Lactobacillus plantarum, bacterial agent and application thereof

Abstract

The application discloses application of Lactiplantibacillus plantarum in at least one of the following: a1, application in improving silage quality; a2, application in preparing silage; a3, application in preparing silage additive; the strain of the Lactiplantibacillus plantarum is named LB1, is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.33485. The Lactiplantibacillus plantarum isolated for the first time has high acid production and can reduce the content / abundance of sulfonamide antibiotic resistance genes, after adding the Lactiplantibacillus plantarum or a bacterial agent or silage additive thereof, the generation and residue of sulfonamide antibiotic resistance genes in feed can be reduced, the spread of sulfonamide antibiotic resistance genes in the food chain is reduced, the damage to animals, the environment and the human body is reduced, the forage safety is ensured, the animal health level is improved, and the drug resistance of human pathogenic bacteria is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a lactobacillus plantarum, a bacterial agent and application thereof. BACKGROUND

[0002] With the rapid development of animal husbandry, improving the quality of feed has become an important problem in current production. Under the background of global climate change and increasingly severe resource and environmental constraints, Stylosanthes guianensis, as a high-quality legume forage, has superior drought tolerance and poor tolerance, and can grow in harsh environments. And because of its unique nutritional properties, lower fiber content and higher digestibility, it has become an ideal choice for silage raw materials. Lactobacillus plantarum plays a key role in the fermentation process of Stylosanthes guianensis silage. They produce lactic acid by metabolizing soluble carbohydrates, reduce the pH value, and inhibit the growth of harmful microorganisms, thereby improving the preservation quality and nutritional value of Stylosanthes guianensis silage.

[0003] However, in recent years, due to the extensive use of antibiotics, the environmental presence of antibiotic resistance genes (ARGs) and drug-resistant bacteria has increased, further exacerbating the risk of antibiotic resistance transmission. Ruminants also ingest feed containing ARGs for a long time, and their intestinal microbiota integrate drug resistance genes through horizontal gene transfer (HGT), forming a biological enrichment effect of drug-resistant bacteria, ultimately threatening human health through multiple pathways. Therefore, the spread of antibiotic resistance genes in silage has attracted widespread attention from the public and research institutions.

[0004] Stylosanthes guianensis, as an important source of silage forage in animal husbandry, the dynamic changes of microbial community in its fermentation process may become a potential way for the spread of resistance genes. Secondly, the resistance genes carried by microorganisms in silage can also make silage become a reservoir of ARGs. There have been studies detecting common resistance genes in Lactobacillus plantarum, such as sulfonamide resistance genes. Lactobacillus plantarum, as the dominant flora in silage, can reduce the pH value of the silage environment through rapid fermentation and acid production, inhibit the proliferation of spoilage bacteria and potential pathogenic bacteria, and these microorganisms usually carry or spread ARGs through horizontal gene transfer (HGT). Among them, mobile genetic elements (such as intl1, intl2) can mediate horizontal gene transfer of ARGs between different species of bacteria. At the same time, Lactobacillus plantarum occupies the ecological niche through competitive exclusion, reduces the biomass of ARGs host bacteria in the microbial community, and may directly inhibit the activity of drug-resistant bacteria by secreting bacteriocins and other bacteriostatic substances. In addition, the distribution, content and transmission mechanism of resistance genes of Lactobacillus plantarum also need further research.

[0005] In summary, the column flower grass in the process of silage faces the problems of high fresh sample moisture content, low sugar content, few epiphyte Lactobacillus, many harmful bacteria, and high content of antibiotic resistance genes (ARGs), especially sulfonamide resistance genes (SRGs) in the silage feed grass after direct silage, which is harmful to animals, environment and human body. SUMMARY

[0006] In order to solve the problems in the prior art, the present application provides the following technical scheme:

[0007] The first aspect of the present application provides an application of Lactiplantibacillus plantarum in at least one of the following:

[0008] a1, application in improving the silage quality of silage feed;

[0009] a2, application in preparing silage feed;

[0010] a3, application in preparing a microbial agent or a silage feed additive.

[0011] The strain of Lactiplantibacillus plantarum is named LB1, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 33485.

