Compound microbial inoculant, application thereof and mountain leek type grassland forage silage and preparation method thereof

Abstract

The present application relates to the technical field of microbial inoculant, and discloses a compound microbial inoculant, application thereof, and a mountain leek type grassland pasture silage feed and a preparation method thereof.The preparation method comprises the following steps: S1, cutting and wilting the mountain leek type grassland pasture to obtain pretreated pasture; the wilting specifically comprises: airing the cut pasture until the water content is 55%-65%, wherein the percentage is a mass percentage; S2, mixing the compound microbial inoculant with the pretreated pasture to obtain inoculated pasture; the compound microbial inoculant is composed of Pediococcus pentosaceus, Enterococcus faecalis and Lactobacillus plantarum; S3, bundling and wrapping the inoculated pasture, and then performing sealed anaerobic fermentation to obtain the mountain leek type grassland pasture silage feed.The compound microbial inoculant provided by the present application has the abilities of rapid start, tolerance to bacteriostatic substances and continuous acid production, and when applied to the preparation of the mountain leek type grassland pasture silage feed, the mountain leek type grassland pasture silage feed with high value can be produced.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and particularly to compound inoculants and their applications, as well as silage for wild leeks and its preparation method. Background Technology

[0002] Wild leeks ( Allium senescens L. (Allium chinense) is a perennial herbaceous plant belonging to the Amaryllidaceae family and the Allium genus. Wild leeks are valuable natural pastures, rich in various minerals, vitamins, and bioactive substances such as natural sulfides from wild leeks, making them highly valuable forage. Currently, most silage technologies used in animal husbandry (such as baling silage) are for cultivated, single-variety forages (such as corn and alfalfa) or relatively simple cultivated mixed forages. Existing silage technologies typically include the following steps: after harvesting the forage, adding commercially available general-purpose lactic acid bacteria starter cultures (such as general-purpose fermented Lactobacillus plantarum and Lactobacillus brunelli); followed by sealed, anaerobic fermentation; the added general-purpose lactic acid bacteria starter cultures are usually designed for forages with high sugar content and lacking naturally occurring strong antibacterial substances.

[0003] The following technical challenges exist in the silage process of wild leek-type pasture: First, fermentation initiation is difficult and the failure rate is high. Wild leek itself contains natural broad-spectrum antibacterial components that, while inhibiting putrefactive and pathogenic bacteria, also strongly inhibit the growth and activity of exogenously added common lactic acid bacteria. This delays or even causes fermentation to fail. Second, silage quality is unstable. Wild leek-type pastures are mixed plant communities with fluctuating water-soluble carbohydrate (WSC) content, often insufficient to support stable and sufficient fermentation with a single lactic acid bacteria inoculant. Simultaneously, due to the inhibition of beneficial bacteria, harmful microorganisms (such as butyric acid bacteria and yeast) proliferate more easily, leading to a slow or insufficient decrease in silage pH, high butyric acid content, severe protein degradation (manifested as a high ammonia nitrogen / total nitrogen ratio), poor feed quality, and an off-odor. Third, functional components cannot be retained. Existing silage inoculants and silage technologies are not compatible with the unique fermentation microenvironment created by wild leeks as the dominant species. They are difficult to maximize the preservation of unique functional active substances such as natural sulfides from wild leeks while effectively preserving basic nutrients.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a compound microbial agent and its application in silage for mountain leek-type pastures and its preparation method, aiming to solve the problems of low success rate and unstable feed quality in the silage technology for mountain leek-type pastures.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing silage from wild leek-type pasture, comprising the following steps: S1. Cut and wither the grass in the mountain leek-type grassland to obtain pre-treated grass; Specifically, the withering process includes: drying the cut forage until the moisture content is 55%-65%, where % is a mass percentage; S2. Mix the compound microbial agent with the pretreated forage to obtain the inoculated forage; The compound microbial agent is composed of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum. S3. The inoculated forage is bundled, wrapped, and then sealed for anaerobic fermentation to obtain the mountain leek-type pasture silage.

[0007] Optionally, the ratio of viable counts of *Pediococcus pentosaceus*, *Enterococcus faecalis*, and *Lactobacillus plantarum* is (1 × 10⁻⁶). 10 -1×10 11 ): (1×10 10 -1×10 11 ): (1×10 10 -1×10 11 ).

[0008] Optionally, the ratio of viable counts of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum is 1:1:1.

