Lactococcus lactis for inhibiting fermentation of clostridium sporogenes and application thereof

Abstract

The application discloses a lactococcus lactis for inhibiting clostridium bryantii fermentation and application thereof. The technical problem is solved by improving the growth of clostridium bryantii and improving the fermentation quality of alfalfa. The lactococcus lactis is lactococcus lactis S0, and the preservation number of the lactococcus lactis S0 in the China General Microbiological Culture Collection Center is CGMCC No. 30594. The strain or fermentation product thereof can inhibit the growth of clostridium bryantii, and the strain can be used for the fermentation of alfalfa containing clostridium bryantii to improve the fermentation quality of alfalfa and can be used for production.

Description

Technical Field

[0001] This invention relates to a strain of lactic acid cactus that inhibits Clostridium fermentation in alfalfa silage and its applications. Background Technology

[0002] Alfalfa, a perennial herbaceous plant belonging to the legume family and the genus *Alfalfa*, is known as the "King of Forage." As a high-quality forage resource, it has high crude protein and crude fiber content. However, harvesting often faces the problem of simultaneous rain and heat, resulting in high moisture content (>70% fresh weight). High-moisture alfalfa, due to its low soluble sugar content, low number of epiphytic lactic acid bacteria, and high buffering energy, is highly susceptible to *Clostridium* fermentation, leading to reduced silage quality and palatability. *Clostridium* is difficult to control due to its broad substrate and anaerobic characteristics, and currently lacks effective methods for inhibiting it. Bacteriocins synthesized by microorganisms have targeted antibacterial properties and are safe and highly effective, making them very promising *Clostridium* inhibitors. Therefore, using bacteriocin-producing microorganisms to inhibit *Clostridium* growth during silage production can effectively prevent *Clostridium* fermentation, reduce production losses, and improve the fermentation and nutritional quality of alfalfa silage, providing a theoretical basis for the large-scale promotion and safe application of alfalfa silage. How to provide a method for inhibiting *Clostridium* and improving the fermentation quality of alfalfa remains a problem for researchers in this field. Summary of the Invention

[0003] The technical problem solved by this invention is to provide a way to inhibit Clostridium and improve the fermentation quality of alfalfa.

[0004] To address the above problems, the present invention provides Lactococcus lactis.

[0005] The lactococcus is Lactococcus lactis ( Lactococcus lactis S0, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 30594.

[0006] To address the aforementioned problems, the present invention also provides a microbial agent.

[0007] The bacterial agent contains the aforementioned Lactococcus lactis.

[0008] The microbial agent has at least one of the following functions: 1) inhibiting and / or slowing down the growth of Clostridium sporogenes, 2) killing Clostridium sporogenes, and 3) improving the fermentation quality of Clostridium fermented alfalfa silage.

[0009] The alfalfa mentioned above may be alfalfa.

[0010] As mentioned above, Clostridium sporogenes was present in the environment where alfalfa fermentation took place.

[0011] In the above text, the ratio of Clostridium sporogenes to Lactococcus lactis in the alfalfa fermentation environment can be 1:10.

[0012] In the above text, the bacterial count of Clostridium sporogenes can be no higher than 1×10⁻⁶. 5 CFU Clostridium sporogenes / g alfalfa.

[0013] In the above text, the bacterial count of Clostridium sporogenes can be 1×10⁻⁶. 5 CFU Clostridium sporogenes / g alfalfa.

[0014] The active ingredient in the above-mentioned bacterial agent may be Lactococcus lactis ( Lactococcus lactis S0 or / and Lactococcus lactis ( Lactococcus lactis The active ingredients of the above-mentioned microbial agent may also contain other biological or non-biological components, and the other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art based on the effect of the microbial agent.

[0015] The aforementioned microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0016] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0017] In addition to the active ingredients, the aforementioned microbial agents may also include excipients such as water, carbon sources, and / or ammonia sources. Carbon sources are nutrients for microbial growth; they are carbon-containing compounds, including sugars, oils, organic acids and esters, and small-molecule alcohols, which are both readily available and slowly available carbon sources. Nitrogen sources refer to substances that provide the nitrogen element required for microbial nutrition, including readily available and slowly available nitrogen sources such as peanut meal, soybean meal, yeast powder, peptone, ammonia, ammonium salts, and nitrates.

[0018] In addition to the active ingredients, the above-mentioned microbial agents may also contain 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 maltodextrin, corn flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol. The liquid carrier may be vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane. In the microbial agent, the active ingredients may exist 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 microbial agent may be various, such as liquid, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0019] To address the aforementioned problems, the present invention also provides pathogen inhibitors.

[0020] The pathogen inhibitor is obtained by culturing the above-mentioned Lactococcus lactis and / or the above-mentioned bacterial agent in a culture medium, collecting the fermentation broth, removing the bacterial cells from the fermentation broth, and obtaining the active ingredient of the pathogen inhibitor.

[0021] The pathogen mentioned above may be Clostridium sporogenes (Clostridium sporogenes) Clostridium sporogenes ).

[0022] In the above text, the diameter of the effective components in the fermentation broth can be less than 0.22 μm.

[0023] The culture medium mentioned above may be a liquid culture medium.

[0024] In the above text, the fermentation temperature can be 37℃. The fermentation period can be 1-2 days.

[0025] To address the aforementioned problems, the present invention also provides an alfalfa fermentation agent.

[0026] The inhibitor contains the above-mentioned Lactococcus lactis, the above-mentioned bacterial agent, and / or the above-mentioned pathogen inhibitor.

[0027] In the above text, the bacterial agent may be a bacterial suspension of the lactococcus lactis.

[0028] The bacterial suspension of Lactococcus lactis described above was prepared as follows: The glycerol-freezing SO was removed, and a disposable inoculation loop was used to pick up the bacterial suspension and streak it onto GM17 solid medium. The suspension was then anaerobically cultured at 37°C. After colonies grew, a single colony was picked and inoculated into 3 mL of GM17 liquid medium. After anaerobic culture at 37°C for 1 day, the culture was expanded to the desired bacterial suspension volume using a 3% inoculation rate. The suspension was then anaerobically cultured at 37°C for 1-2 days to obtain the bacterial suspension.

[0029] In the above text, the viable count of the lactococcus lactis in the bacterial suspension is 10. 8 -10 10 CFU / mL.

[0030] To address the aforementioned problems, the present invention also provides a method for fermenting alfalfa.

[0031] The method includes the step of fermenting alfalfa with the above-mentioned lactococcus lactis and / or the above-mentioned inoculant to obtain alfalfa fermentation products.

[0032] To address the aforementioned problems, the present invention also provides the application of the aforementioned Lactococcus lactis.

