A silage fermentation additive, its preparation method and application

Abstract

The application provides a silage fermentation additive and a preparation method and application thereof, and belongs to the technical field of silage feed. The silage fermentation additive comprises allicin and a compound microbial inoculum, and the compound microbial inoculum comprises lactobacillus acidophilus, lactobacillus plantarum and lactobacillus buchneri. The allicin is compounded with the compound microbial inoculum with a specific composition, and is used for preparing silage feed, so that the aerobic stability of the silage feed can be significantly improved, the growth of spoilage bacteria such as molds and yeasts can be effectively inhibited, the content of various mold toxins such as aflatoxin, zearalenone, T-2 toxin and vomitoxin can be reduced, the fermentation quality can be improved, the protein decomposition can be reduced, and the retention rate of dry matter and water-soluble total sugar can be improved. The application can completely replace chemical preservatives such as potassium sorbate and ammonium propionate, has the characteristics of naturalness, safety and high efficiency, and is suitable for the large-scale production of silage corn, fresh corn straw and other silage feeds.

Description

Technical Field

[0001] This invention belongs to the field of silage preparation technology, specifically relating to a silage fermentation additive containing allicin, its preparation method, and its application. Background Technology

[0002] Silage is an effective way to preserve the nutritional value of green forage. Through anaerobic fermentation by lactic acid bacteria, lactic acid is produced, lowering the pH value and inhibiting the growth of harmful microorganisms, thus achieving long-term preservation of the feed. However, in actual production, the silage process involves competitive growth of harmful microorganisms, affecting fermentation quality. Furthermore, silage is prone to secondary fermentation after being exposed to air, leading to the proliferation of molds and yeasts. This not only causes the feed to mold and spoil but also produces large amounts of mycotoxins, severely impacting fermentation quality. Traditionally, to address these problems, chemical preservatives such as ammonium propionate and potassium sorbate are added to inhibit mold and aerobic bacteria, reduce nutrient loss, improve the aerobic stability of the feed, and extend its shelf life. However, the improper use of chemical additives can cause environmental problems and affect animal health. With the promotion of green and ecological farming concepts, the development of natural, safe, and efficient silage additives has become an industry demand.

[0003] Allicin is the main active ingredient in garlic, possessing broad-spectrum antibacterial and antifungal properties. Current research has explored the use of allicin or lactic acid bacteria as fermentation additives in silage, but the effects of a reasonable combination of these two ingredients on inhibiting mycotoxins in silage, particularly specific application schemes for bulk raw materials such as silage corn, have not yet been reported. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a silage fermentation additive that, by combining allicin with a specific compound microbial agent, can effectively inhibit the production of mycotoxins during silage fermentation and ensure fermentation quality.

[0005] This invention provides a silage fermentation additive, comprising allicin and a compound microbial agent; the mass ratio of allicin to the compound microbial agent is (20-100):(1-2); the compound microbial agent comprises Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli; the viable count ratio of Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli is (0.8-1.2):(1.6-2.4):(1.6-2.4).

[0006] Preferably, the viable count of Lactobacillus acidophilus in the compound microbial agent is ≥1×10⁻⁶. 7 CFU / g, viable count of *Lactobacillus plantarum* ≥ 2 × 10⁻⁶ 7 CFU / g, viable count of Lactobacillus bruneri ≥2×10 7 CFU / g.

[0007] This invention provides an application of a silage fermentation additive in the preparation of silage.

[0008] This invention provides an application of a silage fermentation additive in any of the following silage fermentation processes: ① Improve aerobic stability; ② Reduce mycotoxin content; ③ Improve the nutritional level of fermented feed; ④ Improve fermentation quality.

[0009] Preferably, the mass of the silage fermentation additive is 0.01% to 0.05% of the fresh weight of the silage raw material.

[0010] Preferably, the mycotoxin includes at least one of aflatoxin B1, zearalenone, T-2 toxin, vomitoxin, deoxynivalenol, fumonisin B1, and fumonisin B2.

[0011] The present invention provides a fermentation method for silage, comprising anaerobic fermentation of silage raw materials and the silage fermentation additive.

[0012] Preferably, the compound microbial agent in the silage fermentation additive accounts for 0.0005% to 0.001% of the fresh weight of the silage raw material, and the allicin accounts for 0.01% to 0.05% of the fresh weight of the silage raw material.

[0013] Preferably, the silage raw material includes crop straw.

[0014] Preferably, during the anaerobic fermentation, the initial moisture content of the fermentation system is 55% to 65%. The anaerobic fermentation is carried out under light-protected conditions; The temperature for the anaerobic fermentation is 20℃~35℃; The anaerobic fermentation time is 40-50 days.

