Compound microbial agent and application thereof in silage

Abstract

The application discloses a kind of compound microbial inoculant and its application in silage feed, belong to the field of microbial technology.The compound microbial inoculant is by Enterococcus faecalis ( Enterococcus faecalis )JM263 and Pentoecoccus pentosus ( Pediococcus pentosaceus )ZRS217 is compounded according to different proportions.The application is inoculated in corn stalk containing 65%~70% water content by different proportions of compound microbial inoculant, after 60 days of silage fermentation, the fermentation quality and nutritional value of silage feed are significantly improved.The compound microbial inoculant of the application has synergistic acid production and fiber degradation capacity, can effectively improve the fermentation quality of silage feed, improve the nutritional value and digestibility of silage feed, provides a new technical scheme for the high-quality silage preparation of straw roughage.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound microbial agent and its application in silage. Background Technology

[0002] Corn is one of my country's main food crops, producing a large amount of corn stalks every year. Currently, the main methods for treating corn stalks include direct return to the field, burning, and utilization as animal feed. Among these, silage is one of the important ways to utilize corn stalk resources. Silage is made by fermenting fresh plant material through microorganisms into feed that is easy to preserve and digest, and it has advantages such as high nutritional value, good palatability, and long shelf life.

[0003] However, traditional corn stalk silage technology still has many problems: (1) Low degradation efficiency. The cell wall structure of corn stalks is dense and rich in cellulose, hemicellulose and lignin, forming a natural anti-degradation barrier, resulting in low degradation efficiency under natural fermentation conditions; the stalks are difficult to soften and decompose effectively, limiting the digestion and absorption of nutrients by animals. (2) Natural silage relies on native microorganisms in the environment, whose types and quantities are unstable, resulting in uncontrollable fermentation process, long cycle and large quality fluctuations. Problems such as slow pH decrease and insufficient lactic acid accumulation often occur during fermentation, affecting the stability of silage. (3) Existing single microbial agents (such as adding only lactic acid bacteria) can improve the fermentation effect to a certain extent, but their ability to destroy the stalk fiber structure is limited, making it difficult to achieve efficient degradation and high-quality fermentation at the same time. In particular, the decomposition effect on lignocellulose components is weak, resulting in the silage still having high levels of neutral detergent fiber (NDF), acid detergent fiber (ADF) and lignin, and the nutritional value is not significantly improved. Meanwhile, during the silage process, if fermentation starts slowly or acid production is insufficient, it may lead to excessive consumption of soluble carbohydrates (WSC), reducing the energy value of the feed.

[0004] In recent years, compound microbial agents have gradually become a research hotspot in silage technology. Compound agents can leverage the synergistic effects of different strains to improve fermentation efficiency and feed quality. Currently, some studies have attempted to combine different lactic acid bacteria for straw silage, but the actual application results have not been ideal. Most compound agents struggle to simultaneously achieve effective destruction of straw fiber structure and significant improvement in fermentation quality during the silage process. On the one hand, while some strain combinations can promote lactic acid accumulation and lower pH, their degradation effect on straw cell walls is limited, resulting in still high ADF and lignin content in the silage. On the other hand, some strain combinations with strong fiber degradation capabilities have insufficient acid production efficiency, leading to slow fermentation and low lactic acid yield, affecting the stability and palatability of the silage.

[0005] Therefore, developing a highly efficient, safe, and stable compound microbial agent for the degradation of corn stalks and the preparation of silage remains one of the problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a compound microbial agent and its application in silage, which solves the problems of low fermentation efficiency, large loss of nutritional value, insufficient degradation of straw fiber, and unstable fermentation quality in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a compound microbial agent, including Enterococcus faecalis ( Enterococcus faecalis JM263 and Pediococcus pentosaceus ( Pediococcus pentosaceus ZRS217; The accession number of Enterococcus faecalis JM263 is CGMCC No. 38960, and the accession number of Pediococcus pentosaceus ZRS217 is CGMCC No. 38958; both were deposited on May 29, 2026 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The ratio of viable Enterococcus faecalis JM263 to Pediococcus pentosus ZRS217 is 1:3 to 3:1.

[0008] Preferably, the ratio of viable bacteria of Enterococcus faecalis JM263 to Pediococcus pentosus ZRS217 is 1:2, 1:1, or 2:1.

[0009] Preferably, the ratio of viable bacteria of Enterococcus faecalis JM263 to Pediococcus pentosus ZRS217 is 2:1.

[0010] Preferably, the composite microbial agent is a liquid agent, a solid agent, or a microcapsule agent.

[0011] The present invention also discloses the application of the above-mentioned composite microbial agent in the degradation of corn stalks.

[0012] This invention also discloses the application of compound microbial agents in the preparation of silage.

