Strain combination and its application in corn silage at low temperature

Abstract

The application discloses a strain combination and application thereof in corn ensiling at low temperature, and belongs to the field of biological feed fermentation.The technical problem solved by the application is how to carry out corn ensiling at low temperature.The strain combination disclosed by the application is composed of Lactobacillus plantarum 6H2, Lactobacillus plantarum Q39 and Pediococcus pentosaceus GN16.Experiments prove that after corn ensiling by using the strain combination, the pH is reduced to 3.80, the lactic acid content is 7.14%, the ammonia nitrogen content is 1.87%, and no butyric acid is detected; in terms of nutritional quality, the crude protein content is increased to 8.16%, the NDF and ADF contents are reduced to 53.00% and 21.97% respectively, and the fiber degradation and protein preservation effects are better.It is proved that the strain combination can promote rapid acidification of whole corn ensiling at low temperature and improve the nutritional preservation effect, and can be used as an optimal combination of whole corn ensiling composite microbial agent in low-temperature areas.

Description

Technical Field

[0001] This invention belongs to the field of biological feed fermentation, specifically relating to a combination of strains and its application in corn silage at low temperatures. Background Technology

[0002] Whole-plant maize silage is an indispensable high-quality roughage source in modern livestock farming, characterized by high biomass yield, relatively balanced nutritional structure, and good palatability, making it widely used in ruminant production. However, in northern and high-altitude regions, the long cold season and insufficient accumulated temperature make silage raw materials susceptible to low-temperature stress during processing and storage. This leads to delayed silage initiation, limited lactic acid bacteria proliferation, and reduced acidification rates, thereby increasing dry matter loss and protein risk, ultimately reducing the nutritional value and feed safety of the silage. Therefore, improving the fermentation efficiency and preservation effect of whole-plant maize silage under low-temperature conditions has become an urgent problem to be solved in the development of herbivorous livestock farming in high-altitude and cold regions.

[0003] Lactic acid bacteria are the core functional microorganisms in silage fermentation. They decompose soluble carbohydrates to produce organic acids, thereby rapidly acidifying the silage raw materials, inhibiting the growth of harmful microorganisms, and ensuring the fermentation stability of the silage. Different lactic acid bacteria strains have different functions in rapidly lowering pH, improving nutrient retention, and enhancing aerobic stability. The dosage and amount of compound lactic acid bacteria preparations made from different strains affect the number of lactic acid bacteria, mold levels, and organic acid profile, thus altering fermentation quality and some nutritional indicators. This is a key variable for the promotion of microbial agents. In conclusion, constructing effective compound microbial agents has become an important way to improve silage quality. Especially under low-temperature conditions, single strains often struggle to achieve both rapid start-up and sustained fermentation, while multi-strain combinations hold promise for improving fermentation efficiency through functional complementarity. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to carry out corn silage at low temperatures.

[0005] To address the aforementioned technical problems, the present invention first provides a strain combination, wherein the strain combination consists of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ) 6H2, Lactobacillus plantarum ( Lactiplantibacillus plantarum Q39 and Pediococcus pentosaceus ( Pediococcus pentosaceus It consists of GN16.

[0006] Specifically, in the strain combination, the number of viable cells in each strain is equal.

[0007] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent contains the combination of strains.

[0008] The aforementioned microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, plant material, or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane. In the microbial agent, the active ingredient may be present in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate. The dosage form of the composition may be various, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0009] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0010] The microbial agent can be used in at least one of the following: Y1) Corn silage; Y2) Low-temperature corn silage; Y3) Improves the nutritional quality of corn silage at low temperatures; Y4) Improve the sensory quality of corn silage at low temperatures; Y5) Improves the fermentation quality of corn silage at low temperatures.

