Lactobacillus plantarum S23Y and application thereof in feed mulberry silage

Abstract

The invention belongs to the technical field of biology, and particularly relates to lactobacillus plantarum S23Y and application thereof in feed mulberry silage. The invention provides a lactobacillus plantarum S23Y, and the preservation number of the lactobacillus plantarum S23Y is CCTCC (China Center for Type Culture Collection) NO: M2023646. The lactobacillus plantarum S23Y provided by the invention has relatively good growth speed and acid production capacity and also has remarkable acid resistance, and when the strain is used for silage fermentation, the condition that the pH of high-protein feed silage is relatively slow to decrease can be solved, the pH can be rapidly reduced to the standard of high-quality silage, meanwhile, the production of ammoniacal nitrogen in the silage process can be greatly reduced, and the yield of the silage is improved. The protein loss is reduced, and the fermentation time is shortened.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a strain of Lactobacillus plantarum S23Y and application thereof in silage of mulberry forage. Background Art

[0002] Silage is a storage method that converts the fermentation substrate in the raw materials into acidic substances such as lactic acid through the proliferation of lactic acid bacteria, maintains an acidic anaerobic environment, and is conducive to the long-term preservation of silage crops. Silage is yellow-green in color, sour and fragrant, soft and juicy, and has good palatability. It is widely used in animal husbandry production, especially in ruminant breeding, and has become an important source of high-quality roughage. There are many microorganisms in the silage process, such as lactic acid bacteria (LAB), fungi, clostridium and some common spoilage bacteria. To give full play to the advantages of silage technology and ensure the effect of silage, the most important thing is lactic acid bacteria. Silage fermented feed with lactic acid bacteria as the main microorganism has high-quality energy and protein, which has an effect on the growth of livestock and improving their immunity.

[0003] Mulberry leaves have high protein content (15% to 30% crude protein), a variety of amino acids (containing 18 kinds of amino acids), and a variety of natural active substances and their derivatives (such as sitosterol, isoquercetin, lutein, lutein, etc.). Its nutritional value is comparable to that of alfalfa, which is known as the "king of forage grass". Mulberry has the characteristics of good palatability and high digestibility as livestock and poultry feed. Whether in improving the quality and flavor of livestock and poultry products or in improving animal immunity, it has advantages that other grass species cannot match, so it has a high utilization value. However, due to the low carbohydrate content and high protein content of mulberry leaves, it is difficult to ferment them into high-quality silage. When using existing lactic acid bacteria to ensilage feed mulberry, the pH drops slowly, ammonia nitrogen is produced more, fermentation cannot be completed quickly, and protein loss is high during the fermentation process. Summary of the invention

[0004] The invention aims to provide a strain of Lactobacillus plantarum S23Y and application thereof in silage of mulberry forage, so as to solve the problem that the pH value of high-protein feed silage decreases slowly, quickly reduce the pH value to the standard of high-quality silage, and at the same time greatly reduce the generation of ammonia nitrogen in the silage process, reduce protein loss, and shorten the fermentation time.

[0005] In order to achieve the above object, the present invention provides a strain of Lactobacillus plantarum S23Y, with a preservation number of CCTCC NO: M2023646.

[0006] The present invention also provides a bacterial agent, comprising the Lactobacillus plantarum S23Y described in the above technical solution or the fermentation liquid of the Lactobacillus plantarum S23Y.

[0007] The present invention also provides the use of the Lactobacillus plantarum S23Y or the bacterial agent described in the above technical solution in silage.

[0008] Preferably, the application in silage includes one or more of promoting pH reduction during silage, inhibiting the generation of ammonia nitrogen during silage, and inhibiting protein decomposition during silage.

[0009] Preferably, the silage raw material includes fodder mulberry.

[0010] Preferably, the ingredients of the feed mulberry include: 24.12-24.40wt.% dry matter, 37.42-39.60wt.% neutral detergent fiber, 23.97-26.73wt.% acid detergent fiber, 2.64-3.16wt.% crude fat, 8.42-8.68wt.% crude ash, 23.05-23.73wt.% crude protein and 6.24-6.58wt.% soluble carbohydrates.

[0011] The present invention also provides a silage feed, the raw materials for preparing the silage feed include a first raw material and a second raw material;

[0012] The first raw material includes mulberry, a feed material; and the second raw material includes the plant lactobacillus S23Y or the bacterial agent described in the above technical solution.