[0013] This invention also provides a method for preparing silage, comprising the following steps: (1) Chop fresh corn stalks into 1-2cm pieces and adjust the moisture content to 65%-70%; (2) Inoculate the above compound microbial agent into the corn stalks obtained in step (1) at an inoculation rate of 1-10%; (3) The corn stalks after inoculation in step (2) are put into a sealed container and anaerobic fermented at 20~35℃ for 40~80 days to obtain silage.

[0014] Preferably, the inoculation amount of the compound microbial agent in step (2) is 5%.

[0015] Preferably, the fermentation temperature in step (3) is 30°C and the fermentation time is 60 days.

[0016] The present invention also provides a silage, which is prepared by the above method.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The compound microbial agent provided by this invention (composed of Enterococcus faecalis JM263 and Pediococcus pentosus ZRS217 in a certain proportion) shows a significant synergistic effect in the degradation of corn stalks and the preparation of silage.

[0018] The experimental results showed that in the straw degradation experiment, the degradation rate of the compound microbial agent treatment group (up to 21.83%) was significantly higher than that of the single-strain treatment group (17.68%~19.56%) and the blank control group (7.29%). In the silage fermentation experiment, the pH value (lowest 3.67) and lactic acid content (highest 9.91%) of the compound microbial agent treatment group were better than those of the single-strain treatment group (pH 4.40~4.60, lactic acid 7.19~7.54%) and the blank control group (pH 4.83, lactic acid 6.26%).

[0019] Meanwhile, the compound microbial agent effectively degraded fiber components, reducing the content of acid detergent fiber (ADF) to 36.30% (compared to 40.07% in the control group), and also significantly reducing the content of neutral detergent fiber (NDF), with lignin content decreasing to 9.07%–9.17% (compared to 12.73% in the control group). Furthermore, the soluble carbohydrate (WSC) content in the compound microbial agent-treated group remained at a relatively high level of 8.77–9.00 g / 100g (compared to 9.57 g / 100g in the control group), indicating that the compound microbial agent of this invention can effectively degrade fibers while preserving soluble nutrients. Scanning electron microscopy images showed that the compound microbial treatment group (especially the 2:1 ratio) caused the most significant damage to the surface structure of straw, manifested as numerous cracks, pores, and loose fibers, which is beneficial for the release and absorption of nutrients. Attached Figure Description

[0020] Figure 1 This is a morphological diagram of Enterococcus faecalis JM263 of the present invention; Figure 2 This is a Gram-stained optical micrograph of Enterococcus faecalis JM263, as described in this invention. Figure 3This is a scanning electron microscope (SEM) image of Enterococcus faecalis JM263 of the present invention; Figure 4 This is a morphological diagram of Pediococcus pentosaceus ZRS217, which is the present invention. Figure 5 Gram-stained optical micrograph of Pediococcus pentosaceus ZRS217, as described in this invention; Figure 6 The image shown is a scanning electron microscope (SEM) image of Pediococcus pentosaceus ZRS217 of this invention. Figure 7 This is a bar chart comparing the degradation rates of corn straw in different treatment groups according to the present invention. Figure 8 These are scanning electron microscope (SEM) images of the surface microstructure of corn stalks from different treatment groups in this invention. Figure 9 The pH values ​​of corn stalk silage from different treatment groups in this invention were measured. Figure 10 The results of lactic acid content determination in corn stalk silage from different treatment groups of this invention; Figure 11 The results of neutral detergent fiber (NDF) content determination in corn stalk silage from different treatment groups of this invention; Figure 12 The results show the determination of acid detergent fiber (ADF) content in corn stalk silage from different treatment groups in this invention. Figure 13 The results of lignin content determination in corn stalk silage from different treatment groups of this invention; Figure 14 The results show the determination of soluble carbohydrate (WSC) content in corn stalk silage from different treatment groups in this invention. Figure 15 These are scanning electron microscope (SEM) images of the surface structure of corn stalk silage from different treatment groups according to the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments: Example 1 This example describes the screening, isolation, purification, identification, and preservation of Pediococcus pentosaceus ZRS217.

[0025] 1.1 Isolation and Identification of Enterococcus faecalis JM263 (1) Source of strain: Enterococcus faecalis JM263 was isolated from the intestinal contents of healthy yaks in the Qinghai-Tibet Plateau region.

[0026] (2) Separation and purification methods: Take approximately 1.0 g of fresh yak small intestine contents, add 9.0 mL of sterile physiological saline, and thoroughly shake to mix to prepare 10 -1 Diluent, serially diluted 10-fold to 10 -6 Take 10 -5 and 10 -6 100 μL of each diluent was spread onto MRS solid medium plates and anaerobically incubated at 37°C for 48 h. Single colonies of typical lactic acid bacteria with smooth surfaces, regular edges, and milky white color were picked and purified by repeated streaking 3-5 times to obtain pure cultures, which were named JM263.

[0027] (3) Morphological characteristics: Colony morphology (e.g.) Figure 1 On MRS agar plates, it forms round, raised, smooth, moist, and opaque milky-white colonies with neat edges, without pigment production or hemolytic zones; the colonies are uniform and dense in texture, consistent with the typical phenotypic characteristics of Enterococcus.