[0009] Optionally, in step S1, the harvesting period is from the initial flowering stage to the full flowering stage of the wild leek.

[0010] Optionally, in step S2, the amount of compound microbial agent added is 1×10 based on the weight of the pretreated forage. 6 CFU / gram of hay.

[0011] Optionally, in step S3, the baling density is 450-550 kg / m³. 3 ; and / or, the number of layers of the wrapping is 4-6.

[0012] In a second aspect, the present invention provides a wild leek-type pasture silage prepared by the above-described preparation method.

[0013] Compared with existing technologies, the advantages of this invention are as follows: First, the compound microbial agent provided by this invention possesses the ability to start up quickly, tolerate antibacterial substances, and continuously produce acid. When this compound microbial agent is used for silage of wild leek-type pasture, it solves the problem of delayed or failed fermentation start-up caused by the antibacterial properties of wild leek in wild leek-type pasture, significantly improving the silage success rate. Second, when the compound microbial agent is applied to the preparation of wild leek-type pasture silage, it can achieve rapid and stable lactic acid fermentation. The resulting wild leek-type pasture silage has a pH value below 4.3, extremely low butyric acid content, and well-preserved protein, resulting in excellent quality wild leek-type pasture silage. At the same time, it also fully preserves the biodiversity of wild leek-type pasture and its rich natural functional active ingredients in the feed, producing high-value wild leek-type pasture silage. Attached Figure Description

[0014] Figure 1 The results of bacterial community detection (phylum level) for the silage of the raw material group, comparative examples 1-3 and example 1 are shown in the figure.

[0015] Figure 2 The results of bacterial community detection for the raw material group, comparative examples 1-3 and example 1 are shown in the figure (at the level). Detailed Implementation

[0016] This invention provides a compound microbial agent and its application, as well as a method for preparing silage for wild leek-type pastures. To make the purpose, technical solution, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0017] To address the technical problems of existing silage inoculants and silage technologies in the application of wild leek-type pasture silage, such as difficulties in fermentation initiation, unstable quality, and difficulty in retaining functional components, this invention provides a compound inoculant for wild leek-type pasture silage, which is composed of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum.

[0018] It should be noted that the *Lactobacillus plantarum* can be obtained by isolating from well-fermented natural forage silage or by commercial purchase; *Pediococcus pentosus* and *Enterococcus faecalis* can be obtained by isolating from animal intestines or feces or by commercial purchase.

[0019] This invention uses *Pediococcus pentosaceus* as the main fermentation bacteria, leveraging its rapid growth, tolerance to low pH, and high osmotic pressure to quickly initiate fermentation and secure its ecological niche during the silage process of wild allium-type pastures. *Enterococcus faecium* is used as the core bacteria, exhibiting relatively good tolerance to sulfides in *Allium* species, allowing it to grow well in the presence of wild alliums. *Lactobacillus plantarum* is used as a synergist, acting as a high-acid-producing bacterium to enhance acidification in the later stages of fermentation.

[0020] The compound microbial agent provided by this invention is suitable for pasture grasses containing antibacterial plants, such as Allium genus pastures and Artemisia argyi pastures; preferably, the compound microbial agent is used for Allium chinense type pasture grasses.

[0021] In some embodiments, the ratio of viable counts of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum is (1 × 10⁻⁶). 10 -1×10 11 ): (1×10 10 -1×10 11 ): (1×10 10 -1×10 11 ).

[0022] In some embodiments, the ratio of viable counts of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum is 1:1:1.

[0023] Based on the above-mentioned compound microbial agent, this invention provides a method for preparing silage from wild leek-type pasture, comprising the following steps: S1. Cut and wither the grass in the mountain leek-type grassland to obtain pre-treated grass; Specifically, the withering process includes: drying the cut forage until the moisture content is 55%-65%; S2. Mix the above-mentioned compound microbial agent with the pretreated forage to obtain the inoculated forage; The compound microbial agent is composed of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum. S3. The inoculated forage is bundled, wrapped, and then sealed for anaerobic fermentation to obtain the mountain leek-type pasture silage.

[0024] In this embodiment, firstly, the initial inhibitory intensity of the natural antibacterial components of *Allium chinense* in the forage is reduced by wilting the forage. Secondly, a compound microbial agent is used in conjunction with the forage, taking advantage of its ability to quickly start up, tolerate antibacterial substances, and continuously produce acid. Finally, baling and sealing are combined to create a stable and efficient anaerobic fermentation environment for the forage.

