A lactic acid bacteria combination suitable for green bean silage and a preparation method and application thereof

Abstract

This invention discloses a lactic acid bacteria composition suitable for sesame silage, its preparation method, and its application, relating to the field of microbiology. The lactic acid bacteria composition is *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum M141 and Enterobacter sieboldii ( Weissella paramesenteroides M143, compared to using *Lactobacillus plantarum* M141 or *Enterobacter sieboldii* M143 alone or commercial lactic acid bacteria preparations, the combination of the two lactic acid bacteria (addition amount 5.0 × 10⁻⁶) is more effective. 6 The cfu / g FM ratio yields the highest lactic acid, significantly reduces pH and ammonia nitrogen content, effectively inhibits the growth of harmful microorganisms (yeast), and improves the fermentation quality of sesame silage.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a combination of lactic acid bacteria suitable for sesame silage, its preparation method, and its application. Background Technology

[0002] Soil salinization and water scarcity are major factors restricting the sustainable development of agriculture. Saline-alkali land has low productivity, and conventional forage harvesting, processing, and storage technologies cannot meet the needs of the saline-alkali land forage industry, resulting in poor quality and low yield of forage products and low efficiency in the conversion and utilization of high-quality forage. This severely restricts the development of the forage industry in Xinjiang's saline-alkali land. Introducing and breeding drought-resistant and salt-tolerant forage varieties is one of the effective ways to solve this problem. (Senecio scandens) Sesbania cannabina ) belongs to the genus Sesbania in the family Leguminosae. Sesbania Sesbania sesbania is an annual, multi-purpose herbaceous plant that is salt-tolerant, flood-tolerant, tolerant of poor soil, and has strong resistance to diseases and pests. It has a well-developed root system, abundant root nodules, and strong nitrogen-fixing ability, making it a pioneer plant for improving and restoring saline-alkali soils in tidal flats, as well as an excellent summer green manure. Furthermore, sesbania sesbania has a high content of protein and vitamins, and its roots, stems, leaves, flowers, and fruits contain abundant flavonoids, polyphenols, and other bioactive components. Its stems and leaves are soft, tender, and juicy, with abundant foliage, making it a valuable feed source to enrich regional forage resources and address the current shortage of protein feed. However, when using legumes such as sesbania sesbania as the sole silage material, direct ensiling is difficult to produce high-quality silage due to the high water content, low soluble carbohydrate content, and limited number of surface lactic acid bacteria. It is also prone to Clostridium perfringens infection. Clostridium Fermentation (spp.) leads to a significant loss of nutrients, primarily crude protein.

[0003] Lactic acid bacteria can purposefully regulate the microbial community during silage fermentation, promoting the conversion of polysaccharides and crude fiber, thereby controlling the fermentation effect. Compared with exogenous commercial lactic acid bacteria, epiphytic lactic acid bacteria have a better effect on improving silage quality. However, the population of epiphytic lactic acid bacteria is not always large enough or has a suitable composition to promote effective fermentation. Therefore, it is necessary to isolate and screen epiphytic lactic acid bacteria in sesame silage to obtain lactic acid bacteria strains that can improve silage fermentation quality and provide a reference for the feed utilization of sesame. Summary of the Invention

[0004] The purpose of this invention is to provide a lactic acid bacteria combination suitable for sesame silage, its preparation method, and its application, in order to solve the problems existing in the prior art. This invention screens lactic acid bacteria strains (Lactobacillus plantarum M141 and Enterobacter vesiculosus M143) that can improve the fermentation quality of sesame silage, providing a reference for the feed utilization of sesame.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a lactic acid bacteria blend suitable for sesame silage, the lactic acid bacteria blend comprising *Lactobacillus plantarum* (… Lactiplantibacillus plantarum M141 and Enterobacter sieboldii ( Weissella paramesenteroides M143; The preservation number of the Lactobacillus plantarum M141 is CGMCC No. 33056; The accession number of the Enterobacter vesiculosus M143 is CGMCC No. 33057.

[0006] Preferably, the effective viable count ratio of the *Lactobacillus plantarum* M141 to the *Enteromorpha vesiculosus* M143 is 5:2~3.

[0007] This invention provides the application of the above-mentioned lactic acid bacteria combination in the preparation of microbial agents for sesame silage.

[0008] The present invention provides a microbial agent for sesame silage, the microbial agent comprising the above-mentioned combination of lactic acid bacteria.

[0009] More preferably, the microbial agent also includes excipients.

