] M K2Cr2O7 —Molar mass of potassium dichromate

[0075] V Na2S2O3 —Volume of sodium thiosulfate consumed

[0076] 3. Test Results

[0077] 3.1 Dissolution rate of 9 samples at pH=2.6

[0078] Under pH=2.6 conditions, intuitive analysis and variance analysis of the orthogonal experiment revealed the following: at 30 min, the data for the nine samples were similar, with no significant differences; at 60 min, samples 2, 4, and 5 had lower dissolution rates, significantly lower than the other six samples (p<0.05); at 90 min, samples 2 and 4 had lower dissolution rates, significantly lower than the other seven samples (p<0.05). Considering the dissolution rates at all three time points, samples 2 and 4 had even lower dissolution rates under simulated pig stomach pH conditions.

[0079] Table 4. Average dissolution rate of samples at pH=2.6 (unit: %)

[0080]

[0081] Note: Different lowercase superscripts in the same column indicate significant differences between data (p<0.05).

[0082] Note: Superscripts with different lowercase letters in the same column indicate significant differences between the data (p<0.05).

[0083] 3.2 Dissolution rate of 9 samples at pH=6.2

[0084] Table 5. Average dissolution rate of samples at pH 6.2 (unit: %)

[0085]

[0086] Note: Different lowercase superscripts in the same column indicate significant differences between data (p<0.05).

[0087] Note: Superscripts with different lowercase letters in the same column indicate significant differences between the data (p<0.05).

[0088] Under pH=6.20 conditions, intuitive analysis and analysis of variance revealed the following: At 30 min, samples 1, 4, and 5 showed relatively high dissolution rates, but without significant differences (p>0.05); at 60 min, samples 1, 4, 5, 6, 7, and 9 exhibited relatively high dissolution rates, significantly different from other samples (p<0.05); at 90 min, samples 4, 6, and 7 showed relatively high dissolution rates, significantly different from other samples (p<0.05); at 120 min, samples 1, 4, and 6 showed relatively high dissolution rates, significantly different from other samples (p<0.05); at 150 min, samples 1 and 4 showed relatively high dissolution rates, significantly different from other samples (p<0.05). Considering the dissolution rates at all five time points, samples 1, 4, and 6 showed relatively high dissolution rates under simulated pig intestinal pH conditions.

[0089] In summary, under simulated pig stomach pH (pH=2.6) and pig small intestine pH (pH=6.2), the material prepared by combining 65% detoxifying agent and 35% composite carrier (2% tributyl citrate and 98% Eudragit L100) at 60℃ and 40r / min, namely sample 4, meets the requirements of this experiment.

[0090] Example 2: Using a skeleton-type vomitoxin antidote to remove DON from moldy corn

[0091] 1. The materials and instruments used in Example 2 are shown in Tables 6 and 7.

[0092] Table 6 Test Materials

[0093]

[0094] Table 7 Test Instruments

[0095]

[0096] Preparation of test solution:

[0097] (1) Phosphate buffer:

[0098] 0.1 mol / L disodium hydrogen phosphate: Weigh 35.8 g of disodium hydrogen phosphate, dissolve it in distilled water, and make up to 1000 mL in a volumetric flask.

[0099] 0.1 mol / L sodium dihydrogen phosphate: Weigh 15.6 g of sodium dihydrogen phosphate, dissolve it in distilled water, and make up to 1000 mL in a volumetric flask.

[0100] 0.1 mol / L pH 6.0 phosphate buffer: Accurately measure 12.3 mL of 0.1 mol / L disodium hydrogen phosphate solution into a 100 mL volumetric flask, and then dilute to volume with approximately 87.7 mL of 0.1 mol / L sodium dihydrogen phosphate solution.

[0101] 0.2 mol / L disodium hydrogen phosphate: Weigh 71.6 g of disodium hydrogen phosphate, dissolve it in distilled water, and make up to 1000 mL in a volumetric flask.

[0102] 0.2 mol / L sodium dihydrogen phosphate: Weigh 31.2 g of sodium dihydrogen phosphate, dissolve it in distilled water, and make up to 1000 mL in a volumetric flask.

[0103] 0.2 mol / L pH 6.8 phosphate buffer: Accurately measure 49 mL of disodium hydrogen phosphate solution into a 100 mL volumetric flask, and then dilute to volume with approximately 51 mL of 0.2 mol / L sodium dihydrogen phosphate solution.