[0033] The application is any one of the following:

[0034] A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes;

[0035] A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes;

[0036] A3) Application in killing Clostridium sporogenes;

[0037] A4) Application in the preparation of products that kill Clostridium sporogenes;

[0038] A5) Improves the quality of alfalfa after fermentation;

[0039] A6) Application in the preparation of products that improve the quality of fermented alfalfa.

[0040] To address the aforementioned problems, the present invention also provides the application of the aforementioned microbial agent.

[0041] The application is any one of the following:

[0042] A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes;

[0043] A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes;

[0044] A3) Application in killing Clostridium sporogenes;

[0045] A4) Application in the preparation of products that kill Clostridium sporogenes;

[0046] A5) Improves the quality of alfalfa after fermentation;

[0047] A6) Application in the preparation of products that improve the quality of fermented alfalfa.

[0048] To address the aforementioned problems, the present invention also provides the application of the aforementioned pathogen inhibitors.

[0049] The application is any one of the following:

[0050] A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes;

[0051] A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes;

[0052] A3) Application in killing Clostridium sporogenes;

[0053] A4) Application in the preparation of products that kill Clostridium sporogenes;

[0054] A5) Improves the quality of alfalfa after fermentation;

[0055] A6) Application in the preparation of products that improve the quality of fermented alfalfa.

[0056] To address the aforementioned problems, the present invention also provides the application of the aforementioned alfalfa fermentation agent.

[0057] The application is any one of the following:

[0058] A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes;

[0059] A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes;

[0060] A3) Application in killing Clostridium sporogenes;

[0061] A4) Application in the preparation of products that kill Clostridium sporogenes;

[0062] A5) Improves the quality of alfalfa after fermentation;

[0063] A6) Application in the preparation of products that improve the quality of fermented alfalfa.

[0064] To address the above problems, the present invention also provides a method for preparing a microbial agent.

[0065] The method includes the step of using the above-mentioned Lactococcus lactis as a component of the bacterial agent to obtain the bacterial agent.

[0066] To address the above problems, the present invention also provides a method for preparing fermentation broth supernatant.

[0067] The method includes fermenting the above-mentioned lactococcus lactis or the above-mentioned bacterial agent to prepare a fermentation broth supernatant.

[0068] In the above text, the diameter of the effective components in the supernatant of the fermentation broth is less than 0.22 μm.

[0069] The product may be a microecological preparation.

[0070] The plant mentioned above may be any of the following:

[0071] B1) Leguminosae (family legumes);

[0072] B2) Alfalfa species;

[0073] B3) Alfalfa.

[0074] Beneficial effects

[0075] The purpose of this invention is to provide a strain of *Lactococcus lactis* capable of inhibiting the fermentation of *Clostridium perfringens* in high-moisture alfalfa silage. Lactococcus lactis S0, this bacterium can inhibit the growth of Clostridium in high-moisture alfalfa silage by secreting bacteriocin Nisin Z, thereby inhibiting the Clostridium fermentation process, improving the fermentation quality of alfalfa silage and ensuring its nutritional quality.

[0076] This invention discloses a strain of *Lactococcus lactis* that inhibits *Clostridium sporogenes* fermentation in alfalfa silage and its application. The technical problem solved is to inhibit the growth of *Clostridium sporogenes* and improve the quality of alfalfa fermentation. Specifically, *Lactococcus lactis* is disclosed, wherein *Lactococcus lactis* (… Lactococcus lactis S0, with its accession number CGMCC No. 30594 at the China General Microbiological Culture Collection Center, shows that its strain or fermentation products can inhibit the growth of Clostridium sporogenes. Using this strain in alfalfa silage fermented with Clostridium sporogenes can also inhibit the growth of Clostridium sporogenes, thus improving the fermentation quality of alfalfa and making it suitable for production.

[0077] Depository Instructions

[0078] Strain name: Lactococcus lactis

[0079] Latin name: Lactococcus lactis

[0080] Strain number: S0

[0081] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0082] Collection institution abbreviation: CGMCC

[0083] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0084] Deposit date: May 10, 2024

[0085] CGMCC Registration Number: CGMCC No. 30594

[0086] Strain name: Clostridium sporogenes

[0087] Latin name: Clostridium sporogenes

[0088] Strain number: FW40A

[0089] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0090] Collection institution abbreviation: CGMCC

[0091] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0092] Deposit date: May 10, 2024

[0093] CGMCC Registration Number: 30595 Attached Figure Description

[0094] Figure 1The in vitro inhibition of S0 on Clostridium epiphytum on the surface of high-moisture alfalfa silage is shown in Figure 1. (A) Inhibitory effect of S0 on Clostridium sporogenes FW40A; (B) Inhibitory effect of S0 supernatant at different volume ratios on FW40A.

[0095] Figure 2 Figure 1: The inhibitory effect of S0 supernatant on FW40A cell membrane; (A) Electron micrograph of normal FW40A cells; (B) FW40A cells after S0 supernatant treatment; (C) Effect of S0 supernatant on FW40A cell membrane permeability as determined by fluorescent dye PI.

[0096] Figure 3 The effect of S0 inoculation on the fermentation quality of high-moisture alfalfa silage fermented by Clostridium difficile; (A) pH value; (B) lactic acid; (C) acetic acid; (D) propionic acid; (E) butyric acid; (F) ammonia nitrogen.

[0097] Figure 4 The effect of S0 on the protein composition of high-moisture alfalfa silage fermented by Clostridium difficile is shown in the figure. (A) Dry matter; (B) Crude protein; (C) Ammonia; (D) Soluble true protein; (E) Insoluble true protein; (F) Fiber-bound protein; (G) Non-degradable protein.

[0098] Figure 5 The effect of S0 on the biogenic amine content of alfalfa silage fermented by Clostridium difficile; (A) total biogenic amines; (B) tyramine; (C) cadaverine; (D) tryptamine.

[0099] Figure 6 Figure 1 shows the effect of S0 on the microbial composition of Clostridium fermentation of high-moisture alfalfa silage; (A) Absolute abundance of bacterial community at the species level; (B) Absolute abundance of Clostridium community at the species level. Detailed Implementation

[0100] 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.

[0101] 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 and reagents used in the following examples are commercially available.

[0102] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used. P <0.05 ( () indicates a significant difference. P <0.01 ( () indicates a highly significant difference. P <0.001 ( () indicates a highly significant difference.

[0103] Example 1 Lactococcus lactis Lactococcus lactis Confirmation of S0 genus

[0104] A strain of *Lactococcus lactis* producing bacteriocin Z was isolated from cow's milk using microbial isolation and culture methods. Lactococcus lactis S0 (hereinafter referred to as S0 or Lactococcus lactis S0).

