Compound feed additive with vomitoxin attenuation and detoxification functions as well as preparation method and application of compound feed additive

Abstract

The invention discloses a compound feed additive with vomitoxin attenuation and detoxification functions as well as a preparation method and application thereof, belongs to the technical field of functional feed additives, and provides a compound feed additive with vomitoxin attenuation and detoxification functions, which comprises modified eggshell powder, coconut shell charcoal, yeast cell walls and a honeysuckle extract. The mass ratio of the modified eggshell powder to the coconut shell charcoal to the yeast cell wall to the honeysuckle flower extract is (1-3): (1-3): 1: 2. Modified eggshell powder (500-CSAE) is compounded with coconut shell biochar, yeast cell walls and a honeysuckle flower extract to obtain the novel compound feed additive, and the compound feed additive has the capabilities of reducing toxicity and detoxifying, and can effectively relieve the negative influence of DON on the intestinal morphology, the body antioxidant capability and the immune function of weaned piglets.

Description

Technical Field

[0001] The invention belongs to the technical field of functional feed additives, and in particular relates to a compound feed additive with vomitoxin attenuation and detoxification function, and a preparation method and application thereof. Background Art

[0002] Deoxynivalenol (DON) is a type B trichothecenes toxin. It is also called vomitoxin because it has the property of causing vomiting in animals. DON is mainly produced by microorganisms such as Fusarium graminearum, Fusarium oxysporum and Fusarium nivalis. It is widely present in feed and feed raw materials, and seriously threatens the health of livestock and poultry. DON has stable physical and chemical properties, is resistant to high temperature and weak acid, and is stable in the processing and storage of feed and food. It is not easy to be degraded, and it is difficult to eliminate its toxicity by heating or ultraviolet radiation. When DON binds to the ribosome, it will induce ribosome toxicity stress, leading to abnormal cell protein synthesis. In addition, this binding will also activate a variety of protein kinases, causing cell signal transduction disorders, resulting in non-programmed cell death. As a pollutant widely present in different food crops, relevant departments have set restrictions on the maximum allowable content of DON in feed and raw materials.

[0003] Excessive intake of DON in crops and their by-products can cause abdominal pain, diarrhea, vomiting, gastroenteritis and leaky intestines in animals. In order to reduce the potential harm of DON to livestock and poultry production, the current main methods of comprehensive reduction and control are adsorbent adsorption and biodegradation. In recent years, yeast cell walls and plant fibers (such as biological activated carbon) have received extensive attention from researchers. The yeast cell wall is mainly composed of mannooligosaccharide layer, glucan layer and protein layer. These components together give it a high affinity with a variety of mycotoxins, so that the adsorbed mycotoxins are fixed on the cell wall, lose their pathogenicity, and are excreted from the body with intestinal peristalsis. This biological adsorption method is environmentally friendly and sustainable, and has certain significance for reducing the content of highly hydrophobic mycotoxins (such as aflatoxin) in feed. Although biological adsorbents have many advantages, they still have deficiencies in the adsorption performance and stability of polyhydroxy mycotoxins (such as DON). The biological / physical adsorption method they provide is difficult to ensure that DON does not undergo secondary dissociation in the intestine. In comparison, additive products that have both DON biological / physical adsorption, multi-hydroxyl bond coupling and detoxification functions are more adaptable to the complex intestinal environment of livestock and poultry, and can effectively compensate for the cytotoxicity problem caused by DON desorption in the intestine. According to surveys, there are not many products on the market that can be effectively applied to multiple adsorption and detoxification of DON, and most of them have problems such as unstable effects and incomplete functions. Therefore, the feed additive industry urgently needs to develop compound, low-cost DON attenuation and detoxification preparations to meet actual application needs. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a compound feed additive with the function of attenuating and detoxifying vomitoxin, and its preparation method and application. Modified eggshell powder (500-CSAE) is compounded with coconut shell biochar, yeast cell wall and honeysuckle extract to obtain a compound feed additive with high adsorption efficiency and detoxification ability for DON. The compound feed additive can effectively alleviate the negative effects of DON in feed on the intestinal morphology, antioxidant capacity and immune function of weaned piglets.

[0005] To achieve the above-mentioned purpose, the present invention provides a compound feed additive with the function of attenuating and detoxifying vomitoxin, comprising the following components: modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract; the mass ratio of the modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract is 1 to 3:1 to 3:1:2.

[0006] Preferably, the modified eggshell powder is prepared by siliconizing and modifying the eggshell powder with acetic acid.

[0007] The present invention also provides a method for preparing the compound feed additive, comprising the following steps:

[0008] (1) calcining eggshell powder to obtain calcined eggshell powder, dispersing the calcined eggshell powder in a mineralizing modifier working solution to obtain a calcined eggshell powder solution, stirring in a water bath, taking a precipitate to obtain a primary product, dispersing the primary product in an acetic acid aqueous solution, keeping a constant temperature water bath, discarding the supernatant to obtain a modified eggshell powder;

[0009] (2) A compound feed additive is obtained by mixing the modified eggshell powder obtained in step (1) with coconut shell biochar, yeast cell wall and honeysuckle extract.

[0010] Preferably, the calcination temperature in step (1) is 500° C., and the calcination time is 1 to 3 hours.

[0011] Preferably, the ratio of the calcined eggshell powder to the mineralization modifier working solution in step (1) is 1 g: 5-10 mL; the mineralization modifier working solution in step (1) is prepared by mixing 3-aminopropyltriethoxysilane and tetraethyl silicate in a volume ratio of 2:1.

[0012] Preferably, the temperature of the water bath stirring in step (1) is 60-70° C., and the time of the water bath stirring is 2-4 h.

[0013] Preferably, the concentration of the acetic acid aqueous solution in step (1) is 1 mmol / L.

[0014] Preferably, the temperature of the constant temperature water bath in step (1) is 60-70° C., and the time of the constant temperature water bath is 3-5 hours.