[0015] This invention provides a silage fermentation additive, comprising allicin and a compound microbial agent; the mass ratio of allicin to the compound microbial agent is (20-100):(1-2); the compound microbial agent comprises *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Lactobacillus brunelli*; the viable count ratio of *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Lactobacillus brunelli* is (0.8-1.2):(1.6-2.4):(1.6-2.4). The combined use of allicin and the compound fermentation agent in this invention is beneficial for inhibiting the generation and metabolism of miscellaneous bacteria in the silage fermentation system. Allicin, the main active ingredient in garlic, has broad-spectrum antibacterial and antifungal properties, and has a good inhibitory effect on putrefactive bacteria such as molds and yeasts, without affecting the reproduction and function of the three lactobacilli in the compound microbial agent; the compound fermentation agent can dominate the silage fermentation process, rapidly producing acid and lowering the pH value; the combined use of the two can achieve a natural replacement for chemical preservatives, while simultaneously possessing the dual functions of antibacterial and fermentation-promoting effects. Based on systematic experimental research, this invention screened the optimal ratio of allicin to a specific compound microbial agent, providing a natural silage additive that can effectively inhibit mold, improve aerobic stability, and enhance fermentation quality. Detailed Implementation

[0016] This invention provides a silage fermentation additive, comprising allicin and a compound microbial agent; the mass ratio of allicin to the compound microbial agent is (20-100):(1-2). In this invention, the preferred mass ratio of allicin to the compound microbial agent is (30-80):(1-2), but it can be (30-50):1 or 40:1. While the compound microbial agent alone can produce a certain antibacterial effect, its ability to inhibit molds and yeasts that invade after the silage is opened is limited. Allicin's broad-spectrum antibacterial properties can effectively kill airborne spoilage bacteria, and the synergistic effect of the two increases aerobic stability by 35%-45%, significantly extending the storage time of feed after the silage is opened.

[0017] In this invention, the composite microbial agent includes *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Lactobacillus bruneri*. The preferred ratio of viable counts of *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Lactobacillus bruneri* is (0.8–1.2):(1.6–2.4):(1.6–2.4), more preferably (1–1.2):(2–2.4):(2–2.4), and can be 1:2:2. The viable count of *Lactobacillus acidophilus* in the composite microbial agent is ≥1×10⁻⁶. 7 CFU / g can be 2×10 7 CFU / g, can also be 5×10 7 CFU / g can also be 1×10 8 CFU / g. The viable count of the *Lactobacillus plantarum* is ≥2×10⁻⁶. 7 CFU / g can be 5×107 CFU / g, can also be 8×10 7 CFU / g can also be 1×10 8 CFU / g. The viable count of the *Lactobacillus bruneri* is ≥2×10⁻⁶. 7 CFU / g can be 5×10 7 CFU / g, can also be 8×10 7 CFU / g can also be 1×10 8 CFU / g. In this embodiment, the compound microbial agent was purchased from Shandong Yihao Biotechnology Co., Ltd. The compound microbial agent can rapidly convert sugars in silage into a large amount of lactic acid, quickly lower the pH value, and create an acidic environment unfavorable to the growth of putrefactive bacteria, thereby significantly improving the aerobic stability of silage, preventing feed from heating and spoiling, and reducing the content of acetic acid and propionic acid, possessing both antibacterial effect and natural safety. The purity of the allicin is ≥25%. In this embodiment, the microbial agent was purchased from Weifang Zhongtian Feed Technology Co., Ltd. The allicin can effectively increase the dry matter, crude protein, and water-soluble total sugar content during silage fermentation, reduce the neutral detergent fiber content, and retain the nutritional components intact; it can also significantly reduce the content of various mycotoxins in silage corn, with effects comparable to chemical preservatives.

[0018] This invention selects allicin and a compound microbial agent as its main components. Allicin is the main active ingredient in garlic, possessing broad-spectrum antibacterial and antifungal properties, and exhibiting good inhibitory effects on spoilage bacteria such as molds and yeasts. The compound microbial agent can dominate the silage fermentation process, rapidly producing acid and lowering the pH value. The combined use of these two ingredients effectively inhibits mold, improves aerobic stability, and enhances fermentation quality, achieving a natural replacement for chemical preservatives while simultaneously possessing the dual functions of antibacterial and fermentation-promoting effects.

[0019] In this invention, the method for preparing the silage fermentation additive preferably involves packaging allicin and the compound microbial agent separately, and mixing them with the silage raw materials according to the mass ratio mentioned above during use, so as to avoid the bactericidal effect caused by long-term contact between allicin and the compound microbial agent; at the same time, after being added to the silage raw materials, allicin is diluted to an extremely low concentration, mainly inhibiting spoilage bacteria, while having no significant effect on the fermentation activity of the dominant strain of lactic acid bacteria, thereby achieving synergistic effect.

