Lactobacillus plantarum, bacterial agent, additive, feed and related methods

Abstract

The present invention discloses a plant lactobacillus, a bacterial agent, an additive, a feed and a related method. Plant lactobacillus (Lactiplantibacillus plantarum) L75, whose deposit number in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee is CGMCC No.29678. It has a wide pH range, strong salt tolerance, high extracellular polysaccharide production, can effectively reduce the pH of silage at 15°C, increase the proportion of lactic acid and beneficial bacteria Lactobacillus, and help improve the quality of silage.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular to a plant lactobacillus, a bacterial agent, an additive, a feed and related methods. Background Art

[0002] The Qinghai–Tibet Plateau (QTP), recognized as the roof of the world, has special geographical and climatic conditions, such as low temperature, low pressure, strong ultraviolet light, and cold environment. Such geographical and climatic conditions make the growing season of forage short and the forage yield low. In winter and early spring, the feed shortage in the Qinghai–Tibet Plateau becomes more serious. Oats (Avena sativa L.) are a valuable livestock feed with high nutritional value, low buffering capacity, and high biomass. In the Qinghai–Tibet Plateau, large-scale oats are harvested in late September to early October, when the average temperature is below 15°C. Under such unfavorable conditions, it is difficult to prepare high-quality oat silage using natural fermentation or conventional lactic acid bacteria inoculants. Summary of the invention

[0003] In view of this, the purpose of the present disclosure is to provide a plant lactobacillus, a bacterial agent, an additive, a feed and related methods.

[0004] Based on the above purpose, the present disclosure provides a Lactobacillus plantarum L75, whose deposit number in the General Microbiological Center of China National Microbiological Culture Collection Administration is CGMCC No. 29678.

[0005] Based on the same inventive concept, the disclosed embodiment further provides a bacterial agent, characterized in that the active ingredient of the bacterial agent is the aforementioned Lactiplantibacillus plantarum L75.

[0006] Based on the same inventive concept, the disclosed embodiment further provides a silage additive, the active ingredient of the silage additive includes the Lactiplantibacillus plantarum L75.

[0007] In some embodiments, the active ingredient further comprises Saccharomyces cerevisiae.

[0008] Based on the same inventive concept, an embodiment of the present disclosure further provides a silage, which includes any of the aforementioned silage additives.

[0009] Based on the same inventive concept, the disclosed embodiments also provide the use of any of the aforementioned Lactobacillus plantarum L75 or bacterial agents in inhibiting fungi, preparing fungal inhibitors, and silage additives.

[0010] Based on the same inventive concept, the disclosed embodiments also provide the use of any of the aforementioned silage additives in the preparation of silage.

[0011] Based on the same inventive concept, the present disclosure also provides a method for preparing the aforementioned silage, comprising:

[0012] The silage raw material and the silage feed additive are mixed, solid fermentation is carried out, and the fermentation product is collected to obtain the silage feed.

[0013] In some embodiments, based on the mass of the silage raw material, the addition of Lactiplantibacillus plantarum L75 and the Saccharomyces cerevisiae are 0.5×10 5 ~9.0×10 5 CFU / g FW.

[0014] In some embodiments, the silage feedstock includes oats.

[0015] From the above, it can be seen that the present disclosure provides a plant lactobacillus, bacterial agent, additive, feed and related methods. The plant lactobacillus L75 has a wide pH range for growth, strong salt tolerance, high extracellular polysaccharide production, can effectively reduce the pH of silage at 15°C, increase the ratio of lactic acid and beneficial bacteria Lactobacillus, and help improve the quality of silage. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1A The growth comparison of Lactobacillus plantarum L75 and MTD / 1 provided in the embodiments of the present disclosure at 25°C is shown;

[0018] Figure 1B The growth comparison of Lactobacillus plantarum L75 and MTD / 1 provided in the embodiments of the present disclosure at 15°C is shown;

[0019] Figure 1CThe figure shows a comparison of the acid production of Lactobacillus plantarum L75 and MTD / 1 at 25°C provided in the embodiments of the present disclosure;

[0020] Figure 1D The figure shows a comparison of the acid production of Lactobacillus plantarum L75 and MTD / 1 at 15°C provided in the embodiments of the present disclosure;

[0021] Figure 2 The comparison results of the extracellular polysaccharide yields in the fermentation broth of Lactobacillus plantarum L75 and MTD / 1 provided in the embodiments of the present disclosure are shown;

[0022] Figure 3 A schematic diagram showing the comparison results of oat silage fermentation quality obtained by different fermentation treatments provided in the embodiments of the present disclosure;

[0023] Figure 4 The diagram shows the structure of oat silage microbial community obtained by different fermentation treatments provided in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0025] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should have the usual meanings understood by people with ordinary skills in the field to which the present disclosure belongs. The words "include" or "comprise" and the like used in the embodiments of the present disclosure mean that the components or substances appearing before the word include the components or substances listed after the word and their equivalents, but do not exclude other components or substances.

