Lactobacillus plantarum and application thereof in forage grass silage

Abstract

The invention discloses lactobacillus plantarum and application thereof in forage grass silage. The lactobacillus plantarum disclosed by the invention is lactobacillus plantarum 6H2, and the preservation number of the lactobacillus plantarum is CGMCC (China General Microbiological Culture Collection Center) No.31240 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms). The lactobacillus plantarum 6H2 shows more excellent fermentation quality and nutritional ingredient optimization effect in the oat and vicia sativa mixed silage at low temperature, and can also improve the quality of the oat silage. The method has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biological agriculture, and relates to a plant lactobacillus and application thereof in forage silage. Background Art

[0002] Silage is a convenient, simple and economical processing and storage method. It is less affected by the climate and environment, and the produced forage has good palatability. At the same time, the nutritional components of the forage are retained as much as possible. Therefore, it is suitable to promote silage processing technology in areas with high altitudes, rainy harvest seasons and low temperatures. Silage refers to high-quality feed with long-term storage and good palatability made by cutting green feed into pieces and placing it in a sealed environment through microbial fermentation. Aromatic acid and good palatability are the characteristics of high-quality silage. During the silage fermentation process, the microorganisms attached to the surface of the forage will consume the soluble sugar in the anaerobic environment to produce organic acids such as lactic acid and acetic acid. These organic acids cause the pH value of the silage to decrease, thereby effectively inhibiting the growth and reproduction of undesirable bacteria such as Escherichia coli, Clostridium and mold, so that the forage can be stored for a long time. However, when there are fewer lactic acid bacteria attached to the surface of the forage, it is difficult for the silage to enter the fermentation process quickly, and additives are often added to increase the fermentation speed.

[0003] Silage additives include microbial preparations, nutritional additives, enzyme preparations and chemical preparations. Commonly used microbial preparations are mainly lactic acid bacteria. Lactic acid bacteria additives can provide a large amount of lactic acid in the early stage of silage fermentation, promote the production of organic acids, quickly reduce pH, inhibit the reproduction of various harmful bacteria such as spoilage bacteria, and reduce the nutritional loss of silage. Nutritional additives can improve the quality of silage by adjusting the nutritional components in silage, reducing the degradation of crude protein, and limiting the activity of harmful microorganisms. Cellulase additives, as a commonly used enzyme preparation, can degrade plant cell walls, convert cellulose into soluble sugars, provide fermentation substrates for lactic acid bacteria, promote the production of organic acids, accelerate the fermentation process, and inhibit the growth of harmful bacteria. Formic acid in chemical additives is used to maintain the protein content in silage, while sorbic acid, benzoic acid, propionic acid and acetic acid are used to enhance the aerobic stability of feed and ensure the quality and storage effect of feed. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to perform silage of forage at low temperature.

[0005] The invention firstly provides a Lactobacillus plantarum 6H2, whose preservation number in the General Microbiological Center of China Microorganism Culture Collection Administration Committee is CGMCC No.31240.

[0006] The invention also provides a bacterial agent, the active ingredient of which is the Lactobacillus plantarum 6H2.

[0007] The bacterial agent can be used for silage of forage.

[0008] In the above-mentioned bacterial agent, the forage grass may be grass grass and / or leguminous grass.

[0009] In the above-mentioned bacterial agent, the bacterial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane. In the bacterial agent, the active ingredient may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the composition may be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0010] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0011] The present invention also provides any of the following applications of the Lactobacillus plantarum 6H2 or the bacterial agent:

[0012] X1. Application in forage silage;

[0013] X2. Application in the preparation of forage silage products;

[0014] X3. Application in rapidly increasing the lactic acid and / or acetic acid content of forage silage;

[0015] X4. Application in the preparation of a product for rapidly increasing the lactic acid and / or acetic acid content of forage silage;

[0016] X5. Application in reducing pH of forage silage;

[0017] X6. Application in the preparation of products for lowering the pH of forage silage;

[0018] X7. Application in increasing the dry matter content and / or crude protein content of forage silage;

[0019] X8. Application in the preparation of products for increasing the dry matter content and / or crude protein content of forage silage;

[0020] X9. Application in improving the acid protease activity and / or acid xylanase activity of forage silage;

[0021] X10. Application in the preparation of products for improving the acid protease activity and / or acid xylanase activity of forage silage.