[0020] This invention provides the application of the silage fermentation additive in the preparation of silage.

[0021] The silage fermentation additive provided by this invention can effectively improve the aerobic stability of silage, inhibit the content of miscellaneous bacteria and their metabolic toxins, extend the storage time and improve the quality of silage. It can completely replace the role of chemical preservatives such as ammonium propionate and potassium sorbate in silage fermentation, avoid environmental problems and animal health risks caused by improper use of chemical additives, and conform to the concept of green ecological farming.

[0022] This invention provides the application of the silage fermentation additive in any of the following silage fermentation processes: ① Improve aerobic stability; ② Reduce mycotoxin content; ③ Improve the nutritional level of fermented feed; ④ Improve fermentation quality.

[0023] In this invention, the improvement in aerobic stability refers to the time required for the temperature of the silage to exceed the ambient temperature by 2°C after opening the bag, preferably ≥184 hours, and can be 222~238 hours. In the embodiments of this invention, after adding the silage fermentation additive, the aerobic stability of fermented silage corn and fermented fresh corn stalks is significantly improved compared with the blank control group. Specifically, after adding allicin at a mass percentage of 0.03% of the fresh weight of the silage raw material, the aerobic stability of the fermented silage is comparable to that of the positive control group using potassium sorbate and ammonium propionate. After adding allicin at a mass percentage of 0.05% of the fresh weight of the silage raw material, the aerobic stability of the fermented silage is further improved.

[0024] In this invention, the mycotoxins preferably include at least one of aflatoxin B1, zearalenone, T-2 toxin, vomitoxin, deoxynivalenol, fumonisin B1, and fumonisin B2. In embodiments of this invention, after adding the silage fermentation additive, compared to the blank control group, the contents of aflatoxin B1, zearalenone, T-2 toxin, vomitoxin, deoxynivalenol, and fumonisin B2 in fermented silage corn and fermented fresh corn stalks all decreased. The inhibition rates against aflatoxin B1, T-2 toxin, vomitoxin, fumonisin B1, and fumonisin B2 all exceeded 70%, demonstrating excellent effects; the inhibition rate against zearalenone was approximately 32%, still showing a significant effect. In this invention, after adding allicin at 0.03% or 0.05% of the fresh weight of the silage raw material, the mycotoxin content in the fermented silage feed was comparable to the positive control group using potassium sorbate and ammonium propionate.

[0025] In this invention, improving the nutritional level of fermented feed preferably includes increasing the content of at least one component among the dry matter, starch, and total water-soluble sugars in fermented silage, and decreasing the content of neutral detergent fiber and / or acid detergent fiber in fermented silage, ensuring balanced and comprehensive nutrition. In the embodiments of this invention, after adding the silage fermentation additive, the dry matter, starch, and total water-soluble sugars of fermented silage corn and fermented fresh corn stalks were all increased compared to the blank control group, while the content of neutral detergent fiber and acid detergent fiber decreased slightly; wherein, after adding allicin at a mass ratio of 0.01%, 0.03%, and 0.05% of the fresh weight of the silage raw materials, the nutritional level indicators of the fermented silage feed were comparable to those of the positive control group using potassium sorbate and ammonium propionate.

[0026] In this invention, the improvement in fermentation quality is preferably achieved by using silage with a low pH, increasing lactic acid content, and decreasing acetic acid and ammonium nitrogen content, while butyric acid content is undetectable. In the embodiments of this invention, after adding the silage fermentation additive, the pH, ammonium nitrogen, and acetic acid content of fermented silage corn and fermented fresh corn stalks decreased compared to the blank control group, while the lactic acid content increased slightly. Compared to the positive control group using potassium sorbate and ammonium propionate, the pH, ammonium nitrogen, and propionic acid content of fermented silage corn and fermented fresh corn stalks decreased. Among them, the experimental groups with added allicin at a mass ratio of 0.01% and 0.03% of the fresh weight of the silage raw materials were significantly better than the positive control group in reducing ammonium nitrogen and propionic acid content.

[0027] This invention provides a fermentation method for silage, comprising anaerobic fermentation of silage raw materials and silage fermentation additives.

[0028] In this invention, the compound microbial agent in the silage fermentation additive accounts for 0.0005% to 0.001% of the fresh weight of the silage raw material, specifically 0.0008% to 0.001% or 0.001%. Allicin accounts for 0.01% to 0.05% of the silage raw material, specifically 0.02%, 0.03%, and 0.04%.

