A compound additive for high-moisture silage and application thereof

Abstract

The present application relates to the technical field of silage, in particular to a compound additive for high-moisture silage and application thereof. The compound additive comprises Lactobacillus plantarum CAU-S7 and clove essential oil; the preservation number of the Lactobacillus plantarum CAU-S7 is CGMCC No.20502. The compound additive can improve the quality of high-moisture silage and reduce the emission of rumen greenhouse gas.

Description

Technical Field

[0001] This invention relates to the field of silage technology, specifically to a compound additive for high-moisture silage and its application. Background Technology

[0002] High-quality forage is the fundamental guarantee for the healthy development of herbivorous animal husbandry. Silage, as the main roughage source for ruminants, occupies an important position in modern animal husbandry. However, global climate change has led to more frequent extreme weather events, increased rainfall in the Huang-Huai-Hai Plain, and high temperatures and torrential rains during the harvest season of forages such as oats, resulting in high moisture content in raw materials (usually exceeding 75%), a large proliferation of putrefactive microorganisms, and accelerated feed spoilage. At the same time, methane and carbon dioxide emissions from ruminants have become major sources of greenhouse gases in agriculture. Therefore, developing a synergistic regulation technology that can both improve the quality of high-moisture silage under high temperature and humidity conditions and reduce rumen greenhouse gas emissions has become an urgent need in the fields of ruminant nutrition and environmental protection.

[0003] Currently, feed additive technology is commonly used in the industry for silage quality control and rumen greenhouse gas emission reduction. In silage quality control, *Lactobacillus plantarum* is widely used as a primary silage additive. Our research group previously screened a *Lactobacillus plantarum* strain with strong acid-producing capabilities. This strain can rapidly produce acid and lower pH in the early stages of silage production and exhibits strong fermentation performance under high-temperature conditions. However, under high-temperature and high-humidity conditions, while single lactic acid bacteria additives can promote high-moisture silage fermentation, harmful bacteria such as enterobacteria and acetic acid bacteria can still survive and metabolize at lower pH levels in the later stages of silage production. After opening the silo or exposure to oxygen, aerobic bacteria and fungi proliferate, leading to protein degradation and quality decline in silage. Relying solely on lactic acid bacteria acid production is insufficient to completely suppress these putrefactive microorganisms. Furthermore, the ability of silage with single lactic acid bacteria additives to reduce rumen methane emissions in ruminants is limited. Currently, there are no reported technologies for simultaneously improving the quality of high-moisture silage and reducing rumen greenhouse gas emissions under high-temperature and high-humidity conditions.

[0004] Plant essential oils possess antibacterial, antioxidant, and microbial regulation functions. Due to their small molecular weight, non-toxicity, lack of residue, and rapid absorption and metabolism in animals, they are hailed as "natural antibacterial agents." Eugenol, the core component of clove essential oil, can disrupt bacterial cell membranes and inhibit enzyme activity, exhibiting broad-spectrum antibacterial and antifungal activity, making it a promising feed additive. Studies have shown that clove treatment can significantly improve silage quality and prolong aerobic stability. Regarding rumen methane reduction, clove essential oil can reduce methane production by regulating the rumen microbiota and inhibiting methanogenic bacteria activity. However, in high-temperature and high-humidity silage environments, the volatile components of clove essential oil are easily lost, significantly shortening the duration of its antibacterial and anti-methane activities, making it difficult to achieve stable regulation throughout the entire process when added alone. Therefore, this invention provides a compound additive for high-moisture silage to address the aforementioned problems. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the first objective of the present invention is to provide a compound additive for high-moisture silage that can improve the quality of high-moisture silage.

[0006] The second objective of this invention is to provide a silage feed with higher quality.

[0007] The third objective of this invention is to provide a simple method for preparing silage.

[0008] The fourth objective of this invention is to provide an application of a compound additive for high-moisture silage, which has broad application prospects.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A compound additive for high-moisture silage includes Lactobacillus plantarum CAU-S7 and clove oil; the preservation number of Lactobacillus plantarum CAU-S7 is CGMCC No.20502.

