High-temperature-resistant and acid-resistant multifunctional compound microbial agent and application thereof in ensiling

Abstract

The invention discloses a high-temperature-resistant and acid-resistant multifunctional compound microbial agent and application thereof in silage, and belongs to the technical field of agricultural microorganisms. The microbial agent is prepared by compounding lactobacillus CYM6 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.36209, bacillus CYC6 with the preservation number of CGMCC No.36210 and Vickers ham yeast CYY6 with the preservation number of CGMCC No.35997 according to the ratio of 8: 1: 1. Through the synergistic effect of the three bacteria, the lactic acid bacteria CYM6 are used for rapidly producing acid to reduce the pH value and inhibit putrefying bacteria; cellulose degrading bacteria CYC6 are used for degrading cellulose, so that the palatability and the digestibility are improved; saccharomycetes CYY6 are used for synthesizing mycoprotein, so that the content of crude protein in the feed is increased. The microbial agent can quickly become a dominant flora in an external high-temperature environment, effectively inhibit spoilage microorganisms, reduce the pH value of silage, degrade the cellulose content of forage grass raw materials, reduce dry matter loss and protein degradation, and significantly improve the fermentation quality and nutritional value of the hybrid pennisetum silage; and an effective technical support is provided for solving the production problem of high-quality silage in southern areas.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a high-temperature and acid-resistant multifunctional composite microbial agent and its application in silage. Background Technology

[0002] Silage is the cornerstone of modern animal husbandry. Through anaerobic fermentation by microorganisms such as lactic acid bacteria, the soluble carbohydrates in fresh forage are converted into organic acids, lowering the pH value and inhibiting the growth of putrefactive bacteria, thus preserving the nutritional value of the forage for a long time. In southern my country, especially in hilly and mountainous areas like Chongqing, due to the characteristics of land resources, high-yield and high-quality warm-season forage—hybrid Napier grass—has become the main forage crop. However, this region has a typical subtropical monsoon climate with long summers and significant characteristics of high temperature and high humidity. These climatic conditions make traditional field drying for haymaking almost impossible. Frequent rainfall and high air humidity easily lead to mold and spoilage of forage during the drying process, resulting in severe nutrient loss. Therefore, silage, especially bale silage which is easy to store and transport, has become the most important technical means to ensure a stable supply of high-quality forage for herbivorous livestock throughout the year in this region.

[0003] However, this high-temperature and high-humidity environment presents a severe challenge to the successful silage production of hybrid Napier grass. Firstly, the high temperature and humidity promote vigorous respiration in the hybrid Napier grass after harvesting, and the surface is covered with a large number of highly active putrefactive bacteria (such as molds, yeasts, and butyric acid bacteria). In the early stages of silage fermentation, if an acidic environment cannot be quickly created and lactic acid bacteria cannot establish dominance, putrefactive bacteria will proliferate, causing the silage to heat up and rot, resulting in low silage yield and significant nutrient loss. Secondly, hybrid Napier grass itself has high water content, high buffering energy, and relatively low soluble carbohydrate content, which further increases the difficulty of rapidly and efficiently initiating lactic acid fermentation.

[0004] Currently, commercial silage inoculants are commonly added to guide and enhance the fermentation process during production. However, commercially available silage inoculants are not very effective. In Chongqing during the summer, the temperature inside the silage pit or bale can easily exceed 45°C in the early stages of fermentation. Under these high temperatures, the activity of conventional inoculants is severely inhibited, their growth and reproduction slow, and they cannot quickly produce acid, thus significantly reducing their effectiveness or even rendering them completely ineffective. Simultaneously, as the silage process progresses, acidity gradually accumulates in the pile. Some microbial strains lack tolerance to low pH environments, resulting in decreased activity in the later stages of silage production, which may lead to secondary fermentation and similarly affect the long-term stability of the silage.

[0005] In summary, the unique hot and humid climate of Chongqing, combined with the inherent characteristics of hybrid Napier grass, creates an extremely demanding environment for silage inoculants. The lack of locally developed, specialized silage inoculants capable of rapidly initiating fermentation under high temperatures and maintaining activity in high-acidity environments is the core technological bottleneck leading to unstable silage quality from hybrid Napier grass in this region. Summary of the Invention