[0012] The isolation of the low-resistance gene Lactiplantibacillus LB1 includes the following steps: taking a certain mass of silage feed sample, adding 9-12 times of sterile normal saline, gradually diluting, using MRS medium for culture, selecting single bacterial colony for isolation, purification and preservation. Take a single strain and activate for 48h, mix, adjust the bacterial concentration with a McFarland turbidimeter, inoculate 1% into MRS broth medium, and culture for 24h. The pH value and absorbance (OD 600) of the culture medium are measured, and the bacteria with high acid production are screened out according to the standards of pH value <4.0 and OD value >1.6.

[0013] The second aspect of the present application provides an application of Lactiplantibacillus plantarum in preparing any one of the following products:

[0014] b1, a silage feed product prepared using the Lactiplantibacillus plantarum;

[0015] b2, a microbial agent or silage feed additive product containing the Lactiplantibacillus plantarum as an active ingredient, prepared using the Lactiplantibacillus plantarum;

[0016] The strain of Lactiplantibacillus plantarum is named LB1, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 33485.

[0017] In some embodiments, the nucleotide sequence of the 16S rDNA of the strain LB1 of Lactiplantibacillus plantarum is set forth in SEQ ID NO: 1.

[0018] SEQ ID NO: 1:

[0019]

[0020] In some embodiments, the silage additive product comprises a bacterial inoculant comprising Lactiplantibacillus plantarum, strain named LB1.

[0021] In some preferred embodiments, the bacterial inoculant comprises reduced sulfonamide antibiotic resistance gene content, and optionally at least one of the following properties:

[0022] (1) reducing the pH of the silage;

[0023] (2) increasing the number of lactic acid bacteria in the silage;

[0024] (3) reducing the content of ammoniacal nitrogen in the silage.

[0025] In some more preferred embodiments, the bacterial inoculant comprises reduced sulfonamide antibiotic resistance gene abundance, and optionally at least two of the following properties:

[0026] (1) reducing the pH of the silage;

[0027] (2) increasing the number of lactic acid bacteria in the silage;

[0028] (3) reducing the content of ammoniacal nitrogen in the silage.

[0029] In some most preferred embodiments, the bacterial inoculant comprises reduced sulfonamide antibiotic resistance gene abundance, and the following properties:

[0030] (1) reducing the pH of the silage;

[0031] (2) increasing the number of lactic acid bacteria in the silage;

[0032] (3) reducing the content of ammoniacal nitrogen in the silage.

[0033] In the above described uses, preferably the ensiled plant in the silage comprises clover.

[0034] A third aspect of the application provides a bacterial inoculant comprising Lactiplantibacillus plantarum LB1 as described above.

[0035] In some preferred embodiments, the bacterial inoculant is a bacterial powder comprising Lactiplantibacillus plantarum LB1.

[0036] In the present application, the bacterial agent can be prepared in a bacterial powder state, or in a bacterial liquid state, or in a bacterial powder state for preparation and in a bacterial liquid state for use. In some embodiments, the bacterial agent containing the Lactiplantibacillus plantarum of the present application is in a bacterial powder state with a water content of 0%, and is in a bacterial liquid state activated after adding water / pure water when silage is added.

[0037] In some embodiments, the bacterial agent can be a bacterial powder prepared by freeze-drying. In some preferred embodiments, the preparation method of the bacterial powder comprises: after the Lactiplantibacillus plantarum LB1 of the present application is activated and enriched, centrifugation, washing the centrifugation twice with sterile physiological saline, adding skimmed milk, pouring into a culture dish, pre-freezing, and then placing in a vacuum freeze-drying machine, the vacuum degree is less than 60 pa, the cold trap temperature is less than -55℃, and the sample temperature reaches room temperature, which is the end of drying. The prepared bacterial powder is frozen and stored.

[0038] The present application does not have special restrictions on the freeze-drying procedure of the bacterial powder. In some preferred embodiments, the freeze-drying procedure of the bacterial powder of the present application comprises: -25℃ drying for 300 minutes, -20℃ drying for 60 minutes, -15℃ drying for 60 minutes, -10℃ drying for 60 minutes, -5℃ drying for 60 minutes, -2℃ drying for 60 minutes, -0℃ drying for 60 minutes, 5℃ drying for 60 minutes, 10℃ drying for 60 minutes, and 20℃ drying to the end point. The end point of freeze-drying is that the sample temperature is consistent with room temperature, and the vacuum degree of the machine during freeze-drying is less than 60 pa, and the cold trap temperature is less than -55℃.

