Composite fermentation agent and application thereof in preparation of silage
By using compound fermentation agents to synergistically ferment silage, the problems of high phytic acid content, low mineral element absorption rate, and poor flavor were solved. This improved the CLA content and selenium utilization rate in the rumen, thereby enhancing the palatability of silage and the health of beef cattle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANCHANG UNIV
- Filing Date
- 2024-07-03
- Publication Date
- 2026-05-29
AI Technical Summary
Existing silage is prone to problems such as high phytic acid content, low mineral absorption rate, poor flavor, poor palatability and spoilage during fermentation. In addition, traditional fermentation agents are difficult to effectively improve CLA content and selenium utilization in the rumen.
A compound fermentation agent composed of Lactobacillus plantarum R6, Saccharomyces cerevisiae, and Bacillus subtilis is used to prepare silage through synergistic fermentation. The high phytase production of Lactobacillus plantarum R6 decomposes phytic acid, Saccharomyces cerevisiae improves the flavor, and Bacillus subtilis creates an anaerobic environment to increase CLA content and the abundance of probiotics in the rumen.
It significantly reduces phytic acid content, improves mineral absorption, enhances the flavor of silage, increases CLA content in the rumen, improves CLA and selenium content in beef, and enhances the immunity and nutrient absorption of beef cattle.
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Figure CN118726160B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a compound fermentation agent and its application in the preparation of silage. Background Technology
[0002] Silage is made by chopping, sealing, and fermenting plant stalks and green forage. It is primarily used to feed ruminants and is more durable for storage than fresh feed, with superior nutritional content compared to dry feed. Phytic acid, also known as inositol hexaphosphate, is a phosphorus-containing compound found in plant tissues; for example, its content in corn stalks can reach as high as 2%. Phytic acid has anti-nutritional effects, such as reducing the absorption of minerals and the digestion of proteins, and high-phosphorus manure exacerbates environmental pollution. To reduce the side effects of phytic acid, phytase is often added to the feed. Furthermore, if fermentation conditions are not properly controlled during the silage fermentation process, it can often result in poor flavor, poor palatability, and even spoilage. Summary of the Invention
[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound fermentation agent and its application in the preparation of silage, specifically adopting the following technical solution:
[0004] In a first aspect, the present invention provides a compound fermentation agent, characterized in that it is composed of *Lactobacillus plantarum* (…). Lactobacillus plantarum R6, brewer's yeast ( Saccharomyces cerevisiae ) and Bacillus subtilis ( Bacillus subtilis The composition includes Lactobacillus plantarum R6, which was deposited on March 22, 2024, at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30096.
[0005] The above-mentioned brewing yeast ( Saccharomyces cerevisiae ) and Bacillus subtilis ( Bacillus subtilis All samples were purchased from the China General Microbiological Culture Collection Center (CGMCC). *Saccharomyces cerevisiae* was deposited at CGMCC on January 1, 1952, at No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 2.216; *Bacillus subtilis* was deposited at CGMCC on March 1, 1972, at No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 1.821.
[0006] The Lactobacillus plantarum provided in this invention ( Lactobacillus plantarumLactobacillus plantarum R6 was isolated from the rumen of sheep. This strain produces linoleic acid isomerase, which can convert linoleic acid into C9,T11-CLA in MRS culture medium, with a fermentation level as high as 1 mg / mL. Simultaneously, this strain produces a high amount of phytase, which can effectively decompose phytic acid and improve the digestibility and absorption rate of minerals in straw. Therefore, this invention uses this strain to ferment silage, utilizing its ability to colonize the rumen to increase the abundance of Lactobacillus plantarum R6 in the rumen. Through biotransformation, the CLA content in the rumen is increased, and through metabolic transport, the CLA content in beef is significantly increased, resulting in beef rich in C9,T11-CLA. This also significantly improves the utilization rate of selenium in selenium-enriched straw, resulting in selenium-rich beef, meeting consumers' nutritional and health needs.