[0006] This invention aims to provide a heat- and acid-resistant, multifunctional compound microbial agent and its application in silage. This agent is a compound of heat- and acid-resistant lactic acid bacteria, cellulose-degrading bacteria, and yeast. Through the synergistic effect of these three components, the quality of silage in hot and humid regions is improved. Specifically, the heat- and acid-resistant lactic acid bacteria are responsible for rapidly leading fermentation, quickly lowering the pH value and inhibiting putrefactive bacteria; the cellulose-degrading bacteria are responsible for degrading the cellulose in the forage raw materials in silage, effectively reducing the cellulose content of the silage and improving palatability and digestibility; and the yeast can synthesize cell protein, thereby increasing the overall protein content of the silage. The above-mentioned microbial strains exhibit strong heat and acid resistance and strong adaptability to the environment.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] This invention provides a high-temperature and acid-resistant, multifunctional composite microbial agent, which is formulated from the following strains:

[0009] (1) Lactobacillus strain CYM6, preservation number CGMCC No.36209;

[0010] (2) Bacillus strain CYC6, preservation number CGMCC No.36210;

[0011] (3) Wickerhamomyces strain CYY6, preservation number CGMCC No.35997;

[0012] The live cell ratio of Lactobacillus CYM6, Bacillus CYC6 and Wickham's yeast CYY6 is 8:1:1.

[0013] Furthermore, the optimal culture medium for Lactobacillus CYM6 is MRS liquid medium, and Lactobacillus CYM6 reaches the logarithmic growth phase after being cultured at 37°C for 24 hours.

[0014] Furthermore, the optimal culture medium for Bacillus CYC6 is LB liquid medium, and Bacillus CYC6 reaches the logarithmic growth phase after being cultured at 37°C for 24 hours.

[0015] Furthermore, the optimal culture medium for the *Bacillus thuringiensis* CYY6 is YPD liquid medium, and the *Bacillus thuringiensis* CYY6 reaches the logarithmic growth phase after being cultured at 30°C for 24 hours.

[0016] This invention also provides the application of the aforementioned compound microbial agent in the preparation of silage under high temperature and high humidity conditions.

[0017] The present invention also provides a method for preparing silage using the aforementioned compound microbial inoculant, comprising the following steps:

[0018] S1. Provide silage raw materials;

[0019] S2. Inoculate the compound silage inoculant according to claim 1 into the silage raw material;

[0020] S3. Seal and ferment the inoculated silage raw materials;

[0021] The inoculation amount of the compound microbial agent is 1×10⁻⁶. 7 CFU / g feed fresh weight.

[0022] Furthermore, the silage material is obtained by intercropping hybrid Napier grass and Labrador bean in a 6:4 ratio, harvesting and then crushing the harvested crop.

[0023] Furthermore, the fermentation conditions are as follows: fermentation for 45 days in a constant temperature incubator at 40°C and 85% relative humidity.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) It has strong environmental adaptability and a high fermentation success rate.

[0026] The three strains selected in this invention all possess extremely strong high-temperature resistance (up to 45°C) and acid resistance (they can still grow in an environment with pH < 4.0), and have strong resistance to environmental stress, ensuring that they can effectively start and complete the fermentation process even under harsh conditions.

[0027] (2) Multifunctional synergy to comprehensively improve silage quality

[0028] This invention achieves perfect functional synergy through the scientific combination of three strains of bacteria: Lactic acid bacteria CYM6 acts as the fermentation engine, rapidly dominating the fermentation process and producing a large amount of lactic acid in a short time, quickly lowering the pH value to 3.78 and effectively inhibiting the activity of all putrefactive microorganisms such as butyric acid bacteria and molds. Cellulose-degrading bacteria CYC6 effectively degrades plant cell walls, significantly reducing the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in silage, thereby improving feed palatability, increasing animal digestibility, and ultimately increasing the relative feed value (RFV) from 86.00 to 111.68. Yeast CYY6 synthesizes cell protein during fermentation, significantly increasing the total crude protein (CP) content of silage and effectively reducing protein loss during silage.

[0029] (3) Stable microbial community structure and long shelf life

[0030] The optimal ratio (8:1:1), selected through high-temperature co-cultivation and weighted comprehensive evaluation, ensures the formation of a stable and efficient microbial community in the silage system by the compound microbial agent. Simultaneously, the acetic acid and propionic acid produced further enhance the stability of the silage after opening, effectively preventing secondary fermentation. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0032] Example 1: Screening and Determination of the Optimal Ratio of Compound Microbial Agents

[0033] 1. Materials and Methods

[0034] (1) Strains: Lactic acid bacteria CYM6 (Lacticaseibacillus sp. CGMCC No.36209), cellulose degrading bacteria CYC6 (Bacillus sp. CGMCC No.36210) and yeast CYY6 (Wickerhamomyces sp. CGMCC No.35997).