[0013] The present invention also provides a method for preparing silage according to the above technical solution, comprising the following steps:

[0014] The second raw material is inoculated into the first raw material, and the silage is obtained through ensilage.

[0015] Preferably, the inoculation amount of the second raw material is calculated based on the number of viable bacteria of Lactobacillus plantarum S23Y, and the inoculation amount is ≥10 5 CFU / g.

[0016] Preferably, the silage temperature is 20-25°C and the silage time is 30-45 days.

[0017] Beneficial effects:

[0018] The present invention provides a strain of Lactobacillus plantarum S23Y, with a deposit number of CCTCCNO: M 2023646. The Lactobacillus plantarum S23Y provided by the present invention has a good growth rate and acid production ability, and also has a significant acid resistance. The strain is used for silage fermentation, which can solve the problem that the pH value of high-protein feed silage decreases slowly, and the pH value can be quickly reduced to the standard of high-quality silage. At the same time, the generation of ammonia nitrogen in the silage process can be greatly reduced, the loss of protein can be reduced, and the fermentation time can be shortened.

[0019] Biological deposit information

[0020] Lactobacillus plantarum S23Y, biologically classified as Lactiplantibacillus Plantarum, was deposited in the China Center for Type Culture Collection on April 27, 2023, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M2023646. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 This is the result of the test of biofilm production ability of the candidate strains; different lowercase letters indicate P < 0.01;

[0023] Figure 2 This is a graph showing the results of testing the acid production ability of the candidate strains;

[0024] Figure 3 This is the result of the acid resistance test of the candidate strains; different lowercase letters indicate P < 0.01;

[0025] Figure 4 This is the colony morphology of the candidate strains;

[0026] Figure 5 This is the result of Gram staining microscopy of the candidate strain colony;

[0027] Figure 6 This is the phylogenetic tree of Lactobacillus plantarum S23Y. DETAILED DESCRIPTION

[0028] The invention provides a plant lactobacillus S23Y, with a preservation number of CCTCCNO: M 2023646.

[0029] The present invention isolates a plant lactobacillus S23Y from various pastures and roughage (including silage, silage raw materials, etc.) in Guangdong Province. The strain has a good growth rate and acid production ability, and also has significant acid resistance. It is used for silage fermentation, can solve the problem that the pH value of high-protein feed silage decreases slowly, can quickly reduce the pH value to the standard of high-quality silage, and can also greatly reduce the generation of ammonia nitrogen in the silage process, reduce protein loss, and shorten the fermentation time.

[0030] The present invention also provides a bacterial agent, comprising the Lactobacillus plantarum S23Y described in the above technical solution or the fermentation liquid of the Lactobacillus plantarum S23Y.

[0031] As an embodiment, the number of viable bacteria of Lactobacillus plantarum S23Y in the fermentation broth of Lactobacillus plantarum S23Y is ≥10 5CFU / mL; As another embodiment, the number of viable bacteria of Lactobacillus plantarum S23Y in the fermentation broth of Lactobacillus plantarum S23Y is 10 5 ~10 7 CFU / mL; As another embodiment, the number of viable bacteria of Lactobacillus plantarum S23Y in the fermentation broth of Lactobacillus plantarum S23Y is 10 6 CFU / mL. As an embodiment, the preparation method of the fermentation liquid of Lactobacillus plantarum S23Y comprises: inoculating the Lactobacillus plantarum S23Y into MRS liquid culture medium, and culturing in a shaking incubator at 37° C. to obtain the fermentation liquid of Lactobacillus plantarum S23Y. The MRS liquid culture medium of the present invention is purchased from MacLean Reagent Company.

[0032] In view of the advantages of Lactobacillus plantarum S23Y provided by the present invention, the application of Lactobacillus plantarum S23Y or the bacterial agent in silage described in the above technical solution also belongs to the protection scope of the present invention. As an embodiment, the application in silage includes one or more of promoting pH decrease during silage, inhibiting the production of ammonia nitrogen during silage, and inhibiting protein decomposition during silage.