[0010] The present invention provides a method for preparing the above-mentioned bacterial agent, including the step of mixing the bacterial solution of *Lactobacillus plantarum* M141 and the bacterial solution of *Enteromorpha vesiculosus* M143.

[0011] The present invention provides the application of the above-described lactic acid bacteria combination or the above-described bacterial agent in any of the following: (1) Sesbania silage; (2) Preparation of sesame silage lactic acid bacteria preparation; (3) Improve the fermentation quality of sesame silage; (4) Prepare a preparation to improve the fermentation quality of sesame silage.

[0012] Preferably, the lactic acid bacteria combination or the inoculant reduces the pH value, ammonia nitrogen content, and yeast count in the sesame silage fermentation product, thereby increasing the lactic acid content and lactic acid bacteria count, thus improving the quality of the silage fermentation product.

[0013] The present invention provides a method for improving the quality of sesame silage fermentation products, including the step of inoculating the above-mentioned lactic acid bacteria combination into sesame for silage fermentation.

[0014] Preferably, the dosage of the lactic acid bacteria combination is 5.0 × 10⁻⁶. 6 cfu / g FM.

[0015] The present invention discloses the following technical effects: This invention employs traditional microbial culture methods to isolate and culture lactic acid bacteria from sesame silage at different fermentation stages. Strains (Lactobacillus plantarum M141 and Enterobacter sieboldii M143) are identified and screened using physiological, biochemical, and 16S rDNA sequence analysis. These strains are then added in combination to sesame raw materials for silage effect observation (addition amount: 5.0 × 10⁻⁶). 6 The changes in nutritional quality, fermentation quality, and the number of major microorganisms in sesame silage were detected using the bagged silage method (cfu / g FM). Results showed that after 60 days of silage, compared with the use of *Lactobacillus plantarum* M141 or *Westernella enteronis* M143 alone, or commercial lactic acid bacteria agents, the combination of two lactic acid bacteria (at a dosage of 5.0 × 10⁻⁶ CFU / g FM) significantly improved silage quality. 6 The cfu / g FM ratio yields the highest lactic acid, significantly reduces pH and ammonia nitrogen content, effectively inhibits the growth of harmful microorganisms (yeast), reduces crude protein loss, and improves the fermentation quality of sesame silage. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Phylogenetic analysis of lactic acid bacteria attached to sesame silage; Figure 2 The growth rate of lactic acid bacteria attached to sesame silage; Figure 3 The acid production rate of lactic acid bacteria attached to sesame silage. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 Plant-based lactic acid bacteria ( Lactiplantibacillus plantarum M141 and Enterobacter sieboldii ( Weissella paramesenteroides Isolation, purification, identification and preservation of M143 1. Materials and Methods 1.1 Test Materials Sesbania was planted in the experimental field of the 11th Company of the 12th Regiment in Alar City, Xinjiang (N 40°32′, E 81°18′). When the sesbania reached the budding stage, the fresh sesbania raw material was harvested and crushed into 2-3cm pieces using a green fodder harvester on July 6, 2024.

[0024] 1.2 Silage Preparation When the sesame seeds reach the budding stage, the chopped sesame raw materials are packed into silage bags (23 cm × 45 cm) with one-way exhaust valves, vacuumed and sealed to make silage. Samples are taken after fermentation at room temperature for 3 days, 7 days, 14 days, 30 days and 60 days for the isolation and identification of lactic acid bacteria.

[0025] 1.3 Isolation and Purification of Lactic Acid Bacteria Prepare diluted solutions from sesame silage at different fermentation stages, and select 3 dilution gradients (10... -5 10 -6 10 -7The strains were plated on MRS agar and anaerobically cultured at 30°C for 48 h before isolation. Single colonies with different colors, sizes, and glosses of clear zones were picked using the four-zone streak method and purified four times consecutively. Subsequently, the purified lactic acid bacteria strains were subjected to morphological observation, Gram staining, and catalase detection to preliminarily determine whether the experimental strains were lactic acid bacteria. Finally, the purified strains were enriched and thoroughly mixed with 50% glycerol at a 1:1 volume ratio in sterile cryovials and stored at -80°C for later use.