[0025] In this embodiment, the withering specifically includes: drying the cut forage until the moisture content is 55%-65%. This reduces the moisture content of the forage to 55%-65%. This moisture content range ensures the moisture required for fermentation, effectively reduces the concentration of wild leek juice, lessens its initial inhibitory effect on the inoculant, and facilitates wrapping and compaction.

[0026] In some embodiments, in step S1, the harvesting period is from the initial flowering stage to the peak flowering stage of the wild leek.

[0027] In some embodiments, in step S2, the amount of compound microbial agent added is 1×10 based on the weight of the pretreated forage. 6 CFU / gram of hay.

[0028] In this embodiment, *Pediococcus pentosaceus* is the main fermenting bacterium in the compound microbial agent. Utilizing its rapid growth, tolerance to low pH, and high osmotic pressure, it can quickly initiate fermentation and secure its ecological niche during the silage process of wild leeks. *Enterococcus faecium* is used as the core bacterium resistant to antimicrobial growth, leveraging its relatively good tolerance to sulfides in *Allium* species to grow well in the presence of wild leeks. *Lactobacillus plantarum* is used as a synergist, acting as a high-acid-producing bacterium to enhance acidification in the later stages of fermentation. Therefore, rapid and stable lactic acid fermentation is achieved, while simultaneously preserving the biodiversity of wild leeks and its rich natural functional active ingredients in the feed, resulting in high-value wild leeks silage.

[0029] In some embodiments, in step S3, the baling density is 450-550 kg / m³. 3 ; and / or, the number of layers of the wrapping is 4-6.

[0030] In this embodiment, due to the diverse types of forage grasses in natural grasslands, with significant differences in stem thickness, hardness, and moisture content, a large amount of air remains inside the baler during baling. If only a standard number of wrapping layers is used, aerobic deterioration can easily occur due to inadequate sealing. Therefore, high-density baling (density of 450-550 kg / m³) is employed. 3 The method of wrapping the bag in multiple layers maximizes the removal of air while ensuring airtightness to prevent oxygen from entering and rainwater from damaging it.

[0031] In some embodiments, the bales should be stacked on a leveled, compacted, and well-drained site, with the ambient temperature maintained between 15°C and 30°C and the relative humidity below 70%. It is essential to ensure the site is free of any sharp objects and to take protective measures against rodents and prolonged direct sunlight. The bales should be stacked in a staggered manner, with layers not too high, and appropriate gaps maintained between bundles. Under these conditions, the lactic acid bacteria inside the bales can carry out efficient anaerobic fermentation at the optimal moisture content of 55%-65%, ultimately producing high-quality silage.

[0032] The present invention provides a wild leek-type pasture silage prepared by the above-described preparation method.

[0033] The present invention will be further described below through specific embodiments.

[0034] Example 1 This embodiment provides a method for preparing silage from wild leek-type pasture, the specific steps of which are as follows: S1. Harvesting: Harvest the wild leeks during their initial flowering period to their peak flowering period. S2. Wilting Treatment: The cut forage is dried on-site in the pasture to reduce the overall moisture content of the forage to 62%, resulting in pre-treated forage. The moisture content of the forage is measured using an electronic moisture meter (Huachao High-Tech).

[0035] S3. Resuscitation and addition of compound microbial agents: A compound microbial agent is provided, which is composed of Pediococcus pentosaceus, Enterococcus faecalis and Lactobacillus plantarum, wherein the ratio of the number of viable bacteria of Pediococcus pentosaceus, Enterococcus faecalis and Lactobacillus plantarum is 1:1:1; The compound microbial agent was inoculated into MRS broth medium (MRS broth, purchased from Beijing Luqiao Technology Co., Ltd., catalog number CM187) under aseptic conditions in a laminar flow hood and cultured at 37℃ for 48 hours. Two generations of subculturing were performed. The viable cell count concentration in the bacterial suspension was calculated using the plate count method, and the required amount of bacterial suspension to be added was determined. The amount of bacterial suspension added should ensure that each gram of pretreated forage contains no less than 1 × 10⁻⁶ viable cells. 6 CFU. The above bacterial suspension was then evenly sprayed onto the pretreated forage according to the calculated addition amount to obtain inoculated forage.