[0033] As an embodiment, the raw material of the silage includes mulberry forage. As an embodiment, the ingredients of the mulberry forage include: 24.12-24.40wt.% dry matter, 37.42-39.60wt.% neutral detergent fiber, 23.97-26.73wt.% acid detergent fiber, 2.64-3.16wt.% crude fat, 8.42-8.68wt.% crude ash, 23.05-23.73wt.% crude protein and 6.24-6.58wt.% soluble carbohydrates; as another embodiment, the mulberry forage includes 24.26wt.% dry matter, 38.51wt.% neutral detergent fiber, 25.35wt.% acid detergent fiber, 2.90wt.% crude fat, 8.56wt.% crude ash, 23.39wt.% crude protein and 6.41wt.% soluble carbohydrates.

[0034] The present invention also provides silage, and the raw materials for preparing the silage include a first raw material and a second raw material; the first raw material includes mulberry, and the second raw material includes the plant lactobacillus S23Y or the bacterial agent described in the above technical solution.

[0035] The present invention also provides a method for preparing silage according to the above technical solution, comprising the following steps:

[0036] The second raw material is inoculated into the first raw material, and the silage is obtained through ensilage.

[0037] As an embodiment, the inoculation amount of the second raw material is calculated based on the number of viable bacteria of Lactobacillus plantarum S23Y, and the inoculation amount is ≥10 5 CFU / g; As another embodiment, the inoculation amount of the second raw material is based on the number of viable bacteria of Lactobacillus plantarum S23Y, and the inoculation amount is 10 5 ~10 7 CFU / mL; As another embodiment, the inoculation amount of the second raw material is based on the number of viable bacteria of Lactobacillus plantarum S23Y, and the inoculation amount is 10 6 CFU / mL. As an embodiment, the silage temperature is 20-25°C; as another embodiment, the silage temperature is 22°C; as an embodiment, the silage time is 30-45 days; as another embodiment, the silage time is 30 days. As an embodiment, the silage is sealed fermentation.

[0038] The present invention utilizes the plant lactobacillus S23Y or the bacterial agent described in the above technical solution to ensilage mulberry, which can quickly reduce the pH value during the ensilage process of the mulberry and reduce the generation of ammonia nitrogen, thereby achieving the purpose of reducing protein loss in ensilage and obtaining high-quality silage.

[0039] To further illustrate the present invention, a strain of Lactobacillus plantarum S23Y provided by the present invention and its application in mulberry silage are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0040] In the following examples, unless otherwise specified, all methods are conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0041] Example 1

[0042] 1. Sample collection and lactic acid bacteria isolation and purification

[0043] Use a sterile sampling bag to collect samples (from various pastures and roughage in Guangdong Province, including silage, silage raw materials, etc.), and store them at 4°C; weigh 10g of the sample and put it into a conical flask, add 90mL of MRS broth medium, and culture it at 37°C to enrich the bacteria; take the enriched bacterial solution for 10 -1 Up to 10 -6 Dilution, 200 μL of each gradient dilution solution is spread on the surface of MRS agar medium containing 1% CaCO3, and cultured upside down under anaerobic conditions at 37°C for 48 hours; after the culture is completed, pick out typical lactic acid bacteria colonies with calcium dissolution rings, and purify them 2 to 3 times until no other colonies appear on the MRS agar plate; screen strains with negative catalase tests as backup strains, mix the backup strain bacterial solution and 30% sterilized glycerol (final glycerol concentration is 15%) in a volume ratio of 1:1, and store them at -80°C for a long time.

[0044] 2. Screening of high-yield biofilm lactic acid bacteria

[0045] Referring to the method of biofilm detection in previous literature and making fine adjustments, the biofilm production ability of the lactic acid bacteria isolated in step 1 was detected. The specific method is as follows: the bacterial solution cultured overnight was adjusted to 0.5 MCF using a bacterial turbidity meter, and the bacterial solution was inoculated into 1 mL of sterile MRS broth medium at an inoculum volume of 1% (v / v) in a 48-well plate. The sterile MRS broth medium was used as a control (Control), and 3 replicate wells were set up and cultured at 37°C for 48 hours; after the culture was completed, the upper liquid was taken and the OD 600 Determine the turbidity of free bacteria; discard the remaining bacterial solution in the well, rinse slowly with sterile PBS 2 to 3 times, and then add 1 mL of 10% (v / v) methanol solution to fix for 30 minutes; discard the upper liquid, rinse the excess methanol solution with PBS, dry it, and then add 1wt.% crystal violet solution to stain for 30 minutes; after staining, wash the free crystal violet with slow water flow, dry it, add 33% (v / v) acetic acid solution for decolorization, transfer the liquid to a 96-well plate, and 600 The absorbance value was measured at Figure 1 shown.

