Use of 4-pentenoic acid in the preparation of silage additives

Abstract

The application belongs to the technical field of livestock science feed processing and forage storage, and specifically discloses application of 4-pentenoic acid in preparation of a silage feed additive, wherein the silage feed additive comprises 5-10% of 4-pentenoic acid in terms of mass percentage. The application adopts the application of 4-pentenoic acid in preparation of the silage feed additive, targets to inhibit growth and reproduction of clostridium in a silage system, reduces abundance of harmful clostridium, reduces protein degradation and ammonia nitrogen accumulation in the silage process, stabilizes silage pH, improves dry matter recovery rate, protects forage nutrition, and improves feeding value. Meanwhile, the additive is green and safe, has low addition amount, controllable cost, is suitable for large-scale forage silage production, supports direct silage of fresh high-moisture forage, does not need wilting and water control, simplifies the silage production process, and provides stable technical support for production of high-quality roughage for ruminants.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry feed processing and forage storage technology, and in particular to the application of 4-pentenoic acid in the preparation of silage additives. Background Technology

[0002] my country has abundant crop straw and forage resources, and silage technology is a core technology for realizing the utilization of forage as feed and ensuring a stable supply of roughage for ruminants. However, legume forage such as alfalfa has the characteristics of high buffering energy, high protein, and high moisture content, making it highly susceptible to contamination by Clostridium in the environment and initiation of Clostridium fermentation during the silage process. On the one hand, Clostridium decomposes and utilizes lactic acid in the silage system, leading to a rise in pH and spoilage of the silage. On the other hand, Clostridium can decompose forage protein to produce large amounts of ammonia nitrogen, amines, and other undesirable substances, resulting in nutrient loss, decreased palatability, and seriously affecting the rumen nitrogen utilization efficiency and production performance of ruminants.

[0003] Existing technologies for controlling silage fermentation have significant limitations: (1) The field wilting water control method is time-consuming and labor-intensive, greatly affected by the weather, and easily causes forage leaves to fall off, nutrients to be lost and secondary pollution; (2) Adding molasses or commercial lactic acid bacteria preparation alone has a very limited inhibitory effect on Clostridium under extremely high moisture conditions where the dry matter content of forage is less than 20%, and cannot effectively prevent the initiation of Clostridium fermentation. (3) Traditional acid inhibitors such as formic acid and propionic acid have broad-spectrum antibacterial activity, which limits feed quality and is prone to residues, affecting the palatability of silage and animal health. (4) Conventional silage additives lack targeted antibacterial properties and cannot protect the activity of beneficial lactic acid bacteria while precisely inhibiting harmful Clostridium, which can easily disrupt the normal fermentation process of silage.

[0004] Therefore, there is an urgent need to develop a new type of silage additive, providing a safe, efficient, targeted, and low-cost preparation method for Clostridium inhibitory silage additives to solve the industry problem of easy spoilage of high-moisture forage silage. Summary of the Invention

[0005] The purpose of this invention is to provide an application of 4-pentenoic acid in the preparation of silage additives, which can target and inhibit the growth and reproduction of Clostridium in the silage system, reduce the abundance of harmful Clostridium, reduce protein degradation and ammonia nitrogen accumulation during silage, protect the nutrition of forage, and improve its feed value. At the same time, this additive is green and safe, has a low dosage, and controllable cost. It is suitable for large-scale forage silage production, supports direct silage of fresh, high-moisture forage without the need for wilting and water control, and simplifies the silage production process.

[0006] To achieve the above objectives, the present invention provides the application of 4-pentenoic acid in the preparation of silage additives, wherein the silage additives comprise 5% to 10% 4-pentenoic acid by mass percentage.

[0007] Preferably, the silage additive comprises, by weight percentage: 5%~10% 4-pentenoic acid; 10% dimethyl sulfoxide; 80%~85% water.

[0008] Preferably, the purity of 4-pentenoic acid is ≥98%. Dimethyl sulfoxide is used as a co-solvent to ensure uniform dispersion of 4-pentenoic acid in aqueous solution.

[0009] The preparation method of silage additives is as follows: Prepare a 10% (w / w) dimethyl sulfoxide aqueous solution according to the specified ratio, stir until homogeneous, add 4-pentenoic acid, and continue stirring until completely dissolved to obtain a homogeneous additive solution with a 4-pentenoic acid concentration of 50~100 mg / mL.