[0039] The fourth aspect of the present application provides a silage, which is prepared by fermenting silage plants with Lactiplantibacillus plantarum LB1 and / or a bacterial agent containing Lactiplantibacillus plantarum LB1; wherein the addition amount of Lactiplantibacillus plantarum is 5x10 5 cfu·g -1 -5x10 6 cfu·g -1 .

[0040] In some preferred embodiments, the addition amount of Lactiplantibacillus plantarum is 6x10 5 cfu·g -1 -3x10 6 cfu·g -1 . In some more preferred embodiments, the addition amount of Lactiplantibacillus plantarum is 10 6 cfu·g -1 .

[0041] A fifth aspect of the present application provides a method for preparing silage, comprising fermenting silage plants using Lactiplantibacillus plantarum LB1 or an inoculant comprising Lactiplantibacillus plantarum LB1.

[0042] In some preferred embodiments, the method for preparing silage comprises adding Lactiplantibacillus plantarum LB1 of the present application or an inoculant thereof or a silage additive thereof or thereof to silage plants, mixing evenly, sealing, storing at room temperature (25-30°C), and fermenting for 30-60 days.

[0043] In some embodiments, the silage is cut into lengths of 2-3 cm before Lactiplantibacillus plantarum LB1 of the present application or an inoculant thereof or a silage additive thereof or thereof is added to the silage.

[0044] In some preferred embodiments, the silage plants in the silage comprise sorghum. In the present application, there is no particular limitation on the variety of sorghum. In the present application, there is also no particular limitation on the growth stage of sorghum.

[0045] In the present application, there is no particular limitation on the process of silage processing of sorghum. In the present application, processes of silage processing including bag sealing, barrel silage, and bale silage can all be applied to the present application. In some embodiments, the process of silage processing is bag sealing.

[0046] The beneficial effects of the present application at least include:

[0047] (1) The present application first isolates Lactiplantibacillus with high acid production and low content / abundance of sulfonamide antibiotic resistance genes, and first proposes adding the Lactiplantibacillus or an inoculant thereof or a silage additive thereof to silage to improve fermentation quality while significantly reducing the content of sulfonamide antibiotic resistance genes.

[0048] (2) Compared with traditional inoculants or silage additives, the inoculant or silage additive comprising the Lactiplantibacillus of the present application can effectively increase the number of lactic acid bacteria in the silage, increase the content of lactic acid, and reduce the pH value. As the number of days of silage increases, the pH value of the treatment group with added Lactiplantibacillus significantly decreases, the number of Lactiplantibacillus significantly increases, the reproduction of undesirable microorganisms is inhibited, and the quality of silage fermentation is effectively improved.

[0049] (3) After adding the Lactiplantibacillus plantarum or the bacterial agent thereof or the silage feed additive thereof of the present application, the number of coliforms and the content of ammonia nitrogen in the silage feed (such as silage feed containing Stylosanthes) can be significantly reduced.

[0050] (4) After adding the Lactiplantibacillus plantarum or the bacterial agent thereof or the silage feed additive thereof of the present application, the number of yeast and mold in the silage feed is less than 10 2 cfu·g -1 .

[0051] (5) After adding the Lactiplantibacillus plantarum or the bacterial agent thereof or the silage feed additive thereof of the present application, the abundance of sulfonamide antibiotic resistance genes (SRGs) in the silage feed can be significantly reduced, indicating that the quality of the silage can be effectively improved while reducing the impact of resistance genes on forage grass.

[0052] (6) The Lactiplantibacillus plantarum of the present application has a simple culture step, is easy to store and transport after freeze-drying, and has a low cost.

[0053] (7) After adding the Lactiplantibacillus plantarum or the bacterial agent thereof or the silage feed additive thereof of the present application, the production and residue of sulfonamide antibiotic resistance genes in the feed can be reduced, the spread of sulfonamide antibiotic resistance genes in the food chain is reduced, the harm to animals, the environment and the human body is reduced, the safety of forage grass is ensured, and the health level of animals is improved while reducing the drug resistance of human pathogenic bacteria.