[0104] (2) 10 mg / mL of 0.1 mol / L HCl pepsin solution:

[0105] Accurately weigh 0.2500g of pepsin (accurate to ±0.0001g), dissolve it in 25mL of 0.1mol / L HCl solution, and add 1mL to each sample.

[0106] (3) 50 mg / mL trypsin phosphate buffer:

[0107] Accurately weigh 1.2500g of trypsin (accurate to ±0.0001g), dissolve it in 25mL of 0.2mol / L PPH6.8 phosphate buffer, and add 1mL to each sample.

[0108] (4) Hydrochloric acid solution:

[0109] 1 mol / L HCl: Accurately measure 8.3 mL of hydrochloric acid into a 100 mL volumetric flask, dilute to 100 mL, and store at room temperature.

[0110] 0.1 mol / L HCl: Take 100 mL of 1 mol / L HCl into a 1000 mL volumetric flask and dilute to volume with distilled water.

[0111] 0.2 mol / L HCl: Take 200 mL of 1 mol / L HCl into a 1000 mL volumetric flask and dilute to volume with distilled water.

[0112] (5) Sodium hydroxide solution:

[0113] 1 mol / L sodium hydroxide: Weigh 4.00 g of sodium hydroxide, dissolve it in distilled water in a beaker, cool it, transfer it to a volumetric flask with a volume of 100 mL, and store it at room temperature.

[0114] 0.6 mol / L sodium hydroxide: Accurately weigh 2.40 g of sodium hydroxide, dissolve it in distilled water in a beaker, cool it, transfer it to a 100 mL volumetric flask, and make a final volume of 100 mL. Store at room temperature.

[0115] (6) Chloramphenicol ethanol solution (0.5 mg / mL ethanol): Weigh 50 mg of chloramphenicol and dissolve it in 100 mL of ethanol. Transfer the solution to a reagent bottle and store at room temperature.

[0116] 2. Experimental methods and procedures

[0117] A single-factor experimental design was used to investigate the detoxification effects of the skeleton-type vomitoxin antidote and the common antidote in the gastric and intestinal phases under in vitro simulated conditions. The addition levels (based on active ingredient) of the skeleton-type enteric-coated DON antidote and the common antidote were 0.1% and 0.5%, respectively. A blank group was also included, with three replicates for each group. The experimental design is shown in Table 8.

[0118] Table 8 Experimental Design

[0119]

[0120] Different deoxynivalenol (DON) antidotes were tested at various concentration levels during simulated gastric and intestinal digestion phases, with three parallel samples for each treatment. At the end of both the gastric and intestinal digestion phases, enzyme-linked immunosorbent assay (ELISA) was used to rapidly detect the DON content in each sample.

[0121] Stomach digestion period:

[0122] (1) Weigh 5g of moldy corn (DON=18045.53 μg / kg) that has been sieved through a 40-mesh sieve (accurate to 0.0001g) and put it into a 250mL Erlenmeyer flask.

[0123] (2) Add 125 mL of phosphate buffer (0.1 mol / L, pH=6.0) and glass beads to the Erlenmeyer flask to simulate the peristaltic function of the stomach.

[0124] (3) Add 10 mL of 0.2 mol / L HCl, and then use 1 mol / L HCl or 1 mol / L NaOH to adjust the pH of the solution to 2.0.

[0125] (4) Add 5 mL of fresh pepsin hydrochloric acid solution, which contains 10 mg of pepsin.

[0126] (5) Seal the Erlenmeyer flask with plastic wrap, place the flask in a constant temperature shaker at 39°C, with a vortex frequency of 80 r / min, and start timing after 5 min. The shaking digestion time is 6 h to simulate the digestion process of feed in the stomach. After digestion, take samples to analyze the DON content.

[0127] Stomach + Intestine Digestion Period:

[0128] (1) After the gastric digestion period is over, add 10 mL of phosphate buffer (0.2 mol / L, pH=6.0) and 5 mL of 0.6 mol / L NaOH solution to the chyme, and adjust the pH to 7.0 with 1 mol / L HCl or 1 mol / L NaOH.

[0129] (2) Add 5 mL of fresh trypsin phosphate buffer (containing 50 mg of trypsin).

[0130] (3) After sealing with plastic wrap, place in a constant temperature shaker at 39°C for digestion. The vortex frequency is 80 r / min. Start timing after 5 min and shake for 18 h to simulate the digestion process of feed in the small intestine. After the entire digestion period (6 h gastric period + 18 h intestinal period) is completed, take samples to analyze the DON content in the digestive fluid.

