A compound antifungal agent derived from chili straw, its preparation method, and its application in silage and silage fermentation.

Abstract

This invention provides a chili straw-derived composite antifungal agent, its preparation method, and its application in silage and yellow silage fermentation, belonging to the field of forage processing additives. Using chili straw as the main raw material, after modification and activation treatment, a high-speed homogenization-assisted composite reaction process is employed to cross-link plant essential oils and chitosan under the action of citric acid. By optimizing the raw material ratio and combining it with vacuum freeze-drying technology, a chili straw-derived composite antifungal agent is obtained, whose antifungal effect is more than twice that of untreated chili straw. This invention is further applied to forage fermentation processing stages such as corn silage and mixed silage with straw, effectively inhibiting mold growth with a mold inhibition rate of over 90%, and significantly controlling the production of mold and mycotoxins. This invention features a scientific process, unique formula, and low cost, and has good prospects for widespread application.

Description

Technical Field

[0001] This invention belongs to the field of forage processing additives, specifically relating to a chili straw-derived compound antifungal agent, its preparation method, and its application in silage and yellow silage fermentation. Background Technology

[0002] Mold contamination and the resulting accumulation of mycotoxins are major hazards affecting the quality and safety of forage. Improper control during processing can easily lead to such problems. Silage / yellow silage fermentation is a type of feed that uses anaerobic fermentation to preserve forage raw materials for long-term storage. Because it can maximize the preservation of the nutritional value of the raw materials, improve palatability, and extend the supply period, it has become a basic dietary source for modern ruminant farming. However, silage is highly susceptible to mold contamination during production, storage, and use. Mold not only consumes large amounts of nutrients in the feed, leading to dry matter loss, but its metabolic mycotoxins, such as zearalenone (ZEA), vomitoxin (DON), and aflatoxin B1 (AFB1), can easily cause reproductive system abnormalities in cattle and sheep, and even lead to embryonic death, resulting in serious economic losses. At the same time, these toxins can also be transferred to livestock products through the food chain, seriously threatening animal health and food safety. Therefore, developing efficient, safe, and economical mold prevention technologies for silage / yellow silage has always been a research hotspot in the forage processing field.

[0003] Currently, commercially available silage / yellow silage additives are mainly divided into three categories: organic acids, microbial preparations, and enzyme preparations. Organic acids (such as propionic acid, formic acid, and sodium benzoate) inhibit mold growth by rapidly lowering the pH value, but they are highly corrosive and irritating, posing hazards to operators and equipment, and long-term use may lead to rumen acidosis in ruminants. Microbial preparations (such as lactic acid bacteria and propionibacterium) regulate the microecological environment through competitive inhibition, but they have stringent storage requirements, unstable strain survival rates, and high costs, making them difficult to promote and apply in small and medium-sized farms. Enzyme preparations mainly work by degrading mold cell walls, but the effect of a single enzyme preparation is limited, often requiring combination with other additives. Furthermore, most of these products are imported, making them expensive and significantly increasing farming costs. Moreover, existing mold inhibitors mostly focus on single functions, lacking composite products that address the entire silage / yellow silage process and combine mold prevention with quality improvement. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide a chili straw-derived composite antifungal agent and its preparation method.

[0005] A second objective of this invention is to provide the application of the aforementioned chili straw-derived compound antifungal agent in the preparation of silage / yellow silage fermented feed.

[0006] A third objective of this invention is to provide a mold-resistant silage and its preparation method.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a chili straw-derived composite antifungal agent, comprising the following steps: (1) Chili straw coarse powder was mixed with NaOH solution with a mass fraction of 5-7% at a solid-liquid ratio of 1g:(25~35)mL, stirred, centrifuged, the solid part was washed until neutral, centrifuged, and dried to obtain activated modified chili straw; (2) Activated and modified chili straw was added to water at a solid-liquid ratio of 1g:(4~6)mL, and homogenized and stirred to prepare a straw suspension; the straw suspension, citric acid solution, chitosan solution and plant essential oil were mixed and emulsified by high-speed shearing to obtain a primary emulsion; (3) The primary emulsion was stirred, freeze-dried under vacuum, and then broken up to obtain a chili straw-based composite antifungal agent.

[0008] Preferably, in step (1), the stirring temperature is 75~85℃ and the time is 50~70min; the centrifugation speed is 4000~6000rpm and the time is 5~15min.

[0009] Preferably, in step (2), the citric acid solution is prepared by mixing citric acid and water at a solid-liquid ratio of 1g:(4~6)mL, and the amount of citric acid added is 2.5~3.5% of the mass of the activated and modified chili straw; the chitosan solution is prepared by mixing chitosan and water at a solid-liquid ratio of 1g:(4~6)mL, and the amount of chitosan added is 4.5~5.5% of the mass of the activated and modified chili straw; the plant essential oil is a mixture of Litsea cubeba essential oil and cinnamaldehyde in equal mass ratio, and the amount of plant essential oil added is 1.8~2.2% of the mass of the activated and modified chili straw.

[0010] Preferably, in step (2), the speed of homogenization stirring is 800~1200 rpm and the time is 15~25 min; the speed of high-speed shear emulsification is 800~1200 rpm and the time is 4~6 min.

[0011] Preferably, in step (3), the temperature of the stirring reaction is 70~90℃ and the time is 50~70min.

[0012] The present invention also provides a chili straw-derived composite antifungal agent prepared by the above preparation method.