[0011] The present invention also provides at least one of the following applications of the said strain combination or the said microbial agent: X1) Corn silage; X2) Low-temperature corn silage; X3) Improves the nutritional quality of corn silage at low temperatures; X4) Improve the sensory quality of corn silage at low temperatures; X5) Improves the fermentation quality of corn silage at low temperatures; X6) Prepare products for corn silage; X7) Prepare products for low-temperature corn silage; X8) Prepare products for improving the nutritional quality of corn silage at low temperatures; X9) Prepare products for improving the sensory quality of corn silage at low temperatures; X10) is used to prepare products for improving the fermentation quality of corn silage at low temperatures.

[0012] Specifically, the low temperature is 5–15°C. Further, the low temperature is 5–10°C.

[0013] In one embodiment of the present invention, the low temperature is a temperature condition with an annual average temperature of 7°C.

[0014] Specifically, the improvement in nutritional quality is reflected in the increase in crude protein content, the decrease in fiber content, and / or the decrease in soluble carbohydrate content.

[0015] Furthermore, the fiber content is a neutral detergent fiber content and / or an acid detergent fiber content.

[0016] Specifically, the improvement in sensory quality is reflected in improvements in acidity, moisture and / or odor.

[0017] Specifically, the improvement in fermentation quality is reflected in the reduction of pH, the reduction of ammonia nitrogen content, the increase of lactic acid content, the increase of acetic acid content, and / or the reduction of propionic acid content.

[0018] The present invention also provides a method for low-temperature corn silage, the method comprising: adding the strain combination to corn to be silaged, and silaging fermentation at low temperature to obtain silage corn.

[0019] Specifically, the amount of the strain combination added is (0.5–5) × 10⁻⁶. 6 CFU / g fresh weight. Furthermore, the amount of the strain combination added is 1 × 10⁻⁶. 6 CFU / g fresh weight. The amount of the strain combination added is based on the total viable count.

[0020] Specifically, the low temperature is 5–15°C. Further, the low temperature is 5–10°C.

[0021] In one embodiment of the present invention, the low temperature is a temperature condition with an annual average temperature of 7°C.

[0022] In one embodiment of the present invention, the silage time is 45 days.

[0023] In this invention, the corn used for corn silage is whole-plant corn. Specifically, the corn used for corn silage is whole-plant corn at the waxy maturity stage.

[0024] In this invention, the corn silage refers to the silage of whole corn plants.

[0025] Experiments have shown that after ensiling corn using the strain combination of the present invention, the pH decreased to 3.80, the lactic acid content was 7.14%, the ammonia nitrogen content was 1.87%, and butyric acid was not detected. In terms of nutritional quality, the crude protein content increased to 8.16%, while the NDF and ADF contents decreased to 53.00% and 21.97%, respectively, indicating good fiber degradation and protein preservation effects. This demonstrates that the strain combination of the present invention can promote rapid acidification of whole-plant corn silage and improve nutrient retention under low-temperature conditions, and can be considered a preferred combination for whole-plant corn silage compound inoculants in low-temperature regions. Attached Figure Description

[0026] Figure 1 Growth rate at different temperatures.

[0027] Figure 2 Sensory images of whole-plant maize silage from different treatment groups.

[0028] Figure 3 Nutritional quality of whole-plant maize silage in different treatment groups. Note: There are significant differences between data with different lowercase letters (P < 0.05), and no significant differences between data with the same letter (P > 0.05).

[0029] Figure 4 Silage fermentation quality of whole-plant maize in different treatment groups. Note: There are significant differences between data with different lowercase letters (P < 0.05), and no significant differences between data with the same letter (P > 0.05). Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0032] Example 1 1. Materials and Methods 1.1 Overview of the Experimental Site The experimental site is located in Zha'na Village, Qinxu Township, Minxian County, Dingxi City, Gansu Province (103°41′29″~104°59′23″E, 34°07′34″~34°45′45″N). The altitude ranges from 2040 to 3754 m, the average annual temperature is 7℃, the average annual sunshine duration is 2382h, the frost-free period is 144d, and the annual precipitation is 635 mm.