[0010] The steps for using silage additives are as follows: Add 0.1 mL of the silage additive per 1 g of fresh silage sample. Spray the silage additive evenly onto the surface of fresh forage chopped to 1-2 cm. Mix thoroughly and quickly pack into a silage pit or wrap in silage. Seal and allow to anaerobic ferment for more than 60 days before feeding.

[0011] Preferably, the silage additive is used to reduce the relative abundance of Clostridium species in the silage system while promoting the growth of beneficial lactic acid bacteria.

[0012] Preferably, the genus *Clostridium* in the narrow sense includes *Clostridium butyricum* (…). Clostridium tyrobutyricum Clostridium perfringens (old cellar) Clostridium sp.JN500901 The beneficial lactic acid bacteria include *Lactobacillus symbioticae* (…). Companilactobacillus paralimentarius Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and Lactobacillus brunelli ( Levilactobacillus brevis ).

[0013] Preferably, the silage additive is suitable for the direct silage of high-moisture, high-protein forage, including freshly cut alfalfa.

[0014] Preferably, the silage additive is used to inhibit protein degradation during the silage process, reduce dry matter loss, and improve the nutritional quality of silage.

[0015] Preferably, the system meets the following indicators after silage fermentation: The pH value is stable below 4.2, and the relative abundance of Clostridium is <1%. Based on dry matter, the acetic acid content is 10%~18%, the lactic acid content is 2%~10%, and the ratio of ammonia nitrogen to total nitrogen is ≤15%.

[0016] Therefore, the application of the above-mentioned 4-pentenoic acid in the preparation of silage additives in this invention has the following beneficial effects: (1) Targeted antibacterial and stable fermentation: The 4-pentenoic acid of the present invention can precisely act on the Clostridium species in the silage system, significantly inhibiting the growth of harmful Clostridium species such as Clostridium butyricum and Clostridium tumefaciens, so that the relative abundance of Clostridium species after fermentation is reduced to less than 1%; at the same time, it can promote the proliferation of beneficial lactic acid bacteria such as Lactobacillus symbioticis, Lactobacillus plantarum, and Lactobacillus buchneri, optimize the microbial community structure of silage, ensure the stable progress of anaerobic fermentation, and avoid spoilage.

[0017] (2) Quality Improvement and Nutrient Retention: The additive of this invention can effectively inhibit protein degradation and reduce ammonia nitrogen generation during silage. Ammonia nitrogen is a nitrogenous substance produced by the deamination reaction of forage protein after it is broken down into amino acids. Its content can directly reflect the level of protein degradation in the silage system and is positively correlated with the degree of protein degradation. After applying the additive of this invention, the ratio of ammonia nitrogen to total nitrogen in the silage system can be controlled within 15%, significantly reducing protein loss. At the same time, the additive can regulate the composition of fermentation acid. Based on dry matter, the acetic acid content reaches 10%~18%, the lactic acid content is 2%~10%, the pH of the system is stable below 4.2, the dry matter loss rate is reduced by more than 50% compared with the control group, and the nutrient retention rate of forage is greatly improved.

[0018] (3) Simplified process and suitable for scale: The additive of the present invention can support the direct silage of fresh high-moisture forage without the need for traditional field wilting and water control steps. It is not limited by weather conditions, reduces field operation costs and nutrient loss caused by leaf drop and secondary pollution, greatly simplifies the silage production process, and is suitable for large-scale and intensive forage silage production.

[0019] (4) Safe, economical, green and environmentally friendly: The 4-pentenoic acid of the present invention is a food-grade flavor precursor substance that is permitted to be used. It has no harmful residues, does not affect the palatability of silage, and has no negative impact on the health of ruminants and the environment. Moreover, the amount of additive used is low, the preparation process is simple, and the cost is controllable, which meets the development requirements of green feed production.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is a graph showing the influence of microbial composition on alfalfa silage fermentation for 30 days in Example 3 and Comparative Example 1 of the present invention. Figure 2 This is a graph showing the influence of microbial composition on alfalfa silage fermentation for 60 days in Example 3 and Comparative Example 1 of the present invention. Figure 3 This is a graph showing the changes in quality indicators during the alfalfa silage fermentation process of Example 3 and Comparative Example 1 of the present invention, where (a) is the dry matter loss rate, (b) is the pH, (c) is the lactic acid content, and (d) is the acetic acid content. Figure 4 This is a graph showing the change in ammonia nitrogen content during the alfalfa silage fermentation process of Example 3 and Comparative Example 1 of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] This invention sets up four treatment methods: Examples 1-3 and Comparative Example 1 (CK). Fresh alfalfa was used as the experimental material, harvested at the initial flowering stage and chopped to a length of 1-2 cm. Samples were taken after 30 days and 60 days of alfalfa silage fermentation and after opening to determine changes in microbial community types, and fermentation quality indicators during the fermentation process were monitored.