[0007] A second aspect of the present invention provides a method for preparing the above-mentioned compound fermentation agent, comprising the following steps:
[0008] Lactobacillus plantarum R6 strain was inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum R6 bacterial culture;
[0009] Saccharomyces cerevisiae inoculated into malt extract culture medium and cultured at 25°C for 24 h to obtain Saccharomyces cerevisiae culture liquid;
[0010] Bacillus subtilis strain was inoculated into nutrient broth culture medium and cultured at 30℃ for 24 h to obtain Bacillus subtilis bacterial culture;
[0011] A compound fermentation agent was obtained by mixing Lactobacillus plantarum R6 bacterial solution, Saccharomyces cerevisiae bacterial solution and Bacillus subtilis bacterial solution in a volume ratio of 1:1:1.
[0012] As a further preferred embodiment, the cell concentration of the above-mentioned *Lactobacillus plantarum* R6 bacterial suspension, *Saccharomyces cerevisiae* bacterial suspension, and *Bacillus subtilis* bacterial suspension is 1×10⁻⁶. 8 CFU / mL.
[0013] A third aspect of the present invention provides the application of the above-mentioned compound fermentation agent in the fermentation preparation of silage.
[0014] In a fourth aspect, the present invention provides a silage feed obtained by fermentation of the above-mentioned compound fermentation agent.
[0015] A fifth aspect of the present invention also provides a method for preparing the above-mentioned silage, comprising the following steps:
[0016] The above-mentioned compound fermentation agent is evenly sprayed onto selenium-enriched corn stalks, and then fermented until the pH is less than 4.2 to obtain silage.
[0017] As a further preferred embodiment, the selenium-enriched corn stalks are in small segments of 1 cm to 3 cm.
[0018] As a further preferred embodiment, the ratio of the above-mentioned compound fermentation agent to selenium-enriched corn straw is 1:0.1%-1.0%. More preferably, the ratio of the compound fermentation agent to selenium-enriched corn straw is 1:0.2%.
[0019] The beneficial effects of this invention are as follows:
[0020] (1) The present invention provides a compound fermentation agent, wherein Lactobacillus plantarum R6 is isolated from sheep rumen. This strain produces a high amount of phytase, which can effectively decompose phytic acid and improve the digestibility and absorption rate of mineral elements in straw. By fermenting silage with this strain, the phytic acid content in silage can be significantly reduced by more than 50%, and the digestibility and absorption rate of mineral elements in straw can be improved, with the utilization rate of selenium increasing by nearly 55%. By utilizing its ability to colonize the rumen, the abundance of Lactobacillus plantarum R6 in the rumen can be increased. Through biotransformation, the CLA content in the rumen can be increased. Through metabolic transport, the CLA content in beef can be significantly increased to more than 23 mg / g fat, and the content of c9,t11-CLA is >75%, thus obtaining beef rich in c9,t11-CLA. The CLA yield is high, the purity of c9,t11-CLA isomers is high, and the production method is simple and low in cost.
[0021] (2) This invention also provides a method for preparing silage. Through multi-strain synergistic fermentation, the aroma and palatability of the silage are improved under the action of brewer's yeast. Under the action of aerobic Bacillus subtilis, the air in the feed gaps is rapidly reduced in the early stage of fermentation, quickly forming an anaerobic environment, promoting anaerobic fermentation, and effectively avoiding abnormal fermentation and spoilage. The three selected strains of Lactobacillus plantarum, brewer's yeast, and Bacillus subtilis are all probiotics, which can maintain the balance of intestinal flora, improve the immunity of beef cattle, and promote feed digestion and nutrient absorption. Attached Figure Description
[0022] Figure 1 The diagram shows the level of CLA synthesis by Lactobacillus plantarum R6.
[0023] Figure 2 The diagram shown is a compositional analysis of the CLA isomer synthesized by Lactobacillus plantarum R6.
[0024] Figure 3 The figure shows the effect of multi-strain synergistic fermentation on the phytic acid content in corn silage;
[0025] Figure 4 The figure shows the effect of multi-strain synergistic fermentation of corn silage on the CLA content in beef fat;
[0026] Figure 5 The image shows the effect of multi-strain synergistic fermentation of corn silage on the selenium content in beef. Detailed Implementation
[0027] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.