[0105] 16S rDNA sequence detection:

[0106] Lactococcus lactis Lactococcus lactis S0 strain was inoculated into GM17 medium (Coolaber, catalog number: PM0712-250g) and incubated statically at 37°C for 13 hours. Lactococcus lactis was then collected. Lactococcus lactis S0 bacterial cells were used to extract whole-genome DNA, which was randomly fragmented. The desired DNA fragment length was recovered by electrophoresis, and adapters were added for cluster preparation. HiSeq 4000 sequencing was performed, and the sequencing results were used for SOAP de novo assembly. Based on the assembly results, gene information analysis was conducted, and the bacteria were identified as *Lactococcus lactis*. Lactococcus lactis Its 16S rDNA sequence is shown in Sequence 1 of the sequence listing, and the specific sequence is as follows:

[0107]

[0108] This strain (Lactococcus lactis) Lactococcus lactis S0) is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC: 30594 and deposit date of May 10, 2024.

[0109] Example 2 Lactococcus lactis Lactococcus lacti In vitro inhibition of Clostridium epiphytidum on the surface of high-moisture alfalfa silage by fermentation supernatant of S0

[0110] Clostridium sporogenes Clostridium sporogenes The most abundant Clostridium species isolated from the surface of high-moisture alfalfa silage can influence the fermentation quality of high-moisture alfalfa silage by increasing pH, decreasing lactic acid content, and increasing acetic acid, propionic acid, ammonia nitrogen, and amine content. In vitro antibacterial experiments verified the effect of S0 on Clostridium sporogenes, a Clostridium sporogenes species found on the surface of high-moisture alfalfa silage. Clostridium sporogenes FW40A, this strain (Clostridium sporogenes) Clostridium sporogenes FW40A (hereinafter referred to as FW40A) is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC: 30595, and deposit date of May 10, 2024.

[0111] Lactococcus lactis is a commonly used fermentation strain in the fermentation industry, widely found in dairy and plant products. It grows rapidly, has a relatively simple metabolism, and while its genome is small, it contains sufficient biological information, making it a good substrate for gene manipulation. Lactococcus lactis MG1363 is a widely studied and commonly used chassis strain that can be used as an expression vector for various bioactive substances. It has been widely applied in the pharmaceutical and food industries, and is associated with Lactococcus lactis. Lactococcus lactis Compared to strain S0, it lacks the Nisin Z biosynthesis gene cluster in its genome, thus it cannot produce Nisin Z and perform its function, making it a preferred control strain.

[0112] Preparation of FW40A bacterial culture:

[0113] Clostridium sporogenes FW40A was inoculated into liquid BHI medium (OXOID, catalog number: CM1135B) and anaerobic incubated at 37 °C for 24 h. The culture was then diluted, spread, and counted to obtain the FW40A bacterial suspension for later use. The FW40A bacterial suspension contained 10% Clostridium sporogenes FW40A. 8 CFU / mL.

[0114] Preparation of Lactococcus lactis S0 bacterial culture:

[0115] Lactococcus lactis Lactococcus lactis S0 was inoculated into GM17 medium and cultured at 37°C until OD. 600 =1, obtain Lactococcus lactis S0 bacterial solution (also known as S0 bacterial solution), for later use.

[0116] Preparation of S0 aseptic fermentation broth supernatant:

[0117] The S0 bacterial culture was centrifuged at 8000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the supernatant of the S0 sterile fermentation broth.

[0118] Preparation of Lactococcus lactis MG1363 bacterial suspension:

[0119] Lactococcus lactis MG1363 (described in the following published literature: Resident TP712 Prophage of Lactococcus lactis Strain MG1363 Provides Extra Holin Functions to the P335 Phage CAP for Effective Host Lysi, the name of Lactococcus lactis MG1363 in this literature) was inoculated into GM17 medium and cultured at 37°C until OD100. 600 =1, obtain MG1363 bacterial culture, for later use.

[0120] 100ug / ml Nisin: Weigh 1 mg of Nisin powder (Macklin, catalog number: N874940-5g) into a 15mL centrifuge tube, add 10 mL of sterile water, and mix thoroughly until the powder is completely dissolved to obtain 100ug / ml Nisin.

[0121] 1) Qualitative determination of antibacterial activity using agar diffusion method

[0122] Take 1 mL of the S0 bacterial culture cultured above, centrifuge at 8000 rpm for 5 min, collect the supernatant and filter it through a 0.22 μm filter membrane to obtain the supernatant of S0 sterile fermentation broth.

[0123] Take 1 mL of the MG1363 bacterial culture cultured above, centrifuge at 8000 rpm for 5 min, collect the supernatant and filter it through a 0.22 μm filter membrane to obtain the supernatant of MG1363 sterile fermentation broth.

[0124] Take 1 mL of the 100 ug / ml Nisin solution prepared above and filter it through a 0.22 μm filter membrane to obtain sterile 100 ug / ml Nisin.

[0125] The above FW40A bacterial solution was prepared to a final concentration of 10. 6 CFU / mL was added to the TSC medium (Haibo, catalog number: HB0253-9) that was about to solidify, and mixed well to obtain the FW40A indicator plate.

[0126] The experiment was divided into SO bacterial culture group, MG1363 bacterial culture group, S0 supernatant group, MG1363 supernatant group, Nisin group (100ug / ml) and GM17 group.

[0127] The S0 bacterial culture group was operated as follows:

[0128] Make holes in the FW40A indicator plate using a hole punch (hole diameter 7.8±0.2mm), add 100 μL of S0 bacterial solution to the holes, and anaerobic incubate at 37 ℃ for 24 h. Observe the presence and size of the transparent zone.

[0129] The MG1363 bacterial culture group was operated on as follows:

[0130] Use a punch to make holes in the FW40A indicator plate (hole diameter 7.8±0.2mm), add 100 μL of MG1363 bacterial solution to the holes, and anaerobic culture at 37 ℃ for 24 h. Then observe the presence or absence of the transparent zone and the ratio (transparent zone diameter / hole diameter).

[0131] The S0 supernatant group performs the following operations:

[0132] Make holes in the FW40A indicator plate using a hole punch (hole diameter 7.8±0.2 mm), add 100 μL of the supernatant of the above S0 sterile fermentation broth to the holes, and anaerobic culture at 37 ℃ for 24 h. Observe the presence and size of the transparent zone.

[0133] The MG1363 supernatant group performed the following operations:

[0134] Make holes (7.8±0.2 mm in diameter) in the FW40A indicator plate using a hole puncher. Add 100 μL of the supernatant of the above-mentioned MG1363 sterile fermentation broth to the holes. After anaerobic incubation at 37 ℃ for 24 h, observe the presence and size of the transparent zone.

[0135] The Nisin group (100ug / ml) underwent the following procedures:

[0136] Make holes (7.8±0.2 mm in diameter) in the FW40A indicator plate using a hole puncher. Add 100 μL of sterile 100ug / ml Nisin to the holes. After anaerobic incubation at 37 ℃ for 24 h, observe the presence and size of the transparent zone.