[0036] S4. High-density baling: The inoculated hay is baled using a baler into bales approximately 1.2 meters in diameter, 1.4 meters in height, and weighing 989 kg, ensuring that the bales are tightly packed and that air is removed to the maximum extent possible.

[0037] S5. Multi-layer wrapping: Four layers of high-performance stretch film are wrapped using a wrapping machine to form a sealed package, ensuring airtightness and preventing oxygen from entering and rainwater from eroding.

[0038] S6. Fermentation and Storage: The bales are stacked on a leveled, compacted, and well-drained site, with the ambient temperature maintained between 15℃ and 30℃ and the relative humidity below 70%. The fermentation time is 120 days. It is essential to ensure the site is free of any sharp objects and to take protective measures against rodents and prolonged direct sunlight. The bales should be stacked in a staggered manner, with layers not too high, and appropriate gaps maintained between bundles. Under these conditions, the compound microbial agent inside the bales can undergo efficient anaerobic fermentation at the optimal moisture content of 55%-65%, yielding silage from wild leeks-type pasture.

[0039] Comparative Example 1 This comparative example provides a method for preparing silage from mountain leek-type pasture. The only difference from Example 1 is that in step S3, the compound microbial agent of Example 1 is replaced with an equal amount of distilled water.

[0040] Comparative Example 2 This comparative example provides a method for preparing silage from wild leek-type pasture. The only difference from Example 1 is that in step S3, the compound microbial agent of Example 1 is replaced with an equal amount of Lactobacillus plantarum.

[0041] Comparative Example 3 This comparative example provides a method for preparing silage from wild leek-type pasture. The only difference from Example 1 is that in step S3, the compound microbial agent of Example 1 is replaced with an equal amount of Lactobacillus buchneri.

[0042] Test Example 1 This test case aims to verify the effects of different microbial agents on the quality of silage from wild leek-type pastures. The fermentation quality and nutritional quality of the silage from Example 1 and Comparative Examples 1-3 are compared as follows: In this test case, the raw material was unensiled wild leek-type pasture.

[0043] After opening the silage wrapping, mix the sample well, take 10g and put it into a No. 5 self-sealing bag, add 90mL of physiological saline, pat and soak for 10min on both sides, and take the supernatant for serial dilution for viable count determination. After opening the silage bales, the sample was mixed thoroughly. 10g of the mixture was placed in a No. 5 resealable bag, and 90mL of sterile water was added. The mixture was soaked for 10 minutes, then blended for 1 minute using a juicer. The filtrate was filtered through No. 12 filter paper, and the filtrate was placed in a 15mL centrifuge tube. The pH, ammonia nitrogen, and flavonoid content were measured. Ammonia nitrogen was determined colorimetrically. The flavonoid content was determined using a flavonoid kit. 2mL of the filtrate was filtered through a syringe filter, and organic acids were determined using high-performance liquid chromatography (HPLC). The results are shown in Table 1.

[0044] Table 1. Effects of compound microbial agents on the silage fermentation quality of wild leek-type pasture.

[0045] Wherein, mg / g DM refers to the number of milligrams of the target substance contained in each gram of dry matter sample.

[0046] Experimental results showed that, compared with the raw material group and Comparative Example 1, the silage prepared in Example 1 exhibited the best silage fermentation quality: it had the highest number of lactic acid bacteria (6.98 lg CFU / g), the lowest pH value (4.17), and a significantly increased lactic acid yield (102 g / kg DM). At the same time, it completely inhibited harmful microorganisms such as yeast and Escherichia coli, and the protein was better preserved (ammonia nitrogen content 4.26 g / kg DM). It could also more effectively retain the functional component flavonoids (44.7). The overall fermentation effect of Example 1 was also significantly better than that of Comparative Example 2 and Comparative Example 3.

[0047] Nutritional components: Dry matter was determined using the drying method. 100g samples were placed in an envelope and dried in an oven at 65℃ for 48 hours. After drying, the samples were pulverized, passed through a 40-mesh sieve, and placed in resealable bags for nutrient analysis. Crude protein was determined using the Kjeldahl method, and acid detergent fiber and neutral detergent fiber were determined using the Panthen method. The results are shown in Table 2.

[0048] Table 2. Effects of compound microbial agents on the nutrient composition of forage in mountain leek-type pastures for silage wrapping.