[0026] 1.4 Sequencing of 16S rDNA of Lactic Acid Bacteria Following the kit instructions, DNA was extracted from the 24-hour culture medium of the test strain. PCR amplification was performed using primers FA-27F: 5'-GCAGAGTTCTCGGAGTCACGAAGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and RA-1495R: 5'-AGCGGATCACTTCACACAGGACTACGGGTACCTTGTTACGA-3' (SEQ ID NO.2). The PCR products were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for forward and reverse sequencing. The 16S rDNA sequence of the isolated strain was compared and identified with 16S rDNA sequences in GenBank using BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST) for lactic acid bacteria strain identification. A phylogenetic tree was constructed using MEGA 7 software.

[0027] 1.5 Detection of physiological and biochemical characteristics of lactic acid bacteria A bacterial suspension of the test strain was prepared using a 24-hour culture medium (i.e., the culture medium obtained by anaerobic culture at 30°C for 24 hours in MRS broth). This suspension was inoculated into MRS broth at a 3% inoculum. The growth of the strain was assessed at different temperatures (4°C, 10°C, 30°C, and 45°C), different NaCl concentrations (4% and 6.5%), and different pH values ​​(3.0, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, and 9.0). The absorbance (OD value) at 600 nm was measured using a microplate reader. 600nm <0.5 is recorded as no growth (-), OD 600nm A value between 0.5 and 1.0 is denoted as weak growth (w), OD 600nm A value between 1.0 and 1.5 is considered normal growth (+), OD 600nm A value >1.5 is considered good growth (++).

[0028] The bacterial suspension of the test strain was inoculated into a bacterial micro-biochemical identification tube (Qingdao Haibo Biotechnology Co., Ltd.) and cultured anaerobically at 30°C for 48 h. The utilization of 20 carbon sources by the lactic acid bacteria strain was judged based on the color change of the identification tube.

[0029] The bacterial suspension of the test strain was inoculated at a 3% inoculation rate into MRS broth and M17 broth with initial pH values ​​of 5.84 and 6.96, respectively. The OD value (600 nm) and pH value were measured at 0-24 h (measured every 3 h), and the growth curve and acid production rate curve were recorded and plotted.

[0030] 2 Results and Analysis 2.1 16S rDNA gene sequence analysis of lactic acid bacteria strains The 16S rDNA gene sequence alignment results of the two lactic acid bacteria strains (Table 1) showed that the sequence similarity between the two strains and the corresponding standard strains in GenBank was over 99%. (Phylogeny tree) Figure 1 The results showed that M141 and Lactobacillus plantarum ( Lactiplantibacillus plantarum It showed the highest homology and was identified as *Lactobacillus plantarum*; M143 was similar to *Westernella enterica* (…). Weissella paramesenteroides It had the highest homology with ) and was identified as Enterobacter sieboldii.

[0031] Table 1. Results of 16S rDNA gene sequence analysis of lactic acid bacteria strains 2.2 Physiological and biochemical characteristics of lactic acid bacteria strains The physiological and biochemical characteristics of the two lactic acid bacteria strains are shown in Table 2. The results showed that both strains were Gram-positive, catalase-negative, and produced acid but not gas during glucose fermentation, indicating they were homofermentative lactic acid bacteria. Regarding growth at different temperatures, strain M143 could not grow at 4℃, while both strains grew normally or well between 10℃ and 45℃. At pH 3.0, except for M143, both strains grew normally or well between pH 4.0 and 9.0. Both strains also grew normally or well under 4% NaCl and 6.5% NaCl conditions.

[0032] The results of the sugar fermentation experiment are shown in Table 3. The results showed that strain M141 could utilize amygdalin, arabinose (d), cellobiose, fructose, maltose, galactose, lactose, mannose, mannitol, glucose, melitriose, raffinose, salicin, sorbitol, sucrose, xylose, and trehalose, but could not utilize rhamnose, consistent with the model strain of *Lactobacillus plantarum*. Strain M143 could utilize galactose, maltose, melitriose, sucrose, trehalose, and xylose (d), consistent with the model strain of *Enterobacter esculenta*, but could not utilize arabinose, cellobiose, and raffinose, which differed from the model strain.

[0033] Table 2 Growth characteristics of lactic acid bacteria attached to sesame silage Note: "-" indicates no growth, "w" indicates weak growth, "+" indicates growth, and "++" indicates good growth.

[0034] Table 3 Sugar fermentation characteristics of attached lactic acid bacteria in sesame silage Note: "+" indicates positive, "-" indicates negative, and "d" indicates that 80%-90% of the strains are positive.