[0010] This invention combines lactic acid bacteria and clove oil under high temperature and humidity conditions and applies the mixture to high-moisture silage. The lactic acid bacteria ensure rapid acid production in the early stages of fermentation, establishing a stable acidic environment; while the clove oil inhibits acid-resistant harmful microorganisms that lactic acid bacteria cannot effectively control. The two complement and synergistically enhance each other's functions, thus ensuring the quality of high-moisture silage fermentation and aerobically exposed feed throughout the entire process, and reducing rumen greenhouse gas emissions.

[0011] Furthermore, the *Lactobacillus plantarum* CAU-S7 exists in the form of live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0012] Furthermore, the *Lactobacillus plantarum* CAU-S7 also includes a carrier; the carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material or a biological material.

[0013] Furthermore, the mineral material is selected from at least one of peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is selected from at least one of various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal excrement; and the liquid carrier may be water.

[0014] Furthermore, the composite additive also includes surfactants, binders, stabilizers (such as antioxidants), pH adjusters, etc.; the surfactant is Tween 20 or Tween 80.

[0015] Furthermore, the amount of live bacteria *Lactobacillus plantarum* CAU-S7 added to the silage raw material is (1×10⁻⁶). 6 -4×10 6 The clove oil content is 0.5-1% of the silage raw material by mass (cfu / g).

[0016] Furthermore, the purity of the clove essential oil is 85%.

[0017] Furthermore, the silage raw material is high-moisture forage.

[0018] Furthermore, the high-moisture forage refers to forage with a moisture content greater than 70%.

[0019] Furthermore, the high-moisture forage is oats.

[0020] A silage feed includes silage raw materials and the above-mentioned compound additives.

[0021] The above-mentioned method for preparing silage includes the following steps: The silage is obtained by mixing the raw materials with compound additives and then anaerobic fermentation.

[0022] Furthermore, the anaerobic fermentation temperature is 15-35℃, and the time is 40-60 days.

[0023] The application of the above-mentioned compound additives for high-moisture silage includes at least one of the following: (1) Application in reducing greenhouse gas emissions from the rumen; (2) Application in improving the quality of silage; (3) Application in the preparation of silage.

[0024] Furthermore, the reduction of rumen greenhouse gas emissions includes at least one of the following: B1. Reduce methane production during in vitro rumen fermentation of silage; B2. Reduce carbon dioxide production during in vitro rumen fermentation of silage; B3. Increase the total volatile fatty acid content during the in vitro rumen fermentation of silage; B4. Increase the propionic acid content during the in vitro rumen fermentation of silage.

[0025] Furthermore, the improvement of silage quality includes at least one of the following: A1. Lower the pH of the feed after silage fermentation and / or aerobic exposure; A2. Reduce the ammonia nitrogen content of silage after fermentation and / or aerobic exposure; A3. Reduce the butyric acid content in silage fermentation and / or aerobic exposure feed; A4. Reduce the number of E. coli in silage fermentation and / or aerobic exposure feed; A5. Reduce the neutral detergent fiber content of silage fermentation and / or aerobic exposure feed; A6. Increase the lactic acid content of silage after fermentation and / or aerobic exposure; A7. Increase the dry matter content of silage after fermentation and / or aerobic exposure; A8. Increase the soluble carbohydrate content of silage fermentation and / or aerobic exposure feed.

[0026] The beneficial technical effects of this invention are as follows: 1. This invention provides a compound additive for high-moisture silage, comprising Lactobacillus plantarum CAU-S7 and clove oil. Lactobacillus plantarum CAU-S7 rapidly produces acid to establish an acidic environment, while clove oil inhibits acid-resistant harmful bacteria and methanogenic bacteria. The two components complement and synergistically enhance each other, simultaneously improving quality and reducing greenhouse gas emissions throughout the silage fermentation process and subsequent utilization.