[0033] 1.2 Test Materials Test strains: Lactobacillus plantarum ( Lactiplantibacillus plantarum The core strain is 6H2 (MT525224), which has strong low-temperature resistance and stable acid production performance. This strain is recorded in "Xu Shuangpeng et al., Effects of lactic acid bacteria additives on the quality and enzyme activity of mixed oat and arrowhead pea silage in high-altitude pastoral areas, Acta Grasslandica Sinica, 2025, 33(8):2694". In the article "2702".

[0034] Lactobacillus plantarum ( Lactiplantibacillus plantarum Q39 is a strain that is resistant to low temperatures and has strong antifungal potential. This strain is described in the article "He Tingting et al., Study on the inhibitory effect of Lactobacillus plantarum Q39 on toxin-producing Aspergillus flavus and its active substance characteristics, Grassland and Turf, 2026".

[0035] Leuconostoc mesenteroides ( Leuconostoc mesenteroides GN12 is a heterotrophic lactic acid bacteria that can enhance acid production in the later stages of fermentation. This strain is recorded in the master's thesis "Zhao Yongqi, Screening of low-temperature resistant lactic acid bacteria in silage and the effect of low temperature on the cell membrane of lactic acid bacteria, Gansu Agricultural University, June 2024".

[0036] Pediococcus pentosaceus ( Pediococcus pentosaceus GN16 is a homofermentative lactic acid bacterium with strong acid and salt tolerance. This strain is recorded in the article "Zhao Yongqi, Yin Guoli, Gong Haiqiang, Liang Wenbin, Lei Qin. Study on low temperature growth and cell membrane adaptability of three lactic acid bacteria [J / OL]. Grassland and Turf. 2026-06-01" and the master's thesis "Zhao Yongqi, Screening of low temperature resistant lactic acid bacteria in silage and the effect of low temperature on the cell membrane of lactic acid bacteria, Gansu Agricultural University, June 2024".

[0037] The whole-plant maize variety used in the experiment was "Jinhuang 828", which was sourced from Gansu Seed Industry Co., Ltd. The nutritional composition of the raw material before silage is shown in Table 1.

[0038] Table 1. Nutritional composition of whole-plant corn raw material for silage

[0039] 1.3 Experimental Design The experiment employed a single-factor, completely randomized design, with the bacterial agent combination as the sole treatment factor. Nine treatment groups were established: sterile control (CK); 6H2 single-strain group (W1); 6H2+Q39 (W2); 6H2+GN12 (W3); 6H2+GN16 (W4); 6H2+Q39+GN12 (W5); 6H2+Q39+GN16 (W6); 6H2+GN12+GN16 (W7); and 6H2+Q39+GN12+GN16 (W8), with three replicates for each treatment group. The bacterial strains in each combination treatment were mixed at a 1:1 live bacteria ratio, and the growth rate of each combination treatment group was measured to verify whether antagonistic effects existed between the strains.

[0040] Whole-plant corn was harvested at the waxy maturity stage, chopped to 2-3 cm, with a stubble height of 15 cm, and then inoculated with the above-mentioned nine treatment groups (each group's bacteria were resuspended in distilled water) to obtain the treated raw materials. The total viable count added to each treatment group was 1.0 × 10⁻⁶. 6 CFU / g fresh weight was used, while the control group was sprayed with an equal amount of distilled water. After the raw materials of each treatment were mixed, they were packed into 30×40 cm polyethylene vacuum bags, 500 g per bag, with 3 bags per treatment group. After vacuum sealing, they were placed in the natural low temperature conditions (5-15℃) in Minxian County for silage fermentation for 45 days. After opening the bags, they were used for subsequent index determination and evaluation.