[0029] In this invention, the silage raw material is preferably crop straw, which can be silage corn straw or sweet corn straw, specifically, it can be silage corn at the waxy maturity stage or corn straw remaining after harvesting sweet corn. The silage raw material is processed by cutting and crushing it to 2-3 cm after harvesting. The silage fermentation additive of this invention has achieved good results in improving the nutritional components and fermentation quality of fermented silage corn and sweet corn straw, reducing mycotoxin content, and improving aerobic stability. The additive of this invention is not only suitable for special silage corn, but can also efficiently process sweet corn straw, an important agricultural by-product, and transform it into high-quality silage feed with high safety and high nutritional value.

[0030] In this invention, the anaerobic fermentation is preferably carried out by mixing the silage raw materials and silage fermentation additives evenly and then sealing them in silage fermentation bags. The anaerobic fermentation can inhibit the growth of aerobic putrefactive bacteria such as molds and yeasts, while providing suitable growth conditions for lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli, allowing them to quickly become the dominant bacterial group and dominate the fermentation process.

[0031] In this invention, during anaerobic fermentation, the initial moisture content of the fermentation system is 55%–65%; the anaerobic fermentation is carried out under light-protected conditions; the temperature of the anaerobic fermentation is 20°C–35°C; and the fermentation time is 40–50 days. In this invention, after the silage raw materials and silage fermentation additives are mixed evenly, the initial moisture content of the fermentation system is adjusted to 55%–65%. Specifically, the moisture content can be adjusted to 60%. A moisture content of 55%–65% is the optimal range for the survival and metabolism of lactic acid bacteria. Under these moisture conditions, lactic acid bacteria can rapidly utilize soluble sugars to produce a large amount of lactic acid, quickly lowering the pH value and inhibiting the growth of putrefactive bacteria. Too low a moisture content will inhibit the metabolism of lactic acid bacteria, leading to slow fermentation initiation; too high a moisture content will dilute the concentration of the fermentation substrate, similarly affecting acid production efficiency. In this invention, the silage raw materials and silage fermentation additives are mixed evenly, then placed into silage fermentation bags and sealed. Fermentation is then carried out at room temperature, which can be 20℃~35℃ or 25℃~35℃. This temperature range is the optimal growth temperature for most lactic acid bacteria. Within this range, lactic acid bacteria can rapidly utilize the soluble sugars in the silage raw materials for vigorous anaerobic fermentation, producing a large amount of lactic acid. This causes the pH value to drop rapidly, effectively inhibiting the reproduction of harmful microorganisms such as Escherichia coli, butyric acid bacteria, and molds.

[0032] In this embodiment of the invention, combinations of other common fermentation strains and allicin were used as a control group in silage fermentation experiments. The results showed that, under the same garlic addition conditions, the composite microbial agent formed by *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Lactobacillus bruneri* was superior to the control group in terms of dry matter, crude protein, water-soluble total sugar, and neutral detergent fiber degradation in the fermented silage. Simultaneously, the silage prepared by the composite microbial agent combined with allicin had the lowest pH value and acetic acid content, the highest lactic acid content, and the strongest inhibitory effect on aflatoxin B1, zearalenone, T-2 toxin, vomitoxin, and fumonisin, exhibiting the highest aerobic stability. This indicates that the combination of *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Lactobacillus bruneri* used in this invention is significantly superior to other strains containing *Bacillus subtilis* or yeast in terms of nutrient retention, fermentation quality, mycotoxin inhibition, and aerobic stability. It can effectively retain the nutrients of fermented silage, prevent feed spoilage, and inhibit secondary fermentation after opening the silo.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a silage fermentation additive, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the methods used in the embodiments, comparative examples and application examples of this invention are conventional methods in the art.

[0035] Example 1 I. A method for fermenting silage using silage fermentation additives Silage corn is harvested at the waxy maturity stage and crushed to about 2.0cm for use as silage raw material.

[0036] A compound microbial agent (purchased from Shandong Yihao Biotechnology Co., Ltd.) was prepared by mixing Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli in a live count ratio of 1:2:2, and its addition mass was 0.001% of the fresh weight of the silage raw material. Allicin (purchased from Weifang Zhongtian Feed Technology Co., Ltd., purity ≥25%) was added in different proportions.

[0037] Six treatment groups were set up, with three replicates in each group: blank control group (CK), compound bacteria group (CLAB), compound bacteria + 0.01% allicin group (CLAB + AL0.01, allicin accounting for 0.01% of the fresh weight of silage raw material), compound bacteria + 0.03% allicin group (CLAB + AL0.03), compound bacteria + 0.05% allicin group (CLAB + AL0.05), and potassium sorbate + ammonium propionate group (PS + AP, accounting for 0.1% of the fresh weight of silage raw material).