[0015] The present invention also provides application of the compound feed additive or the compound feed additive prepared by the preparation method in preparing vomitoxin-free feed.

[0016] The present invention also provides a vomitoxin-free feed, wherein the vomitoxin-free feed comprises the compound feed additive.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] The present invention utilizes the modified eggshell powder described in the invention patent with patent number "CN202111068757.0" and titled "Preparation Method of Eggshell Powder Deoxynivalenol Adsorbent", and compounded it with coconut shell biochar, yeast cell wall, and honeysuckle extract to prepare a compound feed additive with high adsorption efficiency for DON, with an adsorption efficiency of up to 98%. Adding the compound feed additive to the feed can effectively alleviate the negative impact of DON in the feed on the intestinal morphology, antioxidant capacity and immune function of weaned piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 It is the DON standard working curve;

[0021] Figure 2 The adsorption rate of DON by the modified eggshell powder and raw eggshell powder prepared in Comparative Examples 1 to 4;

[0022] Figure 3 The adsorption rate of DON by the compound feed additive, commercial adsorbent and activated carbon prepared in Examples 1 to 3;

[0023] Figure 4 The jejunal structure observation diagram of weaned piglets, where a is the NC group, b is the EC-1 group, c is the DON group, and d is the DON-EC-1 group. The scale length in the figure is 200 μm.

[0024] Figure 5 The diagrams of the ileum structure of weaned piglets are shown in Figure 1. a is the NC group, b is the EC-1 group, c is the DON group, and d is the DON-EC-1 group. The scale bar in the figure is 200 μm.

[0025] Figure 6The effects of different treatments on Ki-67(+) cells in the jejunum of weaned piglets, where a is the NC group, b is the EC-1 group, c is the DON group, and d is the DON-EC-1 group. The scale bar in the figure is 50 μm.

[0026] Figure 7 The effects of different treatments on ileal Ki-67(+) cells in weaned piglets, where a is the NC group, b is the EC-1 group, c is the DON group, and d is the DON-EC-1 group. The scale bar in the figure is 50 μm. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The coconut shell biochar described in the present invention is provided by the "Hunan Biochar Engineering Research Center" of Hunan Agricultural University.

[0033] The yeast cell wall of the present invention is provided by the "Hunan Province Fermented Food Engineering Technology Research Center" of Hunan Agricultural University.

[0034] The honeysuckle extract (water extract) of the present invention is provided by the "National Engineering Technology Research Center for Utilization of Plant Functional Ingredients" of Hunan Agricultural University.

[0035] Example 1

[0036] (1) 50 g of discarded eggshells were washed with water, the inner membrane was removed, and the eggshell powder was crushed. The eggshell powder after the above treatment was placed in a crucible, calcined at 500°C in a muffle furnace for 2 h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5 L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3 h, and then centrifuged (speed 120 00rpm, time is 5min), collect the precipitate, dry at 80℃ for 24h to obtain the initial product, disperse the initial product in 500mL of 1mmol / L acetic acid aqueous solution, keep it in a constant temperature water bath at 65℃ for 4h, then slowly add 1mmol / L sodium hydroxide solution to adjust the pH to 6, centrifuge at 12000rpm for 5min, discard the supernatant, wash it with double distilled water for 3 times, dry it at 80℃ to constant weight, and pass it through a 200-mesh sieve to obtain modified eggshell powder;

[0037] (2) Modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract were mixed in a mass ratio of 3:1:1:2 to obtain a compound feed additive EC-1.

[0038] Example 2

[0039] (1) 50 g of discarded eggshells were washed with water, the inner membrane was removed, and the eggshell powder was crushed. The eggshell powder after the above treatment was placed in a crucible, calcined at 500°C in a muffle furnace for 2 h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5 L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3 h, and then centrifuged (speed 120 00rpm, time is 5min), collect the precipitate, dry at 80℃ for 24h to obtain the initial product, disperse the initial product in 500mL of 1mmol / L acetic acid aqueous solution, keep it in a constant temperature water bath at 65℃ for 4h, then slowly add 1mmol / L sodium hydroxide solution to adjust the pH to 6, centrifuge at 12000rpm for 5min, discard the supernatant, wash it with double distilled water for 3 times, dry it at 80℃ to constant weight, and pass it through a 200-mesh sieve to obtain modified eggshell powder;

[0040] (2) Modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract were mixed in a mass ratio of 2:2:1:2 to obtain a compound feed additive EC-2.

[0041] Example 3

[0042] (1) 50 g of discarded eggshells were washed with water, the inner membrane was removed, and the eggshell powder was crushed. The eggshell powder after the above treatment was placed in a crucible, calcined at 500°C in a muffle furnace for 2 h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5 L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3 h, and then centrifuged (speed 120 00rpm, time is 5min), collect the precipitate, dry at 80℃ for 24h to obtain the initial product, disperse the initial product in 500mL of 1mmol / L acetic acid aqueous solution, keep it in a constant temperature water bath at 65℃ for 4h, then slowly add 1mmol / L sodium hydroxide solution to adjust the pH to 6, centrifuge at 12000rpm for 5min, discard the supernatant, wash it with double distilled water for 3 times, dry it at 80℃ to constant weight, and pass it through a 200-mesh sieve to obtain modified eggshell powder;

[0043] (2) Modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract were mixed in a mass ratio of 1:3:1:2 to obtain a compound feed additive EC-3.

