A mycotoxin degrading agent and a method for preparing the same

Abstract

The application discloses a kind of mycotoxin degradation agent and preparation method thereof.The mycotoxin degradation agent includes 10-20 parts of quinoa peptide, 30-40 parts of microcrystalline cellulose, 10-30 parts of modified alpha-liponic acid, 10-20 parts of enzyme-containing preparation, 20-40 parts of L-ascorbic acid composition.The application also provides a preparation method thereof.Compared with the prior art, it can better degrade vomitoxin, reduce its stress response to piglets, promote feed nutrient absorption, and improve animal growth efficiency.

Description

Technical Field

[0001] This invention relates to the field of detoxification technology, and in particular to a fungal toxin degrading agent and its preparation method. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by fungi during their growth and reproduction. They are widely found in natural environments, particularly in grain crops such as wheat, rice, and corn. Currently, the mycotoxins that pose a significant threat to global food security include aflatoxins, ochratoxins, vomitoxins, and fumonisins. Vomitoxin, also known as deoxynivalenol (DON), is a mycotoxin produced by certain Fusarium fungi. DON has the highest detection rate among all mycotoxins and is commonly found in wheat, barley, and corn. Consuming food contaminated with vomitoxin can cause diarrhea, vomiting, and gastrointestinal symptoms. Vomitoxin is acid- and heat-resistant, making it difficult to completely remove using traditional cooking and processing methods. Common removal methods include physical degradation, chemical degradation, biodegradation, and synergistic degradation using multiple methods. Employing effective removal technologies can significantly reduce mycotoxin contamination of crops and ensure food safety.

[0003] Doxorubicin (DON) is a relatively weakly lethal mycotoxin, but its growth-inhibiting effect on animals is comparable to or even stronger than other trichothecene toxins. Before being orally ingested, DON comes into contact with a large number of microorganisms in the small intestine of ruminants or poultry. These microorganisms convert DON into the less toxic DOM-1, thus reducing their sensitivity to DON. However, in humans and pigs, the gut microbiota are mainly located in the hindgut. The vast majority of DON is absorbed in the foregut, with only a small portion reaching the hindgut where it is converted into DOM-1. Therefore, pigs and humans are more sensitive to DON. Piglets are even more sensitive to DON. Consuming food contaminated with vomitoxin can cause diarrhea, vomiting, anorexia, and gastrointestinal diseases in piglets, inhibiting their growth and thus affecting weight gain. Therefore, it is necessary to provide a mycotoxin degrading agent to reduce the impact of DON on piglets.

[0004] CN113383860A discloses a vomitoxin antidote and its preparation process. This vomitoxin antidote is composed of Bacillus subtilis, vitamin C, montmorillonite, yeast cell wall, plant extracts, and zinc methionine. The vomitoxin antidote provided by this invention has a significant effect on vomitoxin and can improve feed conversion rate, making it valuable for the feed and aquaculture industries. This invention mainly utilizes the adsorption of vomitoxin by montmorillonite; however, the upper limit of absorption and the effectiveness against vomitoxin may be relatively limited.

[0005] CN116004449A discloses *Lactococcus lactis*, a bacterial agent, a mycotoxin degrading agent, and its application and method for degrading mycotoxins. The *Lactococcus lactis* in this invention can efficiently degrade various mycotoxins. Using *Lactococcus lactis* as a biomaterial for mycotoxin degradation shows great promise in the development of new mycotoxin biodegrading bacterial agents and biodegradable sterile preparations. However, the degradation agent prepared by this invention may have a relatively high preparation cost. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a fungal toxin degrading agent that can better degrade vomitoxin, reduce its stress response in piglets, promote feed nutrient absorption, and improve animal growth efficiency.

[0007] To achieve the above objectives, the present invention provides a method for preparing a fungal toxin degrading agent, which, by weight, is obtained by mixing and sieving 10-20 parts of quinoa peptide, 30-40 parts of microcrystalline cellulose, 10-30 parts of α-lipoic acid, 10-20 parts of enzyme-containing preparation, and 20-40 parts of L-ascorbic acid.