[0137] Group GM17 performed the following operations:

[0138] Make holes (7.8±0.2 mm in diameter) in the FW40A indicator plate using a hole puncher. Add 100 μL of GM17 medium to the holes and anaerobic culture at 37 ℃ for 24 h. Observe the presence and size of the transparent zone.

[0139] Formula for calculating the size of the transparent ring: Ratio = Diameter of transparent ring / Diameter of hole.

[0140] The results are as follows Figure 1 As shown in Table A and Table 1, S0 bacterial suspension is the S0 bacterial suspension group (ratio = 1.67), MG1363 bacterial suspension is the MG1363 bacterial suspension group (ratio = 1), Nisin 100ug / ml is the Nisin group (ratio = 1.89), GM17 is the GM17 group (ratio = 1), S0 supernatant is the S0 supernatant group (ratio = 1.25), and MG1363 supernatant is the MG1363 group (ratio = 1). This ratio is the diameter of the clear zone / pore diameter. Therefore, the larger the ratio, the larger the diameter of the inhibition zone (clear zone) and the stronger the antibacterial ability; the closer the ratio is to 1, the smaller the inhibition zone (clear zone) and the weaker the antibacterial ability; if the ratio is equal to 1, it indicates no antibacterial ability. It is evident that the MG1363 strain, which does not produce Nisin Z, failed to inhibit Clostridium sporogenes FW40A and form an inhibition zone, neither in its bacterial culture nor in its fermentation supernatant. After excluding the influence of GM17 medium, both the S0 bacterial culture and the fermentation supernatant were found to inhibit Clostridium sporogenes FW40A, an epiphytic bacterium on the surface of high-moisture alfalfa silage, in vitro, forming a relatively obvious inhibition zone. Its inhibitory ability against Clostridium was slightly lower than that of 100 μg / mL Nisin (…). Figure 1 (A)

[0141] Table 1

[0142]

[0143] 2) Quantitative determination of antibacterial activity using fermentation broth supernatant method

[0144] 10 6 Preparation of CFU / mL FW40A bacterial suspension: The above FW40A bacterial suspension was diluted with BHI liquid medium, and the viable count was confirmed to be 10⁻⁶ using the spread count method. 6 CFU / mL, that is, to obtain 10 6 CFU / mL FW40A bacterial suspension.

[0145] The experiment was divided into four groups: CK group, Nisin group, S0 group and MG1363 group.

[0146] Group CK shall perform the following operations:

[0147] Take an appropriate amount of 10 6 CFU / mL FW40A bacterial culture was added to 1.5 mL EP tubes, and the volume of GM17 medium was increased to 5% of the final total volume (10). 6 The volume ratio of CFU / mL FW40A bacterial culture to GM17 medium was 95:5, and 10% (10 6 The volume ratio of CFU / mLFW40A bacterial suspension to GM17 medium was 90:10, and 20% (10) 6 CFU / mL FW40A bacterial culture (80:20 volume ratio) was added to EP tubes and incubated anaerobically at 37 °C for 24 h. Then, 200 μL of the bacterial culture was transferred to a 96-well plate, and OD was detected using a multi-mode microplate reader. 600 .

[0148] The Nisin group performed the following operations:

[0149] Take an appropriate amount of 10 6 CFU / mL FW40A bacterial culture was added to 1.5 mL EP tubes, with the volume of 100 ug / mL Nisin accounting for 5% of the final total volume (10). 6 CFU / mL FW40A bacterial culture and 100ug / mL Nisin (volume ratio 95:5), 10% (10 6 CFU / mL FW40A bacterial culture with 100ug / mL Nisin at a volume ratio of 90:10, 20% (10 6 CFU / mL FW40A bacterial culture and 100 μg / mL Nisin (volume ratio 80:20) were added to EP tubes and incubated anaerobically at 37 °C for 24 h. Then, 200 μL of the bacterial culture was transferred to a 96-well plate, and OD was detected using a multi-mode microplate reader. 600 .

[0150] Group S0 performs the following operations:

[0151] Take an appropriate amount of 10 6 CFU / mL FW40A bacterial culture was added to 1.5 mL EP tubes, and the supernatant volume of the S0 aseptic fermentation broth was 5% (10) of the final total volume. 6 The volume ratio of CFU / mL FW40A bacterial culture to S0 aseptic fermentation broth supernatant was 95:5, 10% (10 6 The volume ratio of CFU / mL FW40A bacterial culture to S0 aseptic fermentation broth supernatant was 90:10, 20% (10 6The supernatant of the FW40A bacterial culture (CFU / mL) and the sterile fermentation broth of S0 (volume ratio 80:20) were added to EP tubes and incubated anaerobically at 37 °C for 24 h. Then, 200 μL of the bacterial culture was transferred to a 96-well plate, and the OD was measured using a multi-functional microplate reader. 600 .

[0152] The MG1363 group should perform the following operations:

[0153] Take an appropriate amount of 10 6 CFU / mL FW40A bacterial culture was added to 1.5 mL EP tubes, and the volume of the supernatant from the MG1363 aseptic fermentation broth was 5% (10) of the final total volume. 6 The volume ratio of CFU / mL FW40A bacterial culture to MG1363 aseptic fermentation broth supernatant was 95:5, 10% (10 6 The volume ratio of CFU / mL FW40A bacterial culture to MG1363 aseptic fermentation broth supernatant was 90:10, 20% (10 6 CFU / mL FW40A bacterial culture and MG1363 aseptic fermentation broth supernatant (80:20 volume ratio) were added to EP tubes and incubated anaerobically at 37 °C for 24 h. Then, 200 μL of the bacterial culture was transferred to a 96-well plate, and OD was detected using a multi-functional microplate reader. 600 .

[0154] The results are as follows Figure 1 As shown in Figure B (CK represents the CK group, Nisin represents the Nisin group, S0 represents the S0 group, and MG1363 represents the MG1363 group; 5%, 10%, and 20% represent the volume percentages of GM17 medium, 100ug / ml Nisin, S0 sterile fermentation broth supernatant, and MG1363 sterile fermentation broth supernatant, respectively), compared to CK (control group), when the S0 sterile fermentation broth supernatant reached 20% of the final total volume (10... 6 A CFU / mL FW40A bacterial suspension mixed with the supernatant of S0 sterile fermentation broth at a ratio of 80:20 can significantly inhibit Clostridium sporogenes FW40A, and its effect is comparable to that of the positive control bacteriocin Nisin.

[0155] Example 3 Lactococcus lactis Lactococcus lacti S0's inhibitory mechanism against Clostridium epiphytum on the surface of high-moisture alfalfa silage

[0156] The above results show that the supernatant of the S0 sterile fermentation broth contains antibacterial substances and has a good inhibitory effect on FW40A. However, the mechanism of its antibacterial function is not yet clear. Therefore, scanning electron microscopy and PI staining experiments were used to explore how the S0 supernatant exerts its antibacterial activity. The specific operation methods are as follows:

[0157] FW40A was inoculated into BHI medium and cultured anaerobically at 37°C for 24 h until the OD reached. 600 =1, obtain FW40A OD 600 =1 bacterial solution.