[0026] Explanation of terms

[0027] Extracellular polysaccharides (EPS) of lactic acid bacteria are a kind of bacterial metabolite. This substance has multiple biological activities, including antibacterial, antioxidant, anti-tumor, and immune regulation. It is a mechanism for bacteria to resist extreme environments (cold environments, etc.).

[0028] As described in the background art, conventional lactic acid bacteria inoculants are difficult to meet the needs of oat silage in the Qinghai-Tibet Plateau. In view of this, the present disclosure provides a plant lactobacillus (Lactiplantibacillus plantarum) L75, which has a deposit number of CGMCC No. 29678 in the General Microbiological Center of the China Microbiological Culture Collection Administration.

[0029] The Lactobacillus plantarum L75 was isolated from the silage of Elymus thunbergii in Huangyuan County, Qinghai Province, China. The single colony of the strain has a calcium-dissolving circle in an MRS solid culture medium containing calcium carbonate; there is a wire drawing phenomenon in the process of picking up a single colony on the colony plate, and it is preliminarily judged that the bacteria can produce extracellular polysaccharides; it can grow at pH values ​​of 3.5 to 8.5, indicating that it has strong acid and alkali resistance; it grows well at 3% and 6% NaCl concentrations, indicating that it has strong salt resistance. The strain has strong growth adaptability and can survive and reproduce in extreme environments. Its 16S rDNA sequence is shown in SEQ ID NO.1.

[0030] In some embodiments, the present disclosure further provides a bacterial agent. Exemplarily, the active ingredient of the bacterial agent is Lactobacillus plantarum L75. In addition to the active ingredient, the bacterial agent may also contain auxiliary materials, such as protective agents such as skim milk.

[0031] In some embodiments, the present disclosure further provides a silage additive. Optionally, the active ingredient of the silage additive includes Lactobacillus plantarum L75.

[0032] In some embodiments, the active ingredient of the silage additive also includes Saccharomyces cerevisiae. Saccharomyces cerevisiae is commonly used in ruminant feeding, and its notable feature is its metabolic flexibility, which enables it to switch between complete respiratory sugar dissimilation and alcohol fermentation processes. Both metabolic pathways are accompanied by the generation of ATP, and the former produces a large amount of ATP. However, Ca 2+ -Only a part of the chemical energy released when ATPase hydrolyzes to generate ATP is converted into osmotic energy. The other part is converted into heat, which is used by cells to maintain a constant and high body temperature. The inventors of the present disclosure mixed brewer's yeast and EPS-producing plant lactobacillus as a silage additive and found that it helped the reproduction of plant lactobacillus and promoted silage fermentation. The reason may be that in the early stage of silage, the externally added brewer's yeast will consume the residual oxygen in the silage pit and the carbohydrates of the feed raw materials, producing a large amount of ATP. The heat generated during the chemical energy conversion process (ATP hydrolysis release) will resist the external cold environment of QTP.

[0033] By mixing Lactobacillus plantarum L75 and Saccharomyces cerevisiae, high-quality silage can be produced, which can not only ferment the silage but also produce probiotic effects (such as antioxidant, antibacterial and other effects), which is conducive to the preparation of high-quality oat silage with probiotic effects.

[0034] In some embodiments, the present disclosure further provides a silage. Optionally, the silage contains the aforementioned silage additive.

[0035] In some embodiments, the present disclosure also provides a use of the Lactobacillus plantarum L75 or the bacterial agent in inhibiting fungi, preparing fungal inhibitors, and silage additives. It should be noted that the fungi here can be molds, especially harmful molds, such as Penicillium cameum.

[0036] In some embodiments, the present disclosure also provides the use of the silage additive in preparing silage. Here, the silage can be oat silage.

[0037] In some embodiments, the present disclosure also provides a method for preparing the silage, comprising:

[0038] The silage raw material and the silage feed additive are mixed, solid fermentation is carried out, and the fermentation product is collected to obtain the silage feed.