[0035] 2.3 Acid production rate and growth rate of lactic acid bacteria strains The growth rate curve of lactic acid bacteria is as follows: Figure 2 As shown, strain M141 grew slowly during the first 0-3 hours of culture, entering the lag phase. After 3 hours of culture, it entered the logarithmic growth phase, showing a slow upward trend from 12 to 18 hours, and then began to decline after 18 hours. Strain M143 was in the lag phase from 0 to 6 hours of culture, in the logarithmic growth phase from 6 to 21 hours, and entered the stationary phase after 21 hours.

[0036] The acid production rate curve of lactic acid bacteria is as follows: Figure 3 As shown, strain M141 produced acid at a relatively fast rate from 0 to 12 h, and the rate of pH decrease slowed down after 12 h, with the pH value decreasing to 3.74 by 24 h. Strain M143 produced acid at a relatively fast rate from 0 to 9 h, and the rate of acid production slowed down from 9 to 18 h, with the pH value changing less after 18 h.

[0037] 3. Biological Preservation In this embodiment, two lactic acid bacteria strains isolated and screened from sesame silage were identified by 16S rDNA sequence analysis: M141 is *Lactobacillus plantarum*, and M143 is *Westernella enteritidis*. Both strains can grow under conditions of 4% and 6.5% NaCl, 10℃-45℃, and pH 4-pH 9, exhibiting strong acid production and growth / reproduction capabilities, and good tolerance to different temperatures, NaCl concentrations, and pH environments. Therefore, these two strains were biopreserved.

[0038] In this embodiment, Lactobacillus plantarum numbered M141 is named Lactobacillus plantarum (…). Lactiplantibacillus plantarum M141, this strain is classified as *Lactobacillus plantarum* (M141). Lactiplantibacillus plantarum The sample was deposited on December 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33056.

[0039] In this embodiment, the enteromorphic Weissella var. M143 is named Enteromorphic Weissella var. ( Weissella paramesenteroides M143, this strain is classified as Enterobacter sieboldii ( Weissella paramesenteroides The sample was deposited on December 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33057.

[0040] Example 2: Application of *Lactobacillus plantarum* M141 and *Westernella enterica* M143 1. Materials and Methods 1.1 Test Materials Sesbania was planted in an experimental field of the 11th Company, 12th Regiment, Alar City, Xinjiang (N 40°32′, E 81°18′). When the sesbania reached the budding stage, the fresh sesbania raw material was harvested and crushed into 2-3 cm pieces using a forage harvester on July 6, 2024. The nutrient composition and microbial count of the sesbania before silage are shown in Table 4.

[0041] Table 4. Nutrient composition and microbial count of sesame seeds before silage The commercial lactic acid bacteria agent is a Lactobacillus plantarum agent: Lactobacillus plantarum ≥ 2.0 × 10⁻⁶. 9 CFU / g, purchased from Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd.

[0042] The test bacterial agent was prepared by mixing *Lactobacillus plantarum* M141 bacterial suspension and *Westernella enterica* M143 bacterial suspension at a volume ratio of 1:1. The bacterial suspension was prepared by activating the bacteria and then inoculating the activated *Lactobacillus plantarum* M141 and *Westernella enterica* M143 bacterial suspensions into MRS broth medium at an inoculation rate of 3% and anaerobic culture at 30°C for 12-24 h to obtain *Lactobacillus plantarum* M141 and *Westernella enterica* M143 bacterial suspensions. The bacterial suspension concentration was then adjusted with sterile physiological saline to obtain the bacterial suspension for silage fermentation.

[0043] 1.2 Experimental Design After the sesame seeds were dried to a moisture content of 60%-70%, they were immediately ensiled using a vacuum bag method. The experiment consisted of 5 treatments: (1) CK1 (sprayed with an equal amount of sterile saline); (2) CK2 treatment (added with commercial lactic acid bacteria agent, the amount added was 5×10). 6 (cfu / g); (3) LP treatment (adding Bacillus plantarum M141 bacterial solution, amount added 5×10 6 cfu / g) (4) WP treatment (adding Enterobacter malignant strain M143, amount added 5×10) 6 (cfu / g) (5) LPWP treatment (mixing Bacillus plantarum M141 bacterial solution and Enterobacter sieboldii M143 bacterial solution at a ratio of 5:2~3 of effective viable count, with an addition amount of 5×10 6 cfu / g).