[0028] Example 1
[0029] Lactobacillus plantarum ( Lactobacillus plantarum Screening, culture and identification of R6
[0030] (1) A screening and isolation method for Lactobacillus plantarum R6, comprising the following steps:
[0031] Sample processing: Immediately after slaughter, a suitable amount of rumen fluid was drawn into a pre-prepared sterile, oxygen-free, carbon dioxide-filled sealed glass bottle using a sterile syringe. The sample was then quickly brought back to the laboratory and filtered through sterile gauze in a laminar flow hood.
[0032] Enrichment culture: 0.5 mL of filtered rumen fluid was pipetted into 5.0 mL of MRS culture medium and incubated at 39°C for 48 h.
[0033] Initial screening by streak plating: In a clean bench, the bacterial samples in the MRS culture medium were streaked onto the MRS solid medium using an inoculation loop. After incubation at 37°C for 48 h, single colonies were picked and incubated in the MRS culture medium at 37°C for 24 h. After incubation, the colonies were removed and stored at 4°C for later use.
[0034] Secondary screening: The preserved strain was activated and subcultured once with a 5% inoculum. After incubation at 37°C for 24 h, 1.0 g / L emulsified linoleic acid was added to the fermentation system. After fermentation at 37°C for 24 h, hexane was added for extraction. The extract was collected by centrifugation, and the CLA content was determined. The specific determination method was carried out according to the method in (Xiaohua Liu, Yusheng Cao, Yan Chen. Separation of conjugated linoleic acid isomers by cyclodextrin-modified micellar electrokinetic chromatography. Journal of Chromatography A, 2005, 1095:197-200).
[0035] (2) The culture of Lactobacillus plantarum R6 includes the following steps:
[0036] The R6 bacterial suspension of Lactobacillus plantarum stored at 4℃ was inoculated into MRS culture medium at an inoculation rate of 5% (v / v), and cultured at 37℃ for 24 h. The bacterial suspension was then stored at 4℃ for later use.
[0037] (3) Identification of R6 of Lactobacillus plantarum, including the following steps:
[0038] Morphological characteristics and Gram chromosome observation: Observe the morphological characteristics of bacterial colonies on MRS solid medium, including size, shape, color, edge structure, surface smoothness, and transparency. Gram staining is performed for observation. Colonies are 1-2 mm in diameter, milky white and translucent, with smooth edges and a slightly convex center. The bacteria are Gram-positive and rod-shaped.
[0039] 16S rDNA gene sequencing analysis: Following standard procedures, the strain's genome was extracted. Universal primers for bacterial identification were used for amplification. The strain's genomic DNA was amplified by PCR, and the PCR product was purified and sent to Shanghai Sangon Biotech for sequencing. The obtained 16S rDNA sequence was compared for homology using BLAST in the NCBI database. The 16S rDNA sequence is shown in SEQ ID No: 1.
[0040]
[0041] (4) Lactobacillus plantarum ( Lactobacillus plantarum Fermentation characteristics of R6
[0042] Skim milk powder was diluted with water to prepare a 10% skim milk solution. 5 mL of this solution was added to each test tube and sterilized at 85°C for 30 min. Simultaneously, 5 mL of MRS medium was prepared and sterilized at 121°C for 20 min. After cooling to room temperature, *Lactobacillus plantarum* R6 inoculum was inoculated at a volume fraction of 5%. Then, 0.12% linoleic acid was added to each test tube, mixed thoroughly, and incubated at 37°C. 0.5 mL of the culture was taken every 12 h to determine the CLA content. The specific determination method followed the procedure described in (Xiaohua Liu, Yusheng Cao, Yan Chen. Separation of conjugated linoleic acid isomers by cyclodextrin-modified micellar electrokinetic chromatography. Journal of Chromatography A, 2005, 1095:197-200). The results after 72 h of fermentation are as follows: Figure 1 As shown, the fermentation levels of CLA in the two culture media were comparable, with the CLA content reaching as high as 0.89 mg / mL after 24 h of fermentation. Subsequently, the CLA yield entered a plateau phase, reaching a maximum of 1.11 mg / mL. Capillary electrophoresis analysis confirmed that *Lactobacillus plantarum* (*Lactobacillus*) was the primary culture medium. Lactobacillus plantarum R6 synthesized only one monomer, c9,t11-CLA, as an isomer of CLA in skim milk and MRS medium, as shown in the results. Figure 2 As shown.