[0038] The compound microbial inoculant and allicin were thoroughly mixed with the silage raw materials, and the moisture content was adjusted to about 60%. The mixture was then placed into silage fermentation bags, sealed, and fermented in a dark room. Samples were taken after 45 days of fermentation to determine the nutritional components, fermentation quality, and mycotoxin content. An aerobic stability test was conducted after 60 days of fermentation.

[0039] II. Fermentation Index Determination 1. Nutritional composition determination and results Samples were taken from the silage on day 45 of fermentation. The samples were dried in a 65℃ oven to constant weight for calculating dry matter content (referencing GB / T 6435-2014). The contents of crude protein (GB / T 6432-2018), neutral detergent fiber (GB / T 20806-2022), acid detergent fiber (NY / T 1459-2022), crude ash (GB / T 6438-2007), crude fat (GB / T 6433-2025), total water-soluble sugars (NY / T 3030-2016), starch (GB / T 20194-2018), calcium (GB / T 6436-2018), and phosphorus (GB / T 6437-2018) were also determined. The results are shown in Table 1.

[0040] Compared with the control group, the dry matter, crude protein, and water-soluble total sugar content of silage corn increased to varying degrees in all groups, including those with compound microbial agents and those with a combination of compound microbial agents and allicin, while the neutral detergent fiber content decreased significantly. Among them, the CLAB+AL0.01 group had the highest dry matter content, reaching 34.03±0.67%; the crude protein content of the CLAB+AL0.03 group was 8.71±0.16%, significantly better than the control group and the single compound microbial agent group; the water-soluble total sugar content was also best in the CLAB+AL0.03 group, at 453.32±12.5 mg / g; the neutral detergent fiber content decreased with increasing allicin addition, with the CLAB+AL0.05 group and the PS+AP group showing similar results, at 30.97±2.1% and 30.4±1.01% respectively, both significantly lower than the control group. There were no significant differences in crude fat, crude ash, starch, calcium, phosphorus, and other indicators among the groups, indicating that the nutritional components were well preserved.

[0041] Table 1. Nutritional Analysis of Fermented Feed

[0042] Note: Data in the same row with the same letter above the heading indicates that the difference is not significant. P >0.05), different lowercase letters indicate significant differences ( P <0.05), the same applies below.

[0043] 2. Fermentation quality determination and results A 20 g sample of silage was collected, placed in a beaker, and 180 mL of sterile water was added. The sample was then shaken in a 4℃ constant temperature shaking incubator for 24 hours. After filtration with quantitative filter paper, the pH was determined according to the pH determination method for silage (SCWT / QM03-L-141), the ammonium nitrogen content was determined according to the ammonium nitrogen content determination method for fertilizers (NY / T 1116-2014), and the contents of lactic acid, acetic acid, propionic acid, and butyric acid were determined according to the detailed rules for the determination of organic acids in feed (SCWT / QM03-L-134). The test results are shown in Table 2.

[0044] The pH values ​​of all added groups were lower than those of the control group, with the CLAB+AL0.05 group having the lowest pH at 3.79±0.08, indicating good fermentation acid production. Regarding ammonium nitrogen content, the compound microbial agent group and the compound microbial agent combined with allicin group were significantly lower than the PS+AP group, indicating less protein decomposition and better fermentation quality. Organic acid detection results showed that butyric acid was not detected in any group, indicating no risk of spoilage fermentation; lactic acid content showed no significant difference among groups; acetic acid content decreased significantly with increasing allicin addition, with the CLAB+AL0.03 group having the lowest acetic acid content at 1.05±0.09%; propionic acid content was moderate, combining antibacterial effect with natural safety.

[0045] Table 2. Fermentation Quality Analysis of Fermented Feed

[0046] 3. Mycotoxin Determination The contents of aflatoxin, zearalenone, and T-2 toxin in feed were determined according to the liquid chromatography-tandem mass spectrometry method (NY / T 2071-2011); the contents of fumonisin B1 and fumonisin B2 in feed were determined according to the method for determining fumonisin in feed (NY / T 1970-2010); and the contents of ochratoxin A in feed were determined according to the immunoaffinity column purification-high performance liquid chromatography method (GB / T 30957-2014). The detection results are shown in Table 3.