[0027] 2. The compound additive of the present invention for high-moisture silage can significantly reduce the ammonia nitrogen content, acetic acid content and neutral detergent fiber content of the silage after fermentation, increase the soluble carbohydrate content, inhibit butyric acid formation, reduce pH value and improve fermentation quality.

[0028] 3. The compound additive of the present invention for high-moisture silage can effectively alleviate protein degradation and the proliferation of putrefactive microorganisms, and prolong the aerobic stability of silage.

[0029] 4. The compound additive for high-moisture silage of the present invention can significantly reduce the emissions of methane and carbon dioxide, while increasing the content of total volatile fatty acids and propionic acid, and optimizing the rumen fermentation mode. Attached Figure Description

[0030] Figure 1 The graph shows the results of the antibacterial activity of clove essential oil against Lactiplantibacillus plantarum ATCC14917; where a is the result of clove essential oil treatment and b is the result of sterile water treatment. Figure 2 The graph shows the results of the antibacterial activity of clove essential oil against Enterococcus faecalis ATCC 29212; where a is the result of clove essential oil treatment and b is the result of sterile water treatment. Detailed Implementation

[0031] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0032] The Lactobacillus plantarum CAU-S7 used in this invention has the preservation number CGMCC No.20502, and the clove essential oil was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Preparation of Lactobacillus plantarum CAU-S7: Lactobacillus plantarum CAU-S7 was inoculated into liquid MRS medium (pH 6.5) and cultured at 30°C for 24 h to obtain the bacterial culture of Lactobacillus plantarum CAU-S7, which was then freeze-dried.

[0034] Liquid MRS medium was prepared as follows: 10.0 g peptone, 5.0 g beef meal, 4.0 g yeast extract, 20.0 g glucose, 5.0 g sodium acetate, 2.0 g triammonium citrate, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate. The volume was adjusted to 1000 mL with distilled water, and the pH was set to 6.2. The prepared medium was then sterilized in an autoclave at 121°C for 15 min.

[0035] MRS solid medium is obtained by adding agar to the above liquid MRS medium and then sterilizing it.

[0036] Example 1 This embodiment provides a compound additive for high-moisture silage, comprising *Lactobacillus plantarum* CAU-S7 and clove oil. When this compound additive is used to prepare whole-plant oat silage, the amount of live *Lactobacillus plantarum* CAU-S7 added to the whole-plant oat silage is 1×10⁻⁶. 6 cfu / g, clove essential oil accounts for 0.5% of the total mass of oat silage.

[0037] This embodiment also provides a silage feed, including the above-mentioned compound additive and whole-plant oat silage. The whole-plant oat silage is prepared by the following method: after harvesting the whole-plant oats, the undried oats (moisture content >75%) are cut into short pieces of about 2cm and mixed evenly.

[0038] This embodiment also provides a method for preparing the above-mentioned silage, including the following steps: Whole-plant oat silage is prepared by mixing it with compound additives and anaerobic fermentation at 25°C for 60 days.

[0039] Example 2 This embodiment provides a compound additive for high-moisture silage, comprising *Lactobacillus plantarum* CAU-S7 and clove oil. When this compound additive is used to prepare whole-plant oat silage, the amount of live *Lactobacillus plantarum* CAU-S7 added to the whole-plant oat silage is 1×10⁻⁶. 6 cfu / g, clove essential oil accounts for 1% of the total mass of oat silage.

[0040] This embodiment also provides a silage feed, including the above-mentioned compound additive and whole-plant oat silage. The whole-plant oat silage is prepared by the following method: after harvesting the whole-plant oats, the undried oats (moisture content >75%) are cut into short pieces of about 2cm and mixed evenly.

[0041] This embodiment also provides a method for preparing the above-mentioned silage, including the following steps: Whole-plant oat silage is prepared by mixing it with compound additives and anaerobic fermentation at 25°C for 60 days.