[0039] Alternatively, the silage material may be oats wilted under natural aeration conditions. The wilting degree may be ≤400 g / kg (FW), such as 386.25 g / kg FW.

[0040] Optionally, the silage additive comprises Lactobacillus plantarum L75. Optionally, the silage additive comprises Lactobacillus plantarum L75 and Saccharomyces cerevisiae. Optionally, the silage additive does not contain harmful bacteria inhibitors and / or antioxidants.

[0041] In some embodiments, after the silage raw material and the silage feed additive are mixed, they are placed in a silage bag for vacuum silage. Optionally, the silage bag can be a polyethylene bag. Optionally, anaerobic fermentation occurs during the silage process.

[0042] Furthermore, based on the mass of the silage raw material, the Lactobacillus plantarum L75 is 0.5×10 5 ~9.0×10 5 CFU / g FW, e.g. 0.5×10 5 CFU / g FW, 1.0×10 5 CFU / g FW, 2.5×10 5 CFU / g FW, 5.8×10 5 CFU / g FW, 7.2×10 5 CFU / g FW, 9.0×10 5 CFU / g FW.

[0043] Furthermore, based on the mass of the silage raw material, the addition amount of brewer's yeast is 0.5×10 5 ~9.0×10 5 CFU / g FW, e.g. 0.5×10 5 CFU / g FW, 1.0×10 5 CFU / g FW, 2.5×10 5 CFU / gFW, 5.8×10 5 CFU / gFW, 7.2×10 5 CFU / g FW, 9.0×10 5 CFU / g FW.

[0044] In some embodiments, the ratio of the Lactobacillus plantarum L75 to the Saccharomyces cerevisiae is 1:0.8 to 1:1.3, for example, 1:0.8, 1:1, 1:1.1, 1:1.3.

[0045] In order to make the technical solution of the present disclosure clearer and easier to understand, the multifunctional optical sensor and the preparation method thereof provided by the present disclosure are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0046] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0047] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the examples of the present disclosure should have the common meanings understood by people with ordinary skills in the field to which the present disclosure belongs.

[0048] Example 1 Isolation and Identification of Lactobacillus plantarum L75

[0049] 1. Isolation and screening of strain L75

[0050] Take an appropriate amount of silage of Elymus thunbergii from Huangyuan County, Qinghai Province, China, add it to sterile water for shaking, absorb the liquid and dilute it and apply it to MRS solid medium with calcium carbonate added for cultivation, pick up the suspected lactic acid bacteria single colony with calcium dissolution circle, expand the culture, and number one of the bacteria as L75. It should be noted that in the process of picking up the single colony, it was found that there was a wire drawing situation, so it was believed that the bacteria would produce extracellular polysaccharides, and the extracellular polysaccharide production was subsequently quantitatively detected. Here, the water solvent in the MRS medium is used as the benchmark, and the amount of calcium carbonate added is 0.5%.

[0051] 2. Identification of strain L75

[0052] 1. Identification of physiological and biochemical characteristics

[0053] Carbon source utilization

[0054] API 50CH was used to test the carbon source utilization of strain L75 and Lactobacillus plantarum strain MTD-1 purchased from Vita Plus (Madison, Wisconsin, USA). The results are shown in Table 1.

[0055] Table 1 Carbon source utilization of exopolysaccharide-producing Lactobacillus plantarum

[0056]

[0057] Note: - indicates that the strain cannot utilize the carbon source; + indicates that the strain can utilize the carbon source.

[0058] As can be seen from Table 1, the carbon source utilization range of the exopolysaccharide-producing strain L75 is similar to that of the commercial strain MTD / 1. Compared with MTD / 1, L75 can utilize raffinose.

[0059] Extracellular polysaccharide production

[0060] The strain L75 and the commercially purchased Lactobacillus plantarum strain MTD-1 were cultured in MRS liquid medium at 37°C for 24 hours, and the exopolysaccharide production in the fermentation supernatant was detected by the phenol-sulfuric acid method. Figure 2 As shown. Figure 2 It can be seen that the extracellular polysaccharide production of L75 in 1L MRS fermentation broth was significantly higher than MTD / 1, greater than 150 mg / ml, and close to 160 mg / mL.

[0061] 2. 16S rDNA sequence homology analysis

[0062]

[0063] The homology comparison of SEQ ID NO.1 in the NCBI database revealed that the sequence similarity between SEQ ID NO.1 and Lactobacillus plantarum was as high as 99.99%.