[0046] OD 600 The larger the absorbance value measured at the position, the stronger the biofilm production ability of the candidate strain. Figure 1 OD of strains numbered 48-1, 28, 14-1, 21, 31-141, and 48-2 600 The absorbance value measured at the strain was significantly higher than that of other strains, and the strain numbered 48-1 was significantly higher than the strains numbered 28, 14-1, 21, 31-141 and 48-2. The strain numbered 48-1 had the highest biofilm production ability.

[0047] 3. Screening of high acid-producing lactic acid bacteria

[0048] Use a bacterial turbidity meter to adjust the overnight culture solution to 0.5 MCF. In a 24-well plate, inoculate the bacterial solution with a 1% (v / v) inoculum into 2.5 mL / well of sterile MRS broth medium. Sterile MRS broth medium is used as a control (Control). Set up 3 replicate wells and culture at 37°C. Measure the pH of the bacterial solution at 0h, 6h, 12h, 24h, and 36h of culture. The results are as follows: Figure 2 shown.

[0049] according to Figure 2 It can be seen that the acid production of the tested strains basically stagnated from 24h to 36h, among which 13-1, 14-1 and 48-1 lactic acid bacteria produced the fastest acid.

[0050] 4. Screening of highly acid-resistant lactic acid bacteria

[0051] Use 1mol / L hydrochloric acid and 1mol / L NaOH solution to adjust the pH of MRS broth to 1.5, 2.5, 3.5, 4.5 and 5.5 respectively for use; use a bacterial turbidity meter to adjust the overnight culture of each bacterial solution to 0.5 MCF, and inoculate the bacterial solution into MRS broth with different pH values ​​at an inoculum volume of 1% (v / v), set up 3 technical replicates, and use MRS broth without pH adjustment as a control. After culturing at 37°C for 24 hours, at OD 600 The growth of each strain was measured at Figure 3 shown.

[0052] according to Figure 3 It can be seen that under the conditions of pH = 1.5 and pH = 2.5, all the reserve lactic acid bacteria strains could not grow ( Figure 3 In a), under the condition of pH=3.5, the growth of strain 48-1 was significantly better (P<0.01) than that of other strains ( Figure 3 (b)

[0053] 3. Morphological Detection

[0054] The strains numbered 48-1, 28, 14-1, 21, 31-141 and 48-2 were inoculated on MRS agar medium, the colony morphology was observed, and Gram staining was performed. Figure 4 ), the colonies of the 7 reserve lactic acid bacteria were all round, medium-sized, convex, slightly white, moist and with neat edges, with typical lactic acid bacteria colony characteristics. Gram staining microscopy results showed that ( Figure 5 ), among the 7 reserve lactic acid bacteria, except for No. 28 which was a coccus, the other 6 strains were all bacilli. Based on the conclusion of step 2, the strain No. 48-1 was determined for subsequent experiments and named S23Y.

[0055] 4.16s rDNA identification

[0056] (1) The overnight cultured S23Y bacterial suspension was centrifuged and the bacterial cells were collected. The genomic DNA of the S23Y strain was extracted using a bacterial DNA extraction kit (column centrifugation method) according to the instructions and used as a PCR template. The 16s rDNA sequence of the S23Y strain was amplified by PCR (forward primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1; reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2); the amplification system was 1 μL of template, 0.4 μL of forward primer 27F (10 μM), 0.4 μL of reverse primer 1492R (10 μM), 10 μL of 2×TaqPCR StarMix (Dye), and dd H2O to 20 μL; the amplification program was pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 90 s, 32 cycles; final extension at 72°C for 10 min.

[0057] (2) The amplified product of step (1) was sent to Sanger sequencing of the strains in the NCBI database. The sequencing requirement was "test-through". The sequencing results were compared using the NCBI BLAST function (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and then the phylogenetic tree was drawn using the MEGA software (V11) to determine the bacterial species. The BLAST comparison showed that the sequence similarity between the S23Y strain and the strains in the NCBI database was above 98% ( Figure 6 ), L. plantarum (Lactobacillus plantarum), the 16s rDNA nucleotide sequence of the S23Y strain is shown in SEQ ID NO. 3. According to the International Microbiological Classification and Nomenclature, it was renamed Lactobacillus plantarum S23Y and biologically preserved.