[0044] Comparative Example 1

[0045] 50g of discarded eggshells were washed with clean water, the inner membrane was removed, and the eggshell powder after the above treatment was put into a crucible, placed in a muffle furnace and calcined at 500°C for 2h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate were mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3h, and then centrifuged (the speed was 12000rpm, The precipitate was collected and dried at 80 °C for 24 h to obtain the initial product, which was dispersed in 500 mL of 1 mmol / L acetic acid aqueous solution and kept in a constant temperature water bath at 65 °C for 4 h. Then, 1 mmol / L sodium hydroxide solution was slowly added dropwise to adjust the pH to 6. The mixture was centrifuged at 12000 rpm for 5 min, the supernatant was discarded, and the mixture was washed three times with double distilled water, dried at 80 °C to constant weight, and passed through a 200-mesh sieve to obtain modified eggshell powder 500-CSAE.

[0046] Comparative Example 2

[0047] 50g of discarded eggshells were washed with clean water, the inner membrane was removed, and the eggshell powder after the above treatment was put into a crucible, placed in a muffle furnace and calcined at 700°C for 2h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate were mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3h, and then centrifuged (the speed was 12000rpm, The precipitate was collected and dried at 80 °C for 24 h to obtain the initial product, which was dispersed in 500 mL of 1 mmol / L acetic acid aqueous solution and kept in a constant temperature water bath at 65 °C for 4 h. Then, 1 mmol / L sodium hydroxide solution was slowly added dropwise to adjust the pH to 6. The mixture was centrifuged at 12000 rpm for 5 min, the supernatant was discarded, and the mixture was washed three times with double distilled water, dried at 80 °C to constant weight, and passed through a 200-mesh sieve to obtain modified eggshell powder 700-CSAE.

[0048] Comparative Example 3

[0049] 50g of discarded eggshells were washed with clean water, the inner membrane was removed, and the eggshell powder after the above treatment was put into a crucible, placed in a muffle furnace and calcined at 500°C for 2h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate were mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3h, and then centrifuged (rotating speed was 12000rpm). , time is 5min), the precipitate is collected, and dried at 80℃ for 24h to obtain the initial product. The initial product is dispersed in 500mL of 1mmol / L propionic acid solution, kept in a constant temperature water bath at 65℃ for 4h, and then 1mmol / L sodium hydroxide solution is slowly added dropwise to adjust the pH to 6. The mixture is centrifuged at 12000rpm for 5min, the supernatant is discarded, and then washed with double distilled water for 3 times, dried at 80℃ to constant weight, and passed through a 200-mesh sieve to obtain modified eggshell powder 500-CSPE.

[0050] Comparative Example 4

[0051] 50g of discarded eggshells were washed with clean water, the inner membrane was removed, and the eggshell powder after the above treatment was put into a crucible, placed in a muffle furnace and calcined at 700°C for 2h, cooled, crushed again, and passed through a 300-mesh sieve to obtain calcined eggshell powder; the calcined eggshell powder was dispersed in 0.5L of a mineralizing modifier working solution (3-aminopropyltriethoxysilane and tetraethyl silicate were mixed in a volume / mass ratio of 1:2), stirred in a water bath at 65°C for 3h, and then centrifuged (speed was 12000rpm). , time is 5min), the precipitate is collected, and dried at 80℃ for 24h to obtain the initial product. The initial product is dispersed in 500mL of 1mmol / L propionic acid solution, kept in a constant temperature water bath at 65℃ for 4h, and then 1mmol / L sodium hydroxide solution is slowly added dropwise to adjust the pH to 6. The mixture is centrifuged at 12000rpm for 5min, the supernatant is discarded, and then washed with double distilled water for 3 times, dried at 80℃ to constant weight, and passed through a 200-mesh sieve to obtain modified eggshell powder 700-CSPE.

[0052] Experimental Example 1

[0053] 1 Materials and methods

[0054] 1.1 Test materials

[0055] 1.1.1 Adsorbent

[0056] The compound feed additives EC-1 to EC-3 prepared in Examples 1 to 3 and the modified eggshell powder 500-CSAE, 700-CSAE, 500-CSPE and 700-CSPE prepared in Comparative Examples 1 to 4 were obtained.

[0057] 1.1.2 Experimental reagents and instruments

[0058] Table 1 Experimental reagents

[0059] name factory DON pure Guangzhou Shuopu Biotechnology Co., Ltd., China DONtestHPLC immunoaffinity column Shimadzu Corporation, Japan hydrochloric acid Beijing Reagent Co., Ltd., China Sodium hydroxide Beijing Reagent Co., Ltd., China Potassium dihydrogen phosphate Beijing Reagent Co., Ltd., China Acetonitrile Merck Biotech, Germany Methanol Merck Biotech, Germany Pepsin Maclean, China Trypsin Maclean, China

[0060] Table 2 Experimental Instruments 1

[0061] name factory High performance liquid chromatography Maclean, China Refrigerated high speed centrifuge Eppendorf, Germany Nitrogen blowing instrument Shanghai Bingyue Electronic Instrument Co., Ltd., China

[0062] 1.2 Test methods

[0063] 1.2.1 Preparation of artificial gastric juice and artificial intestinal juice

[0064] Artificial gastric juice: Dissolve 3.84 mL of 36.5% hydrochloric acid in 800 mL of water, add 100 g of pepsin, shake well, dilute with water to 1000 mL, mix well, and successfully prepare artificial gastric juice that meets the requirements.

[0065] Artificial intestinal fluid: Dissolve 7g potassium dihydrogen phosphate in 500mL water, and adjust the pH value to 7 with 0.1mol / L sodium hydroxide solution. Add 10g pancreatic enzyme, add appropriate amount of water to dissolve, mix the two liquids, dilute with water to 1000mL, shake and mix, and successfully prepare artificial intestinal fluid that meets the requirements.

[0066] 1.2.2 Preparation of DON working solution

[0067] The DON standard was prepared into a stock solution with a concentration of 10 mg / mL using methanol, and the stock solution was further diluted to a DON working solution of 500 ng / mL using two simulated digestive fluids, artificial gastric juice and artificial intestinal juice.