[0028] Gram staining (e.g.) Figure 2 After standard Gram staining, the bacteria are Gram-positive (purple), spherical or oval, mostly arranged in pairs or short chains, occasionally single; no spores, flagella or capsule structures were observed.

[0029] Scanning electron microscopy observation (e.g.) Figure 3 The bacteria are typically spherical to oval in shape, with a smooth and dense surface and tight intercellular connections. Some cells exhibit a typical "beaded" chain arrangement, which is highly consistent with the morphology of Enterococcus faecalis reported in the literature.

[0030] (4) Molecular biological identification: Genomic DNA was extracted from JM263 and amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') to obtain a 16S rRNA gene fragment of 1411 bp (SEQ ID NO.1).

[0031] The sequence was submitted to the NCBI database for comparative analysis, and the results showed that the sequence was similar to... Enterococcus faecalis The 16S rRNA gene sequence showed 99.65% homology. Based on its colony morphology, Gram staining characteristics, and physiological and biochemical properties, it was identified as Enterococcus faecalis (…). Enterococcus faecalis ), named Enterococcus faecalis ( Enterococcus faecalis JM263.

[0032] 1.2 Isolation and Identification of Pediococcus pentosaceus ZRS217 (1) Source of strain: Pediococcus pentosus ZRS217 was isolated from naturally fermented yak yogurt by farmers in Nagqu City, Tibet Autonomous Region.

[0033] (2) Separation and purification methods: Take 1.0g of naturally fermented yak yogurt sample, add it to 9.0mL of sterile physiological saline, and mix thoroughly by shaking to prepare 10 -1 Prepare diluents and serially dilute them 10-fold. Spread 100 μL of the appropriate dilution onto MRS solid medium plates and incubate at 37°C for 24–48 h. Select single colonies with regular morphology, neat edges, smooth surfaces, and a milky white or grayish white color, which are suspected to be lactic acid bacteria. Repeat streak purification 3–5 times to obtain a pure culture, which is named ZRS217.

[0034] (3) Morphological characteristics: Colony morphology (e.g.) Figure 1 ): Forms round or nearly round colonies on MRS solid medium. The colony surface is smooth and moist, with neat edges and opacity. Gram staining (e.g.) Figure 2 Gram-positive bacteria, appearing spherical or nearly spherical under a microscope, mostly arranged singly, in pairs, or in short chains, and do not form spores. Scanning electron microscopy observation (e.g.) Figure 3 The bacteria are spherical or ellipsoidal in shape, with relatively uniform cell morphology and a smooth surface, consistent with the typical morphological characteristics of Pediococcus strains. (4) Molecular biological identification: Genomic DNA was extracted from strain ZRS217, and its 16S rRNA gene sequence was amplified using the universal bacterial primers 27F 5'-AGAGTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3', yielding a 1427bp 16S rRNA gene sequence (SEQ ID NO.2).

[0035] The sequence was submitted to the NCBI database for BLAST comparison, and the results showed that strain ZRS217 was similar to... Pediococcus pentosaceus The 16S rRNA gene sequence similarity reached 99.72%. Based on colony morphology, microscopic morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis, strain ZRS217 was identified as *Pediococcus pentosaccharis*, and named *Pediococcus pentosaccharis*. Pediococcus pentosaceus ZRS217.

[0036] 1.3 Preservation of bacterial strains The two strains mentioned above were deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Enterococcus faecalis ( Enterococcus faecalis JM263, accession number: CGMCC No. 38960; Pediococcus pentosaceus ( Pediococcus pentosaceus ZRS217, accession number: CGMCC No.38958.

[0037] Example 2 This example demonstrates the preparation of a compound microbial inoculant.

[0038] 2.1 Preparation of single-strain seed culture: (1) Preparation of Enterococcus faecalis JM263 seed culture: The preserved JM263 strain was inoculated into MRS liquid medium and anaerobic cultured at 37℃ for 24 h to obtain the seed culture. The viable cell concentration was determined to be 1×10⁻⁶. 8 CFU / mL.

[0039] (2) Preparation of seed culture of Pediococcus pentosaceus ZRS217: The preserved strain ZRS217 was inoculated into MRS liquid medium and anaerobically cultured at 30℃ for 48 h to obtain the seed culture. The viable cell concentration was determined to be 1×10⁻⁶. 8 CFU / mL 2.2 Preparation of compound microbial inoculants: Compound microbial agents were prepared according to different ratios of live bacteria counts: (1) 1:2 ratio compound microbial agent: Take 1 part of JM263 seed liquid and 2 parts of ZRS217 seed liquid and mix them evenly; (2) 1:1 ratio compound microbial agent: Take 1 part of JM263 seed liquid and 1 part of ZRS217 seed liquid and mix them evenly; (3) 2:1 ratio compound microbial agent: Take 2 parts of JM263 seed liquid and 1 part of ZRS217 seed liquid and mix them evenly; The final viable cell concentration of each compound microbial agent was 1×10⁻⁶. 8 CFU / mL.