[0064] Among them, the bacteria-specific primers 27F: 5'-AGRGTTYGATYMTGGCTCAG-3'——SEQ ID NO.2; and 1492R: 5'-RGYTACCTTGTTACGACTT-3'——SEQ ID NO.3.

[0065] Based on the above physiological and biochemical characteristics and the results of 16S rDNA sequence homology analysis, strain L75 was identified as Lactiplantibacillus plantarum and was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101) on January 19, 2024. Its preservation number is CGMCC No. 29678, and its classification name is Lactiplantibacillus plantarum.

[0066] Example 2 Growth of Lactobacillus plantarum L75 at different pH and salt concentrations

[0067] 1. Growth of Lactobacillus plantarum L75 and MTD / 1 at different pH

[0068] The initial pH of the MRS liquid culture medium was adjusted to 3.5, 4.0, 4.5, 7.5, 8.0, and 8.5 with 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide, respectively. L75 and MTD / 1 were cultured at a constant temperature of 37°C for 24 h. The acid was not adjusted during the culture process. The absorbance at 600 nm (OD600) was measured with a UV-visible spectrophotometer to observe the growth of Lactobacillus plantarum L75 and MTD / 1 at different pH values. The results are shown in Table 2.

[0069] Table 2 pH and sodium chloride tolerance of Lactobacillus plantarum producing extracellular polysaccharides

[0070]

[0071] Note: -, no growth (OD600nm < 0.5); W, weak growth (OD600nm range is 0.5 ~ 1); +, growth (OD600nm range is 1 ~ 2); ++, good growth (OD600nm range is 2 ~ 3); +++, vigorous growth (OD600nm range is greater than 3).

[0072] As can be seen from Table 2, compared with the commercial strain MTD / 1, the exopolysaccharide-producing Lactobacillus plantarum L75 grows in a wider pH range.

[0073] 2. Growth of Lactobacillus plantarum L75 and MTD / 1 under different salt concentrations

[0074] Activated plant lactobacillus L75 and MTD / 1 were inoculated into MRS liquid medium (pH 6.8) containing 3%, 6%, 9% and 12% NaCl by mass percentage, respectively, and cultured at 37°C for 24 h. The absorbance at 600 nm (OD600) was measured with a UV-visible spectrophotometer to observe the growth of plant lactobacillus L75 and MTD / 1 under different salt concentrations. The results are shown in Table 2.

[0075] As can be seen from Table 2, compared with the commercial strain MTD / 1, the exopolysaccharide-producing Lactobacillus plantarum L75 grew better under the same salt concentration.

[0076] 3. Growth and acid production of Lactobacillus plantarum L75 and MTD / 1 at different temperatures

[0077] The activated Lactobacillus plantarum L75 and MTD / 1 were inoculated into MRS liquid culture medium (pH 6.8) respectively, and cultured at 25°C for 36 h and 15°C for 132 h. The absorbance at 600 nm (OD600) was measured by UV-visible spectrophotometer to observe the growth and acid production of Lactobacillus plantarum L75 and MTD / 1 at different temperatures. The results are as follows: Figure 1A to Figure 1D shown.

[0078] Figure 1A The growth comparison of Lactobacillus plantarum L75 and MTD / 1 provided in the embodiments of the present disclosure at 25°C is shown; Figure 1B The growth comparison of Lactobacillus plantarum L75 and MTD / 1 provided in the embodiments of the present disclosure at 15°C is shown; Figure 1C The figure shows a comparison of the acid production of Lactobacillus plantarum L75 and MTD / 1 at 25°C provided in the embodiments of the present disclosure; Figure 1D The figure shows a comparison of the acid production of Lactobacillus plantarum L75 and MTD / 1 at 15°C provided in the embodiments of the present disclosure. Figure 1A to Figure 1D It can be seen that the growth performance and acid production capacity of L75 are better than those of MTD / 1 at both 25°C and 15°C.

[0079] It should be noted that the present disclosure has no particular limitation on the culture method of Lactobacillus plantarum and Saccharomyces cerevisiae that produce extracellular polysaccharides. Unless otherwise specified, the conventional culture method of Lactobacillus plantarum and Saccharomyces cerevisiae in the art can be used.