[0043] Example 2
[0044] A method for preparing a compound fermentation agent specifically includes the following steps:
[0045] Lactobacillus plantarum ( Lactobacillus plantarum R6) CGMCC 30096 strain was inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum culture; Saccharomyces cerevisiae (R6) was added. Saccharomyces cerevisiae CGMCC2.126 strain was inoculated into malt extract culture and cultured at 25°C for 24 h to obtain brewer's yeast culture; Bacillus subtilis ( Bacillus subtilis CGMCC 1.821 bacterial strain was inoculated into nutrient broth and cultured at 30℃ for 24 h to obtain Bacillus subtilis bacterial suspension; the bacterial cell concentration of the above three bacterial suspensions was adjusted to 1×10⁻⁶. 8CFU / mL; then, Lactobacillus plantarum culture, Saccharomyces cerevisiae culture and Bacillus subtilis culture were mixed in a volume ratio of 1:1:1 to obtain a compound fermentation agent.
[0046] Example 3
[0047] A method for preparing silage includes the following steps:
[0048] Harvested selenium-enriched whole-plant corn (total selenium content of 385 µg / kg) is cut into 2 cm long stalk pieces. 2 kg of compound fermentation agent is evenly sprayed onto 1000 kg of corn stalks and mixed thoroughly. After being packed with silage using a silage baler, it is naturally fermented for more than 15 days until the pH is <4.2 to obtain selenium-enriched corn silage.
[0049] In this embodiment, a control group was set up in which no compound fermentation agent was added during the above preparation process, and selenium-enriched corn silage was obtained without the addition of fermentation agent.
[0050] The effects of two fermentation methods (selenium-enriched corn silage and selenium-enriched corn silage without added fermenting agent) on the phytic acid content in corn silage were determined. The specific detection method followed the method described in (Luo Kun et al., Effects of high-phytase-producing lactic acid bacteria fermentation on protein quality and baking characteristics of black bean bread, Food Science, 2021, 42(06): 111-117). The results are as follows: Figure 3 As shown, and by Figure 3 It was found that after 15 days of multi-strain synergistic fermentation, the phytic acid content in corn silage decreased by more than 50%, while the control group decreased by less than 15%. At the same time, the corn silage fermented by multi-strain synergistic fermentation had a richer aroma and no abnormal fermentation occurred.
[0051] Selenium-enriched corn silage was used for fattening feeder cattle. The cattle were fed conventionally during the early and middle stages, and their daily diet for the later 60-120 days consisted of 6 kg of concentrate, 1 kg of alfalfa hay, and 2 kg of silage. The concentrate consisted of 60% corn, 15% soybean meal, 11% distillers' grains, 4% soybeans, 4% rapeseed meal, 2% limestone powder, 1% baking soda, 1% salt, 0.9% slow-release urea, 0.6% dicalcium phosphate, and 0.5% molasses. The control group received selenium-enriched corn silage without any fermentation agent. Observations showed that the selenium-enriched corn silage with the compound fermentation agent had better palatability and a significantly reduced incidence of gastrointestinal diseases in beef cattle.
[0052] The effects of two types of selenium-enriched corn silage (selenium-enriched corn silage and selenium-enriched corn silage without added leavening agent) on the CLA content in beef were determined. The specific determination method followed the procedure described in (Xiaohua Liu, Yusheng Cao, YanChen. Separation of conjugated linoleic acid isomers by cyclodextrin-modified micellar electrokinetic chromatography. Journal of Chromatography A, 2005, 1095: 197-200). The results are as follows: Figure 4 As shown, and by Figure 4 It was found that after 45 days of feeding with corn silage fermented by multiple microorganisms, the CLA content in beef increased to as high as 23.6 mg / g fat, while the CLA content in beef fed with traditional silage was 7.5 mg / g fat. Furthermore, the former contained 75.4% of the c9,t11-CLA isomer, while the latter contained only 50.6%. The selenium content in beef was determined according to the National Food Safety Standard for the Determination of Selenium in Food (GB5009.93—2017), and the results are as follows... Figure 5 As shown, and by Figure 5 It was found that after feeding beef with selenium-enriched corn silage fermented by multiple microorganisms for 45 days, the total selenium content in beef reached 88 µg / kg, and the organic selenium content was 80 µg / kg. In contrast, beef fed with selenium-enriched silage without added fermenting agents had a total selenium content of 57 µg / kg and an organic selenium content of 51 µg / kg. By using the multi-strain co-fermentation method of corn silage to feed beef cattle according to this invention, beef rich in C9,T11-CLA can be obtained, with high CLA yield, high purity of C9,T11-CLA isomers, and a simple and low-cost production method.