[0044] The bacterial agent (sterile water) used in each treatment was 5×10 6 cfu / g FM (i.e., 5×10) 6 The total dose of CFU (1g of sesbania) was mixed evenly with sesbania. Then, 1 kg of sample was weighed and vacuum-sealed in a polyethylene anaerobic bag with a one-way exhaust valve. The bag was placed at 18-23℃ for natural fermentation. On the 60th day of silage, 3 bags of silage were randomly selected from each treatment group, mixed, and sampled to determine the nutritional content, fermentation indicators, and microbial count.

[0045] 1.3 Determination of silage nutritional quality, fermentation quality, and the number of major microorganisms Nutritional quality indicators were determined for dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (CP), crude fat (EE), and soluble carbohydrates (WSC). DM content was determined by drying method; CP content was determined by Kjeldahl method; NDF and ADF contents were determined by Van der Waals detergent fiber analysis method; EE content was determined by Soxhlet extraction method; and WSC was determined by anthrone-sulfuric acid method.

[0046] Fermentation characteristic parameters, including pH, ammonia nitrogen (NH3-N), lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA), were determined. pH was measured directly using a pH meter; NH3-N content was determined using the phenol-sodium hypochlorite colorimetric method; LA content was determined using high-performance liquid chromatography (HPLC); and the contents of AA, PA, and BA were determined using gas chromatography (GC).

[0047] The microbial count was determined using the dilution pour plate method for Escherichia coli (E. coli). Es-cherichia coliThe number of bacteria, including yeasts, molds, and lactic acid bacteria (LAB), was determined. Escherichia coli was cultured on eosin methylene blue medium at 30°C for 48 h before counting; yeasts and molds were cultured on potato dextrose agar (PDA) medium at 30°C for 48 h before counting; and LABs were cultured anaerobically on MRS medium at 30°C for 48 h before counting.

[0048] 2 Results and Analysis 2.1 Effects of epiphytic lactic acid bacteria on the nutritional quality of sesame silage As shown in Table 5, after 60 days of silage fermentation, there were no significant differences in NDF and ADF contents among the treatment groups. P >0.05), the DM content in CK2 and LP treatments was significantly lower than that in CK1, WP and LPWP treatments ( P <0.05); CP content in CK1, CK2 and WP treatments was significantly lower than that in LPWP treatment ( P <0.05); the EE content in WP and LPWP treatments was significantly lower than that in CK2 and LP treatments ( P <0.05); the WSC content in CK2, LP, and LPWP treatments was significantly lower than that in CK1 and WP ( P <0.05).

[0049] Table 5. Effects of epiphytic lactic acid bacteria on the nutritional quality of sesame silage. Note: Different lowercase letters in the superscript of data in the same column indicate significant differences. P <0.05, the same or no letter indicates no significant difference ( P >0.05), the same as in the table below.

[0050] 2.2 Effects of epiphytic lactic acid bacteria on the fermentation quality of sesame silage As shown in Table 6, the pH values ​​of the LP and LPWP treatments were significantly lower than those of the CK1, CK2, and WP treatments. P <0.05); the NH3-N content in the LP and LPWP treatments was significantly lower than that in the CK1 treatment ( P <0.05); LA content in CK2, WP, and LPWP treatments was significantly lower than in LP treatment ( P <0.05), CK1 treatment was significantly lower than LP treatment ( P <0.01); the AA content in CK1 and LP treatments was significantly lower than that in CK2 treatment ( P <0.05); there was no significant difference in PA content among the treatment groups ( P >0.05), no BA was detected in any of the treatments.

[0051] Table 6. Effects of epiphytic lactic acid bacteria on the fermentation quality of sesame silage As shown in Table 7, the number of LABs in the CK1, CK2, and WP treatments was significantly lower than that in the LP and LPWP treatments. P <0.05); the number of yeasts in the LP and LPWP treatments was significantly lower than that in the CK1 and WP treatments ( P <0.05); only CK1 and WP treatments detected small amounts of Molds, while no E. coli was detected in any of the treatment groups.

[0052] Table 7. Effects of epiphytic lactic acid bacteria on the abundance of major microorganisms in sesame silage. Based on Examples 1 and 2, this invention combines traditional microbial culture methods with 16S rDNA sequencing identification technology to isolate and screen two lactic acid bacteria strains from various stages of sesame silage fermentation: one is *Lactobacillus plantarum*, and the other is *Westernella enteritidis*. Both strains can grow normally under pH conditions of 4.0-9.0, with *Lactobacillus plantarum* M141 even growing at pH 3.0, demonstrating good acid resistance. Both strains maintained normal life activities under 4% NaCl and 6.5% NaCl conditions and grew well at 10-45℃. *Lactobacillus plantarum* M141 also grew at 4℃, indicating that this strain has a certain adaptability to both low and high temperature conditions and a wide growth temperature range.