[0042] Example 3 This embodiment provides a compound additive for high-moisture silage, comprising *Lactobacillus plantarum* CAU-S7 and clove oil. When using this compound additive to prepare whole-plant oat silage, the amount of live *Lactobacillus plantarum* CAU-S7 added to the whole-plant oat silage is 2 × 10⁻⁶. 6 cfu / g, clove essential oil accounts for 0.8% of the total mass of oat silage.

[0043] This embodiment also provides a silage feed, including the above-mentioned compound additive and whole-plant oat silage. The whole-plant oat silage is prepared by the following method: after harvesting the whole-plant oats, the undried oats (moisture content >75%) are cut into short pieces of about 2cm and mixed evenly.

[0044] This embodiment also provides a method for preparing the above-mentioned silage, including the following steps: Whole-plant oat silage is prepared by mixing it with compound additives and anaerobic fermentation at 35°C for 40 days.

[0045] Example 4 This embodiment provides a compound additive for high-moisture silage, comprising *Lactobacillus plantarum* CAU-S7 and clove oil. When this compound additive is used to prepare whole-plant oat silage, the amount of live *Lactobacillus plantarum* CAU-S7 added to the whole-plant oat silage is 4 × 10⁻⁶. 6 cfu / g, clove essential oil accounts for 0.8% of the total mass of oat silage.

[0046] This embodiment also provides a silage feed, including the above-mentioned compound additive and whole-plant oat silage. The whole-plant oat silage is prepared by the following method: after harvesting the whole-plant oats, the undried oats (moisture content >75%) are cut into short pieces of about 2cm and mixed evenly.

[0047] This embodiment also provides a method for preparing the above-mentioned silage, including the following steps: Whole-plant oat silage is prepared by mixing it with compound additives and anaerobic fermentation at 15°C for 55 days.

[0048] Comparative Example 1 The difference between this comparative example and Example 1 is that an equal volume of sterile water is used instead of the composite additive.

[0049] Experimental Example 1 The antibacterial activity of clove essential oil was tested using the Oxford cup double-layer plate method. The specific experimental procedure is as follows: Cool the nutrient medium containing 1.6% agar to 50°C, and pour 10 mL of the medium into sterile petri dishes to form the lower plate. Prepare an indicator culture medium containing 0.8% agar, cool it to about 40°C, and add bacterial suspensions of Lactiplantibacillus plantarum ATCC14917 and Enterococcus faecalis ATCC 29212 at an inoculation rate of 1%, respectively. Mix well and use it as the upper culture medium. Add 5 mL of the upper culture medium to the lower plate, shake well, spread evenly, and allow to cool and solidify. Using sterile forceps, gently place the Oxford cup onto the plate, add clove oil to the Oxford cup, and incubate the plate at 37°C for 24 hours. Observe whether an inhibition zone appears. Sterile water was used as a control group. The results are as follows: Figure 1 As shown.

[0050] Figure 1 The graph shows the results of the antibacterial activity of clove essential oil against Lactiplantibacillus plantarum ATCC14917; where a is the result of clove essential oil treatment and b is the result of sterile water treatment. Figure 2 This is a graph showing the antibacterial activity of clove essential oil against Enterococcus faecalis ATCC 29212; where a represents the results of clove essential oil treatment and b represents the results of sterile water treatment. Observation Figure 1-2 It can be seen that clove essential oil did not produce obvious inhibition zones against Lactobacillus plantarum and Enterococcus faecalis, indicating that clove essential oil has a weak or no inhibitory effect on lactic acid bacteria, and is suitable for use in silage.