[0053] Example 4
[0054] A method for preparing silage includes the following steps:
[0055] Harvested selenium-enriched whole-plant corn (total selenium content 385 µg / kg) was chopped into 2 cm long stalks, and alfalfa was chopped into 5 cm long pieces. 600 kg of corn stalks and 200 kg of alfalfa were mixed in a 3:1 ratio, and simultaneously sprayed with 1.6 kg of compound fermentation agent. After thorough mixing, the mixture was baled using a silage baler and allowed to ferment naturally for at least 15 days until the pH reached <4.2, yielding selenium-enriched corn-alfalfa silage. It was found that the silage fermented with multiple microbial strains had a richer aroma and showed no abnormal fermentation.
[0056] Selenium-enriched corn silage was used for fattening feeder cattle. The cattle were fed conventionally during the early and middle stages, and their diet for the later 60-120 days consisted of 6 kg of concentrate, 1 kg of alfalfa hay, and 2 kg of silage. The concentrate consisted of 60% corn, 15% soybean meal, 11% distillers' grains, 4% soybeans, 4% rapeseed meal, 2% limestone powder, 1% baking soda, 1% salt, 0.9% slow-release urea, 0.6% dicalcium phosphate, and 0.5% molasses. The control group received selenium-enriched corn and alfalfa silage without any fermentation agent. Observations showed that the corn silage fermented with the compound fermentation agent had better palatability and a significantly reduced incidence of gastrointestinal diseases in beef cattle. After 45 days of feeding with silage fermented with the compound fermentation agent, the CLA content in beef increased to as high as 22.8 mg / g fat, while the CLA content in beef fed with conventional (control group) silage was 7.8 mg / g fat. Furthermore, the former contained 75.1% of the c9,t11-CLA isomer, while the latter (control group) contained only 51.2%. By fermenting corn and alfalfa silage with the compound fermentation agent of this invention and feeding it to beef cattle, beef rich in c9,t11-CLA can be obtained. The yield of CLA is high, the purity of c9,t11-CLA isomer is high, and the production method is simple and low in cost.
[0057] Example 5
[0058] A method for preparing silage includes the following steps:
[0059] Harvested selenium-enriched whole-plant corn (total selenium content of 385 µg / kg) is cut into 2 cm long stalk pieces. 2 kg of compound fermentation agent is evenly sprayed onto 1000 kg of corn stalks and mixed thoroughly. After being packed with silage using a silage baler, it is naturally fermented for more than 15 days until the pH is <4.2 to obtain selenium-enriched corn silage.
[0060] In this embodiment, two control groups were set up. During the preparation process, only Lactobacillus plantarum bacterial solution was added, or a mixture of Saccharomyces cerevisiae and Bacillus subtilis bacterial solution was added, respectively, to obtain selenium-enriched corn silage with two different fermentation agents.
[0061] The effects of three fermentation methods on the phytic acid content in selenium-enriched corn silage were determined. After 15 days of fermentation with a multi-strain compound inoculant or a single strain of Lactobacillus plantarum, the phytic acid content in selenium-enriched corn silage decreased by more than 50%, while the phytic acid content in the mixed fermentation group of Saccharomyces cerevisiae and Bacillus subtilis was less than 15%. Furthermore, the selenium-enriched corn silage fermented with the multi-strain compound inoculant exhibited a richer aroma and showed no abnormal fermentation.
