Leuconostoc mesenteroides for highland barley silage fermentation and application thereof

Abstract

The application belongs to the technical field of sorghum silage, and particularly relates to a leuconostoc mesenteroides for sorghum silage fermentation and application thereof. Leuconostoc mesenteroides The leuconostoc mesenteroides for sorghum silage fermentation (R-12) is a strain with a preservation number of CGMCC No. 37525 and a preservation date of January 26, 2026. The R-12 is obtained by separation in the sorghum silage, and the strain can rapidly colonize and regulate the silage flora in the early stage of silage, and improve the quality of silage feed. The strain has certain tolerance to artificial gastric juice and artificial intestinal juice. The strain is suitable for low-sugar raw materials, such as sorghum ears and whole-plant sorghum silage, can improve the palatability and nutritional value of feed, and prolong the storage time of silage.

Description

Technical Field

[0001] This invention belongs to the field of sorghum silage technology, specifically relating to a Leuconostoc mesenteroides strain for sorghum silage fermentation and its application. Background Technology

[0002] Sorghum is an ideal feed crop to replace corn. However, sorghum has a relatively high content of anti-nutritional factors, resulting in relatively low nutritional value of sorghum silage. Adding lactic acid bacteria fermentation agents during silage preparation can improve the quality of sorghum silage and extend its storage time. Lactic acid bacteria inoculants can promote homo- or hetero-lactic acid fermentation, rapidly accumulating lactic acid and lowering the pH to 3.8-4.2, thereby effectively inhibiting the growth of putrefactive microorganisms such as Enterobacteriaceae, yeasts, and molds, thus improving silage quality and reducing dry matter and nutrient loss.

[0003] Currently, existing lactic acid bacteria fermentation agents used for silage, such as *Lactobacillus plantarum*, *Pediococcus pentosaceus*, *Enterococcus faecalis*, and *Lactobacillus brunelli*, are suitable for corn silage systems. However, sorghum differs significantly from corn in terms of nutritional composition, buffering capacity, and the content of plant phenolic substances. Therefore, existing lactic acid bacteria fermentation agents often suffer from weak colonization, delayed acid production, or unstable antibacterial effects in sorghum silage. Thus, there is a need to find lactic acid bacteria fermentation agents suitable for sorghum silage. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides Leuconostoc mesenteroides for sorghum silage fermentation and its applications.

[0005] To facilitate understanding of this invention, the materials used in this invention and their abbreviations are listed below: Dry matter, abbreviated as DM. Crude fat, abbreviated as EE. Crude protein, abbreviated as CP. Neutral detergent fiber, abbreviated as NDF. Acid detergent fiber, abbreviated as ADF. Acid detergent lignin, abbreviated as ADL. Crude ash, abbreviated as Ash. Non-cellulose carbohydrates, abbreviated as NFC. Water-soluble carbohydrates, abbreviated as WSC. Ammonia nitrogen, abbreviated as NH3-N. Total nitrogen, abbreviated as TN. Lactic acid, abbreviated as LA. Acetic acid, abbreviated as AA.

[0006] The first objective of this invention is to provide a Leuconostoc mesenteroides strain for sorghum silage fermentation, wherein the Leuconostoc mesenteroides strain is R-12, with accession number CGMCC No. 37525 and accession date of January 26, 2026.

[0007] A second object of the present invention is to provide a fermentation agent for sorghum silage fermentation, made from the R-12 strain and auxiliary materials. Alternatively, it may be made from the fermentation products of the R-12 strain and auxiliary materials.

[0008] Preferably, the excipient is one or more of the following: carrier, diluent, protectant, nutrient aid, dispersant, solubilizer, granulator, excipient, or microbial agent.

[0009] Preferably, the diluent is selected from one or more of bentonite, starch, maltodextrin, and Tween-80.

[0010] The protective agent is selected from one or more of skim milk powder, glucose, and glycerin.

[0011] The granulating agent is selected from one or more of starch and maltodextrin.

[0012] The co-solvent is selected from one or more of water and physiological saline.

[0013] Preferably, the viable count of strain R-12 in the fermentation agent is 1×10⁻⁶. 7 CFU / g ~ 1×10 9 CFU / g.