[0040] 2.3 Preparation of bacterial agent formulations (1) Liquid microbial agent: The above compound microbial agent is used directly as a liquid microbial agent. (2) Solid microbial agent: Mix the compound microbial agent with wheat bran carrier at a ratio of 1:10, dry at 30℃ until the moisture content is ≤10% and the viable count is ≥1×10⁻⁶. 6 CFU / g (3) Microencapsulated bacterial agent: Microcapsules were prepared by sodium alginate encapsulation method, with a particle size of 200~500μm and a viable count ≥1×10⁻⁶. 7 CFU / g.

[0041] Example 3 This example is a degradation test of corn stalks.

[0042] 3.1 Experimental Materials and Methods (1) Preparation of culture medium: Hutchison medium (NaCl 0.1g, NaNO3 2.5g, CaCl2 0.1g, FeCl3 0.01g, KH2PO4 1g, MgSO4·7H2O 0.3g, distilled water 1000mL), sterilized at 121℃ for 30min.

[0043] (2) Experimental design: 6 groups were set up, as follows.

[0044] CON group: 5% sterile water treatment (blank control); Group S1: Inoculated only with Enterococcus faecalis JM263; Group S2: Inoculated only with Pediococcus pentosaceus ZRS217; Group S3: JM263:ZRS217 = 1:2; S4 group: JM263:ZRS217 = 1:1; S5 group: JM263:ZRS217 = 2:1; (3) Test method: Accurately weighed corn stalks were added to 75 mL of Hutcheson's medium, sterilized at 120°C for 30 min, cooled to room temperature, and under aseptic conditions, inoculated with the corresponding bacterial strain seed culture at a 5% inoculation rate (1×10⁻⁶). 8The straw was incubated at 30℃ for 15 days (CFU / g) and each treatment was tested in triplicate. 5% sterile water was used as a blank control to observe the degradation of straw and measure the degradation index.

[0045] 3.2 Test Results (1) Degradation rate determination After the above fermentation treatment was completed, the corn stalk samples from each treatment group were taken out, gently rinsed with distilled water to remove the attached bacteria and culture medium residue, and then dried in a 60℃ oven to constant weight. The dry weight after fermentation was recorded as W1 (g); the mass of the dried stalks before fermentation was recorded as W0 (g). The degradation rate (D) of the stalks was calculated according to the formula below.

[0046] Straw degradation rate formula: D (%) = ; Wherein, D: straw degradation rate (%), W0: straw dry weight before fermentation (g), and W1: straw dry weight after fermentation (g).

[0047] Figure 7 The bar chart comparing the degradation rates of corn stalks shows that: CON group 7.29%, S1 group 19.56%, S2 group 17.68%, S3 group 17.54%, S4 group 21.17%, and S5 group 21.83%. Group S5 had the highest degradation rate at 21.83%, significantly higher than the blank control CON group and other treatment groups, followed by group S4. The single-strain treatment groups S1 and S2 were both significantly higher than the CON group; the degradation rate of group S3 was not significantly different from that of group S2. These results indicate that the compound microbial agent of this invention has excellent degradation ability for corn stalks.

[0048] Figure 8 These are scanning electron microscope (SEM) images of the surface structure of corn stalks from different treatment groups. The images show that: The surface of the CON group straw was generally flat and dense with regular texture. Only a few shallow cracks, depressions and scattered particles were observed, indicating that the structure of the corn straw remained basically intact under the condition of no bacterial inoculation and the degree of natural degradation was low.

[0049] Group S1 (Enterococcus faecalis JM263 single-strain treatment group): The straw surface was significantly rougher than that of Group CON. Under low magnification, more depressions, holes and irregular attachments were visible. Under high magnification, flaky peelings, granular debris and local surface damage were observed, suggesting that JM263 can have a certain erosive effect on the surface structure of corn straw.

[0050] Group S2 (Pediococcus pentosaceus ZRS217 single-strain treatment group): The straw surface still retains relatively obvious longitudinal texture and arrangement structure, but shallow grooves, wrinkles, fine cracks and a small number of holes appear locally. Compared with CON, the surface integrity of Group S2 is reduced, but the overall degree of damage is relatively limited, indicating that ZRS217 treatment alone can cause a certain degree of surface loosening and microstructural damage.

[0051] Group S3 (JM263:ZRS217 compound treatment group with a ratio of 1:2): The straw surface showed obvious wrinkling, lamellar peeling and irregular cracks. Under high magnification, the surface roughness was significantly increased, accompanied by the adhesion of fine particulate degradation products, indicating that this compound ratio can further damage the surface structure of the straw.