[0058]

[0059] Example 2

[0060] Silage experiment

[0061] 1. Silage raw materials

[0062] The silage experiment was carried out using fodder mulberry as raw material. The main chemical components of fodder mulberry are shown in Table 1.

[0063] Table 1 Main chemical components of mulberry before ensilage (%)

[0064]

[0065] Note: The content of dry matter is based on fresh weight, and the rest is based on dry weight.

[0066] 2. Experimental group design

[0067] Three treatment groups were set up as follows:

[0068] Group 1 (control group): no fermentation agent was added;

[0069] The second group (S23Y group): using the plant lactobacillus S23Y obtained by screening in Example 1 as the silage starter;

[0070] The third group (common bacteria group): common Lactobacillus plantarum (provided by the Herbivore Research Laboratory of South China Agricultural University, purchased from Zhongke Yijia, JYLP-002) was used as the fermentation agent.

[0071] 3. Silage experiment

[0072] (1) Lactobacillus plantarum S23Y and common Lactobacillus plantarum were inoculated into MRS liquid culture medium respectively, and cultured on a shaker at 37° C. The number of lactic acid bacteria was determined by a plate counting method to obtain Lactobacillus plantarum S23Y bacterial liquid and common Lactobacillus plantarum bacterial liquid.

[0073] (2) Use a grinder to crush the mulberry leaves into 2-3 cm long pieces. According to the experimental group design in step 2, add the corresponding bacterial solution based on the weight of the mulberry leaves so that the concentration of live bacteria added to the system is 10 6 CFU / g, add the same amount of sterile distilled water to the control group; after mixing evenly, put about 500g of samples from each group into sealed bags, use a vacuum machine to extract the air and seal them, and store them normally at room temperature (20-25℃). Each group was tested three times, and a total of 9 groups were set up.

[0074] (3) On the 30th day of ensiling, the nutritional characteristics and fermentation characteristics of the mulberry trees in each treatment group were analyzed. The specific items and analysis methods are as follows. The analysis results are shown in Tables 2 and 3.

[0075] Dry matter (DM): GBT6435-2014;

[0076] Neutral detergent fiber (NDF): GB / T20806-2006;

[0077] Acid detergent fiber (ADF): NY / T1459-2022;

[0078] Crude fat: GBT6433-2006;

[0079] Crude ash: GBT6438-2007;

[0080] Crude protein: GBT6432-2018;

[0081] Soluble carbohydrates (WSC): GOST 26176-2019;

[0082] pH: pB-7890 (Sartorius) pH meter;

[0083] Ammonia nitrogen: phenol-sodium hypochlorite colorimetric method (Wen Yali Gao Min. Research progress on lactic acid bacteria metabolism in silage and its influence on silage quality [J]. Animal Husbandry and Feed Science, 2011, 32(Z1):152-154.);

[0084] Acetic acid: Gas chromatograph Agilent 7890B (Wang Junhong, Gao Ge, Zhang Yuanyuan, et al. Determination of volatile fatty acids in ruminant rumen fluid by headspace-gas chromatography [J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(05): 667-672.)

[0085] Table 2 Nutritional characteristics of mulberry in each treatment group after silage for 30 days

[0086] project Control group S23Y group Common bacteria group DM(%) <![CDATA[24.02±0.27 ab ]]> <![CDATA[24.42±0.16 b ]]> <![CDATA[23.68±0.15 a ]]> NDF(%) 20.91±1.54 22.80±1.28 21.77±1.30 ADF(%) 29.96±1.94 32.43±1.99 31.65±1.85 Crude fat (%) 4.23±0.71 3.35±0.13 3.23±0.37 Crude ash (%) 9.02±0.46 8.78±0.06 9.33±0.53 Crude protein (%) <![CDATA[23.83±0.25 a ]]> <![CDATA[24.28±0.18 b ]]> <![CDATA[24.68±0.05 b ]]> WSC(%) 1.89±0.08 2.33±0.40 2.14±0.22

[0087] Note: Different letters indicate significant differences and P < 0.001, the same letters or no letters indicate no significant differences, the same below.