[0014] Preferably, the viable count of strain R-12 in the fermentation agent is 1.5 × 10⁻⁶. 8 CFU / g.

[0015] A third objective of this invention is to provide the application of Leuconostoc mesenteroides or a fermentation agent for sorghum silage fermentation in the preparation of silage, comprising at least one of the following: (1) Increase the soluble sugar content of silage.

[0016] (2) Improve the palatability of silage.

[0017] (3) Reduce the ammonia nitrogen / total nitrogen ratio in silage to reduce protein loss.

[0018] Preferably, the silage is wet sorghum ear silage or whole sorghum silage.

[0019] Bioinformatics Preservation Instructions The strains involved in this invention are as follows: The described Leuconostoc mesenteroides strain is R-12, deposited on January 26, 2026, at the China General Microbiological Culture Collection Center, and the recommended classification name is Leuconostoc mesenteroides. Leuconostoc mesenteroides The collection number is CGMCC No.37525, and the collection address is No.1, Beichen West Road, Chaoyang District, Beijing.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The *Leuconostoc mesenteroides* strain R-12 used in sorghum silage fermentation of this invention has the preservation number CGMCC No. 37525 and the preservation date of January 26, 2026. The pH of strain R-12 can drop below 4.5 after 8 hours of fermentation and reach 4.2 after 24 hours. It can rapidly accumulate lactic acid in the early stages of silage, quickly creating an acidic environment that inhibits the growth of harmful microorganisms such as *Enterobacteria* and yeast, effectively preventing secondary fermentation and reducing dry matter and nutrient loss. During sorghum silage fermentation, strain R-12 can effectively preserve or increase the soluble sugar content, providing ruminants with more directly usable energy substrates and improving the nutritional value of the feed.

[0021] The R-12 strain of this invention significantly improves the quality of silage fermentation. When used in sorghum silage, the R-12 strain maintains a consistently low NH3-N / TN ratio throughout the silage process, effectively inhibiting protein enzymatic hydrolysis and deammoniation, and reducing ammonia nitrogen production. Simultaneously, the silage achieves an excellent sensory quality score, exhibiting a pleasant aromatic fruity flavor, effectively improving the palatability and feed value of the silage.

[0022] The R-12 strain of the present invention has a certain tolerance to artificial gastric juice and artificial intestinal juice, and can effectively reach the animal intestine in a live state, promoting the digestion and absorption of nutrients in silage by ruminants, thereby obtaining more nutrients for growth and production, and improving the animal's feed utilization efficiency.

[0023] The R-12 strain of this invention does not have bile salt tolerance and cannot survive and colonize in the environment for a long time after being excreted with animal feces. It will not disturb the soil microbial ecology, has high biosafety, and is environmentally friendly. Attached Figure Description

[0024] Figure 1 This is a Gram staining diagram of R-12 of the present invention.

[0025] Figure 2 This is a colony morphology diagram of R-12 of the present invention on MRS agar medium.

[0026] Figure 3 This is a growth curve diagram of R-12 of the present invention.

[0027] Figure 4 This is a growth curve diagram of R-12 of the present invention at different temperatures.

[0028] Figure 5 The diagram shows the acid production rate curve of R-12 in this invention.

[0029] Figure 6 This is a diagram showing the relative abundance of horizontal microbial communities during the wet storage of sorghum ears in Example 3 of the present invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0032] The API 20NE kit used in this invention was purchased from bioMérieux, France, catalog number CN2005010. The ZYM kit was purchased from bioMérieux, France, catalog number 25200.

[0033] The sorghum used in this invention is the Jinnuo No. 3 variety, planted in the experimental field of the Dongbai Experimental Base of the Sorghum Research Institute of Shanxi Agricultural University, and managed normally until maturity. At the waxy ripening stage, the entire sorghum plant was harvested, leaving a stubble height of 10cm, to obtain the whole sorghum plant. Sorghum ears were collected at the waxy ripening stage to obtain the sorghum ears.