[0047] The combination of compound microbial inoculants and allicin significantly reduced the content of various mycotoxins in silage corn. Specifically, the aflatoxin B1 content in the CLAB+AL0.03 and CLAB+AL0.05 groups was 3.33±1.76 μg / kg and 3.07±1.76 μg / kg, respectively, comparable to the PS+AP group. Zearalenone was lowest in the CLAB+AL0.05 group (40.3±0.64 μg / kg); T-2 toxin decreased to 2.16±0.15 μg / kg in the CLAB+AL0.05 group; and vomitoxin was 13.34±2.19 μg / kg in the CLAB+AL0.05 group. Furthermore, the contents of deoxynivalenol, fumonisin B1, and fumonisin B2 all decreased significantly with increasing allicin addition.

[0048] Table 3 Mycotoxin Analysis of Fermented Feed

[0049] 4. Aerobic stability analysis On day 60 of fermentation, the bag was opened and a thermometer was inserted into the center of the fermentation bag. The temperature was measured and recorded every 2 hours until the sample temperature exceeded room temperature by 2°C. The results are shown in Table 4.

[0050] The aerobic stability of silage increased in a gradient with increasing allicin addition. Specifically, the control group had a stability of only 97 hours, the compound microbial agent group had 164 hours, the CLAB+AL0.01 group had 184 hours, the CLAB+AL0.03 group had 222 hours, and the CLAB+AL0.05 group had 238 hours. The aerobic stability of the CLAB+AL0.03 and CLAB+AL0.05 groups was not significantly different from that of the PS+AP group (222 hours), and both were significantly better than the control group and the single compound microbial agent group, indicating that they can effectively inhibit secondary fermentation after opening the silo.

[0051] Table 4. Aerobic stability analysis of fermented feed

[0052] Based on four core indicators—comprehensive nutritional level, fermentation quality, mycotoxin inhibition effect, and aerobic stability—the combination of compound microbial agent and allicin in this invention is judged as follows: the dry matter, crude protein, and water-soluble total sugar content are all within the optimal range; the degradation effect of neutral detergent fiber is significant; there are no nutritional deficiencies; and the overall feed value is high. The pH value is relatively low; the ammonium nitrogen and acetic acid content are both at low levels; butyric acid was not detected; and the fermentation efficiency is high and the safety is strong.

[0053] Example 2 A method for preparing silage by fermenting fresh corn stalks using silage fermentation additives. I. The silage fermentation steps are the same as in Example 1, except that the silage raw material is replaced with fresh corn stalks.

[0054] Three treatment groups were set up: a blank control group (CK), a compound bacteria + 0.03% allicin group (CLAB + AL0.03, allicin accounting for 0.03% of the fresh weight of the silage raw material), and a potassium sorbate + ammonium propionate group (PS + AP). Each group had three replicates.

[0055] The fermentation products of silage corn stalks were analyzed for nutritional components, fermentation quality, mycotoxins, and aerobic stability. The detection and analysis methods were the same as in Example 1.

[0056] II. Determination and Results of Fermentation Indicators 1. Nutritional composition analysis The results of nutritional composition analysis are shown in Table 5. Compared with the blank control group, the dry matter, crude protein, and water-soluble total sugar contents of the CLAB+AL0.03 group and the PS+AP group were increased to varying degrees, while the contents of neutral detergent fiber and acid detergent fiber were decreased. Specifically, the dry matter content of the CLAB+AL0.03 group was 22.03±0.63%, the crude protein content was 10.15±0.29%, the water-soluble total sugar content was 185.48±16.7 mg / g, and the starch content was 77.67±4.68 g / kg, all of which were better than the control group and the chemical preservative group; the neutral detergent fiber content was 51.63±1.54%, and the acid detergent fiber content was 30.77±0.78%, which were not significantly different from the blank control group. There were no significant differences in crude fat, crude ash, calcium, phosphorus, and other indicators among the groups, and the nutritional components were well preserved.

[0057] Table 5. Nutritional composition analysis results

[0058] 2. Fermentation quality analysis The fermentation quality analysis results are shown in Table 6. The pH values ​​of all added groups were lower than those of the control group, with the CLAB+AL0.03 group having the lowest pH at 3.73±0.01, indicating good acid production during fermentation. Regarding ammonium nitrogen content, the compound bacteria + 0.03% allicin group had an ammonium nitrogen content of 0.14±0.01%, which was not significantly different from the control group (0.15±0.01%). However, the PS+AP group had a significantly lower ammonium nitrogen content (0.11±0.01%) than both groups, indicating that the chemical preservative group produced less protein decomposition. Butyric acid was not detected in any group, indicating no risk of spoilage or fermentation. The lactic acid content was highest in the CLAB+AL0.03 group, at 15.17±0.37%, which was significantly better than the control group (14.6±0.62%) and the chemical preservative group (14.5±0.28%). As for the acetic acid content, the compound bacteria + 0.03% allicin group and the PS+AP group were both 1.54%, which was significantly lower than the control group (1.91%), indicating that adding allicin or chemical preservatives can effectively reduce acetic acid accumulation.