[0131] Collection of digestive fluids and enzyme-linked immunosorbent assay (ELISA) of vomitoxin:

[0132] (1) After the gastric / (gastric+intestinal) digestion period, take 50 μL of sample solution from each replicate (each Erlenmeyer flask) of each treatment and put it into a centrifuge tube. Add distilled water to 5 mL and centrifuge for 5 min.

[0133] (2) After centrifugation, take 5 μL of the centrifuged sample solution from the centrifuge tube into the reaction well of the microplate, and then add 45 μL of diluent to the microplate.

[0134] (3) Continue to add 50 μL of antibody-enzyme conjugate to the microplate and place it in an air bath at 37°C for 30 min.

[0135] (4) Shake the microplate dry, rinse it 5 times with washing solution, drain the water, and finally gently pat it with clean absorbent paper to remove the liquid from the wells.

[0136] (5) Add 50 μL of colorimetric solution A and 50 μL of colorimetric solution B to the reaction plate, gently shake the reaction plate to mix thoroughly, and place it in an air bath at 37°C for 10 min.

[0137] (6) Add 50 μL of stop solution to the reaction wells of the ELISA plate, mix well, and allow it to react fully.

[0138] (7) Detect absorbance at a wavelength of 450 nm and read the results within 5 min.

[0139] DON clearance rate calculation:

[0140] In this experiment, the DON content was obtained through a standard curve, and then the corresponding clearance rate for each sample was calculated. Specifically, the clearance rate can be obtained by dividing the DON content of the blank group by the DON content of the sample.

[0141] Statistical analysis of the data:

[0142] The experimental data were first preliminarily organized and processed using Excel. Then, one-way ANOVA was performed using IBM SPSS Statistics 20 to test the significance of different time periods and different levels of skeletal SMBS addition on the DON scavenging effect in moldy maize. Results are expressed as mean ± standard deviation. A p-value less than 0.05 was considered statistically significant.

[0143] 3. Test Results

[0144] (1) Drawing the standard curve

[0145] The kit includes standard vomitoxin samples at six concentrations: 0.0, 0.1, 0.3, 0.9, 2.7, and 8.1 ppb. The absorbance was measured in two wells (OD=450 nm), and the results are shown in Table 9.

[0146] Table 9 Absorbance values ​​of standard solutions or sample solutions (OD=450nm)

[0147]

[0148] Table 10 DON content standard curve data

[0149]

[0150] Tables 9 and 10 show the absorbance (OD) values ​​of a series of DON standard solutions (0, 0.1, 0.3, 0.9, 2.7, 8.1 ppb). The logarithmically transformed data were analyzed using logistic regression, and a standard curve was plotted accordingly (see Table 9). Figure 1 Linear regression analysis showed that the relationship between the logarithm of DON concentration and relative absorbance can be expressed by the equation y = 0.85260 + (0.10427 - 0.85260) / (1 + e^(-5.03762x + -2.01854)) (R^ 2 The value of 0.99190 indicates a significant positive correlation between the two.

[0151] (2) DON release amount in digestive fluid

[0152] Divide the measured absorbance value of each sample by B0 to obtain the corresponding percentage absorbance value. Substitute this value into the standard equation to obtain the logarithmic value of the concentration. Then, calculate the antilogarithmic value. After processing, the DON content can be obtained. The results of the average value of each group are shown in Tables 11 and 12. The calculation results are rounded to two decimal places.

[0153] Table 11. DON release from samples at different dosages during the gastric period (unit: ppb)

[0154]

[0155] Table 12. DON release from samples at different dosages during the gastric and intestinal phases (unit: ppb)

[0156]

[0157] (3) Clearance rate of DON in the gastric stage

[0158] As shown in Table 13, at the 0.1% skeletal SMBS addition level, one-way ANOVA showed that P > 0.05, indicating no significant difference in DON clearance rates between the control and experimental groups. At the 0.5% skeletal SMBS addition level, 0.01 < P < 0.05, and the one-way ANOVA results showed a significant difference.