[0035] (2) Culture media: MRS liquid medium, used for activation and co-culture of lactic acid bacteria; LB liquid medium, used for activation of cellulose-degrading bacteria; YPD liquid medium, used for activation of yeast.

[0036] (3) Ten ratios were set up, with the volume ratios of lactic acid bacteria:cellulose degrading bacteria:yeast being 8:1:1, 7:2:1, 7:1:2, 6:3:1, 6:2:2, 6:1:3, 5:4:1, 5:3:2, 5:2:3, and 5:1:4, respectively. At the same time, separate culture groups for each bacterial species were set up as controls.

[0037] (4) Preparation of bacterial suspensions: The three strains were activated and cultured in their respective optimal media to the logarithmic growth phase (lactic acid bacteria 37°C 24h; cellulose-degrading bacteria 37°C 24h; yeast 30°C 24h). The bacterial cells were collected by centrifugation (8000 rpm, 10 min), resuspended in sterile physiological saline, and the initial concentration of each bacterial suspension was uniformly adjusted to 1×10⁻⁶ under a microscope using a hemocytometer. 8 CFU / mL.

[0038] (5) High-temperature co-cultivation: Prepare the compound bacterial solution according to the above ratio, with a total volume of 10 mL for each treatment group, and inoculate it into an Erlenmeyer flask containing 20 mL of fresh MRS liquid medium. Place all treatment groups in a 45°C constant temperature shaker and incubate at a speed of 150 rpm for 48 hours.

[0039] (6) Coating count: After co-culture, the culture medium of each treatment group was counted 10 times. -3 10 -4 10 -5 Three dilutions were plated, with five replicates for each dilution. After incubation, plates with colony counts between 30 and 300 were selected for counting. The plates were then plated on MRS, LB, and YPD media and incubated at 37°C for 48 hours.

[0040] (7) Determining the optimal ratio:

[0041] 1) Concentration Calculation and Averaging: For each bacterium at each ratio, calculate its 10-1. -3 10 -4 10 -5 The colony concentration (CFU / mL) at three dilutions is calculated, and then the three concentration values ​​are added together and the arithmetic mean is taken as the final concentration (C_final) of the bacterial strain at that dilution. The calculation formula is: C_final = (C_(-3) + C_(-4) + C_(-5)) / 3.

[0042] 2) Data normalization: In order to eliminate the impact of the differences in the growth rate of different bacterial species on the overall evaluation, the C_final of each bacterial species was normalized at all 10 proportions.

[0043] ① Find the minimum concentration value (C_min) and maximum concentration value (C_max) of the bacterial strain in 10 proportions.

[0044] ② Calculate the normalized value (N) of the strain at each ratio. Calculation formula: N = (C_final - C_min) / (C_max - C_min).

[0045] 3) Calculate the overall score:

[0046] Different weighting coefficients are assigned to each strain based on its functional importance in silage fermentation.

[0047] Lactic acid bacteria (L): As the core strain that dominates fermentation and rapidly produces acid, it is assigned the highest weight of 1.5, with a weighted score of N_L × 1.5; Cellulose-degrading bacteria (C): They are crucial for improving feed quality and are assigned a weight of 1.2, with a weighted score of N_C × 1.2; Yeast (Y): Their function is to enhance protein content, and they are assigned a baseline weight of 1.0, with a weighted score of N_Y × 1.0.

[0048] For each proportion, calculate its weighted total score = (N_L × 1.5) + (N_C × 1.2) + (N_Y × 1.0). Calculate the standard deviation (SD) of the three weighted scores for that proportion. Subtract the standard deviation from the weighted total score to obtain the final score for that proportion. The formula is: Final Score = [(N_L × 1.5) + (N_C × 1.2) + (N_Y × 1.0)] – SD(N_L × 1.5, N_C × 1.2, N_Y × 1.0).

[0049] 4) Ranking: The final scores of all proportions are ranked, and the highest score is the optimal ratio, indicating that it has the strongest synergistic growth ability under high temperature and strong acid conditions. This means that in silage production, this microbial agent combination has a greater competitive advantage and can quickly establish and maintain a dominant microbial community.