[0059] Table 6. Fermentation quality analysis results

[0060] 3. Mycotoxin Analysis The results of mycotoxin analysis are shown in Table 7. The CLAB+AL0.03 group significantly reduced the content of multiple mycotoxins in fresh corn stalks. Compared with the blank control group, the levels of aflatoxin B1 in the CLAB+AL0.03 group decreased from 14.82±0.83 μg / kg to 3.65±1.35 μg / kg, zearalenone from 59.91±1.21 μg / kg to 39.47±1.2 μg / kg, T-2 toxin from 10.7±0.32 μg / kg to 3.24±1.03 μg / kg, vomitoxin from 52.69±5.85 μg / kg to 14.87±2.41 μg / kg, fumonisin B1 from 0.20±0.04 mg / kg to 0.04±0.01 mg / kg, and fumonisin B2 from 0.17±0.04 mg / kg to 0.01±0.01 mg / kg. The reduction effect was comparable to or better than that in the PS+AP group.

[0061] Table 7. Results of Mycotoxin Analysis

[0062] 4. Aerobic stability analysis The results of the aerobic stability analysis are shown in Table 8. The aerobic stability of silage significantly improved with the use of additives. The control group had a stability of only 89 hours, the CLAB+AL0.03 group reached 186 hours, and the PS+AP group reached 184 hours. There was no significant difference between the two groups, and all were far superior to the control group, indicating that the additives of this invention can effectively inhibit secondary fermentation of fresh corn stalk silage after opening the silo.

[0063] Table 8 Results of Aerobic Stability Analysis

[0064] In summary, the silage fermentation additive of this invention has achieved good results in improving the nutritional composition and fermentation quality of fresh corn stalks during silage fermentation, reducing mycotoxin content, and enhancing aerobic stability. This demonstrates that the additive is not only suitable for silage corn but can also efficiently process fresh corn stalks, an important agricultural byproduct. It is particularly effective in increasing crude protein content, promoting lactic acid fermentation, and inhibiting mycotoxins, transforming corn stalks into high-safety, high-nutritional-value, high-quality silage feed, and has significant potential for widespread application.

[0065] Comparative Example 1 The silage raw materials and fermentation steps are the same as in Example 1, except that the compound microbial agent in the silage fermentation additive is replaced with other combinations of microorganisms.

[0066] Six treatment groups were set up: a blank control group, a compound microbial agent group (Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus bruneri + 0.03% allicin, with allicin accounting for 0.03% of the fresh weight of the silage raw material), compound microbial agent group A (Lactobacillus plantarum, Bacillus subtilis, and Lactobacillus acidophilus + 0.03% allicin), compound microbial agent group B (Bacillus subtilis + Lactobacillus bruneri and Lactobacillus acidophilus + 0.03% allicin), compound microbial agent group C (Lactobacillus plantarum + Lactobacillus bruneri and yeast + 0.03% allicin), and compound microbial agent group D (Lactobacillus plantarum, Bacillus subtilis, and yeast + 0.03% allicin). Each group had three replicates.

[0067] The components of the compound microbial inoculant group were the same as those of the CLAB+AL0.03 group in Example 1. The viable cell ratio of each species in compound microbial inoculant group A was *Lactobacillus plantarum*: *Bacillus subtilis*: *Lactobacillus acidophilus* = 2:2:1; the viable cell ratio of each species in compound microbial inoculant group B was *Bacillus subtilis*: *Lactobacillus buchneri*: *Lactobacillus acidophilus* = 2:2:1; the viable cell ratio of each species in compound microbial inoculant group C was *Lactobacillus plantarum*: *Lactobacillus buchneri*: *Yeast* = 2:2:1; and the viable cell ratio of each species in compound microbial inoculant group D was *Lactobacillus plantarum*: *Bacillus subtilis*: *Yeast* = 2:2:2. The weight of the inoculant in each experimental group accounted for 0.001% of the fresh weight of the silage raw material.

[0068] II. Fermentation Indicators Detection and Results According to the detection method in Example 1, the nutritional components, fermentation quality, mycotoxins and aerobic stability of fermented silage corn were determined, and the test results are shown in Tables 9 to 12.