[0158] 1) Observation using scanning electron microscopy (SEM)

[0159] Take the above FW40A OD 600 =1 bacterial culture 2 mL, centrifuged at 8000 rpm for 3 min and the supernatant was discarded to obtain FW40A cells. The cells were resuspended in 200 μL of sterile phosphate buffer (Gibco, catalog number: 14190144) to obtain FW40A bacterial suspension 1. To observe a significant antibacterial effect, a high concentration of fermentation broth supernatant was selected for the experiment. The supernatant of S0 sterile fermentation broth was added to FW40A bacterial suspension 1 at a final total volume ratio of 25% (the volume ratio of FW40A bacterial suspension 1 to S0 sterile fermentation broth supernatant was 3:1) to obtain FW40A-S0 sterile fermentation broth supernatant bacterial suspension 1, and anaerobic incubated at 37 ℃ for 40 min.

[0160] After incubation, the bacterial cells were centrifuged to obtain a precipitate. A 2% glutaraldehyde (Acmec, catalog number: AP1126-500ml) solution was added, and the cells were gently mixed and incubated overnight at 4°C to fix the cells. The cells were then centrifuged at 8000 rpm for 3 min to remove the glutaraldehyde. The precipitate was washed multiple times with ultrapure water. The cells were then dehydrated stepwise with 70%, 80%, 95%, and 100% ethanol (Macklin, catalog number: E809056-500ml) for 15 min each time. The precipitate was then pipetted onto a sample slide, which was then dried in an HCP-2 critical point desiccator for 2 h. After drying, the slide was mounted on the stage and sputter-coated with gold. The cell surface was then observed using a field emission scanning electron microscope.

[0161] 2) PI staining experiment

[0162] 10 μg / mL PI dye: Weigh 100 μg of PI dye (Bioss, catalog number: D-9103) powder, add 10 mL of sterile water, mix thoroughly to obtain 10 μg / mL PI dye.

[0163] Take the above FW40A OD 600 =1. Centrifuge 700 μL of bacterial suspension at 8000 rpm for 3 min to obtain bacterial cell pellet. Wash twice with 200 μL of sterile phosphate buffer (Gibco, catalog number: 14190144) and resuspend to obtain FW40A bacterial suspension 2.

[0164] The experiment was divided into four groups: CK group, S0 group, NIS-60 group, and NIS-100 group.

[0165] Group CK shall perform the following operations:

[0166] Take 2640 μL of the above FW40A bacterial suspension and add 160 μL of GM17 liquid culture medium (the volume ratio of FW40A bacterial suspension 2 to GM17 culture medium is 4:1). Mix well and anaerobic at 37℃ for 30 min. After treatment, centrifuge at 8000 rpm for 3 min and remove the supernatant to obtain bacterial cells. Add 700 μL of 10 μg / mL PI dye and incubate at 37℃ in the dark for 15 min. Centrifuge again and resuspend the bacterial cells in 700 μL of sterile phosphate buffer. Take 200 μL of the resuspended bacterial solution into an opaque 96-well microplate and measure the fluorescence intensity at excitation light: 535 nm and emission light: 615 nm using a microplate reader.

[0167] Group S0 performs the following operations:

[0168] Take 2640 μL of the above FW40A bacterial suspension and add 160 μL of S0 sterile fermentation broth supernatant (the volume ratio of FW40A bacterial suspension 2 to S0 sterile fermentation broth supernatant is 4:1). Mix well and anaerobic at 37℃ for 30 min. After treatment, centrifuge at 8000 rpm for 3 min and remove the supernatant to obtain bacterial cells. Add 700 μL of 10 μg / mL PI dye and incubate at 37℃ in the dark for 15 min. Centrifuge again and resuspend the bacterial cells in 700 μL of sterile phosphate buffer. Take 200 μL of the resuspended bacterial solution into an opaque 96-well microplate and measure the fluorescence intensity at excitation light: 535 nm and emission light: 615 nm using a microplate reader.

[0169] The NIS-60 group shall perform the following operations:

[0170] Take 2640 μL of the above FW40A bacterial suspension and add 160 μL of sterile Nisin with a concentration of 60 ug / ml (the volume ratio of FW40A bacterial suspension 2 to 60 ug / ml sterile Nisin is 4:1). Mix well and anaerobic at 37℃ for 30 min. After treatment, centrifuge at 8000 rpm for 3 min and remove the supernatant to obtain bacterial cells. Add 700 μL of 10 μg / mL PI dye and incubate at 37℃ in the dark for 15 min. Centrifuge and resuspend the bacterial cells in 700 μL of sterile phosphate buffer. Take 200 μL of the resuspended bacterial solution into an opaque 96-well microplate and measure the fluorescence intensity at excitation light: 535 nm and emission light: 615 nm using a microplate reader.

[0171] The NIS-100 group performs the following operations:

[0172] Take 2640 μL of the above FW40A bacterial suspension and add 160 μL of sterile Nisin with a concentration of 100 ug / ml (the volume ratio of FW40A bacterial suspension 2 to 100 ug / ml sterile Nisin is 4:1). Mix well and anaerobic at 37℃ for 30 min. After treatment, centrifuge at 8000 rpm for 3 min and remove the supernatant to obtain bacterial cells. Add 700 μL of 10 μg / mL PI dye and incubate at 37℃ in the dark for 15 min. Centrifuge and resuspend the bacterial cells in 700 μL of sterile phosphate buffer. Take 200 μL of the resuspended bacterial solution into an opaque 96-well microplate and measure the fluorescence intensity at excitation light: 535 nm and emission light: 615 nm using a microplate reader.

[0173] Scanning electron microscopy observation results as follows Figure 2 As shown in A and B ( Figure 2 Image A shows an electron micrograph of normal FW40A cells, and image B shows FW40A cells after incubation with the supernatant of the S0 sterile fermentation broth. Normal FW40A cells have a full and intact cell structure, a rough surface, and a spindle-shaped appearance. Figure 2 (A); while FW40A cells incubated with the supernatant of S0 aseptic fermentation broth showed cell surface shrinkage, partial cell rupture, and leakage of contents, exhibiting a significant difference from normal cells. Figure 2 (B)

[0174] PI staining experiment results as follows Figure 2 As shown in Figure C, compared with the CK group, the fluorescence intensity of cells in the SO group was significantly enhanced. The NIS-60 and NIS-100 groups also showed significantly enhanced fluorescence intensity. This indicates that the S0 supernatant and treatment with different concentrations of Nisin increased the cell membrane permeability of FW40A cells, allowing more PI dye to enter the FW40A cells, thus increasing fluorescence intensity. Furthermore, with the same added volume, the effect of the S0 supernatant on the cell membrane permeability of FW40A cells was greater than that of the 60 ug / ml Nisin solution. This, along with the scanning electron microscopy observations, demonstrates that S0 can secrete antibacterial substances that act on FW40A cells, causing cell surface shrinkage, increased cell membrane permeability, and cytoplasmic leakage, thereby exerting an antibacterial function.