[0041] 1.4 Test Methods 1.4.1 Determination of growth rate and antagonism Growth rate determination: Four lactic acid bacteria strains were activated and inoculated into MRS liquid medium at a relative volume of 2%. They were cultured at 5℃, 10℃, 15℃ and 25℃ for 24 h. Samples were taken every 2-4 h, and three parallel samples were taken from each strain. The OD value of each sample was measured at a wavelength of 600 nm.

[0042] Antagonism test: Each strain was streaked across MRS solid plates using a pairwise co-culture method. The presence of an inhibition zone in the cross-section was observed. If there was a clear inhibition zone or the colony growth was inhibited, antagonism existed, indicating incompatible strains. If there was no inhibition zone, the colonies could cross or merge and grow, indicating incompatible strains with no antagonism.

[0043] 1.4.2 Sensory evaluation of silage Referring to commonly used sensory grading and evaluation methods for silage in China, a comprehensive sensory evaluation was conducted on whole-plant corn samples after silage was completed: color (20 points), odor (25 points), texture (10 points), moisture (20 points), and pH value (25 points). The comprehensive score was calculated to determine the grade of the silage. The specific evaluation criteria are as follows:

[0044] 1.4.3 Determination of Nutritional Quality of Silage After 45 days of ensiling, samples were taken from the bags, surface samples were removed, and the mixture was thoroughly mixed. Approximately 250 g of sample was used for nutritional component determination. The sample was subjected to enzyme inactivation at 105℃ for 15 min, then dried at 65℃ to constant weight, pulverized, and passed through a 40-mesh sieve for later use. Crude protein (CP), dry matter (DM), crude fat (Ether Extract, EE), and crude ash (Ash) contents were determined using the AOAC standard analytical method; neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined using the Van der Waals method; and soluble carbohydrates (WSC) were determined using anthrone... Determination by sulfuric acid colorimetric method.

[0045] 1.4.4 Determination of Fermentation Quality of Silage Take 20 g of silage sample from each treatment group, add 180 mL of distilled water, mix well, let stand for 24 h, then homogenize for 1 min using a tissue homogenizer, filter through 4 layers of gauze to remove coarse fiber residue, and then filter through qualitative filter paper to obtain silage extract. The pH value of the extract was determined using a pH meter; the ammonia nitrogen (NH3-N) content was determined using the phenol-sodium hypochlorite colorimetric method; and the contents of lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were determined using high performance liquid chromatography (HPLC).

[0046] 1.5 Data Processing and Analysis Experimental data were compiled using Excel. One-way ANOVA was performed using SPSS 26.0 software, with the significance level set at [value missing]. P < 0.05. The plot was generated using Origin 2025 software.

[0047] Grey relational analysis was used to screen treatment groups with excellent silage quality. The optimal values ​​of each indicator were used as ideal standard reference values, denoted as [X0(k)]( k =1,2,3... n ),in k Representative indicators n As a processing group, the indicators for each processing group will be... X i ( k ) and ideal standard X 0( k Perform dimensionless data processing on the indicators for each processing group. i Grey relational analysis was performed between the index and the ideal index.

[0048] The formula for grey relational degree is: .

[0049] in, For the first i Grey relational degree of each processing group For the first i The difference between each treatment group and the standard value p =0.5 is the correlation coefficient. k =1,2,3,....

[0050] The degree of equal weight correlation is: .

[0051] in, n To measure the index number, For the first i Grey relational degree of the processing group.

[0052] The weighting coefficients are: .