[0013] This invention also provides the application of the above-mentioned chili straw-derived compound antifungal agent in the preparation of silage / yellow silage fermented feed.

[0014] Preferably, the amount of the chili straw-derived compound antifungal agent used in silage fermentation feed is 0.8-1.2% of the mass of the silage raw material, and the amount used in yellow silage fermentation feed is 1.8-2.2% of the mass of the yellow silage raw material.

[0015] This invention also provides a method for preparing anti-mold feed, comprising the following steps: mixing the chili straw-derived compound anti-mold agent with water at a mass ratio of 1:(20~30) to obtain an anti-mold agent solution; using a layered laying process, spraying the anti-mold agent solution at a dosage of 1.8~2.2L per ton of raw material for every 25~35cm thick layer of raw material, compacting after spraying, expelling air from the gaps between the raw materials, repeating the operation, spraying the remaining anti-mold agent solution on the top surface, covering with a film, and sealing with soil; When the anti-mold feed is silage, the amount of chili straw-derived compound anti-mold agent is 0.8-1.2% of the raw material mass; when the anti-mold feed is yellow silage, the amount of chili straw-derived compound anti-mold agent is 1.8-2.2% of the raw material mass.

[0016] The present invention also provides anti-mold feed prepared by the above preparation method.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention provides a chili straw-based composite antifungal agent and its preparation method. By modifying and activating chili straw, it is combined with chitosan, citric acid, and plant essential oils to form an antifungal agent with slow-release properties. This achieves the dual utilization of chili straw resources and the exploitation of endogenous active ingredients, and constructs a multi-target antifungal system with synergistic endogenous and endogenous sources, realizing slow-release and time-series regulation.

[0018] The chili straw-based compound anti-mold agent described in this invention can be applied to silage and yellow silage fermentation. It adopts a process that combines layered application with surface reinforcement, providing a method for preparing anti-mold silage. It achieves a three-in-one technical effect of "anti-mold + quality improvement + cost reduction", providing a new solution for green processing of silage.

[0019] The process of this invention is green and has significant cost advantages. The raw materials are all from natural sources and there are no toxic or harmful solvents. The preparation cost is 15-20 yuan / ton, which is more than 50% lower than that of commercially available organic acid additives. Attached Figure Description

[0020] Figure 1 Comparative study on the sustained-release effect of plant essential oils in chili straw antifungal agents. Detailed Implementation

[0021] This invention provides a method for preparing a chili straw-derived composite antifungal agent, comprising the following steps: (1) Chili straw coarse powder was mixed with NaOH solution with a mass fraction of 5-7% at a solid-liquid ratio of 1g:(25~35)mL, stirred, centrifuged, the solid part was washed until neutral, centrifuged, and dried to obtain activated modified chili straw; (2) Activated and modified chili straw was added to water at a solid-liquid ratio of 1g:(4~6)mL, and homogenized and stirred to prepare a straw suspension; the straw suspension, citric acid solution, chitosan solution and plant essential oil were mixed and emulsified by high-speed shearing to obtain a primary emulsion; (3) The primary emulsion was stirred, freeze-dried under vacuum, and then broken up to obtain a chili straw-based composite antifungal agent.

[0022] In step (1) of this invention, the preparation method of the coarse chili straw powder includes: the chili straw is ultrasonically cleaned, dried, crushed, and sieved to obtain coarse chili straw powder; the ultrasonic cleaning time is preferably 20-30 min; the chili straw is ultrasonically treated to remove surface dirt, surface impurities, and some pollutants; the drying is carried out to constant weight; the crushing is preferably done with a crusher, and the sieving is preferably done through a 20-mesh sieve.

[0023] In step (1) of this invention, the solid-liquid ratio of the coarse chili straw powder and NaOH solution is preferably 1g:26mL, 1g:27mL, 1g:28mL, 1g:29mL, 1g:30mL, 1g:31mL, 1g:32mL, 1g:33mL, or 1g:34mL; the mass fraction of the NaOH solution is preferably 6%; the stirring temperature is 75~85℃, preferably 80℃, and the time is 50~70min, preferably 60min; the solid part is preferably washed 2~3 times with deionized water until the washing liquid is neutral (pH 6.8~7.2); the centrifugation speed is 4000~6000rpm, preferably 5000rpm, and the time is 5~15min, preferably 10min; the drying is preferably carried out at 40℃ by forced air drying until constant weight.

[0024] This invention achieves the simultaneous removal of lignin and hemicellulose to expose the fiber structure and the destruction of cell walls to release endogenous capsaicin-like substances through specific alkaline heat treatment. This successfully promotes the release of endogenous capsaicin, resulting in modified straw that functions as both an adsorbent carrier and an active ingredient. This significantly enhances the effectiveness of single chili straw as a fermentation and anti-mold agent, increasing the anti-mold effect by more than 100% compared to single straw.