[0159] Table 13. DON clearance rate during gastric digestion (unit: %)

[0160]

[0161] (4) Clearance rate of DON in the gastric and intestinal phases

[0162] After the enteric digestion phase, DON release from the samples was more complete. The DON release in the blank control group was higher than that during the gastric digestion phase; therefore, it was necessary to compare the clearance effects during the enteric digestion phase separately. Table 14 shows the DON elimination rates in the control and experimental groups after the enteric digestion phase. At the 0.1% skeletal SMBS addition level, P > 0.05, and the difference was not significant in one-way ANOVA. At the 0.5% skeletal SMBS addition level, P > 0.05, and the difference was not significant in one-way ANOVA.

[0163] Table 14. DON clearance rate during gastric and intestinal digestion (unit: %)

[0164]

[0165] In summary, the optimal parameters for evaluating the in vitro clearance effect of the skeleton-type vomitoxin antidote on DON in moldy corn are: under simulated porcine small intestine pH conditions (pH=7.0), the skeleton-type vomitoxin antidote exhibits the best coating effect at a 0.1% addition level of SMBS, with the DON content in the sample decreasing from 18045.53 ppb to 16323.55 ppb after simulated digestion in vitro (P>0.05).

[0166] Example 3: Using a skeleton-type vomitoxin antidote to remove DON from corn ethanol residue contaminated with mycotoxins.

[0167] 1. The experimental materials and instruments used in Example 3 are as follows:

[0168] Experimental materials:

[0169] DDGS: Initial DON content ≥3000ppb; Skeletal type vomitoxin antidote: active ingredient content 65%, manufacturing process parameters 60℃, 40r / min; domestically produced analytical grade SMBS; DON rapid detection kit (ELISA method, Shanghai Hengyuan Biotechnology Co., Ltd., No. 23-1222-H561).

[0170] Preparation of test reagents:

[0171] Phosphate buffer was prepared using domestically produced analytical grade disodium hydrogen phosphate and sodium dihydrogen phosphate; pepsin and trypsin were from Sigma-Aldrich, USA; and domestically produced analytical grade sodium hydroxide and hydrochloric acid were used as reagents.

[0172] Test instruments and equipment:

[0173] Analytical balance (sensitivity 0.0001g): Mettler ME104E; Leici PHS-3C pH meter: Shanghai Instrument & Electronics Scientific Instruments Co., Ltd. - 600408N0020121954; Constant temperature water bath shaker: Xuri SHA-BA; Enzyme-linked immunosorbent assay (ELISA) analyzer: Thermo Fisher Multiskan FC.

[0174] 2. Experimental methods and procedures

[0175] A single-factor experimental design was used to investigate the detoxification effects of the skeleton-type vomitoxin antidote and the common antidote in the gastric and intestinal phases under in vitro simulated conditions. The skeleton-type enteric-coated DON antidote (based on active ingredient) was added at levels of 0.2% and 0.4%, respectively. A control group of the common antidote was also included, with addition levels of 0.2% and 0.4%. The experimental design is shown in Table 15.

[0176] Table 15 Experimental Design (Unit: %)

[0177]

[0178] For each addition level of different DON antidotes in the in vitro simulated gastric and intestinal phases, three replicates were set up. The DON content in each group of samples was determined by a DON rapid detection kit (ELISA method) after the gastric digestion phase and after the intestinal digestion phase.

[0179] Sample processing:

[0180] Pour the pre-prepared DDGS (DON=3200μg / kg) into a grinder to simulate its powdery state in feed. Accurately weigh 5g portions (accurate to ±0.0001g) using weighing paper and store them in Erlenmeyer flasks. Simultaneously, accurately weigh the enteric-coated DON antidote and SMBS in a preheated analytical balance, and add the enteric-coated DON antidote and SMBS according to different experimental groups. Store these in Erlenmeyer flasks at room temperature for later use.

[0181] Preparation of digestive juices:

[0182] Artificial gastric digestive fluid: Add 125 mL of phosphate buffer (0.1 mol / L, pH=6.0) to a 250 mL Erlenmeyer flask, and add glass beads to simulate gastric peristalsis. Simultaneously add 10 mL of 0.2 mol / L HCl, and adjust the pH to 2.0 with 1 mol / L HCl or 1 mol / L NaOH. Then add 5 mL of fresh pepsin hydrochloric acid solution (containing 50 mg of pepsin). To prevent microbial growth, add 0.5 mL of bacterial growth inhibitor (0.5 mg / mL chloramphenicol ethanol solution) to the digestive fluid. Finally, refrigerate the treated digestive fluid for later use.