[0034] Example 1 Obtaining Leuconostoc mesenteroides for sorghum silage fermentation comprises the following steps: 1. Preparation of whole-plant sorghum silage Whole sorghum plants were coarsely crushed to 2.5cm using a chaff cutter and mixed thoroughly to obtain whole sorghum silage material. 4kg of whole sorghum silage material was then packed into 5L silage-specific plastic buckets equipped with screw caps. During filling, the silage was compacted every 1 / 3 of the way to expel air from the gaps between the materials. After filling, the top of the material was compacted, resulting in a final compacted density of 760kg / m³. 3 After filling, immediately tighten the lid to ensure the inside of the silage is in an anaerobic, sealed state. Store the sealed silage bins indoors in a dark environment. After 60 days, open the bins and take samples to obtain sorghum silage samples.

[0035] 2. Screening of silage strains Under aseptic conditions, 10g of sorghum silage sample was weighed and added to 90mL of sterile physiological saline. The sample was then placed on a shaker at 4℃ and 150rpm for 2 hours and shaken at low temperature. The mixture was filtered to obtain the silage liquid. The silage liquid was then diluted 10-fold with sterile physiological saline, with dilutions of 100, 100, and 100% being used as dilutions. -1 and 10 -2 100 μL of each dilution solution was evenly spread on MRS solid culture medium plates and incubated upside down in a 37℃ constant temperature incubator for 48 h to obtain silage plates.

[0036] Select morphologically typical and well-grown single colonies from silage plates and streak them onto MRS solid medium plates for purification, incubating overnight at 37°C. Repeat the streak purification process three times to obtain single purified colonies with consistent morphology. Inoculate each purified single colony into 5 mL of MRS broth and incubate at 37°C and 150 rpm for 2 days with shaking to allow for sufficient enrichment of the strain, thus obtaining the silage strain.

[0037] The bacterial culture of the silage strain was divided into two portions. One portion was used for 16S rRNA gene identification, and the other portion was mixed with physiological saline containing 50% glycerol at a volume ratio of 1:1 and stored in a refrigerator at -80°C.

[0038] 3. Microbiological characteristics of silage strains (1) Colony morphology: The silage strain was spread on MRS solid medium and incubated at 37°C for 24 hours. The single colony was round, with a diameter of 1 mm to 2 mm, milky white, opaque, moist and smooth, and with neat and regular edges.

[0039] (2) Growth characteristics: The silage strain was inoculated in MRS liquid medium and cultured at 37°C. The strain entered the logarithmic growth phase 2 hours after inoculation and the growth tended to stabilize after 8 hours.

[0040] 4. Molecular biological identification: Total DNA was extracted from the silage strain using a bacterial genomic DNA extraction kit. Using the total DNA as a template, PCR amplification was performed using universal bacterial primers 27F and 1492R to obtain PCR products. The nucleotide sequence of primer 27F was 5΄-AGAGTTTGATCCTGGCTCAG-3΄, and the nucleotide sequence of 1492R was 5΄-GGTTACCTTGTTACGACTT-3΄. After verification by agarose gel electrophoresis, the PCR products were subjected to Sanger sequencing to obtain the 16S rDNA sequence. The 16S rDNA sequence was compared and analyzed in the NCBI database. The results showed that the 16S rDNA sequence of the silage strain shared >99% homology with that of *Leuconostoc mesenteroides* strain 4316, thus identifying the isolated strain as *Leuconostoc mesenteroides*, named R-12.

[0041] Example 2 The biological activity identification of Leuconostoc mesenteroides for sorghum silage fermentation includes the following steps: 1. The form of the R-12 Single colonies of R-12 were selected and smeared for Gram staining: primary staining with crystal violet for 1 min, mordanting with iodine solution for 1 min, destaining with 95% ethanol for 30 s, and counterstaining with safranin for 1 min. After rinsing with deionized water and drying, the morphology of R-12 cells was observed under a 100x oil immersion microscope. The morphology of R-12 cells is as follows. Figure 1 As shown in the figure. The results showed that R-12 cells were short rod-shaped or coccobacillus-shaped and Gram-positive.

[0042] Use an inoculation loop to streak one loopful of activated R-12 bacterial solution onto MRS agar medium. Incubate at 37°C for 24 hours, then observe the colony morphology. The colony morphology of R-12 is shown below. Figure 2 As shown in the figure. The results showed that the R-12 colonies were round, 1 mm to 2 mm in diameter, milky white, opaque, with a moist and smooth surface and regular edges.