[0080] Example 3 Effect of exopolysaccharide-producing Lactobacillus plantarum L75 on the fermentation quality of oat silage

[0081] The silage raw material is oats in the filling stage (Baiyan No. 7). The manually harvested oats are wilted to 386.25g / kg FW under natural ventilation conditions and crushed into small pieces of 1 to 2 cm for later use.

[0082] In the Lactobacillus plantarum L75 treatment group (denoted by L), the addition amount of Lactobacillus plantarum L75 was 1×10 5 CFU / g FW, mixed evenly and vacuum packed into polyethylene bags, and stored for 60 days at room temperature and the temperature during the harvest period of the Qinghai-Tibet Plateau.

[0083] The brewer's yeast treatment group (denoted by S) was added at a rate of 1×10 5 CFU / gFW, mixed evenly and packed into polyethylene bags, and stored for 60 days at room temperature and the temperature during the harvest period of the Qinghai-Tibet Plateau.

[0084] The Lactobacillus plantarum L75 and Saccharomyces cerevisiae treatment group (expressed as LS) had an addition amount of 1×10 5 CFU / g FW, the amount of Saccharomyces cerevisiae added was 1×10 5 CFU / g FW, mixed evenly and packed into polyethylene bags, and stored for 60 days at room temperature and the temperature during the harvest period of the Qinghai-Tibet Plateau. Among them, brewer's yeast was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.

[0085] The control group (denoted by C) differs from the group treated with Lactobacillus plantarum L75 in that Lactobacillus plantarum L75 is not added.

[0086] On the 3rd, 14th and 60th day of fermentation, 20 grams of forage samples were mixed with 180 mL of sterile water, homogenized by a high-speed juicer for half a minute, and then filtered through four layers of cheesecloth. The above filtrate was used to evaluate the fermentation quality of silage. The pH value of the filtrate was measured with a pH meter with a glass electrode (PHSJ-3F, CANY, Shanghai, China), and the content of lactic acid, acetic acid and propionic acid was determined by high performance liquid chromatography. Among them, the conditions of high performance liquid chromatography are: the chromatographic column is Carbomix H-NP10: 8% column, Sepax Technologies, Inc.; column temperature: 55°C; eluent: 2.5 mM H2SO4; flow rate: 0.6 mL / min; detector: differential refractive index detector RID.

[0087] The measurement results are as follows Figure 3 shown. Figure 3 middle,1 C, control group; L, group treated with Lactobacillus plantarum L75 producing extracellular polysaccharide; S, group treated with Saccharomyces cerevisiae; LS, group treated with a mixture of Lactobacillus plantarum and Saccharomyces cerevisiae; DM, dry matter. 2 SEM, standard error of the mean. 3 I, inoculant; T, temperature; D, fermentation days; I×T, interaction between inoculant and temperature; I×D, interaction between inoculant and fermentation days; T×D, interaction between temperature and fermentation days; I×T×D, interaction between inoculant, temperature and fermentation days. Lowercase ac indicates significant analysis between different inoculant treatment groups. Uppercase AB indicates significant analysis between the same treatments at different temperatures.

[0088] Depend on Figure 3 It can be seen that the pH value of the LS mixed inoculant treatment group decreased significantly as early as the third day of ensiling, and the lactic acid concentration increased significantly (P<0.05). At 7-60 days of ensiling, the pH of the L inoculated group was the lowest, the lactic acid content was the highest, and the LA (lactic acid) / AA (acetic acid) ratio was the highest (P<0.05). Whether under the temperature simulated during the harvest period of the Qinghai-Tibet Plateau or under normal temperature fermentation conditions, the exopolysaccharide-producing Lactobacillus plantarum L75 can effectively improve the fermentation quality of oat silage.

[0089] Example 4 Effect of exopolysaccharide-producing Lactobacillus plantarum L75 on the antioxidant activity of oat silage The silage method and treatment method are the same as those in Example 3 and will not be described in detail.

[0090] The total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) activities in the 60-day filtrate in Example 3 were determined using an antioxidant index determination kit (purchased from Nanjing Jiancheng Bioengineering Institute, China). The results are shown in Table 3.

[0091] Table 3 Antioxidant activity of oat silage stored for 60 days

[0092]

[0093] Note: 1 T-AOC, total antioxidant capacity; SOD, superoxide dismutase; CAT, catalase.

[0094] 2 C, control group; L, group treated with Lactobacillus plantarum L75 producing extracellular polysaccharide; S, group treated with Saccharomyces cerevisiae; LS, group treated with a mixture of Lactobacillus plantarum and Saccharomyces cerevisiae; DM, dry matter.