[0049] As shown in Table 2, compared with the raw material group and Comparative Example 1, Example 1 showed better nutritional quality: its dry matter retention rate (36.7%) was higher than that of Comparative Example 1, its crude protein content (13.2% DM) was significantly higher than that of Comparative Example 1 and closer to the raw material level, and its neutral detergent fiber (55.9% DM) and acid detergent fiber (33.8% DM) content were the lowest. This indicates that the treatment of adding compound microbial agent in Example 1 can better preserve nutrients and improve fiber structure.

[0050] Test Example 2 Bacterial community analysis was performed on the raw material group (using unensiled wild leek-type pasture forage as the raw material group), comparative examples 1-3, and example 1's silage. The results are as follows: Figure 1 , Figure 2 As shown.

[0051] In this experiment, the microbial community of the raw material group was dominated by Proteobacteria and Actinobacteriota at the phylum level, while exhibiting a complex composition at the genus level, including Pantotheca (…). Pantoea ) and Serratia ( Serratia Various environmental microorganisms, including *Lactobacillus*, were observed. In Comparative Example 1, after the addition of an equal volume of distilled water, the relative abundance of Firmicutes at the phylum level significantly increased, while the abundance of naturally fermenting lactic acid bacteria genera, such as *Lactobacillus*, increased at the genus level. Lactiplantibacillus ) and Leuconostoc genus ( Weissella ) began to dominate. Comparative Example 2, inoculated with a single commercial microbial agent, showed that *Lactobacillus* genus (*Lactobacillus*) dominated at both the phylum and genus levels. Lactiplantibacillus In Example 1, the absolutely dominant bacterial genus was Firmicutes, as observed by inoculation with a compound bacterial agent. Comparative Example 3 showed a significantly dominant genus of *Lactobacillus bruneri*, with a highly homogeneous community structure. At the phylum level, Firmicutes was the absolutely dominant group when inoculated with the compound bacterial agent, consistent with the fact that all the functional bacteria inoculated belonged to this phylum. At the genus level, the dominant bacterial group clearly corresponded to the added compound bacterial agent components, mainly including *Lactobacillus* (*Lactobacillus* genus). Lactiplantibacillus ), Pediococcus (Pediococcus ) and Enterococcus spp. ( Enterococcus This study achieved synergistic regulation and targeted reshaping of the fermentation microecology. The community structure indicates that the exogenously inoculated compound microbial agent successfully colonized the fermentation system and became the core functional microbial community. Through synergistic effects such as rapid acid production and niche competition, it effectively suppressed inherent contaminants in the raw materials and other wild lactic acid bacteria (such as Leuconostoc) that appeared in the control group. Weissella The growth of microorganisms (such as bacteria) drives the entire microbial community to evolve in a predetermined direction dominated by specific functional species, thus achieving precise control over the fermentation process.

[0052] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing silage from wild leek-type pasture, characterized in that, Includes the following steps: S1. Cut and wither the grass in the mountain leek-type grassland to obtain pre-treated grass; Specifically, the withering process includes: drying the cut forage until the moisture content is 55%-65%, where % is a mass percentage; S2. Mix the compound microbial agent with the pretreated forage to obtain the inoculated forage; The compound microbial agent is composed of Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus plantarum. S3. The inoculated forage is bundled, wrapped, and then sealed for anaerobic fermentation to obtain the mountain leek-type pasture silage.

2. The preparation method according to claim 1, characterized in that, The viable count ratio of *Pediococcus pentosaceus*, *Enterococcus faecalis*, and *Lactobacillus plantarum* was (1×10⁻⁶). 10 -1×10 11 ): (1×10 10 -1×10 11 ): (1×10 10 -1×10 11 ).

3. The preparation method according to claim 2, characterized in that, The ratio of viable counts of *Pediococcus pentosaceus*, *Enterococcus faecalis*, and *Lactobacillus plantarum* is 1:1:

1.

4. The preparation method according to claim 1, characterized in that, In step S1, the harvesting period is from the initial flowering stage to the peak flowering stage of wild leeks.

5. The preparation method according to claim 1, characterized in that, In step S2, the amount of compound microbial agent added is 1×10 based on the weight of the pretreated forage. 6 CFU / gram of hay.

6. The preparation method according to claim 1, characterized in that, In step S3, the baling density is 450-550 kg / m³. 3 ; and / or, the number of layers in the wrapping is 4-6.

7. Wild leeks-type pasture silage prepared by the preparation method according to any one of claims 1-6.

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