[0025] Example 1 An application of 4-pentenoic acid in the preparation of silage additives: an additive solution containing 5% 4-pentenoic acid, 10% dimethyl sulfoxide, and 85% deionized water by mass percentage is prepared. 0.1 mL of the additive solution is sprayed evenly on each 1 g of fresh alfalfa sample and mixed thoroughly.

[0026] After processing and mixing, the alfalfa samples were placed in polyethylene silage bags, vacuum sealed, and placed in a constant temperature and light-proof environment at 25°C for anaerobic fermentation for 60 days. After opening, samples were taken to determine the microbial community and fermentation quality indicators.

[0027] Example 2 An application of 4-pentenoic acid in the preparation of silage additives: an additive solution containing 8% 4-pentenoic acid, 10% dimethyl sulfoxide, and 82% deionized water by mass percentage is prepared. 0.1 mL of the additive solution is sprayed evenly on each 1 g of fresh alfalfa sample and mixed thoroughly.

[0028] After processing and mixing, the alfalfa samples were placed in polyethylene silage bags, vacuum sealed, and placed in a constant temperature and light-proof environment at 25°C for anaerobic fermentation for 60 days. After opening, samples were taken to determine the microbial community and fermentation quality indicators.

[0029] Example 3 An application of 4-pentenoic acid in the preparation of silage additives: an additive solution containing 10% 4-pentenoic acid, 10% dimethyl sulfoxide, and 80% deionized water was prepared by mass percentage. 0.1 mL of the additive solution was sprayed evenly on each 1 g of fresh alfalfa sample and mixed thoroughly.

[0030] After treatment and mixing, the alfalfa samples were placed in polyethylene silage bags, vacuum sealed, and placed in a constant temperature and light-proof environment at 25°C for anaerobic fermentation for 60 days. After opening, samples were taken to determine the microbial community and fermentation quality indicators, which were recorded as 4P.

[0031] Comparative Example 1 Spray 0.1 mL of 10% dimethyl sulfoxide aqueous solution (containing 10% dimethyl sulfoxide and 90% deionized water by mass percentage) evenly on each 1 g of fresh alfalfa sample, and mix thoroughly.

[0032] After treatment and mixing, the alfalfa samples were placed in polyethylene silage bags, vacuum sealed, and placed in a constant temperature and light-proof environment at 25℃ for anaerobic fermentation for 60 days, which was recorded as CK.

[0033] Experimental results: The fermentation quality indicators in Example 3 achieved the expected results: like Figure 1 and Figure 2 As shown, high-throughput sequencing technology was used to analyze the microbial community structure in silage. The results showed that after 30 days of fermentation, the CK group exhibited pre-rotation characteristics of "dominant miscellaneous bacteria, Clostridium initiation, and weak lactic acid bacteria." Miscellaneous bacteria maintained a high abundance, continuously consuming nutrients and accelerating protein degradation, leading to silage spoilage. The microbial community structure of the 4P group with added 4-pentenoic acid was optimized after 30 days, exhibiting benign fermentation characteristics of "dominant lactic acid bacteria, suppressed Clostridium, and significantly reduced miscellaneous bacteria." The abundance of spoilage bacteria was significantly reduced, with the vast majority suppressed to extremely low levels. The fermentation process and abundance of lactic acid bacteria were much higher than those of the control group.

[0034] After 60 days of fermentation, the Clostridium fermentation in the CK group continued to deteriorate. In the 4P group, the abundance of Clostridium butyricum and Clostridium tumefaciens decreased to below 0.5%, while the abundance of digestive symbiotic lactobacillus, Lactobacillus plantarum, and Lactobacillus brunelli increased to over 25%, 14%, and 20%, respectively, verifying the targeted and long-lasting antibacterial effect of 4-pentenoic acid and its stability in promoting lactic acid bacteria fermentation.