[0025] In step (2) of this invention, the solid-liquid ratio of activated modified chili straw to water is preferably 1g:5mL, the stirring speed is 800~1200rpm, preferably 1000rpm, and the time is 15~25min, preferably 20min; the citric acid solution is prepared by mixing citric acid and water at a solid-liquid ratio of 1g:(4~6)mL, preferably 1g:5mL, and the amount of citric acid added is 2.5~3.5% of the mass of activated modified chili straw, preferably... The chitosan solution is prepared by mixing chitosan and water at a solid-liquid ratio of 1g:(4~6)mL, preferably 1g:5mL. The amount of chitosan added is 4.5~5.5% of the mass of the activated and modified chili straw, preferably 5%. The degree of deacetylation of the chitosan is preferably ≥95%. The plant essential oil is a mixture of Litsea cubeba essential oil and cinnamaldehyde in an equal mass ratio (1:1). The amount of plant essential oil added is 1.8~2.2% of the mass of the activated and modified chili straw, preferably 2%. This invention synergistically combines exogenous citric acid, chitosan, plant essential oil, and endogenous capsaicin to effectively overcome the problem of drug resistance easily generated by single components.

[0026] In step (2) of this invention, the activated and modified chili straw is added to water and homogenized by stirring to hydrate and swell the straw fibers, forming a uniform straw suspension. Preferably, citric acid and chitosan are dissolved separately in water to prepare citric acid and chitosan solutions. Preferably, the citric acid and chitosan solutions are added sequentially to the straw suspension, followed by the addition of plant essential oils, and then emulsified by high-speed shearing to form a primary emulsion. The high-speed shearing emulsification is performed at a speed of 800-1200 rpm, preferably 1000 rpm, for a time of 4-6 minutes, preferably 5 minutes.

[0027] This invention utilizes the chemical reaction between citric acid and chitosan to efficiently encapsulate plant essential oils. Combined with freeze-drying technology, it forms a gradient release characteristic, resolving the contradiction of traditional plant essential oil additives that "excessively inhibit bacteria in the early stage and cause secondary mold growth due to failure in the later stage".

[0028] In step (3) of the present invention, it is preferable to transfer the primary emulsion to a reaction vessel and stir it for reaction. The temperature of the stirring reaction is 70~90℃, preferably 80℃, and the time is 50~70min, preferably 60min. The present invention uses stirring reaction to enable the carboxyl groups of citric acid to cross-link with the amino groups of chitosan, thereby attaching them in situ to the surface of the modified straw and embedding the plant essential oil inside the modified straw.

[0029] In step (3) of this invention, the vacuum freeze-drying is preferably performed by pre-freezing the composite solution after stirring and reaction, and then freeze-drying it to dryness under conditions of cold trap temperature -80℃ and vacuum degree 10Pa. The pre-freezing temperature is preferably -20℃, and the time is preferably 12h. After drying, the powder is dispersed to obtain a chili straw-based composite antifungal agent powder with slow-release properties (i.e., chili straw-derived composite antifungal agent). The dispersion is preferably performed by gently dispersing it with a pulverizer.

[0030] The present invention also provides a chili straw-derived composite antifungal agent prepared by the above preparation method.

[0031] The present invention also provides the application of the above-mentioned chili straw-derived compound antifungal agent in the preparation of silage / yellow silage fermented feed, wherein the amount of the chili straw-derived compound antifungal agent in the silage fermented feed is 0.8-1.2% of the mass of the silage raw material, preferably 0.9%, 1% or 1.1%; and the amount of the chili straw-derived compound antifungal agent in the yellow silage fermented feed is 1.8-2.2% of the mass of the yellow silage raw material, preferably 1.9%, 2% or 2.1%.

[0032] The chili straw-based compound antifungal agent of this invention is suitable for various forage fermentation raw materials such as corn / wheat straw and silage corn. It has a mold inhibition rate of over 90%, reduces dry matter loss to below 3% after application, increases crude protein retention by over 5%, and reduces the detection rate of major mycotoxins by over 60%.

[0033] The present invention also provides a method for preparing anti-mold feed, comprising the following steps: mixing the chili straw-derived compound anti-mold agent with water at a mass ratio of 1:(20~30) to obtain an anti-mold agent solution; using a layered laying process, spraying the anti-mold agent solution at a dosage of 1.8~2.2L per ton of raw material for every 25~35cm thick layer of raw material, compacting after spraying, expelling air from the gaps between the raw materials, repeating the operation, spraying the remaining anti-mold agent solution on the top surface, covering with a film, and sealing with soil.

[0034] The preparation method described in this invention can be used to prepare silage or yellow silage. When the anti-mold feed is silage, the dosage of the chili straw-derived compound anti-mold agent is 0.8-1.2% of the raw material mass, preferably 0.9%, 1%, or 1.1%. Preferably, the silage is made from fresh, mold-free green corn stalks, chopped to 2-3 cm in length using a knife or a small shredder. The moisture content of the raw material should be controlled between 60% and 65% before fermentation. When the anti-mold feed is yellow silage, the dosage of the chili straw-derived compound anti-mold agent is 1.8-2.2% of the raw material mass, preferably 1.9%, 2%, or 2.1%. Preferably, the yellow silage is made from corn stalks, chopped to 2-3 cm in length using a knife or a small shredder. The moisture content of the raw material should be controlled between 65% and 70% before fermentation.

[0035] The preferred mass ratio of the chili straw-derived composite antifungal agent to water in this invention is 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, or 1:29. After mixing the chili straw-derived composite antifungal agent with water, it is preferably allowed to stand for 25-35 minutes to activate it, obtaining an antifungal agent solution. The preferred layer thickness in this invention is 30 cm per layer; the preferred covering is a plastic film; the preferred amount of antifungal agent solution added during layering is 50% of the total antifungal agent solution, with the remaining 50% sprayed onto the top layer surface. During the fermentation process in this invention, a composite fermentation agent is preferably added, which can be selected from a mixture of lactic acid bacteria, yeast, and Bacillus.