[0054] (8) The Lactiplantibacillus plantarum or the bacterial agent thereof or the silage feed additive thereof of the present application is particularly suitable for silage feed containing Stylosanthes, which can effectively increase the forage resources and help solve the seasonal forage shortage problem of herbivorous animals. The present application provides a new idea for silage feed and can help solve the problem of silage feed in China to a certain extent.

[0055] The Lactiplantibacillus plantarum LB1 of the present application, the bacterial agent containing the strain, the feed containing the strain or the bacterial agent thereof, and the silage feed additive with the active ingredient being the strain have very important practical application value and application prospect.

[0056] Other features and advantages of the present application will be described in detail in the following specific embodiments.

[0057] Biological preservation instructions

[0058] The Lactiplantibacillus plantarum LB1 of the present application is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Haidian District, Beijing, with a postal code of 100101, with a preservation number of CGMCC No. 33485, and a preservation time of January 21, 2025. DETAILED DESCRIPTION

[0059] In order to make the objectives, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below in conjunction with examples. It should be understood that the following specific examples of the present application are merely exemplary descriptions of specific embodiments of the present application and are intended to explain the present application, but do not constitute a limitation on the present application.

[0060] The endpoints of the ranges and any values disclosed in the present application are not limited to the precise values stated. The endpoints of the ranges are included in the range. Any numerical value, however, can inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, the endpoints of the ranges and the numerical values are approximations which are obtained with a tolerance of ±3% in their respective testing measurements. These endpoints and values of any range, therefore, are not to be "aimed" or "construed" as a limitation.

[0061] The experimental methods in the following examples are all conventional methods or are carried out according to the kit instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0062] General Description:

[0063] In the examples of the present application, the Eupatorium odoratum was collected in the Qilinbei test field of South China Agricultural University, and no watering and fertilization treatment was performed during the planting process of the material.

[0064] The freeze-drying procedure involved in the examples of the present application is as follows: drying at -25℃ for 300 minutes, drying at -20℃ for 60 minutes, drying at -15℃ for 60 minutes, drying at -10℃ for 60 minutes, drying at -5℃ for 60 minutes, drying at -2℃ for 60 minutes, drying at -0℃ for 60 minutes, drying at 5℃ for 60 minutes, drying at 10℃ for 60 minutes, and drying at 20℃ to the end point. The end point of freeze-drying is that the sample temperature is consistent with the room temperature, the vacuum degree of the machine during freeze-drying is less than 60 pa, and the cold trap temperature is less than -55℃.

[0065] The ensiling process adopted in the examples of the present application is the bag sealing method.

[0066] Example 1: Screening of Lactiplantibacillus plantarum with high acid production

[0067] 1. Obtaining of high-acid-producing Lactobacillus plantarum strains

[0068] Strains were isolated from silage. A 20 g sample of silage was added to 180 mL of sterile physiological saline, and 10 -2 -10 -7 1 mL of gradient diluent was mixed with MRS medium, and after incubation at 37°C for 2 days, single colonies were selected for isolation and purification. A total of 226 single strains were obtained and stored by freezing in glycerol. The single strains were activated for 48 h, mixed, and adjusted to the appropriate concentration using a Macalaster turbidimeter. After inoculation into MRS broth at a ratio of 1%, the broth was incubated for 24 h, and the pH and OD 600 values were measured. The strains were selected based on a pH value < 4.0 and an OD value > 1.6. A total of 47 high-acid-producing strains were selected. After activation, the strains were incubated at 37°C and 140 rpm for 12 h, 1 mL of the bacterial solution was centrifuged at 12,000 rpm, the supernatant was discarded, and the precipitate was used for DNA extraction using a TIANamp Bacterial DNA Extraction Kit. After DNA extraction, the PCR amplification product was subjected to 1.5% agarose electrophoresis, and the target gene fragment was observed. If a clear band appeared, it indicated that the target gene was present in the strain, and if not, it indicated that the target gene was not present. The 4th strain without SRGs was prepared into a bacterial powder (as shown in Tables 1 and 2).