[0043] 2. Growth curve The concentration of the activated R-12 bacterial culture was adjusted to 1×10⁻⁶. 8 CFU / mL was inoculated into MRS broth medium at a volume fraction of 1%, and incubated statically at 37°C. Samples were taken at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 20h, 24h, 28h, 32h, 36h, and 48h after inoculation. The absorbance of the MRS broth medium at OD600 was measured using a microplate reader. Triple replicates were performed at each time point. The growth curve of R-12 is shown below. Figure 3 As shown in the figure. The results indicate that R-12 enters the logarithmic growth phase after 2 hours and reaches the plateau phase after 8 hours.

[0044] 3. Growth curves at different temperatures The concentration of the activated R-12 bacterial culture was adjusted to 1×10⁻⁶. 8 CFU / mL was inoculated into MRS broth medium at a volume fraction of 1%, and the culture was statically incubated at 5℃, 15℃, 25℃, and 35℃ for 24 h. The OD600 value of the fermentation broth was measured, and three replicates were set up for each treatment. The growth curves of R-12 at different temperatures are shown below. Figure 4 As shown in the figure. The results indicate that strain R-12 grows most vigorously at 25℃.

[0045] 4. Acid production rate curve The concentration of the activated R-12 bacterial culture was adjusted to 1×10⁻⁶. 8CFU / mL was inoculated into MRS broth medium at a volume fraction of 1%, and incubated statically at 37°C. The pH of the MRS broth medium was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, and 48h after inoculation. The acid production rate curve of R-12 is shown below. Figure 5 As shown in the figure. The results showed that the pH value dropped below 4.5 8 hours after R-12 inoculation and reached 4.2 after 24 hours, indicating that the strain has a fast acid production rate, which is conducive to the rapid formation of an acidic environment in the early stage of silage.

[0046] 5. Carbon source analysis can be utilized. The carbon source utilization ability of strain R-12 was detected using the API 20NE kit. The specific method is as follows: a bacterial suspension was prepared with sterile physiological saline and inoculated into each well of the reagent strip. After incubation at 37°C, the results were determined based on natural color development or color changes after the addition of reagents. The results are shown in Table 1. Strain R-12 can utilize various sugars such as glucose, mannitol, and mannose to produce acid, but cannot utilize carbon sources such as arabinose, maltose, and potassium gluconate. It can hydrolyze aesculin and urea, but cannot reduce nitrates and does not produce indole.

[0047] Table 1 Physicochemical characteristics of strain R-12 In the table, "+" indicates a positive result, "-" indicates a negative result, and " / " indicates that the item is not specified.

[0048] 6. Enzyme production characteristics The enzyme production characteristics of R-12 were detected using the API ZYM kit. Bacterial suspensions with turbidity equivalent to McFarland No. 5-No. 6 were prepared. 65 μL of the bacterial suspension was added to each reaction vessel of the reagent strip, and the mixture was incubated at 37℃ for 4 h. Then, one drop each of ZYM A and ZYM B reagents were added, and the colorimetric reactions were observed. The results are shown in Table 1. Strain R-12 exhibited multiple enzyme activities, including alkaline phosphatase, esterase, lipoesterase, leucine aromatic aminoaminase, valine aromatic aminoaminase, naphthol-AS-BI-phosphatase, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase, and β-glucosidase, but lacked β-uronic acid aglycone, trypsin, chymotrypsin, α-mannosidase, N-acetyl-glucosaminease, and β-fucosidease activities.

[0049] 7. Test for resistance to artificial gastric fluid The concentration of the activated R-12 bacterial culture was adjusted to 1×10⁻⁶. 7CFU / mL, 1 mL was inoculated into 9 mL of artificial gastric juice medium with a pH of 1.2, and incubated at 37°C for 1 hour. The viable bacterial count was then determined using the plate spread method. Five replicates were set up. The artificial gastric juice medium was prepared according to the following steps: 2 g / L sodium chloride was added to MRS liquid medium, the pH was adjusted to 1.2 with concentrated hydrochloric acid, and the medium was autoclaved at 121°C for 15 min. Before use, 1 g / L of filtered and sterilized pepsin was added. The results showed that, compared to 0 h, the survival rate of strain R-12 after 1 h of treatment in artificial gastric juice was 15.48% ± 1.73%, indicating that this strain possesses a certain degree of tolerance to artificial gastric juice.