[0036] The present invention also provides anti-mold feed (silage or yellow silage) prepared by the above preparation method.

[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1 A method for preparing a chili straw-derived compound antifungal agent: (1) The chili straw was ultrasonically treated for 20 minutes to remove surface dirt, surface impurities and some pollutants. After drying to constant weight, it was crushed by a pulverizer and passed through a 20-mesh sieve to obtain coarse chili straw powder. Chili straw coarse powder was mixed with 6% NaOH solution at a solid-liquid ratio of 1g:30mL, stirred at 80℃ for 60min, centrifuged at 5000rpm for 10min, the alkali solution was discarded, the solid part was washed three times with deionized water until neutral, centrifuged again at 5000rpm for 10min, and the solid part was dried at 40℃ with forced air until constant weight to obtain activated modified chili straw.

[0041] (2) Activated and modified chili straw was added to water at a solid-liquid ratio of 1g:5mL and homogenized at 1000rpm for 20min at room temperature to prepare a straw suspension; Weigh 3% of the mass of activated and modified chili straw containing citric acid, add it to deionized water at a solid-liquid ratio of 1g:5mL and stir until dissolved to obtain a citric acid solution; weigh 5% of the mass of activated and modified chili straw containing chitosan (degree of deacetylation ≥95%), add it to deionized water at a solid-liquid ratio of 1g:5mL and stir until dissolved to obtain a chitosan solution; weigh 2% of the mass of activated and modified chili straw containing plant essential oil (a mixture of Litsea cubeba essential oil and cinnamaldehyde in equal mass ratios).

[0042] Citric acid solution and chitosan solution were added to the straw suspension in sequence, followed by plant essential oil. The mixture was then emulsified at 1000 rpm for 5 minutes to form a primary emulsion.

[0043] (3) Transfer the primary emulsion to the reaction vessel and stir at 80°C for 60 min. After the reaction is completed, place the composite solution in a -20°C freezer for 12 h to freeze the composite system completely. Quickly transfer the frozen sample to a vacuum freeze dryer and freeze dry to dry at a cold trap temperature of -80°C and a vacuum degree of 10 Pa. Take out the freeze-dried solid product and gently break it up with a pulverizer to obtain the chili straw-derived composite antifungal agent.

[0044] Example 2 A method for preparing a chili straw-derived compound antifungal agent: (1) The chili straw was ultrasonically treated for 30 minutes to remove surface dirt, surface impurities and some pollutants. After drying to constant weight, it was crushed by a pulverizer and passed through a 20-mesh sieve to obtain coarse chili straw powder. Chili straw coarse powder was mixed with 7% NaOH solution at a solid-liquid ratio of 1g:35mL, stirred at 85℃ for 50min, centrifuged at 6000rpm for 5min, the alkali solution was discarded, the solid part was washed three times with deionized water until neutral, centrifuged again at 6000rpm for 5min, and the solid part was dried at 40℃ with forced air until constant weight to obtain activated modified chili straw.

[0045] (2) Activated and modified chili straw was added to water at a solid-liquid ratio of 1g:6mL and homogenized at 1200rpm for 15min at room temperature to prepare a straw suspension; Weigh 3.5% of the activated and modified chili straw by weight of citric acid, add it to deionized water at a solid-liquid ratio of 1g:6mL and stir until dissolved to obtain a citric acid solution; weigh 5.5% of the activated and modified chili straw by weight of chitosan (degree of deacetylation ≥95%), add it to deionized water at a solid-liquid ratio of 1g:6mL and stir until dissolved to obtain a chitosan solution; weigh 2.2% of the activated and modified chili straw by weight of plant essential oil (a mixture of Litsea cubeba essential oil and cinnamaldehyde in equal mass ratio).

[0046] Citric acid solution and chitosan solution were added to the straw suspension in sequence, followed by plant essential oil. The mixture was then emulsified at 1200 rpm for 4 minutes to form a primary emulsion.

[0047] (3) Transfer the primary emulsion to the reaction vessel and stir at 90°C for 50 min. After the reaction is complete, place the composite solution in a -20°C freezer for 12 h to freeze the composite system completely. Transfer the frozen sample to a vacuum freeze dryer and freeze dry to dry at a cold trap temperature of -80°C and a vacuum of 10 Pa. Take out the freeze-dried solid product and gently break it up with a pulverizer to obtain the chili straw-derived composite antifungal agent.

[0048] Example 3 A method for preparing a chili straw-derived compound antifungal agent: (1) The chili straw was ultrasonically treated for 20 minutes to remove surface dirt, surface impurities and some pollutants. After drying to constant weight, it was crushed by a pulverizer and passed through a 20-mesh sieve to obtain coarse chili straw powder. Chili straw coarse powder was mixed with 5% NaOH solution at a solid-liquid ratio of 1g:25mL, stirred at 75℃ for 70min, centrifuged at 4000rpm for 15min, the alkali solution was discarded, the solid part was washed twice with deionized water until neutral, centrifuged again at 4000rpm for 15min, and the solid part was dried at 40℃ with forced air until constant weight to obtain activated modified chili straw.