[0052] Group S4 (JM263:ZRS217 in a 1:1 ratio): The straw surface showed extensive roughening and loosening of structure, with local cracks, grooves and exposed fiber bundle boundaries; under 3000x magnification, the surface showed fine granular and honeycomb-like roughness, indicating that the equal ratio of the two strains had a significant degradation effect on the surface structure of the straw.

[0053] Group S5 (JM263:ZRS217 in a 2:1 ratio): The surface damage to the straw was most pronounced. Under low magnification, numerous scratches, cracks, granular deposits, and localized loose fibers were visible. Under 1000x magnification, many aggregated particles and debris deposits were observed. Under 3000x magnification, the straw surface exhibited significant roughness, looseness, and fragmentation, with obvious pores, granular fragments, and detached structures. These results indicate that the 2:1 ratio of JM263 to ZRS217 has a stronger destructive effect on the surface structure of corn straw, potentially promoting the exposure and degradation of straw cell wall components.

[0054] The above results show that the corn stalks in the inoculation treatment groups all exhibited varying degrees of surface roughening, increased cracking, pore formation, and lamellae peeling. Among them, the degree of damage in the compound treatment group was generally stronger than that in the single-strain treatment group, especially in the S5 group, which was the most obvious. This indicates that the combination of Enterococcus faecalis JM263 and Pediococcus pentosaceus ZRS217 can enhance the destruction and degradation effect on the surface structure of corn stalks.

[0055] Example 4 This example is a corn stalk silage fermentation experiment.

[0056] 4.1 Test Materials The corn stalks used in the experiment were freshly harvested. After removing soil, mold, and obvious impurities, they were chopped to about 1-2 cm using a chaff cutter. The chopped corn stalks were thoroughly mixed, and their initial moisture content was measured. The moisture content was then adjusted to 65%-70% as needed for use as silage raw material.

[0057] The strains used include Enterococcus faecalis ( Enterococcus faecalis JM263 and Pediococcus pentosaceus ( Pediococcus pentosaceus ZRS217. The two bacterial strains were inoculated into their respective liquid culture media for activation and culture. After culture, the bacterial cells were collected by centrifugation, washed 2-3 times with sterile physiological saline, and resuspended to prepare bacterial suspensions. The concentration of the bacterial suspensions was adjusted to 1×10⁻⁶. 8 CFU / mL was prepared as the inoculum for silage.

[0058] 4.2 Experimental Grouping Six treatment groups were set up, including a blank control group, a single-bacterial inoculation group, and a mixed-bacterial inoculation group. The specific grouping is shown in Table 1 below: Table 1 4.3 Silage Preparation Method Weigh out 2000 g of the mixed corn stalks and place them in clean containers. Spray sterile water or the corresponding bacterial suspension onto each experimental group, stirring thoroughly while spraying to ensure the bacterial solution adheres evenly to the surface of the corn stalks. Pack the treated corn stalks into polyethylene vacuum bags or silage fermentation bags, compact them to remove air, and then seal them using a vacuum sealer. Each treatment should have at least three biological replicates. Place the sealed silage bags in a dark, room-temperature environment for 60 days to ferment. After 60 days, open the bags and take samples for testing.

[0059] 4.4 Detection Indicators and Methods for Silage Samples 4.4.1 Fermentation quality testing (1) pH value measurement The pH value of the silage sample extract was directly measured using a pH meter. 10 g of fresh silage sample was taken, 90 mL of distilled water was added, the mixture was thoroughly mixed, allowed to stand for 30 min, filtered, and the filtrate was measured using a pH meter.

[0060] (2) Determination of lactic acid content The sample was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase was 0.1% phosphoric acid solution-methanol (95:5, v / v), flow rate was 1.0 mL / min, detection wavelength was 210 nm, column temperature was 30 ℃, and injection volume was 20 μL.

[0061] 4.4.2 Nutritional composition testing (1) Neutral detergent fiber (NDF) content Accurately weigh approximately 1 g of sample (m5) into a cylindrical beaker. Add 100 mL of neutral detergent, a few drops of decahydronaphthalene, and 0.5 g of anhydrous sodium sulfite. Gently shake to disperse the sample, preventing reflux and adhesion to the bottom. Heat to boiling within 5 minutes. Add 2 mL of heat-stable α-amylase solution and shake gently. Place the beaker on a condenser on an electric furnace and boil for 5-10 minutes, maintaining a gentle boil for 60 minutes. After boiling, remove the cylindrical beaker and pour the solution into a pre-weighed glass crucible (m6) mounted on a vacuum filtration flask for filtration. Transfer all residue from the beaker to the crucible and rinse with boiling water until the filtrate is neutral. Rinse twice with 20 mL of acetone and filter under vacuum. Place the glass crucible (m7) in a 105°C oven for 2 hours, then cool in a desiccator for 30 minutes and weigh until constant weight is achieved.

[0062] Formula for calculating the fiber content of neutral detergent: .