[0008] Alternatively, it can be obtained by mixing and sieving 10-20 parts of quinoa peptide, 30-40 parts of microcrystalline cellulose, 10-30 parts of modified α-lipoic acid, 10-20 parts of enzyme-containing preparation, and 20-40 parts of L-ascorbic acid.

[0009] The modified α-lipoic acid is a natural cationic polymer modified α-lipoic acid.

[0010] Preferably, 1-10 parts of natural cationic polymer are mixed evenly with 80-120 parts of solvent, and then 1-3 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-1 parts of N-hydroxysuccinimide are added to obtain mixture 1; 1-5 parts of α-lipoic acid are mixed evenly with 10-20 parts of anhydrous ethanol, added to mixture 1, and reacted at 30-50℃ and 800-1200rpm for 20-28h. After cooling to room temperature, 150-250 parts of 70wt% ethanol aqueous solution are added, and the mixture is stirred at 800-1200rpm for 5-15min. After standing, the mixture is centrifuged at 1000-3000rpm for 3-8min, the precipitate is collected, 10-25 parts of water are added, and the mixture is freeze-dried for 20-28h to obtain modified α-lipoic acid.

[0011] More preferably, the solvent is water or a 0.5-2.0 wt% aqueous solution of acetic acid.

[0012] More preferably, the natural cationic polymer is selected from at least one of chitosan, chitosan oligosaccharide, and polylysine.

[0013] As a further explanation of the present invention, lipoic acid contains carboxyl groups and disulfide bonds. Chitosan, chitosan oligosaccharides, and polylysine all contain amino and hydroxyl groups in their structures. These groups may undergo amidation, esterification, disulfide bond exchange, or hydrogen bonding reactions to produce amide bonds, hydrogen bonds, and other functional groups, forming copolymers to prepare modified lipoic acid. The resulting modified lipoic acid exhibits good stability in both acidic and alkaline environments. The present invention discovers that introducing modified lipoic acid into a fungal toxin degrading agent can create a special structure that can kill Fusarium fungi, prevent the production of new toxins, and the contained active functional groups may react with the weakly acidic hydroxyl molecules on the DON molecule or undergo hydrogen bonding to degrade or adsorb DON. Furthermore, it can enhance the intestinal physical barrier, reduce DON permeation and absorption by the intestine, scavenge free radicals to reduce the production of pro-inflammatory factors, reduce the diarrhea rate in piglets, promote feed nutrient absorption, and improve animal growth efficiency.

[0014] Preferably, the enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase.

[0015] More preferably, the mass ratio of Bacillus licheniformis, alkaline protease, and glucose oxidase in the enzyme-containing preparation is 5-7:2-4:5-7.

[0016] Preferably, the sieving is a 40-60 mesh sieve.

[0017] The lipoic acid added in this invention has antioxidant properties, microcrystalline cellulose can improve the texture and stability of the mycotoxin degrading agent, and L-ascorbic acid, as a powerful antioxidant, reduces oxidative stress by scavenging free radicals and protects agricultural products from damage.

[0018] This invention incorporates an enzyme-containing preparation made from Bacillus licheniformis, alkaline protease, and glucose oxidase, which has certain advantages in the degradation of vomitoxin. By competing with pathogenic microorganisms for nutrients and living space, it inhibits the growth and reproduction of the latter, reducing the production of vomitoxin. The extracellular metabolites produced by the microbial metabolism play a role in biodegradation, unlike montmorillonite and other aluminosilicates which require physical adsorption for degradation. This avoids the problem of vomitoxin contamination in grains and feed in actual production. It enhances the protection and stability of agricultural products, reduces their stress response to piglets, promotes feed nutrient absorption, and improves animal growth efficiency.

[0019] Quinoa peptides in the mycotoxin degrading agent of this invention help alleviate physiological discomfort and inflammatory response in animals caused by vomitoxin poisoning. They can help adsorb mycotoxins, separating them from agricultural products, thus purifying and removing vomitoxins, enhancing the degradation effect of the degrading agent on mycotoxins, and synergistically improving the overall performance of the degrading agent when used with enzyme-containing preparations.