[0069] Table 1 Acid-producing ability of lactic acid bacteria screened from alfalfa silage after 30 days

[0070]

[0071]

[0072] Table 2 Detection of resistance genes in Lactobacillus plantarum

[0073] Number intl1 intl2 sul1 sul2 sul3 Number intl1 intl2 sul1 sul2 sul3 1 + + - + + 25 + + - - + 2 + + - + + 26 + + + + + 3 + + + + + 27 + + + + + 4 + + - - - 28 + + - + + 5 + + + + + 29 + + + + + 6 + + + + - 30 + + + - + 7 + + + + - 31 + + + - + 8 + + + - + 32 + + + + + 9 + + + - + 33 + + - - + 10 + + + + + 34 + + + + + 11 + + + + + 35 + + + - + 12 + + - - + 36 + + + - + 13 + + - + + 37 + + + + + 14 + + + + + 38 + + + + - 15 + + - - + 39 + + - + + 16 + + + + + 40 + + - + + 17 + + + + + 41 + + + + - 18 + + + + - 42 + + + + + 19 + + + + - 43 + + + - - 20 + + + - + 44 + + + + + 21 + + + - + 45 + + + - + 22 + + + + - 46 + + - - + 23 + + + + + 47 + + + + + 24 + + + + -

[0074] Note: +, detected; -, not detected.

[0075] The 4th strain shown in Table 1 and Table 2 is preserved and identified: 100 μL of bacterial liquid is taken from the strain screened above, DNA is extracted by bacterial genome DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.), 50 μL of buffer B1 is added, and the mixture is fully mixed by pipetting, vortexed after standing for 5 min, 50 μL of buffer B2 is added, and the mixture is fully mixed by pipetting, vortexed after standing for 5 min, centrifuged at 12500 rpm for 2 min, 2.5 μL of supernatant is taken and added to 50 μL of system for PCR reaction, primers are SEQ ID NO: 2: 27F (5'-AGTTTGATCMTGGCTCAG-3') and SEQ ID NO: 3: 1492R (5'-AAGTCGTAACAAGGTAACC-3'). The PCR reaction system includes: 25 μL of mix enzyme, 1.5 μL of forward primer (27F), 1.5 μL of reverse primer (1492R), 19.5 μL of double distilled water (ddH2O), and 2.5 μL of cDNA (template DNA). The PCR reaction program includes: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C post-extension for 5 min; 4°C termination reaction.

[0076] A gel block is made, 10 μL of mark of 2000 bp is added to the first hole of the gel block, and 10 μL of PCR amplification product is sequentially added. The gel electrophoresis reaction program: current 200 A, voltage 200 U, power 50 P, time 20 min. The clear band is sent to Genesee Biotechnology Co., Ltd. for sequencing. The results obtained after sequencing by the sequencing company are compared by homology by the blast program of NCBI to determine the strain belonging, which is determined as Lactiplantibacillus plantarum by comparison, named LB1, and preserved in China General Microbiological Culture Collection Center (CGMCC) on January 21, 2025, located at No. 1, Beichen West Road, Haidian District, Beijing, with a postcode of 100101, and a preservation number of CGMCC No. 33485.

[0077] 2. Physiological and biochemical experiments of the strain of Lactiplantibacillus plantarum

[0078] (1) Gram staining: typical single colonies in the culture medium are picked up with an inoculation ring in the center of a glass slide, a drop of sterile distilled water is dropped, and it is naturally dried and fixed by alcohol lamp flame; crystal violet solution and iodine solution are soaked for 1 min respectively, and the water is fully washed and removed; 95% alcohol is soaked for 10 s to decolorize, and then sterile distilled water is washed again; it is naturally dried and observed under a microscope. If it is dark purple, it is a gram-positive bacterium, and if it is red, it is a gram-negative bacterium.

[0079] (2) Catalase test: pick a single colony and place it in 3% hydrogen peroxide solution, observe the results after 30 seconds. Positive if bubbles are produced, negative if no bubbles are produced.

[0080] (3) Growth characteristics study: after two generations of inoculating a single lactic acid bacteria into MRS liquid medium, inoculate again into MRS liquid medium at a ratio of 3%, and place in constant temperature incubators at different temperatures (10℃, 15℃, 20℃, 30℃, 40℃, 45℃) for 3-5 days; inoculate the lactic acid bacteria into sterile MRS liquid medium with different pH values (3.0, 3.5, 8.0, 9.0) and different concentrations of NaCl (3%, 6.5%) to perform an acid tolerance test, and place in a constant temperature incubator at 37℃ for 3-5 days to observe the growth of the lactic acid bacteria. The physiological and biochemical identification of the lactic acid bacteria was performed according to the instructions of the lactic acid bacteria physiological and biochemical identification kit purchased from Beijing Luqiao Technology Co., Ltd. The results are shown in Table 3.