[0063] (2) Acid detergent fiber (ADF) content Accurately weigh approximately 1g of sample (m) into a cylindrical beaker, add 100mL of acidic detergent, a few drops of decahydronaphthalene, and 0.5g of anhydrous sodium sulfite. Place the beaker on a condenser on an electric furnace and boil for 5-10 minutes, maintaining a gentle boil for 60 minutes. After boiling, remove the beaker and pour the solution into a pre-weighed glass crucible (m1) mounted on a vacuum filtration flask for filtration. Transfer all residue from the beaker to the crucible and rinse with boiling water until the filtrate is neutral. Rinse twice with 20mL of acetone and filter. Place the glass crucible in a 105℃ oven for 2 hours, then cool in a desiccator for 30 minutes and weigh until constant weight (m2).

[0064] Formula for calculating acid detergent fiber content: .

[0065] (3) Cellulose content Hydrolyze the acid detergent fiber with 10 mL of 72% sulfuric acid for 3 h, add 45 mL of distilled water and leave at room temperature overnight. The next day, filter with a sintered glass funnel until the filtrate is neutral, place it in a 60°C oven to constant weight, place it in a desiccator at room temperature, and weigh it (m3).

[0066] Formula for calculating cellulose content: .

[0067] (4) Lignin content The remaining residue was placed in a muffle furnace and calcined at 550°C for 2.5 hours. When the temperature dropped to about 200°C, it was taken out, placed in a desiccator to room temperature, and weighed (m4).

[0068] Formula for calculating lignin content: .

[0069] (5) Soluble carbohydrate (WSC) content Take an appropriate amount of sample into a 100mL volumetric flask, add water to make up to the volume, and sonicate for 1 hour, then filter. Take 50mL of the filtrate into another 100mL volumetric flask, add 5mL of 6mol / L hydrochloric acid solution, and hydrolyze in a boiling water bath for 30 minutes. Adjust the pH to neutral and make up to 100mL. Take 5mL of alkaline copper tartrate solution A and 5mL of alkaline copper tartrate solution B into an Erlenmeyer flask, add 1mL of the sample hydrolysis solution, add two glass beads, heat to boiling, and titrate with glucose standard solution to the endpoint while maintaining boiling. Perform a blank calibration experiment at the same time.

[0070] The content of water-soluble carbohydrates is calculated using the formula: ; In the formula: X: Water-soluble carbohydrate content in the sample, in g / 100g; C: Concentration of glucose standard solution, in mg / mL; G: The volume of glucose standard solution consumed in the calibration of alkaline copper tartrate solution, in mL; V: The volume of glucose standard solution consumed in the sample titration, in mL; M: Sample weight, in grams; Vsample: The volume of the sample hydrolysate used for titration, in mL (1 mL in this experiment). V_fixed: The final volume of the sample hydrolysate, in mL (100 mL in this experiment).

[0071] This method is based on Fehling's reagent titration (alkaline copper tartrate method), which indirectly reflects the soluble carbohydrate content by determining the reducing sugar content in the sample. After acid hydrolysis, non-reducing sugars in the sample are converted into reducing sugars, and then titrated with a glucose standard solution. The soluble carbohydrate content in the sample is calculated based on the volume of glucose standard solution consumed.

[0072] 4.4.3 Structural Observation Indicators Scanning electron microscopy was used to observe the changes in the surface structure of corn stalks in different treatment groups, including the degree of damage to the epidermal wax layer, the degree of loosening of fiber bundles, changes in pore structure, and cell wall degradation, in order to verify the destructive and degradative effects of the strain on the fiber structure of corn stalks.

[0073] 4.5 Analysis of Experimental Results 4.5.1 Fermentation quality test results: (1) pH measurement results are as follows Figure 9As shown, the pH values ​​were 4.83 for group CON, 4.4 for group S1, 4.6 for group S2, 4.13 for group S3, 3.80 for group S4, and 3.67 for group S5. Compared with the blank control group (CON), the pH values ​​of all treatment groups inoculated with the bacterial strains were significantly lower, indicating that the strains could effectively promote acid production during silage fermentation. Among them, the pH values ​​of the compound bacterial treatment groups (S3, S4, and S5) were lower than those of the single bacterial treatment groups (S1 and S2), indicating that the combination of the two strains had a synergistic acid-producing effect. The results show that the compound microbial agent of the present invention can significantly reduce the pH value of silage, with the 2:1 ratio (group S5) showing the best effect, reducing the pH value to 3.67, which is about 24% lower than that of the blank control group.