[0050] 2. Results

[0051] Table 1. Ranking of comprehensive scores after high-temperature co-cultivation of different proportions of compound microbial agents

[0052] Lactic acid bacteria: Cellulose-degrading bacteria: Yeast Normalized value of lactic acid bacteria (N_L) Normalized value (N_C) of cellulose-degrading bacteria Normalized value of yeast (N_Y) Weighted total score Standard deviation (SD) Final score Ranking 8:1:1 1.3462 1.2000 0.9130 3.4592 0.1799 3.2792 1 7:2:1 0.2795 0.7789 0.6709 1.7293 0.2146 1.5147 3 7:1:2 0.2378 0.6264 0.7562 1.6205 0.2203 1.4003 4 6:3:1 0.2831 0.3343 0.4714 1.0888 0.0795 1.0093 6 6:2:2 1.5000 0.0818 0.2163 1.7981 0.6392 1.1588 5 6:1:3 1.1262 0.0457 0.0000 1.1720 0.5205 0.6515 9 5:4:1 0.0678 0.3386 0.6934 1.0998 0.2562 0.8436 8 5:3:2 0.4916 0.8060 0.5051 1.8026 0.1451 1.6575 2 5:2:3 0.1391 0.1882 0.8553 1.1826 0.3266 0.8559 7 5:1:4 0.0000 0.0000 0.5354 0.5354 0.2524 0.2830 10

[0053] The analysis results are shown in Table 1. The treatment group with a ratio of 8:1:1 (lactic acid bacteria CYM6, cellulose-degrading bacteria CYC6, and yeast CYY6) achieved the highest score of 3.2792, ranking first. This indicates that at this ratio, all three bacteria grew well and the microbial community structure was relatively stable, demonstrating the best synergistic effect. The treatment group with a ratio of 5:3:2 ranked second with a score of 1.6575, and the treatment group with a ratio of 7:2:1 ranked third with a score of 1.5147. Therefore, based on the growth status of the microbial community after high-temperature co-cultivation and using a comprehensive scoring method for evaluation, the optimal ratio of the compound microbial agent was determined to be lactic acid bacteria:cellulose-degrading bacteria:yeast = 8:1:1. This ratio ensures that the compound microbial agent obtains the largest and most stable microbial community quantity under high temperature and strong acid stress, laying a solid foundation for subsequent silage applications.

[0054] Example 2: Application of compound microbial inoculant in the fermentation of hybrid Napier grass-laba soybean mixed silage

[0055] 1. Materials

[0056] Silage raw materials: Hybrid Napier grass and Laba beans are intercropped in a 6:4 ratio. After harvesting, the mixed raw materials are quickly crushed to a length of 1-2 cm using a straw crusher.

[0057] Compound microbial inoculant: Prepared according to the optimal ratio determined in Example 1. Each bacterial strain was first cultured to the logarithmic growth phase, and the cells were collected by centrifugation and resuspended in sterile water to a concentration of 1×10⁻⁶. 8 CFU / mL, then mix according to the ratio, the inoculation amount is 10 mL / kg (fresh weight), that is, the final inoculation amount is 1×10 7 CFU / g.

[0058] 2. Method

[0059] Silage preparation: Weigh 600 g (fresh weight) of the above mixed raw materials and spray them evenly with a compound microbial agent (experimental group) or an equal volume of sterile water (control group) using a sterile sprayer. Each group has 5 replicates. Then, pack the raw materials into silage bags, manually compact them to remove as much air as possible, and finally seal them using a vacuum sealer.

[0060] Fermentation conditions: Fermented for 45 days in a 40°C constant temperature incubator to simulate the typical environmental conditions of silage in high temperature and high humidity regions (relative humidity 85%).

[0061] After fermentation, samples were taken and the following indicators were measured:

[0062] (1) Sensory evaluation: Three experienced personnel will comprehensively score the odor, color and structure of silage (out of 10 points).

[0063] (2) pH value: Take 20 g of sample and add 180 mL of distilled water. After shaking and soaking for 1 hour, filter and measure the pH value of the filtrate with a precision pH meter.

[0064] (3) Fermentation quality: The contents of lactic acid, acetic acid, propionic acid and butyric acid were determined by high performance liquid chromatography (HPLC). Total nitrogen was determined by Kjeldahl method and ammonia nitrogen content was determined by colorimetric method.

[0065] (4) Nutritional composition: The contents of crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were determined using conventional chemical methods. Dry matter (DM) was determined by direct drying, crude protein (CP) was determined by the Kjeldahl method, NDF and ADF were determined by the VanSoest modified Van Soest method, and water-soluble carbohydrate (WSC) was determined by the anthrone-sulfuric acid method. The relative feed value (RFV) was calculated. RFV = 120 / NDF × (88.9 - 0.779ADF) / 1.29.