[0050] 8. Test for resistance to artificial intestinal fluid The concentration of the activated R-12 bacterial culture was adjusted to 1×10⁻⁶. 7 CFU / mL, 1 mL was inoculated into 9 mL of artificial intestinal fluid culture medium with a pH of 7.0, and incubated at 37℃ for 4 h. The viable count was determined using the plate spread method, with 5 replicates. The artificial intestinal fluid culture medium was prepared as follows: 5 g / L sodium chloride was added to MRS liquid medium, the pH was adjusted to 7.0 with sodium hydroxide solution, and the medium was autoclaved at 121℃ for 20 min. Before use, filtered and sterilized trypsin stock solution was added to a concentration of 1 g / L. The results showed that compared to 0 h, the survival rate of R-12 in artificial intestinal fluid was 59.29% ± 1.52%, indicating that R-12 has good tolerance to artificial intestinal fluid.

[0051] 9. Test for tolerance to artificial bile salts The concentration of the activated R-12 bacterial culture was adjusted to 1×10⁻⁶. 7 CFU / mL, 1 mL was inoculated into 9 mL of sterile physiological saline containing 0.3 g / 100 mL porcine bile salts, and incubated at 37 °C for 3 h. The viable bacterial count was determined by plate spread method, with 5 replicates. The results showed that, compared with 0 h, strain R-12 did not exhibit bile salt tolerance at a concentration of 0.3 g / 100 mL bile salts.

[0052] Example 3 The application of Leuconostoc mesenteroides in the preparation of sorghum silage includes the following steps: The sorghum ears were coarsely crushed using a pulverizer and passed through a 1cm sieve to obtain sorghum ear particles. The R-12 bacterial solution was then added at a concentration of 1.5 × 10⁻⁶. 8 The sorghum ears were sprayed with CFU / kg and mixed evenly. They were then packed into polyethylene bags for silage, sealed with a vacuum sealer to remove all air and create an anaerobic environment. The bags were fermented at room temperature in the dark for 112 days to obtain wet sorghum ears, which were designated as the SEL group.

[0053] Example 4 The application of Leuconostoc mesenteroides in the preparation of sorghum silage includes the following steps: The amount of R-12 bacterial solution used in Example 3 was adjusted to 1×10 7 CFU / g, with other conditions remaining the same as in Example 3, to prepare sorghum ears for wet storage.

[0054] Example 5 The application of Leuconostoc mesenteroides in the preparation of sorghum silage includes the following steps: The amount of R-12 bacterial solution used in Example 3 was adjusted to 1×10 9 CFU / g, with other conditions remaining the same as in Example 3, to prepare sorghum ears for wet storage.

[0055] Examples 4 and 5 have similar properties to those of sorghum ears stored in wet conditions as in Example 3.

[0056] Comparative Example 1 The application of Leuconostoc mesenteroides in the preparation of sorghum silage includes the following steps: The R-12 bacterial solution in Example 3 was replaced with water, and the other conditions were kept the same as in Example 3 to prepare sorghum ear wet storage, denoted as SEC.

[0057] To illustrate the beneficial effects of the present invention, the following experiments were also conducted.

[0058] I. Indicator Testing for Wet Storage of Sorghum Ears The following indicators were measured on the sorghum ears prepared in Example 3 and Comparative Example 1 after wet storage on days 0, 3, 14, 56, and 112: (1) Sensory evaluation: The sensory quality of wet-stored sorghum ears was evaluated using the sensory evaluation standards for fermented feed issued by the German Agricultural Association. The specific scoring details are shown in Table 2.