[0020] The beneficial effects of this invention are:

[0021] 1. Compared with the prior art, the present invention obtains a mycotoxin degrading agent by rationally combining the interaction between various substances and optimizing the preparation process. The mycotoxin degrading agent obtained by the present invention has the function of degrading mycotoxins, especially in reducing the incidence of diarrhea caused by vomitoxin stress in piglets. It can also promote the absorption of feed nutrients and improve animal growth efficiency.

[0022] 2. Compared with the prior art, the lipoic acid of the present invention has antioxidant properties and reacts with chitosan, chitosan oligosaccharide and polylysine with certain antibacterial properties to obtain modified lipoic acid, which has both antibacterial and antioxidant properties. It also has good stability in both acidic and alkaline environments and can play an antibacterial role in different digestive scenarios, reducing the impact of vomitoxin on piglets. Detailed Implementation

[0023] The parameters and sources of some raw materials in this embodiment of the invention are as follows:

[0024] Bacillus licheniformis: Latin name Bacillus licheniformis, strain number CGMCC1.10314, commercially available product, sourced from China General Microbiological Culture Collection Center;

[0025] Alkaline protease: Enzyme activity is 300,000 U / g;

[0026] Glucose oxidase: Enzyme activity is 20,000 U / g;

[0027] Catalase: Enzyme activity is 300,000 U / g;

[0028] Quinoa peptide: sourced from a reputable supplier, Xi'an Tongze Biotechnology Co., Ltd., product number TZ-6398745;

[0029] 70wt% chitosan: Deacetylation degree ≥85%, sourced from Zhejiang Jinke Pharmaceutical Co., Ltd.;

[0030] Chitosan oligosaccharide: molecular weight ≤1000Da, sourced from Zhejiang Jinke Pharmaceutical Co., Ltd.;

[0031] Polylysine: CAS No.: 25104-18-1, Product No.: P43050, sourced from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0032] Example 1

[0033] A method for preparing a fungal toxin degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g α-lipoic acid, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0034] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0035] Example 2

[0036] A method for preparing a fungal toxin degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g modified α-lipoic acid, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0037] The preparation method of the modified α-lipoic acid includes the following steps:

[0038] Mix 5g of chitosan oligosaccharide with 100g of water until homogeneous, then add 2g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.5g of N-hydroxysuccinimide to obtain mixture 1; mix 2g of α-lipoic acid with 15g of anhydrous ethanol until homogeneous, add to mixture 1, react at 40℃ and 1000rpm for 24h, cool to room temperature, add 200g of 70wt% ethanol aqueous solution, stir at 1000rpm for 10min, let stand, centrifuge at 2000rpm for 5min, take the precipitate, add 20g of water and freeze dry for 24h to obtain modified α-lipoic acid.

[0039] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0040] Example 3

[0041] A method for preparing a fungal toxin degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g modified α-lipoic acid, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0042] The preparation method of the modified α-lipoic acid includes the following steps:

[0043] Mix 5g of chitosan with 100g of 1.0wt% acetic acid aqueous solution until homogeneous, then add 2g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.5g of N-hydroxysuccinimide to obtain mixture 1; mix 2g of α-lipoic acid with 15g of anhydrous ethanol until homogeneous, add to mixture 1, react at 40℃ and 1000rpm for 24h, cool to room temperature, add 200g of 70wt% ethanol aqueous solution, stir at 1000rpm for 10min, let stand, centrifuge at 2000rpm for 5min, take the precipitate, add 20g of water and freeze dry for 24h to obtain modified α-lipoic acid.

[0044] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0045] Example 4

[0046] A method for preparing a fungal toxin degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g modified α-lipoic acid, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0047] The preparation method of the modified α-lipoic acid includes the following steps:

[0048] Mix 5g of polylysine with 100g of water until homogeneous, then add 2g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.5g of N-hydroxysuccinimide to obtain mixture 1; mix 2g of α-lipoic acid with 15g of anhydrous ethanol until homogeneous, add to mixture 1, react at 40℃ and 1000rpm for 24h, cool to room temperature, add 200g of 70wt% ethanol aqueous solution, stir at 1000rpm for 10min, let stand, centrifuge at 2000rpm for 5min, take the precipitate, add 20g of water and freeze dry for 24h to obtain modified α-lipoic acid.