[0049] (2) Activated and modified chili straw was added to water at a solid-liquid ratio of 1g:4mL and homogenized at 800rpm for 25min at room temperature to prepare a straw suspension; Weigh 2.5% of the activated and modified chili straw by weight of citric acid, add it to deionized water at a solid-liquid ratio of 1g:4mL and stir until dissolved to obtain a citric acid solution; weigh 4.5% of the activated and modified chili straw by weight of chitosan (degree of deacetylation ≥95%), add it to deionized water at a solid-liquid ratio of 1g:4mL and stir until dissolved to obtain a chitosan solution; weigh 1.8% of the activated and modified chili straw by weight of plant essential oil (a mixture of Litsea cubeba essential oil and cinnamaldehyde in equal mass ratio).

[0050] Citric acid solution and chitosan solution were added to the straw suspension in sequence, followed by plant essential oil. The mixture was then emulsified at 800 rpm for 6 minutes to form a primary emulsion.

[0051] (3) Transfer the primary emulsion to the reaction vessel and stir at 70°C for 70 min. After the reaction is complete, place the composite solution in a -20°C freezer for 12 h to freeze the composite system completely. Transfer the frozen sample to a vacuum freeze dryer and freeze dry to dry at a cold trap temperature of -80°C and a vacuum degree of 10 Pa. Take out the freeze-dried solid product and gently break it up with a pulverizer to obtain the chili straw-derived composite antifungal agent.

[0052] Example 4 A method for preparing mold-resistant silage: The chili straw-derived composite antifungal agent of Example 1 was used at a dosage of 1% of the raw material mass. The chili straw-derived composite antifungal agent was mixed with water at a mass ratio of 1:25, stirred, and allowed to stand for 30 minutes to obtain the antifungal agent solution.

[0053] Select fresh, mold-free, green corn stalks and chop them into 2-3 cm lengths using a knife or small shredder. The moisture content of the raw materials should be controlled between 60% and 65%. Add a compound fermentation agent (purchased from Yijiayi Biotechnology Co., Ltd., with approximately 2 × 10⁻⁶ live bacteria) at a ratio of 0.1% of the total material mass. This agent is composed of lactic acid bacteria, yeast, and Bacillus. 10 (CFU / g) Stir thoroughly to ensure the inoculant adheres evenly to the material surface. Use a layered filling process. For every 30cm layer of silage material, spray with 2L of antifungal agent solution per ton of material. Immediately after spraying, compact mechanically to remove air from the gaps between the materials. Repeat this process until the silage pit is full. Spray the remaining amount (approximately 50%) of the antifungal agent solution onto the top layer after filling. Cover with plastic film, press down soil, and seal for fermentation for 45 days.

[0054] Example 5 A method for preparing mold-resistant silage: The chili straw-derived compound antifungal agent of Example 2 was used at a dosage of 1.2% of the silage raw material mass. The chili straw-derived compound antifungal agent was mixed with water at a mass ratio of 1:20, stirred, and allowed to stand for 35 minutes to obtain the antifungal agent solution.

[0055] Select fresh, mold-free, green corn stalks and chop them into 2-3 cm lengths using a knife or small shredder. The moisture content of the raw materials should be controlled between 60% and 65%. Add a compound fermentation agent (purchased from Yijiayi Biotechnology Co., Ltd., with approximately 2 × 10⁻⁶ live bacteria) at a ratio of 0.1% of the total material mass. This agent is composed of lactic acid bacteria, yeast, and Bacillus. 10 (CFU / g) Stir thoroughly to ensure the inoculant adheres evenly to the material surface. Use a layered filling process. For every 25cm layer of silage material, spray with 1.8L of antifungal agent solution per ton of material. Immediately after spraying, compact mechanically to remove air from the gaps between the materials. Repeat this process until the silage pit is full. Spray the remaining amount (approximately 50%) of the antifungal agent solution onto the top layer after filling. Cover with plastic film, press down soil, and seal for fermentation for 45 days.

[0056] Example 6 A method for preparing mold-resistant silage: The chili straw-derived compound antifungal agent of Example 3 was used at a dosage of 0.8% of the silage raw material mass. The chili straw-derived compound antifungal agent was mixed with water at a mass ratio of 1:30, stirred, and allowed to stand for 30 minutes to obtain the antifungal agent solution.

[0057] Select fresh, mold-free, green corn stalks and chop them into 2-3 cm lengths using a knife or small shredder. The moisture content of the raw materials should be controlled between 60% and 65%. Add a compound fermentation agent (purchased from Yijiayi Biotechnology Co., Ltd., with approximately 2 × 10⁻⁶ live bacteria) at a ratio of 0.1% of the total material mass. This agent is composed of lactic acid bacteria, yeast, and Bacillus. 10 (CFU / g) Stir thoroughly to ensure the inoculant adheres evenly to the material surface. Use a layered filling process. For every 35cm layer of silage material, spray with 2.2L of antifungal agent solution per ton of material. Immediately after spraying, compact mechanically to remove air from the gaps between the materials. Repeat this process until the silage pit is full. Spray the remaining amount (approximately 50%) of the antifungal agent solution onto the top layer after filling. Cover with plastic film, press down soil, and seal for fermentation for 45 days.

[0058] Example 7 A method for preparing mold-resistant silage: The chili straw-derived composite antifungal agent of Example 3 was used at a dosage of 2% of the mass of the silage raw material. The chili straw-derived composite antifungal agent was mixed with water at a mass ratio of 1:30, stirred, and allowed to stand for 30 minutes to obtain the antifungal agent solution.