[0035] like Figure 3 and Figure 4As shown, compared to the control group (CK), the dry matter loss rate decreased by 50%, and the core nutrients such as carbohydrates and proteins in the forage were retained to the greatest extent, significantly improving the overall nutrient density and utilization efficiency of the silage. The pH value decreased to below 4.2, forming a stable acidic antibacterial environment that can inhibit the growth and reproduction of most harmful bacteria for a long time, ensuring the quality stability of the silage during long-term sealed storage. The ammonia nitrogen content decreased, and the ratio of ammonia nitrogen to total nitrogen was ≤15%, indicating that protein degradation was significantly inhibited during the silage process, and the crude protein nutrition of the forage was effectively retained, thus improving the feed value of the silage.

[0036] The lactic acid content is 2%~10% (dry matter basis), and the acetic acid content is 14%~18% (dry matter basis), with both acetic acid and lactic acid contents increasing. Sufficient lactic acid ensures rapid acid reduction and suppression of unwanted microorganisms in the early stages of fermentation. The high acetic acid content mainly comes from the metabolism of beneficial lactic acid bacteria such as *Lactobacillus brucellosis*, which not only synergistically enhances the antibacterial effect of lactic acid but also significantly improves the aerobic stability after silage is opened, reducing secondary fermentation losses caused by air entry during the harvesting stage. This makes it suitable for large-scale ranches with multiple harvesting operations.

[0037] In summary, the study confirms that 4-pentenoic acid has a significant effect on Clostridium spp. in silage systems. Clostridium sensu stricto It has a significant targeted inhibitory effect, which can reduce the abundance of Clostridium to less than 1% and reduce the accumulation of ammonia nitrogen. The less ammonia nitrogen is generated, the lower the degree of protein degradation. This indicates that silage additives can inhibit protein degradation during the silage process and have no negative impact on the growth of beneficial lactic acid bacteria, or even promote it. It can reduce dry matter loss and improve the nutritional quality of silage.

[0038] Therefore, this invention utilizes the application of the aforementioned 4-pentenoic acid in the preparation of silage additives to target and inhibit the growth and reproduction of Clostridium in the silage system, reduce the abundance of harmful Clostridium, decrease protein degradation and ammonia nitrogen accumulation during silage, protect forage nutrition, and enhance feed value. At the same time, this additive is green and safe, requires low dosage, and has controllable costs. It is suitable for large-scale forage silage production, supports direct silage of fresh, high-moisture forage without the need for wilting and water control, and simplifies the silage production process.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

Application of 1,4-pentenoic acid in the preparation of silage additives that inhibit Clostridium fermentation.

2. The application of 4-pentenoic acid according to claim 1 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that, The silage additive includes 5% to 10% 4-pentenoic acid by weight percentage.

3. The application of 4-pentenoic acid according to claim 2 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that: The purity of the 4-pentenoic acid is ≥98%.

4. The application of 4-pentenoic acid according to claim 3 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that, The silage additives include, by weight percentage: 5%~10% 4-pentenoic acid; 10% dimethyl sulfoxide; 80%~85% water.

5. The application of 4-pentenoic acid according to claim 1 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that: The silage additive is used to reduce the relative abundance of Clostridium species in the silage system, while promoting the growth of beneficial lactic acid bacteria.

6. The application of 4-pentenoic acid according to claim 5 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that, The genus *Clostridium* in the narrow sense includes *Clostridium butyricum* and *Clostridium laurylonitrile*, and the beneficial lactic acid bacteria include *Lactobacillus symbioticis*, *Lactobacillus plantarum*, and *Lactobacillus bryonicus*.

7. The application of 4-pentenoic acid according to claim 1 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that: The silage additive is suitable for the direct silage of high-moisture, high-protein forage, including freshly cut alfalfa.

8. The application of 4-pentenoic acid according to claim 1 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that: Add 0.1 mL of the silage additive per 1 g of fresh silage sample, spray the silage additive evenly onto the surface of the chopped forage, mix well, and then seal for anaerobic fermentation for more than 60 days.

9. The application of 4-pentenoic acid according to claim 1 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that: The silage additive is used to inhibit protein degradation, reduce dry matter loss, and improve the nutritional quality of silage during the silage process.

10. The application of 4-pentenoic acid according to claim 1 in the preparation of silage additives that inhibit Clostridium fermentation, characterized in that, After silage fermentation is complete, the system must meet the following indicators: The pH value is stable below 4.2, and the relative abundance of Clostridium is <1%. Based on dry matter, the acetic acid content is 10%~18%, the lactic acid content is 2%~10%, and the ratio of ammonia nitrogen to total nitrogen is ≤15%.

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