[0053] After 60 days of ensiling, the CP content in the LPWP treatment was significantly higher than that in CK1, while the ammonia nitrogen content was lower, indicating that the combination of plant lactic acid bacteria and Enterobacter-like Weissella spp. has a significant advantage in nutrient preservation of CP. During ensiling, lactic acid bacteria decompose carbohydrates to produce organic acids, mainly lactic acid, which lowers the pH value, inhibits the growth and reproduction of harmful microorganisms, and prevents nutrient loss due to the utilization of nutrients by harmful microorganisms. After 60 days of ensiling, the WSC content in the LPWP treatment was significantly lower than that in the CK1 treatment, while the LA content was significantly higher than that in both CK1 and CK2 treatments, indicating that epiphytic lactic acid bacteria can fully utilize the fermentation substrate to produce more lactic acid during fermentation. No BA was detected in any group throughout the ensiling process, indicating that no significant butyric acid spoilage occurred during fermentation. After 60 days of ensiling, the number of viable LAB bacteria in the LPWP treatment was higher than that in the CK1 and CK2 treatments, indicating that epiphytic lactic acid bacteria have a stronger growth capacity in sesame silage compared to commercial lactic acid bacteria agents. Excessive yeast viable counts can lead to a pungent alcoholic odor in silage, causing spoilage and posing a risk of nitrite poisoning to animals. Furthermore, yeasts compete with lactobacillus (LAB) for nutrients, resulting in nutrient loss. After silage fermentation, the yeast count in the LPWP treatment was significantly lower than in the CK1 and CK2 treatments, indicating a synergistic effect between *Lactobacillus plantarum* and *Enterobacter siracemi*, effectively inhibiting yeast growth. Molds and *E. coli* were not detected in the LPWP treatment at day 60, indicating that their growth was completely inhibited by the anaerobic environment and the combined inoculation of the two lactobacillus species. Therefore, the combined addition of *Lactobacillus plantarum* M141 and *Enterobacter siracemi* M143 to sesame silage can utilize the limited nutrients in the substrate to produce lactic acid, thereby lowering the pH of the fermentation system and inhibiting protein spoilage and the growth of harmful bacteria.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A lactic acid bacteria combination suitable for sesame silage, characterized in that, The lactic acid bacteria combination includes *Lactobacillus plantarum* (… Lactiplantibacillus plantarum M141 and Enterobacter sieboldii ( Weissella paramesenteroides M143; The preservation number of the Lactobacillus plantarum M141 is CGMCC No. 33056; The accession number of the Enterobacter vesiculosus M143 is CGMCC No. 33057.

2. The lactic acid bacteria combination according to claim 1, characterized in that, The effective viable count ratio of *Lactobacillus plantarum* M141 to *Westernella enterica* M143 is 5:2~3.

3. The use of the lactic acid bacteria combination according to claim 1 or 2 in the preparation of microbial agents for sesame silage.

4. A microbial agent for sesame silage, characterized in that, The microbial agent includes the lactic acid bacteria combination as described in claim 1 or 2.

5. The method for preparing the microbial agent according to claim 4, characterized in that, The step includes mixing the bacterial culture of *Lactobacillus plantarum* M141 and the bacterial culture of *Westernella enterica* M143.

6. The use of the lactic acid bacteria combination of claim 1 or 2 or the microbial agent of claim 4 in any of the following: (1) Sesbania silage; (2) Preparation of sesame silage lactic acid bacteria preparation; (3) Improve the fermentation quality of sesame silage; (4) Prepare a preparation to improve the fermentation quality of sesame silage.

7. The application according to claim 6, characterized in that, The lactic acid bacteria combination or the bacterial agent improves the quality of silage fermentation products by reducing the pH value, ammonia nitrogen content, and yeast count in the sesame silage fermentation products, thereby increasing the lactic acid content and lactic acid bacteria count.

8. A method for improving the quality of sesame silage fermentation products, characterized in that, The step includes inoculating the lactic acid bacteria combination described in claim 1 or 2 into sesame for silage fermentation.

9. The method according to claim 8, characterized in that, The dosage of the lactic acid bacteria combination is 5.0 × 10⁻⁶. 6 cfu / gFM.

Images