[0074] (2) Results of lactic acid content determination are as follows Figure 10 As shown, the lactic acid content was 6.26% in group CON, 7.54% in group S1, 7.19% in group S2, 7.28% in group S3, 9.24% in group S4, and 9.91% in group S5. Compared with the blank control group, the lactic acid content of all inoculated strains was significantly increased. Among the single-strain treatment groups, the lactic acid content of group S1 (inoculated only with JM263) was 7.54%, higher than that of group S2 (7.19%). In the compound microbial treatment groups, the lactic acid content increased with the increase of the proportion of JM263. The results show that the compound microbial agent of the present invention can significantly increase the lactic acid content in silage, with the 2:1 ratio (group S5) showing the strongest acid-producing ability, reaching 9.91%, which is 58.3% higher than the blank control group and 31.4-37.8% higher than the single-strain treatment groups, indicating that the combination of Enterococcus faecalis and Pediococcus pentosaceus has a significant synergistic acid-producing effect.

[0075] 4.5.2 Results of nutrient composition determination (1) Results of neutral detergent fiber (NDF) content determination are as follows Figure 11 As shown, the NDF content was 56.87% in group CON, 47.13% in group S1, 51.17% in group S2, 47.40% in group S3, 45.73% in group S4, and 43.70% in group S5. Compared with the blank control group, the NDF content in all inoculated strain treatment groups was significantly reduced, indicating that the strains can effectively degrade fibrous materials in corn stalks. This demonstrates that the compound microbial agent of the present invention can significantly reduce the neutral detergent fiber content in corn stalks, with the 2:1 ratio (group S5) showing the best degradation effect, reducing the NDF content to 43.70%, which is about 23.2% lower than the blank control group and 7.3-14.6% lower than the single-strain treatment group. This indicates that the compound microbial agent has a significant degradation effect on non-structural carbohydrates such as hemicellulose and pectin, which is beneficial to improving the digestibility and nutritional value of feed.

[0076] (2) Results of Acid Detergent Fiber (ADF) Content Determination Figure 12As shown, the ADF content was 40.07% in group CON, 37.37% in group S1, 38.37% in group S2, 37.13% in group S3, 37.6% in group S4, and 36.3% in group S5. Compared with the blank control group, the ADF content in all inoculated strain treatment groups was reduced, indicating that the compound microbial agent of the present invention can effectively degrade cellulose and lignin components in corn straw. Among them, the 2:1 ratio (group S5) showed the best effect, with the ADF content decreasing to 36.3%, which was 9.4% lower than the blank control group and 2.1-4.6% lower than the single-strain treatment group. This indicates that the compound microbial agent of the present invention has a good degradation ability for structural fibers, can destroy the plant cell wall structure, and improve feed availability.

[0077] (3) Results of lignin content determination are as follows Figure 13 As shown, the lignin content was 12.73% in group CON, 10.17% in group S1, 10.80% in group S2, 9.07% in group S3, 9.13% in group S4, and 9.17% in group S5. Lignin is a difficult-to-degrade component of plant cell walls, and its content directly affects feed digestibility. Compared with the blank control group, the lignin content of all inoculated strains was significantly reduced. Among them, the 1:2 ratio (group S3) showed the best degradation effect, with the lignin content decreasing to 9.07%, a reduction of 28.75% compared with the blank control group and a reduction of 10.8-16.0% compared with the single-strain treatment group. This indicates that Pediococcus pentosaceus (ZRS217) has a unique advantage in lignin degradation, and its dissociation effect on the lignin-carbohydrate complex is more significant when its proportion in the compound inoculant is high.

[0078] (4) Results of soluble carbohydrate (WSC) content determination are as follows Figure 14 As shown, the WSC content was 9.57 g / 100g for group CON, 9.40 g / 100g for group S1, 9.53 g / 100g for group S2, 9.00 g / 100g for group S3, 8.83 g / 100g for group S4, and 8.77 g / 100g for group S5. Compared with the blank control group, the WSC content in the inoculated groups was slightly lower, which is because the strain consumed some carbohydrates during fermentation. The difference in WSC content among the treatment groups was not significant, indicating that the compound microbial agent of this invention utilizes carbohydrates rationally and efficiently, fully utilizing the substrate for lactic acid fermentation without causing excessive carbohydrate consumption that would affect fermentation quality.

[0079] 4.5.3 Scanning electron microscopy observation results The surface microstructure changes of corn straw silage from the different treatment groups were observed using scanning electron microscopy after 60 days of fermentation. The results are as follows: Blank control group (CON): The surface of corn stalks was relatively intact, the epidermal structure was relatively continuous, the fibers were tightly arranged, and only a few cracks, local surface peeling and slight pore structure were visible; indicating that although corn stalks without bacterial inoculation undergo some natural softening and degradation during silage, the overall cell wall structure remains relatively intact.

[0080] Group S1 (Enterococcus faecalis JM263 single-strain treatment group): The surface of corn stalks showed more obvious structural damage than that of Group CON; under low magnification, the epidermis was peeled off and the fiber bundles became loosely arranged; under medium and high magnification, more pores, cracks and cell wall breaks were visible, and some areas showed honeycomb or layered erosion structures, indicating that JM263 can promote the destruction of the epidermis and cell wall structure of corn stalks during silage.