[0095] 3 SEM, standard error of the mean.

[0096] 4 I, inoculant; T, temperature; D, fermentation days; I×T, interaction between inoculant and temperature; I×D, interaction between inoculant and fermentation days; T×D, interaction between temperature and fermentation days; I×T×D, interaction between inoculant, temperature and fermentation days. Lowercase ac indicates significant analysis between different inoculant treatment groups. Uppercase AB indicates significant analysis between the same treatments at different temperatures.

[0097] As can be seen from Table 3, under the temperature simulated during the harvest period of the Qinghai-Tibet Plateau, compared with the control group and the L inoculant treatment group, the mixed inoculation of Lactobacillus plantarum L75 and Saccharomyces cerevisiae significantly increased the T-AOC and SOD activity of oat silage.

[0098] Example 5 Effect of exopolysaccharide-producing Lactobacillus plantarum L75 on the microbial community of oat silage The silage method and treatment method are the same as those in Example 3 and will not be repeated here.

[0099] Microbial community analysis was performed on silage samples fermented for 3, 14 and 60 days. The specific steps are as follows: Mix 15g of forage with 70mL of sterile saline, shake on a shaker at 120rpm / min for 20 minutes, and then pass through four layers of sterile gauze. Centrifuge the filtrate obtained above at 10000rpm / min for 6 minutes to collect microbial cells. According to the instructions of the DNA extraction kit (MPBiomedicals, Solon, USA), DNA was extracted from the microbial cells collected above. The concentration and purity of the extracted DNA were determined using a NanoDrop2000 UV-visible spectrophotometer. PCR amplification, amplicon sequencing and sequencing data analysis were completed by Shanghai Meiji Biomedical Technology Co., Ltd.

[0100] The bacterial specific primers used in this experiment are SEQ ID NO. 2 and SEQ ID NO. 3. The fungal specific primers are ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3' - SEQ ID NO. 4) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3' - SEQ ID NO. 5).

[0101] The microbial data were analyzed using the free online Majorbio cloud platform (www.Majorbio.com). Figure 4 shown.

[0102] from Figure 4It can be seen that the main dominant bacteria before ensilage were Pantoea agglomerans, Weissella confusa and a certain proportion of Curtobacterium. After ensilage, the abundance of Lactiplantibacillus plantarum in the L or LS inoculation group increased rapidly. Whether under the temperature simulated during the harvest period of the Qinghai-Tibet Plateau or under room temperature fermentation conditions, LS mixed inoculation significantly promoted the growth of lactic acid bacteria on the third day of ensilage, and its proportion accounted for more than 65% of the total bacteria. At 14 days of ensilage, Lactobacillus was the main genera in the L and LS treatment groups; while the dominant genera in the control group and the Saccharomyces cerevisiae treatment group were Weissella and Leuconostoc. At 60 days of ensilage, the fungal community composition of the control group and the L treatment group was completely different from that of the yeast treatment group and the LS mixed treatment group. For example, we observed a certain proportion of the mold Penicillium cameum (black arrow) in the control group and the L treatment group. This bacterium is a major mycotoxin producer, and its presence poses a great threat to oat silage and even animal health.

[0103] Using the LS mixed treatment group, there is no need to add additional harmful bacteria inhibitors or antioxidant feed additives before oat silage during the harvest period on the Qinghai-Tibet Plateau, which saves costs. At the same time, the fermentation quality is significantly higher than that of the control group, which is helpful for preparing high-quality oat silage with probiotic effects.

[0104] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0105] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A silage additive, characterized in that: The active ingredients of the silage additive include Lactobacillus plantarum ( Lactiplantibacillus plantarum ) L75 and Saccharomyces cerevisiae; among them, The Lactobacillus plantarum L75 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No. 29678. Based on the mass of the silage raw material, the addition amount of the plant lactobacillus L75 and the brewer's yeast is 0.5×10 5 ~9.0×10 5 CFU / g FW.

2. A silage, characterized in that: The silage comprises the silage additive according to claim 1.

3. Use of the silage additive according to claim 1 in preparing silage.

4. The method for preparing silage according to claim 2, characterized in that: include: The silage raw material and the silage feed additive are mixed, solid fermentation is carried out, and the fermentation product is collected to obtain the silage feed.

5. The preparation method according to claim 4, characterized in that: The silage raw material includes oats.

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