[0053] 2 Results and Analysis 2.1 Growth rate and antagonism of the complex bacteria Table 2 shows the antagonism test results. All pairwise combinations of the four strains 6H2, Q39, GN12, and GN16 showed a positive result ("+"), indicating no inhibition bands. This demonstrates good compatibility among the strains and provides a basis for their combined application. Figure 1 It was found that the growth rates of the treatment groups differed significantly under different temperature conditions, with the differences being more pronounced at low temperatures. At 5℃, the growth of each treatment group was relatively slow from 0 to 6 hours, and then gradually differentiated. Among them, W6 showed a significant increase in growth rate after 8 hours, maintained a high level from 12 to 24 hours, and had the highest OD value at 24 hours; W2 and W4 were generally slightly lower than W6. When the temperature rose to 10℃, the overall proliferation rate of each treatment group accelerated, but the differences between groups were still significant. W6 still had the highest OD value, while W1, W2, and W4 also reached high levels. At 15℃ and 25℃, all treatment groups entered the growth phase relatively quickly and gradually stabilized, with the differences between groups significantly narrowing, but W6 remained at a relatively high level overall; specifically, at 15℃, the growth rate of each treatment group stabilized after 16 hours, and at 25℃, it stabilized after 12 hours. Overall, under low-temperature conditions of 5–15℃, the growth curve of group W6 consistently remained at the top, with OD values ​​at all time points higher than those of the single-strain group and other compound combinations, indicating the highest growth rate. Under incubation conditions of 25℃, the strains in all treatment groups grew rapidly, with no significant difference in growth rate; after 24 h of incubation, the OD values ​​of all treatment groups reached above 2.4.

[0054] Table 2. Strain Antagonism

[0055] Note: "+" indicates that there is no antagonistic effect between the two strains.

[0056] 2.2 Sensory evaluation of whole-plant maize silage Depend on Figure 2 It was found that after 45 days of ensiling, all samples in each treatment group reached the high-quality grade, with total scores ranging from 79 to 88 points. The overall structure of the samples in each treatment group was intact, the stem and leaf outlines were clear, the color was well preserved, and all samples had a sour aroma without the putrid odor produced by butyric acid fermentation. This indicates that the whole-plant corn ensiling fermentation was sufficient under the experimental conditions, and no obvious spoilage or fermentation failure occurred. Table 3 shows that the differences in moisture content, texture, and color among the treatment groups were small, mainly reflected in pH value (i.e., acidity) and odor scores. Among them, group W6 had the highest total score of 88 points, while group CK had the lowest total score of 79 points. The lactic acid bacteria treatment group was generally better than the control, indicating that inoculation with lactic acid bacteria helps improve the sensory quality of low-temperature whole-plant corn ensiling.

[0057] Table 3 Sensory Evaluation Table for Whole-Plant Maize Silage

[0058] 2.3 Nutritional quality of whole-plant maize silage like Figure 3 As shown, each compound microbial treatment group significantly affected the crude protein, NDF, ADF, and WSC content of silage. P < 0.05). Among them, group W6 had the highest crude protein content, at 8.16%, which was 19.8% higher than the control group and significantly higher than the four-strain combination W8 (7.29%) and the single-strain group W1 (7.17%). The NDF and ADF of group W6 decreased to 53.00% and 21.97%, respectively, which were 11.2% and 25.2% lower than the control group, respectively. P < 0.05). The WSC content in each treatment group was significantly lower than that in the control group. Among them, the W2, W4 and W6 groups had the lowest residual amounts, indicating that the addition of lactic acid bacteria promoted the fermentation and utilization of soluble carbohydrates.

[0059] There were no significant differences in DM, Ash, and EE levels among the treatment groups. P >0.05). DM content ranged from 31.93% to 33.75%, with the W4 group having the highest dry matter content at 33.75%. EE content ranged from 2.08% to 3.02% across all treatment groups, with the control group having the highest and the W4 group the lowest. Ash content ranged from 4.17% to 4.98% across all treatment groups, with the control group having the highest and the W4 group the lowest. Overall, the improvement in nutritional quality was mainly reflected in increased crude protein and decreased fiber content, while the impact on DM, EE, and Ash was relatively small.