[0049] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0050] Comparative Example 1

[0051] A method for preparing a fungal toxin degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0052] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0053] Comparative Example 2

[0054] A method for preparing a fungal toxin degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g chitosan oligosaccharide, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0055] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0056] Comparative Example 3

[0057] A method for preparing a fungicide degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g chitosan, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0058] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0059] Comparative Example 4

[0060] A method for preparing a fungicide degrading agent, comprising mixing 15g quinoa peptide, 35g microcrystalline cellulose, 20g polylysine, 15g enzyme-containing preparation, and 30g L-ascorbic acid and passing the mixture through a 50-mesh sieve;

[0061] The enzyme-containing preparation is composed of Bacillus licheniformis, alkaline protease, and glucose oxidase in a mass ratio of 6:3:6.

[0062] Test Example 1

[0063] Diarrhea rate test

[0064] This experiment used 28-day-old weaned Landrace piglets. The experimental period was 21 days. The piglets were randomly divided into 9 groups of 10 piglets each. Examples 1-4, comparative examples 1-4, and a control group were established for the experiment. The control group was fed a basal diet, while Examples 1-4 and comparative examples 1-4 were fed a basal diet supplemented with 500g / ton of the fungicide degrading agent obtained in each experimental group. During the experiment, the piglets were housed in a fully enclosed nursery, with the temperature controlled at 25-27℃, and had free access to feed and water. The basal diet did not contain any antibiotics, and the piglets were immunized according to the standard immunization program.

[0065] Measurement indicators: Production performance of weaned piglets in each treatment group, specifically including the following indicators:

[0066] During the experiment, the condition of piglets' feces was observed and recorded every morning at 10:00 AM, and the diarrhea rate of weaned piglets was calculated using the following formula:

[0067] Diarrhea rate (%) = (cumulative number of days of diarrhea in all piglets during the trial period) / (total number of pigs × number of trial days) × 100%.

[0068] The test results are shown in Table 1 below:

[0069] Table 1 Diarrhea rate

[0070] serial number Diarrhea rate (%) control group 11.90 Example 1 10.0 Example 2 6.67 Example 3 7.14 Example 4 2.86 Comparative Example 1 10.95 Comparative Example 2 9.52 Comparative Example 3 9.52 Comparative Example 4 9.05

[0071] Comparisons of Examples 1-4, Comparative Examples 1-4, and the control group revealed that the diarrhea rate in Comparative Examples 1-4 was lower than that in the control group. Comparisons of Examples 1-4 showed that Example 4 had the lowest diarrhea rate, at only 2.86%. This may be because the α-lipoic acid added in Example 4 was modified with polylysine. Polylysine has a high amino density and more binding sites with the carboxyl groups of α-lipoic acid, resulting in a more complete reaction. Stable covalent bonds are formed through amidation, leading to a more stable modified product structure. The resulting unique structure may kill Fusarium fungi and prevent the production of new toxins. Polylysine-modified α-lipoic acid may have specific adsorption or degradation properties for DON, enhancing the intestinal physical barrier, reducing DON permeation and absorption by the intestine, scavenging free radicals, and reducing the production of pro-inflammatory factors. Modified lipoic acid, enzyme-containing preparations, and quinoa peptides may have a synergistic regulatory effect, further reducing the diarrhea rate. In Examples 2 and 3, the α-lipoic acid was modified with chitosan oligosaccharide and chitosan. The amino density of chitosan and chitosan oligosaccharide is lower than that of polylysine, resulting in lower reactivity and less active functional group content. Consequently, their adsorption or degradation effect on DON is lower than in Example 4. However, the chitosan oligosaccharide in Example 2 has a short-chain structure with less steric hindrance, leading to a higher reactivity than chitosan and easier binding to the intestinal mucosa to enhance the physical barrier. In Example 3, the long-chain chitosan has greater steric hindrance, resulting in a lower reactivity. Although Comparative Examples 2-4 contain natural cationic polymers with antibacterial properties, and chitosan oligosaccharide and chitosan bind to bacterial cell membranes through positive charge, disrupting their integrity, and polylysine interferes with microbial metabolism, potentially killing Fusarium fungi and preventing the production of new toxins, their DON-producing effect and free radical scavenging and reduction of pro-inflammatory factor production are lower than in Examples 2-4.