[0175] Example 4 Lactococcus lactis Lactococcus lacti S0's effect on controlling Clostridium fermentation in high-moisture alfalfa silage

[0176] The above work has demonstrated that Lactococcus lactis Lactococcus lacti S0 can inhibit Clostridium sporogenes FW40A, an epiphytic Clostridium species, on the surface of high-moisture alfalfa silage in vitro. Therefore, the addition of Lactococcus lactis to the actual Clostridium fermentation process was investigated. Lactococcus lacti Whether S0 can achieve a preventive effect, the specific operation method is as follows:

[0177] Harvest the fourth crop of alfalfa at the budding stage. Medicago sativa After the alfalfa is harvested, it is dried until the moisture content is 78%. The withered alfalfa is then shredded into pieces about 1-2 cm in length using a chaff cutter and packed into polyethylene bags (about 500 g per bag) to obtain packaged alfalfa.

[0178] The experiment was divided into three groups: the Lactococcus lactis S0 group and the control group.

[0179] The following procedures were performed on the Lactococcus lactis S0 group:

[0180] According to Lactococcus lactis S0 1×10 6 CFU / g alfalfa and Clostridium sporogenes FW40A 1×10 5 The amount of CFU / g added to alfalfa was determined by spraying S0 and FW40A bacterial solutions onto the alfalfa, mixing thoroughly, and fermenting at room temperature in a sealed container for 60 days. After fermentation, silage was obtained. A sample was taken from the bag, stirred thoroughly, and 10 g of silage sample was added to 90 mL of sterile water. The mixture was homogenized for 2 min using a beater homogenizer. The homogenized liquid was transferred to a homogenization bag (Huankai, BCYD400G1, filter pore size <250 μmol), filtered to remove impurities, and the filtrate was collected and sent to the CVAS Feed Analysis China Service Center for testing its fermentation quality (pH, organic acids, ammonia nitrogen, etc.) and protein composition. pH was measured using a glass electrode pH meter (Mettler, America); ammonia nitrogen content was determined using the phenol-hypochlorite method; and organic acids were determined using gas chromatography. The filtrate was further filtered through a 0.22 μm aqueous microporous membrane (Nalgene, DS0210-4020) in 1.5 mL of water. Accurately add 1 mL of the supernatant and 0.2 mL of 25% metaphosphoric acid solution containing the internal standard 2EB to a centrifuge tube, mix well, freeze overnight at -20 ℃, centrifuge (10,000 rpm × 10 min) to remove protein precipitates from the sample, and collect the supernatant for later use. Detection is performed using a gas chromatograph. The chromatographic conditions are as follows: a Φ 6 mm x 2 m quartz glass packed column (stationary phase 15% FFAP, support 80–100 mesh Chromosorb), with a column temperature of 150 ℃ and an injection port temperature of 220 ℃; an injection volume of 1 μL; an FID detector temperature of 280 ℃; high-purity nitrogen as the carrier gas, with a flow rate of 30 mL / min and a pressure of 200 kPa; hydrogen as the combustion fuel, with a flow rate of 30 mL / min; and air as the oxidizing agent, with a flow rate of 300 mL / min.

[0181] Weigh 100 g of silage sample into a kraft paper bag, place it in a 65 ℃ oven to dry for 48 hours, then transfer it to a 105 ℃ oven to dry to constant weight, record the weight as n, and calculate its dry matter content (DM% = n / 100). After drying and pulverizing the 100% sample, crude protein (CP) was determined according to GB / T 6432-2018, "Method for Determination of Crude Protein in Feed". Soluble protein (SP), acid-detergent-insoluble nitrogen (ADIN), and neutral-detergent-insoluble protein (NDIP) were determined using the Kjeldahl method. According to CNCPS 6.5, protein can be divided into three parts: non-protein nitrogen (NPN, PA), true protein (PB), and non-degradable protein (PC). PA can be further divided into ammonia (PA1) and soluble true protein (PA2), and PB can be divided into poorly soluble true protein (PB1) and fibrous-bound protein (PB2), calculated according to the following formula:

[0182] ;

[0183] ;

[0184] ; ;

[0185]

[0186] The silage samples were then sent to Wuhan Punes Testing Technology Co., Ltd. for analysis of their biogenic amine targeted metabolomics, etc. Biogenic amine sample processing:

[0187] (1) Grind the sample into powder in liquid nitrogen and accurately weigh about 0.5 g of the sample into a test tube;

[0188] (2) Add 500 μL of ammonia and 2 mL of water, vortex for 30 s, sonicate for 10 min, and then let stand for 1 h;

[0189] (3) Add 5 mL of acetonitrile, vortex for 30 s, extract by ultrasonication for 30 min, centrifuge at 5000 rpm for 10 min, take the supernatant, repeat the above operation once for the precipitate, take the supernatant, and finally combine the supernatants.

[0190] (4) Add 0.5 g PSA to the supernatant, vortex for 60 s, centrifuge at 5000 rpm for 10 min, accurately pipette 1 mL of supernatant into a test tube, and blow dry with nitrogen.

[0191] (5) The residue after drying is dissolved in a methanol-water (20:80) solution containing 1% formic acid, filtered through a 0.22 μL organic filter membrane, and then tested on the instrument.

[0192] Chromatographic conditions:

[0193] Column: Agilent Poroshell 120 HILIC-Z (2.1 × 150 mm, 2.7 µm); Column temperature: 35 ℃; Mobile phase A: 0.5% formic acid in water; Mobile phase B: acetonitrile; Flow rate: 0.30 mL / min;

[0194] Injection volume: 2 μL.

[0195] Mass spectrometry conditions:

[0196] Scan type: MRM; Air curtain gas: 35 psi; Spray voltage: +4500 V; Atomizing gas pressure: 55 psi;

[0197] Auxiliary gas pressure: 55 psi; Atomization temperature: 550 ℃;

[0198] The test results are as follows:

[0199] pH=5.81;

[0200] Lactic acid (% dry matter) = 0.45;

[0201] Acetic acid (% dry matter) = 3.03;

[0202] Butyric acid (% dry matter) = 2.15;

[0203] Propionic acid (% dry matter) = 0.57;

[0204] Ammonia nitrogen (% dry matter) = 0.51;

[0205] Dry matter (%) = 22.54;

[0206] Crude protein (% dry matter) = 21.65;

[0207] Ammonia (% crude protein) = 0.0234;

[0208] Soluble protein (% crude protein) = 59.16;

[0209] Insoluble true protein (% crude protein) = 26.03;

[0210] Fiber-bound protein (% crude protein) = 4.68;

[0211] Non-degradable protein (% crude protein) = 7.18;

[0212] Total biogenic amines (μg / g) = 1602.22;

[0213] Tyrosine (μg / g) = 555.44;

[0214] Cadaverine (μg / g) = 625.08;

[0215] Tryptophan (μg / g) = 108.40.