[0060] 2.4 Whole-plant corn silage fermentation quality Depend on Figure 4It can be seen that the fermentation quality of silage differed significantly among the treatment groups after 45 days of ensiling. P < 0.05). All treatment groups had pH values ​​below 4.00, indicating that the overall fermentation reached a good level. Group W6 had the lowest pH value at 3.80, a 4.00% decrease compared to the control group; groups W2, W5, and W7 all had relatively low pH values ​​of 3.84. The control group had the highest ammonia nitrogen content at 2.48%; group W6 had the lowest at 1.87%, a 24.5% decrease compared to the control group. The ammonia nitrogen content from highest to lowest was: CK > W3 > W1 > W7 > W8 > W4 = W5 > W2 > W6. This indicates that treatment W6 was more effective in inhibiting protein decomposition.

[0061] The control group had the lowest lactic acid content at 5.63%, while group W6 had the highest at 7.14%, a 27% increase compared to the control group. Groups W4 and W2 also had high lactic acid content, increasing by 22.0% and 17.4% respectively compared to the control group. The acetic acid content in groups W7 and W8, which contained GN12, was significantly higher than in other treatments. P <0.05), the acetic acid content from highest to lowest was: W7>W8>W3>W5>W1>W2>W4>W6>CK, indicating that the addition of heterotrophic fermentation bacteria enhanced acetic acid production. The propionic acid content in each treatment group was generally low, ranging from 0.18% to 0.21%, with W2 having the lowest content; butyric acid was not detected in any treatment group, indicating that no significant butyric acid fermentation occurred in any group.

[0062] 2.5 Comprehensive evaluation of whole-plant maize silage quality using grey relational analysis Grey relational analysis was conducted based on 12 silage quality indicators. Table 4 shows that group W6 had the highest overall correlation score (0.959), ranking first. Its correlation scores for fermentation quality (pH, NH3-N, LA) all reached 1.000, and for nutritional quality (CP, WSC, NDF, ADF) also reached 1.000. Groups W4 and W2 were next, with overall correlation scores both above 0.900. Groups W1, W3, and the control group had lower overall correlation scores, below 0.800. Group CK had the lowest score. The overall ranking was: W6 > W4 > W2 > W5 > W8 > W7 > W1 > W3 > CK, indicating that group W6 performed best overall in terms of nutritional and fermentation quality.

[0063] Table 4. Grey relational analysis of whole-plant maize silage quality in different treatment groups

[0064] 3. Discussion 3.1 Growth response of different compound treatments to low temperature Rapid proliferation and early colonization of lactic acid bacteria under low-temperature conditions are crucial factors determining the success or failure of silage fermentation. The experimental results of this invention show that all treatment groups can grow at low temperatures of 5–15℃, but the growth rates differ significantly among the different treatment groups. W6 showed a significant increase in proliferation after 8 hours and maintained a high level, with its OD value consistently at the highest level; W2 and W4 followed, while the combinations containing GN12 (W3, W5, W7, W8) showed a more gradual overall proliferation. This further indicates that under low-temperature conditions, treatment groups W6, W2, and W4 are more conducive to the rapid accumulation of bacterial populations and may establish an early dominant bacterial community more quickly, thereby facilitating a rapid decrease in pH, shortening the active time of contaminating bacteria, and reducing the risk of protein hydrolysis. The experiments of this invention demonstrate that the addition of compound bacteria is not a simple additive process, but rather enhances fermentation start-up efficiency through low-temperature proliferation advantages, influencing the establishment of dominant bacterial communities and the efficiency of lactic acid fermentation start-up. Among them, the fermentation combination dominated by 6H2 was more conducive to rapid acid production and cell growth, while the fermentation combination introducing GN12 showed a relatively weak synergistic effect under the experimental conditions of this invention. This may be related to differences in metabolic pathways and limited proliferation capacity under low temperature conditions. Therefore, multi-strain combination may enhance the adaptability of the bacterial community to low-temperature environments through functional complementarity between strains, and the dynamic changes in OD values ​​of each treatment group also support this result to some extent.