[0081] Table 3 Physiological and biochemical characteristics of Lactobacillus plantarum LB1

[0082]

[0083]

[0084] Example 2 Preparation of high-acid-producing Lactobacillus plantarum powder

[0085] The Lactobacillus plantarum LB1 obtained by screening in Example 1 was prepared into a bacterial powder, including the following steps: after enrichment culture of the Lactobacillus plantarum, centrifugation at 4000 rpm for 10 min, washing and centrifuging twice with sterile normal saline, adding 10% sterile skim milk and mixing uniformly, pouring into a culture dish, pre-freezing, and then placing in a vacuum freeze dryer, with a vacuum degree less than 60 pa, a cold trap temperature less than -55℃, and the sample temperature reaching room temperature as the drying ends, and the prepared bacterial powder was stored in a freezer.

[0086] Example 3 Lactobacillus plantarum addition treatment of silage feed

[0087] 1. Collection of columnflower:

[0088] The silage material in the present application is columnflower in the flowering stage. The columnflower is cut at 3-5 cm from the ground with a chopping knife, quickly brought back to the laboratory, crushed into 2-3 cm in length with a rubbing machine, and mixed uniformly to collect three repeated samples to determine the relevant characteristic indexes of the raw materials. The characteristics of the grass and columnflower raw materials are shown in Table 4, wherein DM refers to dry matter and FM refers to fresh sample.

[0089] The gene primers involved in the embodiments of the present application are shown in Table 5.

[0090] Table 4 Chemical properties and microbial numbers of ensiled Eupatorium odoratum before ensiling

[0091] Items Determinations Column flower grass 37.70±0.93 Dry matter (% FM) 66.20±2.68 Neutral detergent fiber (NDF, % DM) Acid detergent fiber (ADF, % DM) 50.80±4.85 Lactic acid bacteria (LAB, log 10 cfu·g -1 FM) 5.74±0.13 Yeast (lg cfu-g -1 FM) 5.61±0.17 Mold (lg cfu·g -1 FM) <2.00 lg cfu·g -1 FM) 6.81±0.06

[0092] Table 5 Primer sequences of resistance genes

[0093]

[0094] 2. Lactobacillus plantarum LB1 addition treatment of ensiled feed, ensilage bag packaging and preservation:

[0095] Eupatorium odoratum ensiling: 500 g of chopped Eupatorium odoratum was mixed with 10 mL of pure water and evenly divided into 3 polyethylene plastic bags as a control treatment (CK). Another 500 g of chopped Eupatorium odoratum was added with 10 mL of pure water, 10 mL of Lactobacillus plantarum LB1, and 10 mL of Bacillus subtilis, and evenly divided into 3 polyethylene plastic bags as a treatment group. 6 cfu·g -1 The Lactobacillus plantarum LB1 group was obtained by activating the bacterial powder described in Example 2 with pure water for 20 min.

[0096] All the prepared ensilage bags were sealed with a miniature vacuum sealer, and the ensilage quality related indicators were measured after opening the ensilage bags at room temperature for 3, 7, 14, and 30 days.

[0097] 3. Measurement of ensilage quality: The ensilage bags were opened at room temperature. The dry matter content, pH value, number of lactic acid bacteria, yeast, mold, and coliform bacteria, organic acid content (lactic acid, acetic acid, propionic acid, butyric acid), ammonia nitrogen content, and resistance gene abundance were measured. The results are shown in Tables 6, 7, and 8. The measurement methods for each item are as follows:

[0098] Dry matter determination: After opening the bag, the ensilage samples were mixed evenly and weighed, and then placed in a constant temperature oven at 65°C for about 48 h until dry. The weight ratio before and after drying was calculated, which was the dry matter content.

[0099] pH value determination: After opening the bag, 20 g of ensilage samples were randomly taken according to the five-point sampling method, 180 mL of distilled water was added, and the mixture was mixed evenly and squeezed for 1 min with a household juicer. The filtrate was obtained by sequentially filtering with 4 layers of gauze and medium speed qualitative filter paper, and the pH value was measured with a pH meter (PHS-3C, Shanghai Lei Magnet).