[0216] The control group underwent the following procedures:

[0217] The only difference between the control group and the Lactococcus lactis S0 group was that the control group used an equal volume of sterile water instead of Lactococcus lactis S0, and the rest of the operation was the same as the Lactococcus lactis S0 group.

[0218] The test results are as follows:

[0219] pH=4.87;

[0220] Lactic acid (% dry matter) = 2.22;

[0221] Acetic acid (% dry matter) = 2.05;

[0222] Butyric acid (% dry matter) = 0.06;

[0223] Propionic acid (% dry matter) = 0.08;

[0224] Ammonia nitrogen (% dry matter) = 0.54;

[0225] Dry matter (%) = 24.03;

[0226] Crude protein (% dry matter) = 23.35;

[0227] Ammonia (% crude protein) = 0.0232;

[0228] Soluble protein (% crude protein) = 61.08;

[0229] Insoluble true protein (% crude protein) = 25.70;

[0230] Fiber-bound protein (% crude protein) = 4.37;

[0231] Non-degradable protein (% crude protein) = 6.52;

[0232] Total biogenic amines (μg / g) = 1372.86;

[0233] Tyrosine (μg / g) = 462.49;

[0234] Cadaverine (μg / g) = 508.11;

[0235] Tryptophan (μg / g) = 22.40.

[0236] The analysis of the above fermentation quality test results is as follows: Figure 3 The ( Figure 3 In the study, S0 represented the Lactococcus lactis S0 group, and FW40A represented the control group. After 60 days of fermentation, the pH value of the Clostridium fermentation group with FW40A was 5.81, while the pH value of the group with both Lactococcus lactis S0 and FW40A was 4.87. This indicates that the pH value decreased significantly after the addition of Lactococcus lactis S0. Figure 3 (Group A); The lactic acid content in the group with FW40A was 0.45% DM, and the lactic acid content increased to 2.22% DM after the addition of Lactococcus lactis S0. The addition of Lactococcus lactis S0 promoted the production of lactic acid. Figure 3 B); In addition, the addition of Lactococcus lactis S0 significantly decreased the contents of acetic acid, propionic acid, and butyric acid, while the contents of ammonia nitrogen showed no significant difference. Figure 3 (C, E, F).

[0237] Nutritional quality test results as follows Figure 4 ( Figure 4 In the diagram (where S0 represents the Lactococcus lactis S0 group and FW40A represents the control group), the addition of Lactococcus lactis S0 can reduce the dry matter loss during Clostridium fermentation. Figure 4 (A), and can increase crude protein content ( Figure 4 (B) Among them, it has no significant effect on the ammonia content ( Figure 4 (C), but it can increase the content of soluble true protein to some extent ( Figure 4 D), reduce the content of non-degradable proteins ( Figure 4 (G), but had no significant effect on the content of insoluble true proteins and fibronectin (G). Figure 4 (E and F in the middle).

[0238] Analysis of the results of targeted metabolomics analysis of downstream amino acid metabolites—biogenic amines (measured by Wuhan Punes Testing Technology Co., Ltd.) is as follows: Figure 5 ( Figure 5 In the figure, S0 represents the Lactococcus lactis S0 group, and FW40A represents the control group. As shown, the addition of S0 can reduce the total amount of biogenic amines in alfalfa silage from 1602.22 μg / g to 1372.86 μg / g. Figure 5 (A), which can significantly reduce the content of tyramine, cadaverine, and tryptamine ( Figure 5 (B, C, and D). The results showed that the addition of S0 could reduce the production of biogenic amines and prevent Clostridium from excessively consuming amino acids in alfalfa silage.

[0239] The above results indicate that during the Clostridium perfringens fermentation process of high-moisture alfalfa silage, the addition of SO can reduce the pH value of the fermentation environment by decreasing the content of acetic acid, propionic acid, and butyric acid and increasing the content of lactic acid, thereby alleviating Clostridium perfringens fermentation and improving the fermentation quality of high-moisture alfalfa silage fermented by Clostridium perfringens. It can also increase the crude protein content by increasing the content of soluble true protein and decreasing the content of non-degradable protein, and reduce the total amount of biogenic amines, the downstream metabolites of amino acids, by decreasing tyramine, cadaverine, and tryptamine, thereby controlling the consumption of nutrients and the generation of adverse products in alfalfa silage by Clostridium perfringens fermentation and ensuring its nutritional quality.

[0240] Example 5 Lactococcus lactis Lactococcus lacti S0 influences the microbial composition of Clostridium fermentation in high-moisture alfalfa silage.

[0241] The above experiments demonstrate that S0 can play a corresponding control role in the actual Clostridium fermentation process of high-moisture alfalfa and ensure that its nutritional quality is not excessively lost. However, how S0 exerts its control effect is still unknown. Therefore, this study explores how S0 exerts its control effect in Clostridium fermentation of high-moisture alfalfa. The specific operation method is as follows:

[0242] Lactococcus lactis S0 group:

[0243] According to Lactococcus lactis S0 1×10 6 CFU / g alfalfa and Clostridium sporogenes FW40A 1×10 5 The amount of CFU / g added to alfalfa was determined by spraying S0 and FW40A bacterial solutions onto the alfalfa and mixing thoroughly. After 60 days of sealed fermentation at room temperature, the bag was opened, and 15 g of silage sample was weighed and placed in a sterile polyethylene bag. 40 mL of sterile phosphate buffer was added, and the surface microorganisms were washed off by shaking on a shaker for 30 min (100 rpm). The sample was then filtered using a sterile homogenizing bag (Sorfa, catalog number: sterile homogenizing bag, 400 mL, filter mesh size less than 250 μm) to remove impurities. The obtained filtrate was centrifuged at 10000 rpm, 4℃, for 3 min, and the supernatant was removed to obtain bacterial precipitate. Genomic DNA of the surface microorganisms was extracted using a kit and sent to Meiji Biotechnology for quality control and absolute 16S quantification of bacteria. The analysis aimed to identify the true differences in bacterial composition in alfalfa silage and explore the control mechanism of Lactococcus lactis S0 in the Clostridium fermentation process.