[0066] 3. Results

[0067] Table 2 Sensory evaluation results of mixed silage

[0068] Group color aroma sour texture evaluate Experimental group 1 yellow-green Aromatic sour taste Strong sour taste Soft and moist, stems and leaves remain intact excellent Experimental group 2 yellow-green Aromatic sour taste Strong sour taste Soft and moist, stems and leaves remain intact excellent Experimental group 3 yellow-green Aromatic sour taste Strong sour taste Soft and moist, stems and leaves remain intact excellent Experimental group 4 yellow-green Aromatic sour taste Strong sour taste Soft and moist, stems and leaves remain intact excellent Experimental group 5 yellow-green Aromatic sour taste Strong sour taste Soft and moist, stems and leaves remain intact excellent Control group 1 yellow Weak aroma Weak sour taste Soft, slightly moist middle Control group 2 light yellow Weak aroma Weak sour taste Soft, slightly moist middle Control group 3 yellow Weak aroma Weak sour taste Soft, slightly moist middle Control group 4 yellow Weak aroma Weak sour taste Soft, slightly moist middle Control group 5 yellow Weak aroma Weak sour taste Soft, slightly moist middle

[0069] As shown in Table 2, after 45 days of ensiling, the ensiling was evaluated in terms of color, aroma, sourness, and texture. The experimental group with added compound microbial inoculant was yellowish-green, had a strong aroma, and a moist texture, and maintained the original shape of the stems and leaves well, and was rated as excellent. The control group without added compound microbial inoculant was slightly yellowish in color, had a slightly weaker aroma, and slightly more moisture, and was rated as medium.

[0070] Table 3. Nutritional composition analysis of mixed silage

[0071] Group CP% NDF% ADF% WSC% RFV experimental group <![CDATA[13.52±0.21 a ]]> <![CDATA[49.89±1.15 b ]]> <![CDATA[37.23±1.61 b ]]> <![CDATA[1.13±0.17 a ]]> <![CDATA[111.68 a ]]> control group <![CDATA[9.47±0.15 b ]]> <![CDATA[57.25±1.56 a ]]> <![CDATA[46.17±2.31 a ]]> <![CDATA[0.89±0.14 b ]]> <![CDATA[86.00 b ]]>

[0072] Note: Different letters in the superscript of data in the same column indicate significant differences (P<0.05), while the same letter or no letter indicates no significant differences (P>0.05); the same applies to the following table.

[0073] Table 3 shows that after 45 days of high-temperature fermentation, compared with the control group without inoculation of the compound microbial agent, the experimental group inoculated with this compound microbial agent showed significant advantages in all key nutritional quality indicators (P < 0.05), fully demonstrating the excellent effect of this compound microbial agent in improving the nutritional value of silage. The crude protein (CP) content of the experimental group reached 13.52%, which was significantly higher than 9.47% of the control group (P < 0.05), representing a relative increase of 42.8%. This result strongly confirms that the yeast CYY6 in the compound microbial agent successfully synthesized cell protein during fermentation, thereby significantly improving the overall protein level of silage. In terms of fiber composition, the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of the experimental group were 49.89% and 37.23%, respectively, both significantly lower than 57.25% and 46.17% of the control group (P < 0.05). This indicates that the cellulose-degrading bacteria CYC6 in the compound microbial agent effectively degraded plant cell walls, reducing the indigestible fiber content in the feed, thus laying the foundation for improving palatability and digestibility. The higher WSC content in the experimental group improved the energy value and palatability of the final silage. The RFV of the experimental group reached 111.68, significantly better than the 86.00 of the control group (P < 0.05). This significant increase in RFV directly proves that the silage treated with this compound microbial agent has a higher potential dry matter intake and energy value, fundamentally optimizing its overall feed quality. The compound microbial agent effectively improved the nutritional quality and feed efficiency of silage.

[0074] Table 4. Fermentation quality analysis of mixed silage (dry matter basis)

[0075] Group pH value Lactic acid% Acetic acid % propionic acid % butyric acid % Ammonia nitrogen / total nitrogen % experimental group <![CDATA[3.78±0.06 b ]]> <![CDATA[12.28±1.03 a ]]> <![CDATA[2.78±0.96 a ]]> <![CDATA[1.36±1.77 a ]]> <![CDATA[0.12±0.39 b ]]> <![CDATA[0.22±0.07 b ]]> control group <![CDATA[4.25±0.08 a ]]> <![CDATA[7.11±2.05 b ]]> <![CDATA[1.13±0.52 b ]]> <![CDATA[0.29±0.72 b ]]> <![CDATA[0.59±0.25 a ]]> <![CDATA[0.43±0.12 a ]]>

[0076] After silage fermentation, the analysis results of the core fermentation quality indicators (see Table 4) showed that the experimental group inoculated with this compound microbial agent was significantly better than the uninoculated control group in terms of fermentation efficiency, substrate preservation and putrefaction inhibition (P < 0.05).