[0062] The three groups of selenium-enriched corn silage were used for fattening feeder cattle. The feeder cattle were fattened using conventional methods in the early and middle stages, while the diet for the later stages (60-120 days) consisted of 6 kg of concentrate, 1 kg of alfalfa hay, and 2 kg of silage. The concentrate consisted of 60% corn, 15% soybean meal, 11% distillers' grains, 4% soybeans, 4% rapeseed meal, 2% limestone powder, 1% baking soda, 1% salt, 0.9% slow-release urea, 0.6% dicalcium phosphate, and 0.5% molasses. Observations showed that the selenium-enriched corn silage fermented with the compound fermentation agent had better palatability and a significantly reduced incidence of gastrointestinal diseases in beef cattle, while the selenium-enriched corn silage fermented with a single strain of *Lactobacillus plantarum* had slightly poorer palatability.
[0063] After 45 days of feeding with selenium-enriched corn silage fermented with a compound fermentation agent, the CLA content in beef increased to as high as 23.6 mg / g fat. Beef fed with selenium-enriched corn silage fermented only with *Lactobacillus plantarum* added had a CLA content of 20.5 mg / g fat, while beef fed with selenium-enriched corn silage fermented with a mixture of *Saccharomyces cerevisiae* and *Bacillus subtilis* had a CLA content of 7.3 mg / g fat. After 45 days of feeding with selenium-enriched corn silage fermented with a compound fermentation agent, the total selenium content in beef reached as high as 88 µg / kg, and the organic selenium content was 80 µg / kg. Beef fed with selenium-enriched corn silage fermented only with *Lactobacillus plantarum* added had a total selenium content as high as 82 µg / kg, and the organic selenium content was 73 µg / kg, while beef fed with selenium-enriched corn silage fermented with a mixture of *Saccharomyces cerevisiae* and *Bacillus subtilis* had a total selenium content of 58 µg / kg and an organic selenium content of 53 µg / kg.
[0064] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.
Claims
1. A compound fermentation agent, characterized in that, Lactobacillus plantarum ( Lactobacillus plantarum R6, brewer's yeast ( Saccharomyces cerevisiae ) and Bacillus subtilis ( Bacillus subtilis The composition includes Lactobacillus plantarum R6, which was deposited on March 22, 2024, at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30096. The *Saccharomyces cerevisiae* was deposited on January 1, 1952, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 2.216; the *Bacillus subtilis* was deposited on March 1, 1972, at the same center, with accession number CGMCC No. 1.
821.
2. A method for preparing the compound fermentation agent according to claim 1, characterized in that, Includes the following steps: Lactobacillus plantarum R6 strain was inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum R6 bacterial culture; Saccharomyces cerevisiae inoculated into malt extract culture medium and cultured at 25°C for 24 h to obtain Saccharomyces cerevisiae culture liquid; Bacillus subtilis strain was inoculated into nutrient broth culture medium and cultured at 30℃ for 24 h to obtain Bacillus subtilis bacterial culture; A compound fermentation agent was obtained by mixing Lactobacillus plantarum R6 bacterial solution, Saccharomyces cerevisiae bacterial solution and Bacillus subtilis bacterial solution in a volume ratio of 1:1:
1.
3. The preparation method according to claim 2, characterized in that, The bacterial concentrations of the *Lactobacillus plantarum* R6 culture, *Saccharomyces cerevisiae* culture, and *Bacillus subtilis* culture were 1×10⁻⁶. 8 CFU / mL.
4. The application of the compound fermentation agent according to claim 1 in the fermentation preparation of silage.
5. A type of silage, characterized in that, It is obtained by fermentation with the compound fermentation agent described in claim 1.
6. A method for preparing silage, characterized in that, Includes the following steps: The compound fermentation agent prepared by the method described in claim 3 is evenly sprayed onto selenium-enriched corn stalks, and then fermented until the pH is less than 4.2 to obtain silage.
7. The preparation method according to claim 6, characterized in that, Selenium-enriched corn stalks are in small segments of 1 cm to 3 cm.
8. The preparation method according to claim 6, characterized in that, The mass ratio of compound fermentation agent to selenium-enriched corn straw is 0.2%:1.