[0068] 1.2.3 HPLC conditions

[0069] Equipped with a UV monitor set at 105 nm and a Diamonsil C18 reverse phase column (2.1×50 mm), the injection volume was 10 μL, acetonitrile-water (5:95, v / v) was used for isopycnic elution, and the flow rate was 1.0 mL / min.

[0070] 1.2.4DON standard working curve preparation

[0071] The DON stock solution was diluted in a gradient to obtain a series of solutions of different concentrations. Subsequently, these solutions were measured according to the detection conditions of high performance liquid chromatography, and the corresponding standard chromatographic peaks were recorded. By analyzing these chromatographic peaks, a standard working curve of peak area and DON content was drawn.

[0072] 1.2.5 Adsorption test

[0073] Weigh 20 mg of each adsorbent in a 2.5 mL centrifuge tube and add 2 mL of DON working solution (DON concentration is 500 ng / mL). The negative control is a 2 mL of DON working solution without adsorbent. Each treatment condition was repeated three times to ensure the accuracy of the results. Subsequently, the centrifuge tubes were placed in a constant temperature oscillator at 37 ° C and continuously shaken for 24 hours to simulate the actual reaction conditions. After the reaction, the centrifuge tubes were taken out and centrifuged at 7000 rpm for 5 minutes to separate the solid particles in the solution from the liquid. Take the supernatant and filter it through a 0.22 μm regenerated cellulose membrane to further remove possible impurities. In order to determine the concentration of DON in the supernatant, two methods, high performance liquid chromatography and enzyme-linked immunosorbent assay kit, were used. Finally, the adsorption rate Y (%) was calculated according to the following formula to evaluate the adsorption effect of the adsorbent on DON:

[0074] Y=(C0-C x ) / C0×100%;

[0075] Where C0 and C x are the concentrations of DON in the negative control sample and the experimental sample (μg / mL), respectively.

[0076] 1.3 Data Statistical Analysis

[0077] All experimental results are expressed as mean ± standard deviation (SD), and the data were analyzed using SPSS25.0 software. One-way analysis of variance was used for comparison between groups, followed by the least significant difference and Tamhane's T2 test. Duncan's multiple comparison was used to test the significance of differences between groups within multiple variables. When the probability was less than 0.05, the result was considered statistically significant (P<0.05).

[0078] 1.4DON standard working curve

[0079] In order to perform quantitative analysis of DON, the DON stock solution was diluted into a series of different concentration gradients, specifically 15.625 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL and 1000 ng / mL. Mix thoroughly to ensure uniform concentration distribution. Pipette 1.0 mL of DON solution and filter through a 0.22 mm regenerated cellulose filter to remove possible impurities and particulate matter. To ensure the accuracy and reliability of the results, three parallel injections were performed. There is a linear relationship between the DON concentration and its peak area (such as Figure 1 ). The equation of the fitted line is:

[0080] Y = 91.412691X + 1064.886046;

[0081] Wherein, Y is the peak area of ​​DON; X is the concentration of DON (ng / mL); the linear correlation coefficient r of the fitting line is 0.99620542.

[0082] 2 Results and analysis

[0083] 2.1 Study on the adsorption effect of adsorbent on DON under different pH conditions

[0084] The adsorption rates of DON by raw eggshell powder (E) and modified eggshell powder 500-CSAE, 700-CSAE, 500-CSPE and 700-CSPE prepared from comparative examples 1 to 4 under different pH conditions are shown in Table 1. Figure 2 Under the conditions of artificial gastric juice and artificial intestinal juice at pH=2.0 and pH=7.0, the modified eggshell powder had a higher DON adsorption effect than the eggshell powder, and the adsorption rate of DON by 500-CSAE and 500-CSPE was higher than that of 700-CSAE and 700-CSPE.

[0085] 2.2 Study on the adsorption effect of compound feed additives on DON under different acid and alkaline conditions

[0086] The adsorption rates of DON by the compound feed additives CE-1 to CE-3 prepared in Examples 1 to 3 and the commercial adsorbent (Utoxin, Shenlite Biotechnology Co., Ltd.) at different pH values ​​are shown in Table 1. Figure 3 The results showed that under the conditions of artificial gastric juice and artificial intestinal juice at pH = 2.0 and pH = 7.0, all compound feed additives had significantly improved DON adsorption effects compared with modified eggshell powder, and the effects had reached the level of commercial adsorbents, with the adsorption efficiency of DON reaching more than 95%.

[0087] Coconut shell biochar, yeast cell wall and honeysuckle extract are used as compound materials and compounded with 500-CSAE with the best adsorption performance. Although plant biochar has a good adsorption effect on DON, the principle of broad-spectrum adsorption makes it also have a certain adsorption effect on most trace elements. Therefore, the coconut shell biochar with the smallest proportion is preferably used for the preparation and application of compound feed additives. The feed additives prepared by this compounding method also have an adsorption effect of more than 95% on DON.

[0088] Experimental Example 2

[0089] 1 Materials and methods

[0090] 1.1 Experimental Materials

[0091] 1.1.1 Adsorbent

[0092] The compound feed additive prepared in Example 1.

[0093] 1.1.2 Experimental instruments

[0094] Table 3 Experimental Instrument II

[0095] name factory Constant temperature fermentation box Shanghai Hetian Scientific Instrument Co., Ltd., China Fully automatic biochemical analyzer Mindray Medical, China Multifunctional ELISA reader Tecan AG, Switzerland

[0096] 1.1.3 Preparation of artificial moldy feed

[0097] The Q2021 strain preserved in liquid PDA was inoculated on a PDA plate and cultured in a shaker at 25°C for 5 days. After the PDA plate was covered with hyphae by naked eye observation, the hyphae were washed off and inoculated into the basic feed for fermentation for 28 days. During this period, 4 fermentation samples from different areas of each group were sampled every 8 days to determine the DON concentration. The DON concentration determination method was tested with reference to the AgraQuant DON enzyme-linked immunosorbent assay kit test instructions. Subsequently, according to the measured concentration of DON in the artificial moldy feed, it was combined with normal feed to prepare a contaminated feed with a DON concentration of 1.09 mg / kg for subsequent experiments.