[0051] Experimental Example 2 The silage obtained in Examples 1-2 and Comparative Example 1 was placed in a container with a density of approximately 350 kg / m³. 3 The silage was stored in individual 1L plastic containers. The containers were covered with gauze to prevent evaporation and contamination, and then aerobically exposed for 6 days at room temperature. Samples were taken after ensiling (at the time of opening) and again after 6 days of aerobic exposure, and the following parameters were measured: (1) pH value: measured using a pH meter; (2) Organic acids: A Shimadzu GC-14 high-performance liquid chromatograph (column: KC-811 column, Shimadzu, Japan), detector: SPD-M10AVP, mobile phase: 3 mmol L⁻¹ perchloric acid, flow rate: 1 mL min⁻¹, column temperature: 50℃, detection wavelength: 210 nm, injection volume: 5 μL. Lactic acid, acetic acid and propionic acid were used as standards for qualitative analysis based on the retention time of the standards and quantitative analysis was performed using the standard curve method (external standard method). (3) Ammonia nitrogen content: determined by the phenol-sodium hypochlorite colorimetric method; (4) Dry matter content: After opening the bag of silage, dry it in an oven at 65℃ for 48 hours, crush it through a 40-mesh sieve, weigh it and calculate the dry matter content; (5) Crude protein content: The crude protein content of the pulverized sample was determined by the Kjeldahl method using a FOSS KJ2300 fully automatic nitrogen analyzer. (6) Soluble carbohydrate content: anthrone-sulfuric acid colorimetric method was used; (7) Neutral detergent fiber content: Van der Waals fiber analysis method was used; (8) Escherichia coli count: After fermentation and 6 days of aerobic exposure, the bag was opened and 20g of oat silage sample was taken from each replicate. The sample was added to 180 mL of sterile distilled water and shaken on a shaker at 150 rpm for 30 min. Then it was diluted and spread onto Escherichia coli BlueLight medium and incubated in a 37℃ incubator for 48 h before counting.

[0052] Experimental data were processed using Excel software, and one-way ANOVA was performed using SPSS software to compare the significant differences between groups (P<0.05). The results are shown in Table 1-2.

[0053] Table 1. Quality of Silage After Fermentation Note: DM represents dry matter; TN represents total nitrogen; data with the same letter in each row indicates no significant difference, data without the same letter indicates a significant difference, and ND indicates not detected.

[0054] Table 2 Quality of life after 6 days of aerobic exposure Table 1 shows that, compared with Comparative Example 1, the silage from Examples 1 and 2 significantly reduced the content of ammonia nitrogen, acetic acid, and neutral detergent fiber (P<0.05), and significantly increased the content of soluble carbohydrates (P<0.05). Specifically, the pH value of Example 2 was significantly lower than that of Comparative Example 1 and Example 1 (P<0.05), and butyric acid was not detected in either Example 1 or Example 2. These results indicate that the composite additive of the present invention can effectively inhibit the activity of harmful microorganisms, reduce protein degradation, retain more soluble carbohydrates, and improve the fermentation quality of high-moisture oat silage.

[0055] Table 2 shows that, compared with Comparative Example 1, the silage from Examples 1 and 2 significantly reduced pH, ammonia nitrogen content, acetic acid content, and Escherichia coli count after 6 days of aerobic exposure (P<0.05), indicating that the compound additive of the present invention can effectively alleviate protein degradation and harmful bacteria proliferation during aerobic exposure. Among them, Example 1 had the highest dry matter content and the lowest neutral detergent fiber content (P<0.05), while Example 2 had a significantly higher lactic acid content than Comparative Example 1 (P<0.05).

[0056] Experimental Example 3 The fermented feeds obtained from Examples 1-2 and Comparative Example 1 were used in in vitro digestion experiments. The specific steps are as follows: (1) Rumen fluid collection and processing: Rumen fluid from healthy dairy cows was collected, filtered through four layers of coarse cloth, and then mixed with artificial saliva at a volume ratio of 1:2 under anaerobic conditions with continuous CO2 flow to prepare rumen culture medium.

[0057] (2) In vitro fermentation: Take an appropriate amount of silage sample and place it in a fermentation bottle. Add rumen culture medium and use an automated in vitro fermentation system (AGRS-Ⅲ) to continuously record the cumulative gas production for 72 hours under constant temperature conditions of 39℃.