[0244] Control group:

[0245] According to 1×10 5Add CFU / g alfalfa, spray FW40A bacterial solution onto alfalfa and mix well, and add an equal amount of sterile water as Lactococcus lactis S0 in the above-mentioned Lactococcus lactis S0 group. After 60 days of sealed fermentation at room temperature, open the bag, weigh 15 g of silage sample, place it in a sterile polyethylene bag, add 40 mL of sterile phosphate buffer, shake on a shaker for 30 min (100 rpm) to wash off surface microorganisms, and filter using a sterile homogenizing bag (Sorfa, product number: sterile homogenizing bag, 400 mL, filter mesh size less than 250 μm) to remove impurities. Centrifuge the obtained filtrate at 10000 rpm, 4℃ for 3 min, remove the supernatant, and obtain bacterial precipitate. Use a kit to extract the genomic DNA of the surface microorganisms, and submit it to Meiji Biotechnology for quality control and 16S absolute quantitative detection of bacteria.

[0246] The results are as follows Figure 6 As shown, the dominant bacterial species in the control group was *Lelloseia rhynchophylla* (Lelloseia rhynchophylla). Lelliottia amnigena 4.7×10 7 copies g -1 FM), Lactobacillus short-lived ( Levilactobacillus brevis 3.5×10 7 copies g - 1 FM) and Weissella esculenta ( Weissella cibaria 1.9×10 7 copies g -1 FM), while Lactococcus plantarum in group S0 ( Lactiplantibacillus plantarum 1.1×10 8 copies g -1 The abundance of FM was significantly increased, and the abundance of short-lived Lactobacillus (FM) was significantly increased. Levilactobacillus brevis 2.2×10 7 copies g -1 Although the abundance of FM has decreased, it is still the dominant bacterial species among them. Figure 6 (A). It can be seen that the addition of Lactococcus lactis S0 changed the microbial composition of Clostridium fermentation of alfalfa silage, reduced the total amount of bacteria in the fermentation environment, and promoted the increase of beneficial bacteria abundance during the fermentation process.

[0247] Analysis of the Clostridium species within them yielded the following results: Figure 6 As shown in Figure B, the dominant microorganism in the high-moisture alfalfa silage fermented by Clostridium difficile inoculated with FW40A was Clostridium perfringens (Clostridium perfringens). Clostridium fermenticellae Clostridium butyricum (C. butyricum) Clostridium tyrobutyricum ) and Clostridium sporogenes ( Clostridium sporogens ),in, Clostridium fermenticellae Abundance can reach 1.3 × 10⁻⁶ 7 copies g -1FM indicates that Clostridium inoculation promoted the growth of native Clostridium on the surface of high-moisture alfalfa; however, after the addition of Lactococcus lactis S0, its abundance decreased sharply, indicating that Lactococcus lactis S0 has a good inhibitory effect on other Clostridium species not limited to FW40A, which can effectively reduce their abundance and inhibit the process of Clostridium fermentation.

[0248] The above results indicate that Lactococcus lactis S0 can alter the microbial composition and structure, reduce the total amount of bacteria in the fermentation environment, promote the abundance growth of beneficial bacteria in the fermentation environment, and inhibit Clostridium in high-moisture alfalfa silage during Clostridium fermentation, thereby playing a role in controlling Clostridium fermentation.

[0249] In summary, Lactococcus lactis Lactococcus lactis S0 can secrete antibacterial substances that act on Clostridium sporogenes. Clostridium sporogens The FW40A cell membrane causes the cell surface to wrinkle, resulting in cytoplasmic leakage, thus exerting an inhibitory effect; during the Clostridium fermentation process, Lactococcus lactis... Lactococcus lactis S0 can lower the pH of the fermentation environment by reducing the content of acetic acid, propionic acid, and butyric acid while increasing the content of lactic acid, thereby improving the fermentation progress of Clostridium fermentation and thus enhancing the fermentation quality of high-moisture alfalfa silage fermented by Clostridium. It can also increase the crude protein content by increasing the content of soluble true protein and decreasing the content of non-degradable protein, and reduce the total amount of biogenic amines, downstream metabolites of amino acids, by decreasing tyramine, cadaverine, and tryptamine, thereby controlling the consumption of nutrients and the generation of adverse products in alfalfa silage by Clostridium fermentation and ensuring its nutritional quality. Furthermore, it can alter the microbial composition of high-moisture alfalfa silage fermented by Clostridium, reducing the total number of bacteria in the fermentation environment, promoting the abundance of beneficial bacteria, and inhibiting Clostridium in high-moisture alfalfa silage, thus playing a role in controlling Clostridium fermentation.

[0250] 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. Lactococcus lactis, characterized in that, The lactococcus is Lactococcus lactis ( Lactococcus lactis S0, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 30594.

2. A microbial agent, characterized in that, The bacterial agent contains the lactococcus lactis as described in claim 1.

3. A pathogen inhibitor, characterized in that, The pathogen inhibitor is the lactococcus lactis of claim 1 or the bacterial agent of claim 2, which is cultured in a culture medium.

4. An alfalfa fermentation agent, wherein the alfalfa fermentation agent contains the lactococcus lactis of claim 1 or the microbial agent of claim 2.

5. The application of the *Lactococcus lactis* according to claim 1, characterized in that, The application is any one of the following: A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes; A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes; A3) Application in killing Clostridium sporogenes; A4) Application in the preparation of products that kill Clostridium sporogenes; A5) Application in improving the quality of alfalfa after fermentation; A6) Application in the preparation of products that improve the quality of fermented alfalfa.

6. The application of the microbial agent according to claim 2, characterized in that, The application is any one of the following: A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes; A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes; A3) Application in killing Clostridium sporogenes; A4) Application in the preparation of products that kill Clostridium sporogenes; A5) Application in improving the quality of alfalfa after fermentation; A6) Application in the preparation of products that improve the quality of fermented alfalfa.

7. The application of the pathogen inhibitor according to claim 3, characterized in that, The application is any one of the following: A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes; A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes; A3) Application in killing Clostridium sporogenes; A4) Application in the preparation of products that kill Clostridium sporogenes; A5) Application in improving the quality of alfalfa after fermentation; A6) Application in the preparation of products that improve the quality of fermented alfalfa.

8. The application of the alfalfa fermentation agent according to claim 4, characterized in that, The application is any one of the following: A1) Application in inhibiting and / or slowing the growth of Clostridium sporogenes; A2) Application in the preparation of products that inhibit and / or slow down the growth of Clostridium sporogenes; A3) Application in killing Clostridium sporogenes; A4) Application in the preparation of products that kill Clostridium sporogenes; A5) Application in improving the quality of alfalfa after fermentation; A6) Application in the preparation of products that improve the quality of fermented alfalfa.

9. A method for preparing a microbial agent, characterized in that, The method includes the step of using the Lactococcus lactis of claim 1 as a component of the bacterial agent to obtain the bacterial agent.

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