[0098] 1.2 Experimental animal grouping and diet composition

[0099] 1.2.1 Experimental animals and groups

[0100] Thirty-two 21-day-old weaned piglets (Du × Chang × Da, Sanyuan pigs) with similar body weight were randomly divided into negative control group (NC), negative control + compound feed additive group (EC-1), DON challenge group (DON), DON challenge + compound feed additive group (DON + EC-1) according to body weight. Each experimental group contained 8 replicates, and each group repeated 1 weaned piglet. The overall experimental period was 14 days. The content of DON in the diet of each experimental group was: negative control group (0.06 mg / kg DON), negative control + compound feed additive group (0.06 mg / kg DON), DON challenge group (1.09 mg / kg DON) and DON challenge + compound feed additive group (1.09 mg / kg DON). The amount of EC-1 added was 0.2% of the diet mass. The piglets were placed in cages and raised individually. The feeding and health conditions of the piglets during the feeding period were recorded. The cages were cleaned every day to keep the experimental environment clean. The grouping of experimental animals is shown in Table 4.

[0101] Table 4 Experimental design

[0102] Group Number of repetitions Feed treatment NC 8 Basic feed EC-1 8 Basic feed + 0.2% EC-1 DON 8 Artificial moldy feed DON-EC-1 8 Artificial moldy feed + 0.2% EC-1

[0103] 1.2.2 Experimental diet

[0104] The experimental diet was formulated according to the NRC2012 standard, and a corn-soybean meal-based basal diet was prepared to meet the nutritional needs of piglets. The composition and chemical composition of the basal diet are shown in Table 5.

[0105] Table 5 Composition and chemical composition of basal diet

[0106]

[0107]

[0108] Note: The nutritional levels in this feed are actual measured values.

[0109] 1.3 Slaughter experiment

[0110] After the feeding experiment, 32 piglets were slaughtered after fasting for 12 h, and were marked and weighed before slaughter.

[0111] 1.4 Experimental records and sample collection

[0112] During the experiment, the daily feed consumption of the piglets was recorded in detail, and the weights were recorded at 08:00 on the 0th and 14th days of the experiment.

[0113] Feces were collected on the 0th, 3rd, 6th, 9th, 12th and 15th days of the experiment, placed in centrifuge tubes, and stored in a -20°C refrigerator until testing.

[0114] After weighing on the 14th day of the experiment, all piglets were subjected to blood sampling from the anterior vena cava on the 14th day of the experiment. The collected blood samples were left to stand to allow the serum to precipitate naturally. The centrifuge was centrifuged at 3000rpm for 10min to better separate the serum. The separated supernatant was packaged and stored in a refrigerator at -20℃ for subsequent testing.

[0115] 1.5 Measurement indicators and methods

[0116] 1.5.1 Intestinal morphology

[0117] The jejunal and ileal segments fixed for 24 hours were removed from the 4% paraformaldehyde and replaced with new 4% paraformaldehyde. The intestinal tissue was rinsed with tap water for 12 hours to wash off the excess paraformaldehyde on the surface of the sample. Dehydrated with ethanol according to the concentration gradient from low to high, and then made transparent with xylene. The transparent sample was embedded in paraffin, and the tissue was sliced ​​into 3-5 μm thick slices, and then stained with hematoxylin-eosin. Observed under a fluorescence microscope, at least 8 complete villi were selected from each sample to measure the villus height and crypt depth, and the villus-crypt ratio was calculated.

[0118] 1.5.2 Serum indicators

[0119] The levels of MPO and DAO in serum were detected by enzyme-linked immunosorbent assay.

[0120] 1.5.3 Relative expression of intestinal tight junction protein mRNA

[0121] In order to study the gene expression in intestinal tissue, an appropriate amount of samples were first cut and ground in a liquid nitrogen mortar, and then RNA was extracted from the intestinal samples using Trizo1 reagent. The concentration of the extracted RNA was accurately measured and quantified using an ultra-micro spectrophotometer. The RNA was reverse transcribed into cDNA using a reverse transcription kit as a template for subsequent PCR reactions. The mRNA expression levels of multiple tight junction protein-related genes (including ZO-1, Occludin, Claudin-1, Claudin-3, and Claudin-4) in the jejunum were determined by real-time fluorescence quantitative PCR.

[0122] 1.5.4 Blood indicators

[0123] Serum IgA, IgG and IgM were detected by enzyme-linked immunosorbent assay. Catalase (CAT), malondialdehyde (MDA), total superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and glutathione (GSH) were detected by enzyme-linked immunosorbent turbidimetry. Inflammatory factors IL-1β, IL-6 and TNF-α were detected by enzyme-linked immunosorbent turbidimetry.

[0124] 1.5.5 Intestinal immunohistochemistry

[0125] For subsequent observation and analysis, paraffin sections were processed. The sections were baked at 60°C for 30 minutes to fix the sections. The sections were dewaxed and rehydrated using xylene and a series of different concentrations of ethanol to restore them to a suitable humidity state. The sections were placed in a sodium citrate solution with a pH of 6, heated to boiling and maintained for 45 minutes to achieve antigen retrieval. After the solution cooled to room temperature, it was washed with deionized water for 5 minutes. The sections were immersed in a 3% H2O2 solution for 5 minutes to eliminate the activity of endogenous peroxidase. The sections were rinsed with PBS solution to remove residual H2O2. In order to block nonspecific binding sites on the sections, the sections were blocked in a 5% BSA solution for 20 minutes. After the blocking was completed, the blocking solution was gently absorbed with absorbent paper, and then the diluted Ki67 primary antibody solution was dropped on the sections and incubated at room temperature for 1 hour to specifically bind to the target antigen. After the primary antibody incubation was completed, the sections were washed with PBS to remove unbound antibodies. Incubate with secondary antibody for 30 minutes to further enhance the signal strength. After the secondary antibody incubation is completed, wash the slices with PBS again to remove the excess secondary antibody. Add color developer to develop the color reaction to visualize the target antigen. After the color development is completed, wash the slices with deionized water to remove the excess color developer. Perform hematoxylin staining to color the cell nuclei. After the staining is completed, wash the slices with deionized water again. In order to make the slices have a better observation effect, use different concentrations of anhydrous ethanol and xylene for dehydration and transparency. Use neutral gum to seal the slices and dry the slices for subsequent observation and analysis.