[0058] (3) Gas collection and measurement: Gas in each fermentation bottle was collected using aluminum foil bags, and the emissions of methane (CH4) and carbon dioxide (CO2) were determined by gas chromatography.

[0059] (4) Determination of dry matter digestibility: After fermentation, the fermentation residue was dried in an oven at 65°C to constant weight, and the in vitro dry matter digestibility (IVDMD) was calculated.

[0060] (5) Determination of volatile fatty acids: The supernatant of the fermentation broth was taken and the total volatile fatty acids (TVFA) and the concentration of each component (acetic acid, propionic acid, butyric acid, etc.) were determined by gas chromatography. The results are shown in Table 3.

[0061] Table 3. In vitro rumen fermentation characteristics of silage Table 3 shows that, compared with Comparative Example 1, the silage obtained in Examples 1 and 2 significantly reduced in vitro rumen carbon dioxide emissions (P<0.05). Specifically, the silage in Example 1 also significantly reduced in vitro rumen methane emissions (P<0.05), while the methane emissions in Example 2 were not significantly different from those in Comparative Example 1. Furthermore, the silage in Examples 1 and 2 significantly increased the total volatile fatty acid content and propionic acid content (P<0.05).

[0062] 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. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A compound additive for high-moisture silage, characterized in that, It includes Lactobacillus plantarum CAU-S7 and clove essential oil; the preservation number of Lactobacillus plantarum CAU-S7 is CGMCC No.20502.

2. The compound additive for high-moisture silage according to claim 1, characterized in that, The amount of live bacteria *Lactobacillus plantarum* CAU-S7 added to the silage raw material was (1×10⁻⁶). 6 -4×10 6 The clove oil content is 0.5-1% of the silage raw material by mass (cfu / g).

3. The compound additive for high-moisture silage according to claim 2, characterized in that, The silage material is high-moisture forage.

4. The compound additive for high-moisture silage according to claim 3, characterized in that, The high-moisture forage is oats.

5. A type of silage, characterized in that, It includes silage raw materials and the compound additives described in any one of claims 1-4.

6. A method for preparing silage according to claim 5, characterized in that, Includes the following steps: The silage is obtained by mixing the raw materials with compound additives and then anaerobic fermentation.

7. The method for preparing silage according to claim 6, characterized in that, The anaerobic fermentation is carried out at a temperature of 15-35℃ for 40-60 days.

8. The application of a compound additive for high-moisture silage according to any one of claims 1-4, characterized in that, Application in at least one of the following: (1) Application in reducing greenhouse gas emissions from the rumen; (2) Application in improving the quality of silage; (3) Application in the preparation of silage.

9. The application of the compound additive for high-moisture silage according to claim 8, characterized in that, The reduction of rumen greenhouse gas emissions includes at least one of the following: B1. Reduce methane production during in vitro rumen fermentation of silage; B2. Reduce carbon dioxide production during in vitro rumen fermentation of silage; B3. Increase the total volatile fatty acid content during the in vitro rumen fermentation of silage; B4. Increase the propionic acid content during the in vitro rumen fermentation of silage.

10. The application of the compound additive for high-moisture silage according to claim 8, characterized in that, Improving the quality of silage includes at least one of the following: A1. Lower the pH of the feed after silage fermentation and / or aerobic exposure; A2. Reduce the ammonia nitrogen content of silage after fermentation and / or aerobic exposure; A3. Reduce the butyric acid content in silage fermentation and / or aerobic exposure feed; A4. Reduce the number of E. coli in silage fermentation and / or aerobic exposure feed; A5. Reduce the neutral detergent fiber content of silage fermentation and / or aerobic exposure feed; A6. Increase the lactic acid content of silage after fermentation and / or aerobic exposure; A7. Increase the dry matter content of silage after fermentation and / or aerobic exposure; A8. Increase the soluble carbohydrate content of silage fermentation and / or aerobic exposure feed.

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