[0072] Test Example 2

[0073] Weight change test

[0074] Healthy 28-day-old weaned Landrace piglets were selected and randomly divided into 10 groups of 20 piglets each. These groups included Examples 1-4, Comparative Examples 1-4, a control group, and a blank control group. The piglets were fed the prescribed feed for 45 days. The control group was fed a feed containing 0.5 mg / kg vomitoxin. Examples 1-4 and Comparative Examples 1-4 were fed a feed containing 0.5 mg / kg vomitoxin, supplemented with a fungicide degrading agent prepared in Examples 1-4 and Comparative Examples 1-4, respectively. The addition ratio was 500 grams of the fungicide degrading agent per ton of feed containing 0.5 mg / kg vomitoxin. The blank control group was fed a normal feed without the addition of vomitoxin or the fungicide degrading agent. During the experiment, the piglets were housed in a fully enclosed nursery at a temperature controlled at 25-27℃, with free access to feed and water. Weights were recorded before and after the experiment, and the average values ​​were taken. The test results are shown in Table 2 below.

[0075] Table 2 Results of weight change

[0076]

[0077]

[0078] Piglets fed a diet containing 0.5 mg / kg of vomitoxin in the control group had an average daily weight gain of 190 g; the control group, fed a diet without vomitoxin or antifungal toxins, had an average daily weight gain of 371 g, indicating that DON significantly affects the weight gain of piglets. Comparisons between Examples 1-4, Comparative Examples 1-4, the control group, and the control group revealed that the daily weight gain in Example 4 was slightly higher than that in the control group. This may be because Example 4 added polylysine-modified α-lipoic acid, whose special structure may kill Fusarium fungi and prevent the production of new toxins. Polylysine-modified α-lipoic acid may also have specific adsorption or degradation properties for DON, enhancing the intestinal physical barrier, reducing DON permeation and absorption, scavenging free radicals, reducing the production of pro-inflammatory factors, and decreasing the impact of DON on piglets. Synergistically with enzyme preparations and quinoa peptides, it can promote nutrient absorption and increase the daily weight gain of piglets to a certain extent.

Claims

1. A method for preparing a mycotoxin degrading agent, characterized in that, By weight: It is obtained by mixing and sieving 10-20 parts quinoa peptide, 30-40 parts microcrystalline cellulose, 10-30 parts modified α-lipoic acid, 10-20 parts enzyme-containing preparation, and 20-40 parts L-ascorbic acid. Among them, the modified α-lipoic acid is a natural cationic polymer modified α-lipoic acid; The preparation method of the modified α-lipoic acid includes the following steps, in parts by mass: Mix 1-10 parts of natural cationic polymer with 80-120 parts of solvent until homogeneous, then add 1-3 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-1 parts of N-hydroxysuccinimide to obtain mixture 1; mix 1-5 parts of α-lipoic acid with 10-20 parts of anhydrous ethanol until homogeneous, add to mixture 1, and react at 30-50℃ and 800-1200rpm for 20-28h; cool to room temperature, add 150-250 parts of 70wt% ethanol aqueous solution, stir at 800-1200rpm for 5-15min, let stand, centrifuge at 1000-3000rpm for 3-8min, take the precipitate, add 10-25 parts of water, and freeze-dry for 20-28h to obtain modified α-lipoic acid; The natural cationic polymer is selected from at least one of chitosan, chitosan oligosaccharide, and polylysine; The mass ratio of Bacillus licheniformis, alkaline protease, and glucose oxidase in the enzyme-containing preparation is 5-7:2-4:5-7.

2. The method for preparing the mycotoxin degrading agent as described in claim 1, characterized in that: The solvent is water or a 0.5-2.0 wt% aqueous solution of acetic acid.

3. The method for preparing the mycotoxin degrading agent as described in claim 1, characterized in that: The sieve used is a 40-60 mesh sieve.

4. A fungicide degrading agent, characterized in that: Prepared by the method described in any one of claims 1-3.

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