[0126] 1.6 Data Statistical Analysis

[0127] All experimental results are expressed as mean ± standard deviation (SD), and the data were analyzed using SPSS25.0 software. One-way analysis of variance was used for comparison between groups, followed by the least significant difference and Tamhane's T2 test. Duncan's multiple comparison was used to test the significance of differences between groups within multiple variables. When the probability was less than 0.05, the result was considered statistically significant (P<0.05).

[0128] 2 Results and analysis

[0129] 2.1 Effects of compound feed additives on growth performance of weaned piglets

[0130] As shown in Table 6, compared with the basic feed group (negative control), the addition of compound feed additive EC-1 had no significant effect on the growth performance of weaned piglets. There were no significant differences in the average daily weight gain, average daily feed intake, feed-to-weight ratio and diarrhea rate between the two groups. When weaned piglets ingested artificial moldy diets (containing 1.09 mg / kg DON), their average daily weight gain was significantly reduced compared with the basic feed group (negative control). At the same time, the feed-to-weight ratio and diarrhea rate of piglets showed a significant upward trend, indicating that artificial moldy diets (containing 1.09 mg / kg DON) had a negative impact on the growth and health of weaned piglets. Compared with the NC group, the average daily weight gain of piglets in the DON group decreased by 22.39% (P<0.05), the average daily feed intake decreased by 3.63% (P<0.05), the feed-to-weight ratio increased by 19.37% (P<0.05), and the diarrhea rate increased by 25% (P<0.05). Adding compound feed additive EC-1 to artificial moldy feed (containing 1.09 mg / kg DON) significantly improved its growth performance and alleviated diarrhea. Compared with the DON group, the average daily weight gain of piglets in the DON-EC-1 group increased by 25.97% (P<0.05), the average daily feed intake increased by 4.23%, the feed-to-weight ratio decreased by 13.21%, and the diarrhea rate decreased by 22.33% (P<0.05).

[0131] Table 6 Effects of compound feed additives on growth performance of weaned piglets

[0132]

[0133] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P<0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group fed with 0.2% EC-1 added to the basal diet; DON represents the challenge group fed with a moldy diet containing 1.09 mg / kg DON prepared with artificial moldy corn; DON-EC-1 represents the experimental group fed with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0134] 2.2 Effects of compound feed additives on blood biochemical parameters of weaned piglets

[0135] As shown in Table 7, the addition of compound feed additive EC-1 to the basal diet did not significantly affect the serum biochemical indices of weaned piglets. However, the intake of artificial moldy diets resulted in a significant increase in the levels of total protein (TP), glucose (GLU), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of weaned piglets. Specifically, compared with the basal diet group (NC group), the serum total protein content of piglets in the artificial moldy diet (containing 1.09 mg / kg DON) group increased by 18.62%. The serum Glu, ALP, ALT and AST contents of piglets in the artificial moldy diet (containing 1.09 mg / kg DON) group were 17.81% (P<0.05), 23.93%, 39.11% (P<0.05) and 201.58% (P<0.05) higher than those in the basal diet group, respectively.

[0136] Adding compound feed additive EC-1 to artificial moldy diet (containing 1.09 mg / kg DON) helps restore blood biochemical indicators such as total protein, glucose, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase in piglet serum to levels comparable to those of the basic feed group (negative control). Specifically, compared with the artificial moldy diet (containing 1.09 mg / kg DON), the serum total protein (TP), glucose (GLU), creatine kinase (CK), alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) contents of weaned piglets in the compound feed additive group (DON-EC-1 group) decreased by 9.05%, 14.62%, 16.98%, 14.59% (P<0.05) and 56.77% (P<0.05), respectively.

[0137] Table 7 Effects of compound feed additives on blood biochemical parameters of weaned piglets

[0138]

[0139] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group fed with 0.2% EC-1 added to the basal diet; DON represents the challenge group fed with a moldy diet containing 1.09 mg / kg DON prepared with artificial moldy corn; DON-EC-1 represents the experimental group fed with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0140] 2.3 Effects of compound feed additives on organ indexes of weaned piglets

[0141] As shown in Table 8, the addition of compound feed additive EC-1 to the basal diet did not have a significant effect on the organ index. Compared with the basal diet group (negative control), the intake of artificial moldy diet (containing 1.09 mg / kg DON) significantly increased the liver index of piglets, and the liver index increased by 12.74% (P<0.05). The addition of EC-1 to the artificial moldy diet (containing 1.09 mg / kg DON) reduced the liver index of piglets, restoring it to a level equivalent to that of the basal feed group (negative control). Compared with the DON group, the liver index of piglets in the DON-EC-1 group decreased by 14.35% (P<0.05).

[0142] Table 8 Effects of compound feed additives on organ indexes of weaned piglets

[0143]

[0144] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the moldy feed containing 1.09 mg / kg DON.

[0145] 2.4 Effect of compound feed additives on DON detoxification in weaned piglets

[0146] As shown in Table 9, DON was detected in the ileum contents, jejunum contents, and feces of piglets in the DON group. Compared with the DON group, the addition of compound feed additive EC-1 to artificial moldy feed (containing 1.09 mg / kg DON) significantly increased the content of DON in the ileum contents, jejunum contents, and feces, indicating that the addition of compound feed additive EC-1 to artificial moldy feed (containing 1.09 mg / kg DON) achieved effective DON adsorption and detoxification in vivo.