[0183] Artificial intestinal digestive fluid: After the gastric digestion period, add 10 mL of phosphate buffer (0.2 mol / L, pH=6.8) and 5 mL of 0.6 mol / L NaOH solution to the chyme, and adjust the pH to 7.0 with 1 mol / L hydrochloric acid or 1 mol / L NaOH. Then add 5 mL of fresh trypsin phosphate buffer. Artificial intestinal digestive fluid should be added after the gastric digestion period to simulate the digestive environment in an animal.

[0184] Indicator evaluation methods:

[0185] To ensure accuracy, a two-well assay was used. The absorbance of the standard samples in the kit was measured at 450 nm. The average absorbance value (B) of each concentration of standard solution and sample was divided by the absorbance value of the 0 ppb standard (B0) and then multiplied by 100% to obtain the percentage absorbance value. A standard curve was plotted with the negative logarithm of DON concentration on the X-axis and the percentage absorbance value on the Y-axis. Based on the sample percentage absorbance value, the x-axis of the corresponding point on the curve was obtained, which is the logarithm of the DON concentration. The antilogarithm was calculated and multiplied by the dilution factor to obtain the DON concentration in the assay solution. One-way ANOVA was used in IBM SPSS Statistics 20 software to test the significance of the effects of different SMBS antidotes and different SMBS addition levels on the DON concentration in the digestion solution. The least significant difference (LSD) method was used for multiple comparisons of significantly different groups. Data are expressed as mean ± standard deviation.

[0186] 3. Test Results

[0187] (1) Drawing the standard curve

[0188] The kit contains standard DON samples with six concentrations of 0, 0.1, 0.3, 0.9, 2.7, and 8.1 ppb. The absorbance was measured in two wells (OD=450nm), and the results are shown in Table 16.

[0189] Table 16 Absorbance of Standard Samples (OD=450)

[0190]

[0191] Enter the data into ELISA Calc. Plot the DON concentration standard curve using the logarithm of the standard sample concentration as the X-axis and the percentage absorbance as the Y-axis. See [link to ELISA Calc]. Figure 2 .

[0192] Standard curve regression coefficient R 2 =0.9919, the simulation effect is good, and the nonlinear regression equation can be obtained as follows: Based on this, the DON content in the sample can be calculated.

[0193] (2) Experimental data results

[0194] Divide the measured absorbance value of each sample by B0 to obtain the percentage absorbance value, substitute it into the standard equation to solve for the negative logarithm of the concentration, and then multiply it by the dilution factor (10000 times). After processing, the DON content is obtained. The results of the average value after repeating each group are shown in Table 17. The calculation results are rounded to two decimal places.

[0195] Table 17 DON content in each group (unit: ppb)

[0196]

[0197] By comparing the DON content data of each experimental group with the blank control group, the DON scavenging rate of each experimental group can be calculated. The calculation results are shown in Table 18.

[0198] Table 18 DON clearance rate in each experimental group (unit: %)

[0199]

[0200] 4. Comparison of detoxification effects among different experimental groups during the gastric digestion period

[0201] One-way ANOVA was performed on the DON concentration data measured in the blank control group and the four experimental groups during the gastric digestion period. The differences were extremely significant. Multiple comparisons were performed on each group using the LSD value method. The results are shown in Table 19.

[0202] Table 19 Multiple comparisons of the effects of different antidotes and different SMBS addition levels on DON concentration during gastric digestion.

[0203]

[0204] Table 19 shows that after the gastric digestion period, the DON content measured in experimental group 3 was the highest, while that in experimental group 2 was the lowest. This indicates that the addition of 0.2% skeleton-type vomitoxin antidote to experimental group 3 resulted in the least amount of SMBS released in the stomach, providing the best protective effect. Comparing experimental group 3 (0.2% skeleton-type SMBS addition level) with experimental group 1, the dissolution rate of experimental group 3 in the stomach was calculated to be 51.20%. Comparing experimental group 4 (0.4% skeleton-type SMBS addition level) with experimental group 2, the dissolution rate of experimental group 4 in the stomach was calculated to be 42.41%. This demonstrates that the 0.4% skeleton-type SMBS addition level of the enteric-coated DON antidote had a better coating effect in the gastric environment, which is more in line with experimental expectations.