[0088] Table 3 Fermentation characteristics of mulberry in each treatment group after silage for 30 days

[0089]

[0090]

[0091] According to Table 2 and Table 3, it can be seen that the dry matter content of the control group, S23Y group and common bacteria group is 24.02%, 24.42% and 23.68% respectively. Among them, the dry matter content of the S23Y group is higher than that of the control group and the common bacteria group, and the loss of dry matter is the least, indicating that the plant lactobacillus S23Y of the present invention can reduce the loss of dry matter during ensiling. The crude protein content of each group is 23.83% DM, 24.28% DM and 24.68% DM respectively, among which the protein content of the control group is the lowest and the protein content of the common bacteria group is the highest. The NDF and ADF of the control group, S23Y group and common bacteria group are 20.91 ± 1.54% DM, 29.96 ± 1.94% DM, 22.80 ± 1.28% DM and 32.43 ± 1.99% DM, 21.77 ± 1.30% DM, 31.65 ± 1.85% DM respectively. Excessive NDF and ADF content will affect the digestibility of animals. To a certain extent, the lower the NDF and ADF, the higher the digestibility of animals. The pH value of the S23Y group was the lowest among all treatment groups, reaching the standard of high-quality silage (pH value <4.2). Although the lactic acid content of the S23Y group was 23.55±1.32g / kgFW, which was lower than that of the ordinary bacteria group 25.17±0.19g / kgFW, it was still significantly higher than that of the control group 22.03±2.01g / kgFW, indicating that the addition of plant lactobacillus S23Y can significantly increase the lactic acid fermentation of the silage fermentation process and increase its lactic acid content. The acetic acid content of the S23Y group was the lowest among the three groups, indicating that WSC was better converted into lactic acid. Among the three treatment groups, the ammonia nitrogen content of the S23Y group was 2.52±0.39%TN, which was significantly lower than that of the other two groups. The ratio of ammonia nitrogen to total nitrogen reflects the degree of protein and amino acid decomposition in silage. The larger the ratio, the more protein decomposition and the poor silage quality. This shows that adding S23Y to silage can reduce the decomposition of protein in silage, thereby improving the quality of silage.

[0092] It can be seen from the above content that the Lactobacillus plantarum S23Y provided by the present invention can quickly reduce the pH to the standard of high-quality silage, greatly reduce the production of ammonia nitrogen during the silage process, reduce protein loss, and shorten the fermentation time.

[0093] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum S23Y, with a deposit number of CCTCCNO: M2023646.

2. A bacterial agent, characterized in that The invention comprises the Lactobacillus plantarum S23Y according to claim 1 or the fermentation liquid of the Lactobacillus plantarum S23Y according to claim 1.

3. Use of the plant lactobacillus S23Y according to claim 1 or the bacterial agent according to claim 2 in silage.

4. The use according to claim 3, characterized in that: The application in silage includes: one or more of promoting pH reduction during silage, inhibiting the generation of ammonia nitrogen during silage, and inhibiting protein decomposition during silage.

5. The use according to claim 3 or 4, characterized in that: The raw material for silage includes mulberry leaves.

6. The use according to claim 5, characterized in that: The ingredients of the feed mulberry include: 24.12-24.40wt.% dry matter, 37.42-39.60wt.% neutral detergent fiber, 23.97-26.73wt.% acid detergent fiber, 2.64-3.16wt.% crude fat, 8.42-8.68wt.% crude ash, 23.05-23.73wt.% crude protein and 6.24-6.58wt.% soluble carbohydrates.

7. A silage, characterized in that: The preparation raw materials include a first raw material and a second raw material; The first raw material includes mulberry forage; the second raw material includes the plant lactobacillus S23Y described in claim 1 or the bacterial agent described in claim 2.

8. The method for preparing silage according to claim 7, characterized in that: The steps include: The second raw material is inoculated into the first raw material, and the silage is obtained through ensilage.

9. The preparation method according to claim 8, characterized in that: The inoculation amount of the second raw material is calculated based on the number of viable bacteria of Lactobacillus plantarum S23Y, and the inoculation amount is ≥10 5 CFU / g.

10. The preparation method according to claim 8, characterized in that: The silage temperature is 20-25° C. and the silage time is 30-45 days.

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