[0065] 3.2 Synergistic effect of Lactobacillus plantarum 6H2 and low-temperature resistant functional strains In this invention, *Lactobacillus plantarum* 6H2 serves as the dominant bacterium, providing a sustained advantage in lactic acid production and proliferation throughout the fermentation process under low-temperature conditions. *Lactobacillus plantarum* Q39, belonging to the same genus as 6H2, significantly improves silage quality when combined with 6H2. The W4 treatment group outperformed some single-strain strains and other combinations in terms of lactic acid levels and fermentation quality, indicating that GN16 is more likely to play an ecological role in early proliferation and site occupation at low temperatures. Furthermore, W6 achieved optimal performance when combined with Q39, suggesting that this combination may constitute a synergistic effect of *Pediococcus pentosaceus* shortening the start-up time, *Lactobacillus plantarum* continuously producing acid, and Q39 inhibiting contaminating bacteria. The combinations containing GN12 (W3, W5, W7, W8) showed relatively weaker overall silage quality, and some treatment groups performed only moderately in acid production efficiency and nutrient retention, indicating that GN12 and 6H2 did not exhibit a significant synergistic effect. Simultaneously, the low-temperature environment may have amplified its dependence on the cell protection system and limited its proliferation rate, and may even have caused substrate diversion to some extent, thereby weakening lactic acid production efficiency.

[0066] 3.3 Effects of different compound microbial treatment groups on the quality of whole-plant maize low-temperature silage 3.3.1 Nutritional quality The core nutritional value of whole-plant maize silage lies in the retention of crude protein and the moderate degradation of fiber. In this invention, the W6 treatment group had the lowest ammonia nitrogen content among all treatment groups, decreasing by 24.5% compared to the control group. This indicates that the W6 compound system can rapidly lower the pH before the peak of protein degradation by quickly producing acid and accelerating the acidification process, thereby inhibiting protease hydrolysis and deamination, promoting the preservation of nitrogen in the form of protein and peptides, and ultimately manifesting as a significant increase in crude protein. The results of this invention show that the degradation rate of neutral detergent fiber in the single-strain treatment group was approximately 5%, showing a more significant decreasing trend compared to the W6 group, indicating that the compound strains may have a certain synergistic advantage in the degradation of structural carbohydrates. Combined with the analysis of the functional characteristics of the strains, this synergistic effect may be related to the stronger substrate utilization ability and the ability of strains such as 6H2 and GN16 to promote plant cell wall softening under low temperature conditions, thus providing ruminants with a more easily digestible carbohydrate source.

[0067] 3.3.2 Fermentation quality A rapid decrease in pH is one of the key indicators of successful silage fermentation. In this invention, the pH of all treatment groups was below 4.00, indicating that the silage fermentation in each treatment group was relatively complete. Ammonia nitrogen content reflects the degree of protein degradation. The ammonia nitrogen content of the W6 treatment group was 1.87%, which was about 24.5% lower than that of the control group (CK), indicating that the W6 treatment group could inhibit protein hydrolysis and deamination through rapid acidification, thereby reducing ammonia nitrogen accumulation and improving fermentation quality. Lactic acid is the most important organic acid in silage fermentation. In this invention, the lactic acid content of the W6 treatment group was 7.14%, significantly higher than the 5.63% of the control group. However, the lactic acid content of the W1 treatment group was only 5.93%, indicating that the lactic acid production capacity of a single strain was weak. For treatment groups containing *Leuconostoc mesenteroides*, such as W7 and W8, the increased acetic acid content reveals the role of heterologous lactic acid bacteria fermentation pathways. Although acetic acid production may slightly reduce the energy utilization rate during silage fermentation compared to homologous fermentation, its strong antifungal activity helps improve the aerobic stability of silage. Under conditions where rapid acidification at low temperature is the primary objective, the W6 compound system, dominated by homologous fermentation, clearly exhibits higher efficiency. The formation of propionic and butyric acids is generally considered an undesirable byproduct of silage fermentation, especially butyric acid, whose formation is closely related to Clostridium activity and is usually regarded as a significant indicator of poor silage fermentation. In this invention, the W2 and W4 treatment groups showed lower propionic acid content, indicating that the compound lactic acid bacteria help reduce propionic acid formation. This demonstrates that during silage fermentation, the use of compound lactic acid bacteria can effectively inhibit harmful microbial metabolism and reduce propionic acid formation. Simultaneously, it increases the formation of lactic acid and acetic acid, thereby improving the quality of silage fermentation. No butyric acid was detected in any of the treatment groups in this invention, indicating that no significant butyric acid fermentation occurred in any of the treatment groups, and the overall fermentation status was good.