[0059] Select corn stalks and chop them into 2-3 cm lengths using a knife or small shredder. The moisture content of the raw materials should be controlled between 65% and 70%. Add a compound fermentation agent (purchased from Yijiayi Biotechnology Co., Ltd., with approximately 2 × 10⁻⁶ live bacteria) at a ratio of 0.1% of the total material mass. This agent is composed of lactic acid bacteria, yeast, and Bacillus. 10 (CFU / g) Stir thoroughly to ensure the inoculant adheres evenly to the material surface. Use a layered filling process. For every 35cm layer of silage, spray with 2.2L of antifungal agent solution per ton of material. Immediately after spraying, compact mechanically to remove air from the gaps between the materials. Repeat this process until the silage pit is full. Spray the remaining amount (approximately 50%) of the antifungal agent solution onto the top layer after filling. Cover with plastic film, press down soil, and seal for fermentation for 45 days.

[0060] Comparative Example 1 The only difference from Example 1 is that the addition of plant essential oils and chitosan solution is omitted.

[0061] Comparative Example 2 The only difference from Example 1 is that the addition of plant essential oils is omitted.

[0062] Comparative Example 3 The only difference from Example 1 is that the plant essential oil is cinnamaldehyde.

[0063] Comparative Example 4 The only difference from Example 1 is that the plant essential oil is only Litsea cubeba essential oil.

[0064] Comparative Example 5 The only difference from Example 1 is that the addition of citric acid and chitosan is omitted, and the plant essential oil is only cinnamaldehyde.

[0065] Comparative Example 6 The only difference from Example 1 is that citric acid was omitted and the plant essential oil is cinnamaldehyde only.

[0066] Experimental Example 1 1. Using chili straw as a source of antifungal agents as a selection criterion Cut corn stalks into 2-3 cm lengths and mix thoroughly to obtain the basic mixture. Add water to adjust the moisture content to 65%-70% and let it stand to soften for 2 hours. Then, add 15% of the total material mass of chili stalk powder, sunflower stalk powder, and wheat stalk powder and mix well. Next, add 0.1% of the total material mass of a compound fermentation agent composed of lactic acid bacteria, yeast, and Bacillus (purchased from Yijiayi Biotechnology Co., Ltd., with approximately 2×10⁻⁶ live bacteria). 10 (CFU / g), mix thoroughly to ensure the bacterial agent adheres evenly to the material surface, and control the density of the mixture to 500 kg / m³. 3 After 60 days of fermentation, the number of molds in each treatment was measured to compare the anti-mold effects of different straw resources. The results are shown in the table below.

[0067] Table 1 Comparison of anti-mold effects of different types of straw

[0068] The results showed that when 15% chili straw powder was added, the amount of mold in the product was the lowest after 60 days of fermentation.

[0069] 2. Comparison of the anti-mold effects of adding plant essential oils on chili straw-based compound anti-mold agents Cut corn stalks to 2-3 cm and mix thoroughly to obtain a basic mixture. Add water to adjust the moisture content to 65%-70% and let it stand to soften for 2 hours. Then, add 3% of the total mass of chili straw-derived compound antifungal agent (using the chili straw-derived compound antifungal agent from Comparative Examples 1-4 and Example 1, respectively, denoted as S0, S1, S2, S3, and S4), and mix well. Then, add 0.1% of the total mass of the material a compound fermentation agent composed of lactic acid bacteria, yeast, and Bacillus (purchased from Yijiayi Biotechnology Co., Ltd., with approximately 2×10⁻⁶ live bacteria). 10 (CFU / g), mix thoroughly to ensure the bacterial agent adheres evenly to the material surface, and control the density of the mixture to 500 kg / m³. 3 The anti-mold effect was verified after 60 days of fermentation, and the results are shown in the table below.

[0070] Table 2 Comparison of anti-mildew effects of products made from different raw material compositions

[0071] The results showed that when the raw materials contained plant essential oils, the proliferation of mold in the product could be significantly inhibited. When the plant essential oils were a mixture of Litsea cubeba essential oil and cinnamaldehyde, the inhibitory effect on mold was further enhanced.

[0072] Experimental Example 2 The chili straw-derived composite antifungal agent prepared in Example 1 was used in the experiment.

[0073] 1. Selection of the minimum addition amount of compound chili straw-derived antifungal agent (1) Minimum addition amount in silage corn fermentation The dosage of the chili straw-based compound antifungal agent was set at 0% (control group), 0.2%, 0.5%, 1%, 3%, and 5% of the silage raw material mass, respectively. The remaining steps were the same as in Example 4, and the mixture was sealed and fermented for 45 days.

[0074] After opening the can, the color and texture were evaluated. The minimum addition amount of chili straw in corn silage fermentation was determined by measuring the total mold count and 17 mycotoxins. The 17 mycotoxins included: aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, T-2 toxin, HT-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, varicellic acid, fumonisin, fumonisin B1, fumonisin B2, fumonisin B3, zearalenone, penicillic acid, and patulin. The results are shown in the table below.

[0075] Table 3. Comparison of the addition amount of chili straw-based mildew inhibitors in silage corn.

[0076] The results showed that when the addition amount was higher than 1%, the inhibition rate against mold reached over 90%, effectively controlling the production of mycotoxin metabolites. Therefore, considering both cost and effectiveness, the appropriate addition amount of this compound antifungal agent is 1%.