[0059] Table 2 Sensory Evaluation Criteria (2) Nutrient determination: Dry matter was determined according to the method in GB / T6435-2014 "Determination of Moisture in Feed". Crude protein was determined according to the method in GB / T6432-2018 "Determination of Crude Protein in Feed - Kjeldahl Method". Crude fat was determined according to the method in GB / T6433-2006 "Determination of Crude Fat in Feed". Neutral detergent fiber was determined according to the method in GB / T20806-2022 "Determination of Neutral Detergent Fiber (NDF) in Feed". Acid detergent fiber was determined according to the method in NYT1459-2022 "Determination of Acid Detergent Fiber in Feed". Acid detergent lignin was determined according to the method in GB / T20805-2006 "Determination of Acid Detergent Lignin (ADL) in Feed". Crude ash was determined according to the method in GB / T6438-2007 "Determination of Crude Ash in Feed". Water-soluble carbohydrates were determined according to the method in GB / T37493-2019 "Grain and Oil Inspection - Determination of Soluble Sugars in Cereals and Legumes - Copper Reduction-Iodometric Method". The content of non-fiber carbohydrates was equal to 1 minus the content of crude protein, crude fat, crude ash, and neutral detergent fiber.

[0060] (3) Fermentation parameters: pH, ammonia nitrogen, ammonia nitrogen / total nitrogen, lactic acid / acetic acid ratio. The ammonia nitrogen content was determined by the phenol-sodium hypochlorite colorimetric method described in GAB, JHK Automated Simultaneous Determination of Ammonia and Total AminoAcids in Ruminal Fluid and In Vitro Media[J]. Journal of Dairy Science, 1980, 63(1): 64-75. DOI: 10.3168 / JDS.S0022-0302(80)82888-8. The organic acid content was analyzed by the high performance liquid chromatography method described in Zhang Yawei, Tian Boya, Wang Yuehong, et al. Simultaneous detection of 8 short-chain organic acids in intestinal contents by high performance liquid chromatography based on ligand exchange column[J]. Journal of Animal Nutrition, 2022, 34(11): 7411-7420.

[0061] (4) Microbial diversity: The relative abundance of bacteria at the genus level was analyzed using 16S rRNA gene amplicon sequencing technology.

[0062] II. Indicators for Wet Storage of Sorghum Ears 1. Sensory quality The sensory qualities of the sorghum ears stored in wet conditions in Example 3 and Comparative Example 1 are shown in Table 3. The results showed that the addition of strain R-12 had no significant effect on the texture and color of the stored sorghum ears, but it significantly improved the sourness of the stored sorghum ears starting from day 3 of wet storage, and produced a pleasant aromatic fruity flavor after day 14. Therefore, strain R-12 can significantly improve the flavor and palatability of stored sorghum ears.

[0063] Table 3 Sensory quality of sorghum ears stored in wet conditions in Example 3 and Comparative Example 1 2. Conventional nutrients The effects of adding Leuconostoc mesenteroides R-12 strain on the conventional nutrients of sorghum ears at different storage times are shown in Table 4. On day 3 of wet storage, the WSC content of the SEL group was 2.43%, significantly lower than that of the control group (6.77%), indicating that the inoculated strain rapidly utilized soluble sugars for lactic acid fermentation in the early stage of wet storage. On days 56 and 112 of wet storage, the WSC content of sorghum ears in the SEL group was 9.21% and 6.67%, respectively, significantly higher than that of the control group (1.88% and 1.22%), indicating that this strain helps maintain a high level of soluble sugars in the middle and later stages of wet storage.

[0064] There were no significant differences in the contents of DM, EE, CP, NDF, ADF, Ash, and NFC among the treatment groups, indicating that inoculation with this strain does not affect the preservation of the basic nutrients in the wet-stored materials. The ADL content differed significantly among the treatment groups, with the ADL in the SEL group decreasing to 1.06% at 112 days, lower than the 1.19% in the control group.

[0065] Table 4. Effects of Mycotoxin R-12 on the conventional nutrients in wet storage of sorghum ears. In the table, uppercase letters indicate significant differences between different time points within the same column. P <0.05.

[0066] 3. Changes in fermentation parameters The effects of strain R-12 on the wet storage fermentation parameters of sorghum ears at different storage times are shown in Table 5. The results showed that, regarding pH, the pH of the SEL group rapidly decreased from the initial 6.86 to 5.16 on the 3rd day of wet storage, significantly lower than the 6.36 of the control group. This indicates that inoculation with this strain can rapidly initiate lactic acid fermentation, thereby lowering the pH and inhibiting the activity of other microorganisms. With prolonged storage, the pH of both groups continued to decrease, reaching 4.37 in the SEL group and 4.18 in the control group at 112 days, both within the pH range suitable for high-quality silage.