[0205] Except for the insignificant differences in DON content between experimental groups 3 and 4, and between experimental groups 4 and 1, all other groups showed highly significant differences. This indicates that the addition levels of 0.2% and 0.4% antidote for vomitoxin resulted in similar SMBS dissolution rates in the stomach, both providing some protection. However, the 0.4% antidote for vomitoxin, due to its higher content of active ingredients, resulted in lower dissolution rates, but the released SMBS content was not significantly different from that in experimental group 1 (where 0.2% antidote for vomitoxin was directly added). Experimental group 2 had even lower DON content than experimental group 1, indicating that as the level of antidote for vomitoxin increases, the DON content in the digestive tract decreases, consistent with experimental expectations.

[0206] 5. Comparison of detoxification effects among different experimental groups during the intestinal digestion period

[0207] One-way ANOVA was performed on the DON concentration data measured in the blank control group and the four experimental groups during the intestinal digestion period. The differences were extremely significant. Multiple comparisons were performed on each group using the LSD value method. The results are shown in Table 20.

[0208] Table 20 Multiple comparisons of the effects of different antidotes and different SMBS addition levels on DON concentration during the enteric digestive period.

[0209]

[0210] Table 20 shows that the highest DON content was measured in experimental group 3, while the lowest was measured in experimental group 2, indicating that the highest DON clearance efficiency (26.84%) was achieved with the addition of 0.4% matrix-type SMBS. The difference between experimental group 4 and experimental group 2 was extremely significant, indicating that the dissolution rate of experimental group 4 in the intestine did not meet expectations. The DON clearance rates of experimental groups 3 and 1 were 7.93% and 9.15%, respectively. The difference between experimental groups 4 and 2 with the 0.4% matrix-type SMBS addition level was extremely significant, indicating that the DON clearance effect at the 0.2% matrix-type SMBS addition level was poor, and the 0.4% matrix-type SMBS addition level was more in line with experimental expectations.

[0211] Except for the insignificant difference in DON content between experimental group 3 and experimental group 1, all other groups showed highly significant differences, indicating that the SMBS dissolution rate in the intestinal environment with the addition of 0.2% skeleton-type vomitoxin antidote was similar to that of experimental group 1 with direct addition of 0.2% skeleton-type SMBS. Calculated 24 hours after the experiment's termination, the dissolution rate of experimental group 3 was 86.67%, and that of experimental group 4 was 78.84%. The higher dissolution rate of experimental group 3 better aligns with the experimental expectation that most of the coating material would dissolve in the intestinal environment.

[0212] In summary, at the addition level of 0.4% skeleton-type vomitoxin antidote, the coating material exhibits good protective effect and high DON removal efficiency, which meets experimental expectations. This provides a theoretical basis and production guidance for the practical application of laboratory-made skeleton-type vomitoxin antidote in actual production.

[0213] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A skeleton-type antidote for vomitoxin, characterized in that: The skeleton-type vomiting toxin antidote consists of 60%–70% Na2S2O5 and 30%–40% composite carrier, wherein the composite carrier is composed of 2% 2-hydroxy-1,2,3-propanetricarboxylic acid tributyl ester and 98% Eudragit L100. The active ingredient in the skeleton-type vomitoxin antidote is skeleton-type Na2S2O5; The skeleton-type vomitoxin antidote was extruded through a hot melt extruder at a sleeve outlet temperature of 60℃~80℃ and a screw speed of 30r / min~50r / min using an L9 (3) extruder. 4 The dissolution rate was obtained through orthogonal experiment screening. Under orthogonal experiment conditions, the dissolution rate was ≤20% under gastric environment conditions and ≥70% under intestinal environment conditions.

2. The application of the skeletal type vomitoxin antidote according to claim 1 in corn-related feed processing.

3. The application according to claim 2, characterized in that: The antidote is used to remove vomitoxin from moldy corn.

4. The application according to claim 3, characterized in that: The optimal efficacy of the skeletal-type vomitoxin antidote is achieved when the level of its active ingredient, skeletal-type Na2S2O5, is 0.1%.

5. The application of the skeleton-type vomitoxin antidote according to claim 1 in the processing of corn alcohol residues-related feed.

6. The application according to claim 5, characterized in that: The antidote is used to remove vomiting toxins from corn ethanol residues contaminated with mycotoxins.

7. The application of the skeleton-type vomitoxin antidote according to claim 6, characterized in that: The optimal efficacy of the skeletal-type vomitoxin antidote is achieved when the level of its active ingredient, skeletal-type Na2S2O5, is 0.4%.

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