[0100] Ammonia nitrogen (NH3-N) determination: The content of ammonia nitrogen was determined by phenol-sodium hypochlorite colorimetry.

[0101] Determination of organic acids: The extract was filtered through a filter membrane with a pore size of 0.22 μm to obtain a filtrate, and the contents of lactic acid and acetic acid were determined using a Shimadzu GC-14 type high performance liquid chromatograph (chromatographic column: Shodex Rspak KC-811s DVB gel column, Japan; detector: SPD-M10AVP), the mobile phase was 3 mmol·L -1 perchloric acid, the flow rate was 1 mL·min -1 ; the column temperature was 50°C; the detection wavelength was 210 nm, and the injection amount was 5 μL.

[0102] Determination of the number of lactic acid bacteria (including Lactobacillus plantarum LB2), yeast, mold and coliform bacteria: 20 g of the sample was randomly sampled according to the five-point sampling method, 180 mL of sterilized normal saline was added, and the mixture was uniformly mixed and then sequentially diluted, and the number of microorganisms was determined by plate counting. Lactic acid bacteria were cultured using MRS agar medium, yeast and mold were cultured using Bengal red (tiger red) agar medium, and coliform bacteria were cultured using crystal violet neutral red bile salt agar medium (Guangzhou Dingguo Biotechnology Co., Ltd.); lactic acid bacteria and coliform bacteria were incubated at 30°C in an incubator for 2 days, and yeast and mold were incubated at 28°C in an incubator for 2 days.

[0103] Determination of antibiotic resistance genes:

[0104] (I) qPCR method: 10 g of the sample was added with 40 ml of normal saline, and shaken on a 140 rpm shaker for 3 h, then filtered through two layers of sterile gauze, and the filtrate was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was discarded after the precipitate was removed. The DNA extraction kit was used to extract DNA from the precipitate. After DNA extraction, the PCR amplification product was subjected to 1.5% agarose electrophoresis, the target gene fragment was observed, the gel was recovered, and the target gene fragment was obtained. The target gene was constructed into a vector, and the plasmid DNA (standard) was extracted. The standard was diluted in 7 gradients as a template for fluorescence quantification, and the instrument generated ct values and a standard curve. The amplification system and conditions refer to the instructions: 2×SYBR Green qPCR Premix (Universal), the sample DNA was subjected to quantitative analysis, and each sample was set with 3 groups of parallel samples.

[0105] (II) Macro-genome analysis method: 10 g of the sample was taken, and the bacteria obtained by the above method were sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for DNA extraction, library construction, sequencing and bioinformatics analysis, and the relative abundance of resistance genes under different treatments was compared.

[0106] Table 6 Fermentation quality and protein composition of column flower grass added with Lactobacillus plantarum silage

[0107]

[0108]

[0109] Note: different lowercase letters indicate differences between the same row, and different capital letters indicate differences between the same column.

[0110] Table 7 Effect of adding Lactobacillus plantarum LB1 on the abundance of column grass silage resistance genes (lg copies·g -1 ) (qPCR method)

[0111]

[0112]

[0113] Note: different lowercase letters indicate differences between the same row, and different capital letters indicate differences between the same column.

[0114] Table 8 Effect of adding Lactobacillus plantarum LB1 on the relative abundance of column grass silage resistance genes (%) (metagenomic analysis method)

[0115]

[0116] Note: different lowercase letters indicate differences between the same row, and different capital letters indicate differences between the same column.

[0117] From the above table, it can be seen that after the method of the present application is used, the silage quality of column grass is significantly improved. With the increase of silage days, the pH values of the control group and the treatment group with high acid-producing Lactobacillus plantarum added are significantly reduced, and the pH value of the treatment group is significantly reduced compared with the control group. Compared with the silage column grass of the control group, the high acid-producing Lactobacillus plantarum added group significantly increased the number of lactic acid bacteria in the silage column grass; and significantly reduced the number of coliform bacteria and the content of ammonia nitrogen in the column grass silage, and reduced the content of ammonia nitrogen. Compared with the control group, the abundance of sulfa antibiotic (SRGs) resistance genes in the treatment group was significantly reduced. The present application has obvious improvement effect on the improvement of column grass silage quality and the preservation of nutritional ingredients, and also has significant effect on reducing SRGs.