[0147] Table 9 DON concentrations in jejunal contents, ileal contents and feces of weaned piglets (day 14) with different dietary intakes (mg / kg)

[0148]

[0149] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the moldy feed containing 1.09 mg / kg DON.

[0150] 2.5 Effects of compound feed additives on intestinal morphology of weaned piglets

[0151] Table 10, Table 11, Figure 4 Middle a, Figure 4 Middle b, Figure 4 Middle c, Figure 4 Middle d, Figure 5 Middle a, Figure 5 Middle b, Figure 5 Medium c and Figure 5 Figure d shows the effects of different treatments on the villus height and crypt depth indicators of the jejunum and ileum of piglets. It can be observed from the tabular data that compared with the NC group, the DON group significantly reduced the jejunal villus length and the ratio of villus height to crypt depth, which were reduced by 17.93% (P<0.05) and 14.40% (P<0.05), respectively. There was no significant difference in the jejunal villus height of piglets in the DON-EC-1 group compared with the DON group, but the ratio of villus height to crypt depth increased significantly by 17.69% compared with the DON group (P<0.05). These results indicate that artificial moldy diets (containing 1.09 mg / kg DON) had a significant negative impact on the morphology of jejunal villi, and the addition of compound feed additive EC-1 alleviated this negative impact.

[0152] Compared with the NC group, the height of the ileal villi of the piglets in the DON group was significantly reduced by 25.51% (P<0.05), and the ratio of the villus height to the crypt depth was significantly reduced by 23.75% (P<0.05). This indicates that the artificial moldy diet (containing 1.09 mg / kg DON) had a negative impact on the morphology of the ileal villi. Compared with the DON group, the height of the ileal villi of the piglets in the DON-EC group increased by 16.1%, and the ratio of the villus height to the crypt depth also increased by 12.26%. These results indicate that the compound feed additive EC-1 alleviates the negative impact of DON on the morphology of the ileal villi.

[0153] Table 10 Effects of different treatments on ileum morphology in weaned piglets

[0154]

[0155] Table 11 Effects of different treatments on jejunal morphology in weaned piglets

[0156]

[0157] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the moldy feed containing 1.09 mg / kg DON.

[0158] 2.6 Effects of compound feed additives on the intestinal mucosal barrier of weaned piglets

[0159] As shown in Table 12, compared with the basic feed group (negative control), the serum DAO content of piglets in the artificial moldy diet group increased significantly by 50.76% (P<0.05), and the MPO content increased significantly by 47.52% (P<0.05). Compared with the DON group, the serum DAO content of piglets in the DON-EC-1 group increased significantly by 26.71% (P<0.05), and the MPO content decreased significantly by 20.1% (P<0.05).

[0160] Table 12 Effects of different treatments on serum DAO and MPO activities in weaned piglets

[0161]

[0162] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the moldy feed containing 1.09 mg / kg DON.

[0163] 2.7 Effects of compound feed additives on the mRNA expression level of intestinal tight junction proteins in weaned piglets

[0164] As shown in Table 13, the intake of artificial moldy diet (containing 1.09 mg / kg DON) significantly reduced the relative mRNA levels of four tight junction proteins, ZO-1, Occludin, Claudin-1 and Claudin-4, in the jejunum of weaned piglets (P<0.05). However, compared with the DON group, the mRNA levels of tight junction proteins in the jejunum of piglets in the DON-EC-1 group showed a significant upward trend.

[0165] Table 13 Effects of different treatments on relative expression levels of tight junction protein mRNA in the jejunum of weaned piglets

[0166]

[0167] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0168] 2.8 Effects of compound feed additives on serum immunoglobulins in weaned piglets

[0169] As shown in Table 14, the intake of artificial moldy feed (containing 1.09 mg / kg DON) changed the level of immunoglobulins in the serum of weaned piglets. Compared with the basal feed group (negative control), the serum IgA content of the DON group increased by 27.5%, and the IgG content also increased by 23.19% (P<0.05). Compared with the DON group, the serum IgA content of piglets in the DON-EC-1 group increased by 17.65%, while the IgG content decreased by 23.15%. This result suggests that the addition of compound feed additive EC-1 affects the immune function of weaned piglets, mainly manifested in the regulation of serum immunoglobulin content.

[0170] Table 14 Effects of different treatments on serum immunoglobulins in weaned piglets

[0171]

[0172] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group fed with 0.2% EC-1 added to the basal diet; DON represents the challenge group fed with a moldy diet containing 1.09 mg / kg DON prepared with artificial moldy corn; DON-EC-1 represents the experimental group fed with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0173] 2.9 Effects of compound feed additives on Ki-67 cells in the jejunum and ileum of weaned piglets

[0174] Figure 6 Middle a, Figure 6 Middle b, Figure 6 Middle c, Figure 6 Middle d, Figure 7 Middle a, Figure 7 Middle b, Figure 7 Middle c, Figure 7 Figure d and Table 15 show the effects of different treatments on the proportion of Ki-67 positive cells in the intestine of piglets. Compared with the basic feed group (negative control), the proportion of Ki-67 positive cells in the jejunum of piglets fed with artificial moldy feed was significantly reduced. Compared with the DON group, the proportion of Ki-67 positive cells in the jejunum of piglets in the DON-EC-1 group showed a significant recovery. This result shows that the addition of compound feed additive EC-1 effectively alleviates the negative impact of DON-exceeding feed on intestinal cell proliferation.