[0077] (2) Minimum addition amount for straw silage fermentation Corn stalks were used, and the dosage of the chili stalk-derived compound antifungal agent was set at 0% (control group), 0.2%, 0.5%, 1%, 2%, and 5% of the weight of the silage raw material, respectively. The remaining steps were the same as in Example 7, and the mixture was sealed and fermented for 45 days.

[0078] After opening the cans, the color and texture were evaluated. The minimum addition amount of chili straw for silage fermentation was determined by measuring the total mold count and 17 mycotoxins. The 17 mycotoxins included: aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, T-2 toxin, HT-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, varistoxin, fumonisin B1, fumonisin B2, fumonisin B3, zearalenone, penicillic acid, and patulin. The results are shown in the table below.

[0079] Table 4 Comparison of the addition amount of chili straw-based antifungal agents in silage fermentation

[0080] The results showed that when the addition amount was higher than 2%, the inhibition rate against mold reached over 95%, effectively controlling the production of mycotoxin metabolites. Therefore, considering both cost and effectiveness, the appropriate addition amount of this compound antifungal agent is 2%.

[0081] 2. Comparison of the effects of compound antifungal agents (1) Comparison of applications using pure chili straw as an additive: Following the same procedures as in Example 4, 1% chili straw-derived compound antifungal agent was added and applied to silage fermentation. A control group of pure chili straw was also set up, replacing the chili straw-derived compound antifungal agent with pure chili straw (preparation method: chili straw was ultrasonically treated for 20 minutes to remove surface soil, impurities, and some contaminants; after drying to constant weight, it was pulverized and passed through a 20-mesh sieve). A blank control group (CK) was also set up. After 45 days of fermentation, the content of molds and mycotoxins in the forage, as well as nutritional indicators such as dry matter and protein, were measured. The results are shown in the table below.

[0082] Table 5 Comparison results with pure chili straw antifungal agent

[0083] The results showed that adding 1% pure chili straw resulted in a 45.2% inhibition rate against mold and a decrease in the detection rate of mycotoxins to 23.5%, indicating that chili straw has a certain anti-mold effect, but the effect is limited. However, adding the chili straw-derived composite anti-mold agent prepared according to this invention resulted in a 90% inhibition rate against mold, and no mycotoxins were detected. This result fully demonstrates that this invention, by releasing endogenous capsaicin from chili straw through alkali treatment and combining it with an exogenous citric acid, chitosan, and plant essential oils to construct a multi-target synergistic anti-mold system, can effectively inhibit mold proliferation and block the production of mycotoxins at the source. Simultaneously, adding pure chili straw increased the dry matter retention rate by 7.5%, while adding the chili straw-derived composite anti-mold agent of this invention increased the dry matter retention rate by 12% compared to the blank control group. Adding pure chili straw increased the crude protein content to 9.5%, while adding the composite anti-mold agent of this invention further increased the crude protein content to 10.3%, an increase of 22.6% compared to the blank control group and 8.4% compared to the pure chili straw group. This indicates that the chili straw-derived compound antifungal agent of the present invention can effectively inhibit the activity of protein-degrading bacteria while inhibiting mold, reducing protein degradation loss, thereby significantly improving the nutritional quality of silage.

[0084] (2) Comparison with commercially available antifungal agents Using sodium benzoate as a control, silage corn was prepared under the same experimental conditions as in Example 4. Fermentation was performed with either 1% chili straw-derived compound antifungal agent or 1% sodium benzoate. A blank control group (CK) was also included. After 45 days of fermentation, the mold and mycotoxin content in the forage was measured. The results are shown in the table below.

[0085] Table 6 Comparison results with commercially available mildew inhibitors

[0086] The results showed that the treatment group with 1% chili straw-derived compound antifungal agent achieved a 90% mold inhibition rate and no detectable mycotoxins; while the treatment group with 1% sodium benzoate showed only a 52% mold inhibition rate and a 23.5% mycotoxin detection rate. Furthermore, the chili straw-derived compound antifungal agent was superior to sodium benzoate in maintaining both crude protein and dry matter content. These results indicate that, under the same addition conditions, the chili straw-derived compound antifungal agent of this invention has significantly better inhibitory effects on mold and control effects on mycotoxin metabolism than commercially available sodium benzoate.

[0087] 3. Comparative Example 1, Comparative Example 5, and Comparative Example 6: The sustained-release effect of plant essential oils. The experimental procedure was as follows: To evaluate the sustained-release effect of the product on compound plant essential oils, sustained-release verification experiments were conducted on the samples obtained from the preparation processes of Example 1, Comparative Example 5, and Comparative Example 6. The experiments were conducted under room temperature drying conditions (average 25°C), with three parallel samples for each product type. Samples were taken on days 0, 3, 5, 7, 10, 13, 15, 18, and 30 to determine the residual content of Litsea cubeba essential oil and cinnamaldehyde in the samples. Using the initial content on day 0 as a baseline (100%), the ratio of the content at each time point to the initial content was calculated as the essential oil retention rate, thereby comprehensively evaluating the sustained-release performance of the product. Since Litsea cubeba essential oil is a mixture, its main component is citral, accounting for 80%. For accurate quantification, citral was used for quantification, and then the result was adjusted back to the Litsea cubeba essential oil content. Gas chromatography was used for quantitative analysis of both citral and cinnamaldehyde.