[0118] The above is only a preferred embodiment of the present application, and it should be noted that the above preferred embodiment should not be regarded as a limitation of the present application.

[0119] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A type of Lactobacillus plantarum ( ‌Lactiplantibacillus plantarum Applications in at least one of the following: a1. Application in improving the quality of silage; a2. Application in the preparation of silage; a3. Application in the preparation of silage additives; The plant lactobacillus ( ‌Lactiplantibacillus plantarum The strain is named LB1 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33485. The silage additive includes a microbial agent, which contains *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum LB1; The bacterial agent possesses the property of significantly reducing the abundance of sulfonamide antibiotic resistance genes, as well as the following properties: (1) Significantly reduces the pH value of silage; (2) Significantly increases the number of lactic acid bacteria in silage; (3) Significantly reduces the content of ammonia nitrogen in silage.

2. A type of Lactobacillus plantarum ( ‌Lactiplantibacillus plantarum Applications in the preparation of any of the following products: b1. Silage products prepared using the aforementioned Lactobacillus plantarum; b2. A silage additive containing Lactobacillus plantarum as an active ingredient, prepared using the Lactobacillus plantarum. The plant lactobacillus ( ‌Lactiplantibacillus plantarum The strain is named LB1 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33485. The silage additive includes a microbial agent, which contains *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum LB1; The bacterial agent possesses the property of significantly reducing the abundance of sulfonamide antibiotic resistance genes, as well as the following properties: (1) Significantly reduces the pH value of silage; (2) Significantly increases the number of lactic acid bacteria in silage; (3) Significantly reduces the content of ammonia nitrogen in silage.

3. The application according to claim 1 or claim 2, characterized in that, The silage plant in the silage includes Stylosanthes.

4. A microbial agent, characterized in that, The microbial agent contains *Lactobacillus plantarum* (… Lactiplantibacillus plantarum LB1 bacterial powder, the Lactobacillus plantarum ( ‌Lactiplantibacillus plantarum The preservation number of LB1 is CGMCC NO.33485. The preparation method of the bacterial powder includes: activating and enriching the Lactobacillus plantarum culture, centrifuging, washing with sterile physiological saline and centrifuging twice, adding skim milk and mixing evenly, pouring into a culture dish, pre-freezing and then placing it in a vacuum freeze dryer with a vacuum degree of less than 60 Pa and a cold trap temperature of less than -55℃. The drying is completed when the sample temperature reaches room temperature. The obtained bacterial powder is then frozen and stored. The freeze-drying procedure for the bacterial powder includes: drying at -25℃ for 300 minutes, drying at -20℃ for 60 minutes, drying at -15℃ for 60 minutes, drying at -10℃ for 60 minutes, drying at -5℃ for 60 minutes, drying at -2℃ for 60 minutes, drying at -0℃ for 60 minutes, drying at 5℃ for 60 minutes, drying at 10℃ for 60 minutes, and drying at 20℃ to the endpoint. The endpoint of freeze-drying is when the sample temperature is the same as room temperature. During the freeze-drying process, the machine vacuum degree is less than 60 Pa, and the cold trap temperature is less than -55℃.

5. A type of silage, characterized in that, The silage is made from Lactobacillus plantarum ( Lactiplantibacillus plantarum LB1 and / or containing Lactobacillus plantarum ( ‌Lactiplantibacillus plantarum Silage is prepared by fermenting LB1 microbial agents with plant growth regulators; the plant growth regulator (LB1) is prepared by fermenting plant growth regulators with plant growth regulators (LB1) ‌Lactiplantibacillus plantarum The accession number for LB1 is CGMCC NO.33485. The amount of *Lactobacillus plantarum* added is 5 × 10⁻⁶ relative to the fresh weight of the silage. 5 cfu·g -1 - 5×10 6 cfu·g -1 .

6. A method for preparing silage, characterized in that, Including the use of Lactobacillus plantarum ( ‌ Lactiplantibacillus plantarum LB1 or containing Lactobacillus plantarum ( Lactiplantibacillus plantarum LB1 microbial agent fermentation of silage plants, wherein the Lactobacillus plantarum ( Lactiplantibacillus plantarum The accession number of LB1 is: CGMCC NO.33485.

7. The silage according to claim 5 or the method for preparing silage according to claim 6, characterized in that, The silage plant in the silage includes Stylosanthes.

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