[0081] Group S2 (Pediococcus pentosaceus ZRS217 single-strain treatment group): obvious longitudinal cracks and transverse fractures appeared on the surface of corn stalks, and local fiber bundles were separated; under high magnification, pores, wrinkled textures and a small amount of bacteria or granular attachments were visible on the surface of the stalks; indicating that ZRS217 treatment can weaken the dense structure of the surface of the stalks, causing the fiber tissue to become loose and ruptured to a certain extent.

[0082] Group S3 (JM263:ZRS217 in a 1:2 ratio of compound microbial treatment): The damage to the surface of corn stalks was further aggravated; under low magnification, large-scale cracking and peeling of the stalk surface could be observed; under medium magnification, obvious crack expansion, epidermal lifting and local collapse were visible; under high magnification, granular aggregates were visible attached to the stalk surface, and the cell wall surface was significantly roughened, indicating that the compound microbial treatment enhanced the destructive effect on the waxy layer of the stalk epidermis and the cell wall structure.

[0083] Group S4 (JM263:ZRS217 in a 1:1 ratio of compound bacteria treatment): The straw surface showed more obvious lamellar peeling and fragmentation characteristics; low and medium magnification observations revealed a large number of flaky fragments and loose fiber structures, and the original surface integrity of the straw was significantly reduced; under high magnification, large pores, fiber bundle separation, and bacterial particle attachment were visible, indicating that the equal ratio of the two strains can significantly promote the loosening of the straw cell wall structure and the formation of pores.

[0084] Group S5 (JM263:ZRS217 in a 2:1 ratio): The microstructure of corn stalks was most significantly damaged; under low magnification, extensive breakage, peeling, and exposure of fiber bundles were visible on the stalk surface; under medium magnification, large amounts of lamellar cell wall fragments accumulated, and the surface structure was significantly loose; under high magnification, numerous pores, depressions, granular attachments, and suspected bacterial structures were visible, and the stalk surface exhibited significant roughening, porosity, and fragmentation. These results indicate that a 2:1 ratio of JM263 to ZRS217 has the strongest destructive effect on the epidermis, fiber bundles, and cell wall structure of corn stalks.

[0085] The above results indicate that inoculation with JM263, ZRS217, and their compound inoculants can all disrupt the surface structure of corn stalks to varying degrees, manifested as rupture of the epidermal wax layer, increased cracks, porosity, loosening of fiber bundles, lamellar peeling, and cell wall fragmentation. The compound inoculant treatment group showed a stronger overall degree of disruption than the single-strain treatment group, with group S5 exhibiting the most significant effect. This suggests that Enterococcus faecalis JM263 and Pediococcus pentosaceus ZRS217 have a synergistic degradation effect during corn stalk silage fermentation, promoting the loosening of the stalk fiber structure and increasing the degree of biodegradation of corn stalks.

[0086] The foregoing has provided a detailed description of the compound microbial agent disclosed in this invention and its application in silage. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. A compound microbial agent, characterized in that, Including Enterococcus faecalis ( Enterococcus faecalis JM263 and Pediococcus pentosaceus ( Pediococcus pentosaceus ZRS217; the accession number of Enterococcus faecalis JM263 is CGMCC No. 38960, and the accession number of Pediococcus pentosaccharis ZRS217 is CGMCC No. 38958; the ratio of viable counts of Enterococcus faecalis JM263 and Pediococcus pentosaccharis ZRS217 is 1:3 to 3:

1.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of viable Enterococcus faecalis JM263 to Pediococcus pentosus ZRS217 is 1:2, 1:1, or 2:

1.

3. The compound microbial agent according to claim 2, characterized in that, The ratio of viable Enterococcus faecalis JM263 to Pediococcus pentosus ZRS217 was 2:

1.

4. The compound microbial agent according to any one of claims 1-3, characterized in that, The compound microbial agent is a liquid agent, a solid agent, or a microcapsule agent.

5. The application of the compound microbial agent as described in any one of claims 1-3 in the degradation of corn stalks.

6. The application of the compound microbial agent as described in any one of claims 1-3 in the preparation of silage.

7. A method for preparing silage, characterized in that, Includes the following steps: (1) Chop fresh corn stalks into 1-2cm pieces and adjust the moisture content to 65%-70%; (2) The compound microbial agent according to any one of claims 1-3 is inoculated into the corn stalks obtained in step (1) at an inoculation amount of 1-10%; (3) The corn stalks after inoculation in step (2) are put into a sealed container and anaerobic fermented at 20~35℃ for 40~80 days to obtain silage.

8. The method for preparing silage according to claim 7, characterized in that, The inoculation amount of the compound microbial agent in step (2) is 5%.

9. The method for preparing silage according to claim 7, characterized in that, The fermentation temperature in step (3) is 30°C and the fermentation time is 60 days.

10. A type of silage, characterized in that, It is prepared by the method described in any one of claims 7-9.

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