[0175] Table 15 Comparison of the proportion of Ki-67(+) cells in the jejunum and ileum of weaned piglets under different treatments

[0176]

[0177] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0178] 2.10 Effects of compound feed additives on the expression levels of serum inflammatory factors in weaned piglets

[0179] As shown in Table 16, compared with the basic feed group (negative control), the levels of inflammatory factors IL-1β, IL-6 and TFN-α in the serum of piglets in the artificial moldy diet group increased significantly. This indicates that the artificial moldy diet triggered an inflammatory response in piglets, leading to increased secretion of inflammatory factors. Compared with the DON group, the levels of IL-1β, IL-6 and TFN-α in the serum of piglets in the DON-EC-1 group were significantly reduced, indicating that the compound feed additive EC-1 effectively reversed the excessive secretion of inflammatory factors caused by DON. Specifically, compared with the NC group, the level of IL-1β in the serum of piglets in the DON group increased by 186.09%, IL-6 increased by 69.32%, and TFN-α increased by 128.4% (P<0.05). Compared with the DON group, the levels of serum IL-1β, IL-6, and TFN-α in piglets in the DON-EC-1 group were reduced by 40.16%, 49.67%, and 36.92% (P<0.05), further indicating that the compound feed additive EC-1 effectively alleviated the inflammatory response caused by DON.

[0180] Table 16 Effects of different treatments on serum inflammatory factor levels in weaned piglets

[0181]

[0182] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0183] 2.11 Effects of compound feed additives on serum antioxidant indexes in weaned piglets

[0184] As shown in Table 17, compared with the basal diet group (negative control), the malondialdehyde (MDA) content in the serum of piglets in the artificial moldy diet group (DON group) was significantly increased, while glutathione (GSH), catalase (CAT) and total antioxidant capacity (T-AOC) were significantly decreased. This indicates that the artificial moldy diet (containing 1.09 mg / kg DON) caused the oxidative stress in weaned piglets to increase and the antioxidant capacity was damaged. However, when the feed additive EC-1 was added to the artificial moldy diet (containing 1.09 mg / kg DON), the situation was significantly improved. Compared with the DON group, the GSH, CAT and T-AOC contents in the serum of piglets in the DON-EC-1 group were significantly increased, while the MDA content was significantly decreased. In summary, artificial moldy feed (containing 1.09 mg / kg DON) has a negative impact on the antioxidant capacity of weaned piglets, while the addition of feed additive EC-1 significantly improved this situation, enhanced the antioxidant defense mechanism of piglets, and maintained a healthy state.

[0185] Table 17 Effects of different treatments on serum antioxidant indexes of weaned piglets

[0186]

[0187] Note: Data with different letters on the shoulders of the same row indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05). NC represents the negative control group fed only with the basal diet; EC-1 represents the experimental group with 0.2% EC-1 added to the basal diet; DON represents the challenge group with 1.09 mg / kg DON moldy diet prepared with artificial moldy corn; DON-EC-1 represents the experimental group with 0.2% EC-1 added to the artificial moldy feed containing 1.09 mg / kg DON.

[0188] 3. Conclusion

[0189] Artificial moldy diet (containing 1.09 mg / kg DON) significantly reduced the average daily weight gain of piglets, reduced feed conversion rate, increased diarrhea rate, and damaged the body's antioxidant and immune functions. Adding compound feed additive EC-1 to the diet effectively alleviated the negative impact of DON on weaned piglets. The mechanism of action of compound feed additive EC-1 mainly includes: (1) chemical coupling between Si-O bonds of modified eggshell powder and polyhydroxyl groups of DON; (2) biological and physical adsorption between the complex pore structure of modified eggshell powder, yeast cell wall, coconut shell biochar and DON; (3) the active ingredients of honeysuckle extract exert antioxidant and anti-inflammatory effects to enhance the piglets' detoxification ability of DON.

[0190] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite feed additive with the function of attenuating and detoxifying vomitoxin, characterized in that: The invention comprises the following components: modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract; the mass ratio of the modified eggshell powder, coconut shell biochar, yeast cell wall and honeysuckle extract is 1-3:1-3:1:

2.

2. The compound feed additive according to claim 1, characterized in that: The modified eggshell powder is prepared by siliconizing and modifying the eggshell powder with acetic acid.

3. The method for preparing the compound feed additive according to claim 1 or 2, characterized in that: The following steps are involved: (1) calcining eggshell powder to obtain calcined eggshell powder, dispersing the calcined eggshell powder in a mineralizing modifier working solution to obtain a calcined eggshell powder solution, stirring in a water bath, taking a precipitate to obtain a primary product, dispersing the primary product in an acetic acid aqueous solution, keeping a constant temperature water bath, discarding the supernatant to obtain a modified eggshell powder; (2) A compound feed additive is obtained by mixing the modified eggshell powder obtained in step (1) with coconut shell biochar, yeast cell wall and honeysuckle extract.

4. The preparation method according to claim 3, characterized in that: The calcination temperature in step (1) is 500° C., and the calcination time is 1 to 3 hours.

5. The preparation method according to claim 3, characterized in that: The ratio of the calcined eggshell powder to the mineralization modifier working solution in step (1) is 1 g: 5-10 mL; the mineralization modifier working solution in step (1) is prepared by mixing 3-aminopropyltriethoxysilane and tetraethyl silicate in a volume ratio of 2:

1.

6. The preparation method according to claim 3, characterized in that: The temperature of the water bath stirring in step (1) is 60-70° C., and the time of the water bath stirring is 2-4 hours.

7. The preparation method according to claim 3, characterized in that: The concentration of the acetic acid aqueous solution in step (1) is 1 mmol / L.

8. The preparation method according to claim 3, characterized in that: The temperature of the constant temperature water bath in step (1) is 60-70° C., and the time of the constant temperature water bath is 3-5 hours.

9. Use of the compound feed additive according to claim 1 or 2 or the compound feed additive prepared by the preparation method according to any one of claims 3 to 8 in preparing vomitoxin-free feed.

10. A vomitoxin-free feed, characterized in that: The vomitoxin-free feed includes the compound feed additive according to claim 1 or 2.

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