[0088] The results are as follows Figure 1 As shown in the figure. The results show that the essential oil in the product of Example 1 has the best sustained-release effect.

[0089] 4. Typical application examples of chili straw-derived compound antifungal agents in livestock farms (1) At Xuxin Farm, whole-plant corn silage was used to produce whole-plant corn silage feed. The silage process was the same as in Example 4. After 50 days of sealed fermentation, the silo was opened for use. After taking out the material, it was immediately covered with plastic sheeting to prevent secondary fermentation. After fermentation, samples were taken for testing. The results showed that the inhibition rate of mold was 90%, the detection rate of mycotoxins was reduced by 50%, the dry matter retention rate was increased by 5%, and the crude protein retention rate was increased by 8%. The annual feed cost was reduced by 30%.

[0090] (2) At the beef cattle farm of Mengxin Planting and Breeding Professional Cooperative in Hangjinhou Banner, corn stalks and oat straw were mixed and silaged to prepare silage feed. The silage process was the same as in Example 7. After 45 days of fermentation, the silage was opened for testing. The results showed that the mixed silage feed had a fragrant aroma, a yellow-green color, and a total mold count of <10. 3 CFU / g. Annual feed costs reduced by 30%.

[0091] In summary, the chili straw-derived compound antifungal agent prepared in this invention has the following significant advantages: excellent antifungal effect, with a mold inhibition rate of over 90%, which is more than 50% higher than that of commercially available organic acid antifungal agents; effective control of mycotoxin production, reducing the detection rate of 17 major toxins such as zearalenone (ZEA), vomitoxin (DON), and aflatoxin B1 (AFB1) by 60% to 70%; strong applicability, widely applicable to various silage raw materials such as silage corn, corn straw, alfalfa, and gramineous forage, and suitable for different seasons and climatic conditions; effectively improves silage quality, reducing dry matter loss by more than 50% and increasing crude protein retention by 8% to 10%; and has a significant cost advantage, with the overall cost reduced by more than 50% compared to commercially available products.

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a chili straw-derived composite antifungal agent, characterized in that, Includes the following steps: (1) Chili straw coarse powder was mixed with NaOH solution with a mass fraction of 5-7% at a solid-liquid ratio of 1g:(25~35)mL, stirred, centrifuged, the solid part was washed until neutral, centrifuged, and dried to obtain activated modified chili straw; (2) Activated and modified chili straw was added to water at a solid-liquid ratio of 1g:(4~6)mL, and homogenized and stirred to prepare a straw suspension; the straw suspension, citric acid solution, chitosan solution and plant essential oil were mixed and emulsified by high-speed shearing to obtain a primary emulsion; (3) The primary emulsion was stirred, freeze-dried under vacuum, and then broken up to obtain a chili straw-based composite antifungal agent.

2. The preparation method according to claim 1, characterized in that, In step (1), the stirring temperature is 75~85℃ and the time is 50~70min; the centrifugation speed is 4000~6000rpm and the time is 5~15min.

3. The preparation method according to claim 1, characterized in that, In step (2), the citric acid solution is prepared by mixing citric acid and water at a solid-liquid ratio of 1g:(4~6)mL, and the amount of citric acid added is 2.5~3.5% of the mass of the activated and modified chili straw; the chitosan solution is prepared by mixing chitosan and water at a solid-liquid ratio of 1g:(4~6)mL, and the amount of chitosan added is 4.5~5.5% of the mass of the activated and modified chili straw; the plant essential oil is a mixture of Litsea cubeba essential oil and cinnamaldehyde in equal mass ratio, and the amount of plant essential oil added is 1.8~2.2% of the mass of the activated and modified chili straw.

4. The preparation method according to claim 1, characterized in that, In step (2), the speed of homogenization stirring is 800~1200 rpm and the time is 15~25 min; the speed of high-speed shear emulsification is 800~1200 rpm and the time is 4~6 min.

5. The preparation method according to claim 1, characterized in that, In step (3), the temperature of the stirring reaction is 70~90℃ and the time is 50~70min.

6. The chili straw-derived composite antifungal agent prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the chili straw-derived compound antifungal agent according to claim 6 in the preparation of silage / yellow silage fermented feed.

8. The application according to claim 7, characterized in that, The compound antifungal agent derived from chili straw is used at a rate of 0.8-1.2% of the mass of the silage raw material in silage fermentation feed and at a rate of 1.8-2.2% of the mass of the yellow silage raw material in yellow silage fermentation feed.

9. A method for preparing anti-mold feed, characterized in that, The process includes the following steps: mixing the chili straw-based composite antifungal agent described in claim 6 with water at a mass ratio of 1:(20~30) to obtain an antifungal agent solution; using a layered laying process, spraying the antifungal agent solution at a dosage of 1.8~2.2L per ton of raw material for every 25~35cm thick layer of raw material, compacting after spraying, expelling air from the gaps between the raw materials, repeating the operation, spraying the remaining antifungal agent solution on the top surface, covering with a film, and sealing with soil; When the anti-mold feed is silage, the amount of chili straw-derived compound anti-mold agent is 0.8-1.2% of the raw material mass; when the anti-mold feed is yellow silage, the amount of chili straw-derived compound anti-mold agent is 1.8-2.2% of the raw material mass.

10. The anti-mold feed prepared by the preparation method according to claim 9.

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