[0077] The pH value of the fermentation broth in the experimental group was as low as 3.78, significantly lower than that of the control group (4.25). This acidification process was due to the extremely significant increase in lactic acid content in the experimental group, which reached 12.28% DM, 1.73 times that of the control group (7.11% DM). This indicates that the thermotolerant and acid-resistant lactic acid bacteria CYM6 in this compound microbial agent successfully colonized and rapidly became the dominant fermentation bacteria, establishing a strong acidic environment for the entire silage system through efficient homolactic fermentation.

[0078] The experimental group had extremely low butyric acid content (0.12% DM) and an ammonia nitrogen / total nitrogen ratio of only 0.22%, both of which were significantly lower than those in the control group (butyric acid: 0.59% DM, ammonia nitrogen / total nitrogen: 0.43%). These two highly synergistic indicators demonstrate that the strongly acidic environment (low pH, high lactic acid) initially established in the experimental group effectively inhibited the growth and activity of putrefactive microorganisms such as butyric acid bacteria (Clostridium), thereby reducing protein decomposition and degradation and maximizing the preservation of the true protein nutritional value of the silage.

[0079] Furthermore, the experimental group also showed significantly higher levels of acetic acid (2.78% DM) and propionic acid (1.36% DM) than the control group. Although the acetic acid content was slightly higher, this is related to vigorous fermentation activity and, as an effective antifungal agent, works in conjunction with propionic acid, indicating that the experimental group's silage exhibits superior aerobic stability when exposed to air, effectively resisting secondary fermentation and mold growth after opening the silo.

[0080] In summary, this compound microbial agent, through the synergistic effect of its core components—lactic acid bacteria CYM6, cellulose-degrading bacteria CYC6, and yeast CYY6—successfully guides silage fermentation towards high lactic acid content, low pH, no butyric acid spoilage, and high protein retention. This result fully validates the application value of this compound microbial agent in solving the fermentation challenges of silage in high-temperature and high-humidity regions, enabling the stable production of high-quality silage.

Claims

1. A multifunctional composite microbial agent resistant to high temperature and acid, characterized in that, It is formulated from the following strains: (1) Lactobacillus strain CYM6, preservation number CGMCC No.36209; (2) Bacillus strain CYC6, preservation number CGMCC No.36210; (3) Wickerhamomyces strain CYY6, preservation number CGMCC No.35997; The live cell ratio of Lactobacillus CYM6, Bacillus CYC6 and Wickham's yeast CYY6 is 8:1:

1.

2. The compound microbial agent according to claim 1, characterized in that, The optimal culture medium for Lactobacillus CYM6 is MRS liquid medium, and Lactobacillus CYM6 reaches the logarithmic growth phase after being cultured at 37°C for 24 hours.

3. The compound microbial agent according to claim 1, characterized in that, The optimal culture medium for Bacillus CYC6 is LB liquid medium, and Bacillus CYC6 reaches the logarithmic growth phase after being cultured at 37°C for 24 hours.

4. The compound microbial agent according to claim 1, characterized in that, The optimal culture medium for the *Bacillus thymosa* CYY6 is YPD liquid medium, and the *Bacillus thymosa* CYY6 reaches the logarithmic growth phase after being cultured at 30°C for 24 hours.

5. The application of the compound microbial agent according to claims 1-4 in the preparation of silage under high temperature and high humidity conditions.

6. A method for preparing silage using the compound microbial agent according to claim 1, characterized in that, Includes the following steps: S1. Provide silage raw materials; S2. Inoculate the compound silage inoculant according to claim 1 into the silage raw material; S3. Seal and ferment the inoculated silage raw materials; The inoculation amount of the compound microbial agent is 1×10⁻⁶. 7 CFU / g feed fresh weight.

7. The method according to claim 6, characterized in that, The silage material is obtained by planting hybrid Napier grass and Laba bean in a 6:4 ratio, harvesting and then crushing the crops.

8. The method according to claim 6, characterized in that, The fermentation conditions were as follows: fermentation for 45 days in a constant temperature incubator at 40°C and 85% relative humidity.