[0067] Table 5. Effects of Mycotoxin R-12 on parameters of wet storage fermentation of sorghum ears. In the table, uppercase letters indicate significant differences between different time points within the same column. P <0.05. Lowercase letters indicate significant differences between different treatment groups at the same time point. P <0.05. “ / ” indicates not detected.

[0068] The NH3-N / TN ratio of the SEL group was consistently lower than that of the control group throughout the wet storage process, with significant differences at 3, 56 and 112 days. This indicates that the addition of this strain can effectively inhibit the enzymatic deammoniation of protein in wet-stored feed and reduce protein loss.

[0069] At day 14, the LA / AA ratio in the SEL group was 7.68, significantly higher than the 5.19 in the control group, indicating that homolactic acid fermentation was the dominant fermentation mode of this strain. The ratios in the two groups were close at day 56. At day 112, the ratio in the SEL group decreased to 3.21, lower than the 4.99 in the control group, possibly related to microbial community succession in the later stages of fermentation.

[0070] 3. Microbial community structure The relative abundance of horizontal microbial communities during the wet storage of sorghum ears in this invention is as follows: Figure 6 As shown in the figure. The results showed that *Leuconostoc* and *Lactobacillus* were dominant throughout the entire wet storage period of sorghum ears. In the SEL group, *Leuconostoc* was the absolutely dominant microorganism throughout the wet storage process, with an average relative abundance of 61.68%, indicating that the exogenously inoculated *Leuconostoc* R-12 could rapidly and successfully colonize and become the dominant microorganism in the early stage of wet storage. In the SEC group, *Pantotheca* was dominant in the early stage of wet storage, while in the middle and late stages it evolved into being dominated by *Leuconostoc* and *Lactobacillus*, with the relative abundance of *Lactobacillus* reaching 27.77% at 112 days. This indicates that the dominant microbial community structure underwent a significant succession under natural fermentation conditions.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

[0072] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention is also intended to include such modifications and variations.

Claims

1. A Leuconostoc mesenteroides strain for sorghum silage fermentation ( Leuconostoc mesenteroides ), characterized in that, The Leuconostoc mesenteroides strain is R-12, with accession number CGMCC No. 37525 and accession date of January 26, 2026.

2. The fermentation agent for sorghum silage fermentation according to claim 1, characterized in that, It is made from the R-12 strain and excipients; or, it is made from the fermentation products of the R-12 strain and excipients.

3. The fermentation agent for sorghum silage fermentation according to claim 2, characterized in that, The excipients are selected from one or more of the following: diluents, protectants, nutrient additives, dispersants, solubilizers, granulators, excipients, or microbial agents.

4. The fermentation agent for sorghum silage fermentation according to claim 3, characterized in that, The diluent is selected from one or more of bentonite, starch, maltodextrin and Tween-80; The protective agent is selected from one or more of skim milk powder, glucose, and glycerin; The granulating agent is selected from one or more of starch and maltodextrin; The co-solvent is selected from one or more of water and physiological saline.

5. The fermentation agent for sorghum silage fermentation according to claim 2, characterized in that, The viable count of strain R-12 in the fermentation agent is 1×10⁻⁶. 7 CFU / g ~ 1×10 9 CFU / g.

6. The fermentation agent for sorghum silage fermentation according to claim 5, characterized in that, The viable count of strain R-12 in the fermentation agent was 1.5 × 10⁻⁶. 8 CFU / g.

7. The application of *Leuconostoc mesenteroides* for sorghum silage fermentation according to claim 1 or the fermentation agent according to claim 2 in the preparation of silage, characterized in that... Includes at least one of the following: (1) Increase the soluble sugar content of silage; (2) Improve the palatability of silage; (3) Reduce the ammonia nitrogen / total nitrogen ratio in silage to reduce protein loss.

8. The application according to claim 7, characterized in that, The silage is either wet sorghum ears or whole sorghum silage.

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