[0068] 4. Conclusion Under natural low-temperature conditions, different combinations of lactic acid bacteria can improve the quality of whole-plant maize silage to some extent, with the W6 treatment, composed of 6H2, Q39, and GN16, showing the best effect. After 45 days of ensiling, the pH of the W6 treatment group decreased to 3.80, lactic acid content increased to 7.14%, crude protein content increased to 8.16%, ammonia nitrogen content decreased to 1.87%, and NDF and ADF contents decreased to 53.00% and 21.97%, respectively, demonstrating good fermentation promotion and nutrient retention effects. Grey relational analysis further showed that the W6 group had the best overall evaluation. Therefore, the W6 treatment group can be regarded as the preferred combination of compound microbial agents for whole-plant maize silage in low-temperature areas.

[0069] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Strains, consisting of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) 6H2, Lactobacillus plantarum ( Lactiplantibacillus plantarum Q39 and Pediococcus pentosaceus ( Pediococcus pentosaceus It consists of GN16.

2. A microbial agent, the active ingredient of which contains the combination of strains as described in claim 1.

3. At least one of the following applications of the strain combination of claim 1 or the microbial agent of claim 2: X1) Corn silage; X2) Low-temperature corn silage; X3) Improves the nutritional quality of corn silage at low temperatures; X4) Improve the sensory quality of corn silage at low temperatures; X5) Improves the fermentation quality of corn silage at low temperatures; X6) Prepare products for corn silage; X7) Prepare products for low-temperature corn silage; X8) Prepare products for improving the nutritional quality of corn silage at low temperatures; X9) Prepare products for improving the sensory quality of corn silage at low temperatures; X10) is used to prepare products for improving the fermentation quality of corn silage at low temperatures.

4. The application according to claim 3, characterized in that: The low temperature is 5–15°C; Furthermore, the low temperature is 5–10°C.

5. The application according to claim 3 or as described above, characterized in that: The improvement in nutritional quality is reflected in the increase in crude protein content, the decrease in fiber content, and / or the decrease in soluble carbohydrate content. Furthermore, the fiber content is a neutral detergent fiber content and / or an acid detergent fiber content.

6. The application according to any one of claims 3-5, characterized in that: The improvement in sensory quality is reflected in improvements in acidity, moisture and / or odor.

7. The application according to any one of claims 3-6, characterized in that: The improvement in fermentation quality is reflected in the decrease in pH, the decrease in ammonia nitrogen content, the increase in lactic acid content, the increase in acetic acid content, and / or the decrease in propionic acid content.

8. A method for low-temperature corn silage, comprising: The strain combination described in claim 1 is added to the corn to be ensiling, and ensiling fermentation is carried out at low temperature to obtain ensiling corn.

9. The method according to claim 8, characterized in that: The amount of the strain combination added is (0.5~5)×10. 6 cfu / g fresh weight; And / or, the low temperature is 5 to 15°C.

10. The method according to claim 8 or 9, characterized in that: The amount of the strain combination added was 1×10⁻⁶. 6 cfu / g fresh weight; And / or, the low temperature is 5 to 10°C.

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