[0069] 1. Nutritional components The composite microbial agent group described in this invention is superior to other microbial combinations in terms of dry matter, crude protein, total water-soluble sugar, and degradation of neutral detergent fibers, with values ​​of 32.97%, 8.71%, 453.32 mg / g, and 33.4%, respectively. Other combinations have dry matter content of 31.03%–31.73%, crude protein content of 6.67%–8.08%, and total water-soluble sugar content of 242.69%–391.5 mg / g, all lower than the combination described in this invention.

[0070] Table 9 Nutritional Composition Analysis

[0071] 2. Fermentation quality analysis The compound microbial agent combination of this invention has the lowest pH value (3.85%), the highest lactic acid content (8.00%), the lowest acetic acid content (1.05%), and no butyric acid was detected. Other combinations all had pH values ​​higher than 4.05, lactic acid contents ranging from 6.87% to 7.17%, and significantly lower fermentation efficiency than the combination of this invention.

[0072] Table 10 Fermentation Quality Analysis

[0073] 3. Mycotoxin Inhibition Analysis The combination of strains in this invention exhibits the strongest inhibitory effect against aflatoxin B1, zearalenone, T-2 toxin, vomitoxin, and fumonisin. For example, the aflatoxin B1 concentration in the combination of this invention is 3.33 μg / kg, while other combinations range from 5.12 to 10.53 μg / kg; the T-2 toxin concentration in the combination of this invention is 2.97 μg / kg, while other combinations range from 5.57 to 6.07 μg / kg; and the vomitoxin concentration in the combination of this invention is 14.87 μg / kg, while other combinations range from 19.55 to 22.55 μg / kg. This indicates that the synergistic antibacterial effect of the strain combination of this invention combined with allicin is the most significant.

[0074] Table 11 Mycotoxin Analysis of Fermented Feed

[0075] 4. Aerobic stability analysis The combination of the present invention exhibits the highest aerobic stability, reaching 222 hours, far superior to other combinations. Specifically, the aerobic stability of compound microbial agent group A is 134 hours, compound microbial agent group B is 156 hours, compound microbial agent group C is 145 hours, and compound microbial agent group D is 153 hours. This indicates that the combination of the present invention can most effectively inhibit secondary fermentation after the fermentation pit is opened.

[0076] Table 12 Aerobic stability analysis

[0077] In summary, under the same allicin addition conditions, the combination of Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli selected in this invention is significantly superior to other strains containing Bacillus subtilis or yeast in terms of nutrient retention, fermentation quality, mycotoxin inhibition, and aerobic stability. This indicates that the combination of these three specific lactic acid bacteria is the key to achieving excellent silage results.

[0078] 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 silage fermentation additive, characterized in that, Including allicin and compound microbial agents; The mass ratio of allicin to the compound microbial agent is (20-100):(1-2); The compound microbial agent includes Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli; the ratio of viable Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus brunelli is (0.8-1.2):(1.6-2.4):(1.6-2.4).

2. The silage fermentation additive according to claim 1, characterized in that, The viable count of Lactobacillus acidophilus in the compound microbial agent is ≥1×10⁻⁶. 7 CFU / g, viable count of *Lactobacillus plantarum* ≥ 2 × 10⁻⁶ 7 CFU / g, viable count of Lactobacillus bruneri ≥2×10 7 CFU / g.

3. The application of the silage fermentation additive according to claim 1 or 2 in the preparation of silage.

4. The application of the silage fermentation additive according to claim 1 or 2 in any of the following silage fermentation processes: ① Improve aerobic stability; ② Reduce mycotoxin content; ③ Improve the nutritional level of fermented feed; ④ Improve fermentation quality.

5. The application according to claim 4, characterized in that, The amount of the silage fermentation additive added is 0.01% to 0.05% of the fresh weight of the silage raw material.

6. The application according to claim 4 or 5, characterized in that, The mycotoxins include at least one of aflatoxin B1, zearalenone, T-2 toxin, vomitoxin, deoxynivalenol, fumonisin B1, and fumonisin B2.

7. A method for fermenting silage, characterized in that, The silage raw materials are mixed with the silage fermentation additives described in claim 1 or 2 and then subjected to anaerobic fermentation.

8. The fermentation method according to claim 7, characterized in that, The compound microbial agent in the silage fermentation additive accounts for 0.0005% to 0.001% of the fresh weight of the silage raw material, and the allicin accounts for 0.01% to 0.05% of the fresh weight of the silage raw material.

9. The fermentation method according to claim 7, characterized in that, The silage raw materials include crop straw.

10. The fermentation method according to any one of claims 7 to 9, characterized in that, During the anaerobic fermentation, the initial moisture content of the fermentation system is 55%~65%; The anaerobic fermentation is carried out under light-protected conditions; The temperature for the anaerobic fermentation is 20℃~35℃; The anaerobic fermentation time is 40-50 days.

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