[0022] In the above application, the forage grass may be grass grass and / or leguminous grass.

[0023] The present invention also provides a method for silaging forage grass, which comprises: adding the Lactobacillus plantarum 6H2 to the forage grass for silaging, thereby completing the silaging of the forage grass.

[0024] In the above method, the amount of Lactobacillus plantarum 6H2 added can be 1×10 6 cfu / g forage.

[0025] In the above method, the ensiling can be carried out at a temperature of -6°C to 18°C ​​(such as -6°C to 15°C or 1°C to 12°C).

[0026] And / or, the ensiling time may be 7-90 days (such as 14 days, 30 days, 60 days, etc.).

[0027] In the above method, the forage grass may be grass grass and / or leguminous grass.

[0028] The present invention separates a low-temperature resistant Lactobacillus plantarum 6H2. When the strain is used to ensilage oats and peas at low temperature, the lactic acid and acetic acid contents can be increased, which helps to reduce dry matter loss and improve the nutritional value of feed. The strain can also decompose protein in the silage through deamination under low temperature conditions, provide microorganisms with nutrients required for growth and reproduction, and promote the fermentation of silage. When Lactobacillus plantarum 6H2 is used to ensilage oats at low temperature, it is found that Lactobacillus plantarum 6H2 can significantly increase the lactic acid content, quickly reduce pH, consume crude protein content, and have the lowest soluble sugar content. In summary, lactic acid bacteria 6H2 improves the nutritional components, fermentation quality and enzyme activity of mixed silage of oats and saguaro peas to a certain extent, and low-temperature-resistant lactic acid bacteria Lactobacillus plantarum 6H2 (Lactobacillus plantarum 6H2) shows more superior fermentation quality and nutritional component optimization effect in mixed silage of oats and saguaro peas at low temperatures. When oats are silaged, the quality of oats silage can be improved by adding Lactobacillus plantarum 6H2 (Lactobacillus plantarum 6H2). The present invention has a good application prospect.

[0029] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0030] Description of biological material deposit

[0031] Abbreviation of depository unit: CGMCC

[0032] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0033] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0034] Deposit date: July 9, 2024

[0035] CGMCC Registration Number: CGMCC No.31240

[0036] Classification name: Lactobacillus plantarum

[0037] Strain ID: 6H2 BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Effects of additives on dry matter of oat silage.

[0039] Figure 2 The effect of additives on soluble sugar in silage oats.

[0040] Figure 3 The effect of additives on crude protein in oat silage.

[0041] Figure 4 Effect of additives on crude fat content of oat silage.

[0042] Figure 5 Effects of additives on neutral detergent fiber of oat silage.

[0043] Figure 6 Effects of additives on acid detergent fiber of oat silage.

[0044] Figure 7 Effects of additives on crude ash content of oat silage.

[0045] Figure 8 Effects of additives on pH of oat silage fermentation.

[0046] Fig. 9 Effects of additives on lactic acid during oat silage fermentation.

[0047] Fig.10 Effects of additives on acetic acid during oat silage fermentation.

[0048] Fig.11 Effects of additives on propionic acid during oat silage fermentation.

[0049] Fig.12 Effects of additives on ammonia nitrogen during oat silage fermentation.

[0050] Figure 1-Figure 12 In the figure, different lowercase letters indicate significant differences at the 0.05 level (P < 0.05), CK represents the control treatment, H represents the treatment with the addition of Lactobacillus plantarum 6H2, X represents the treatment with the addition of cellulase, F represents the treatment with the addition of the compound bacterial agent Zhuanglemei, H+X represents the treatment with the addition of Lactobacillus plantarum 6H2 + cellulase, and F+X represents the treatment with the addition of the compound bacterial agent Zhuanglemei + cellulase.

[0051] Fig.13 Characteristics of microbial communities at the phylum level.

[0052] Fig.14 Characteristics of microbial communities at the genus level.

[0053] Fig.15Correlation between microbial communities and nutritional quality.

[0054] Fig.16 The correlation between microbial community and fermentation quality.

[0055] Figure 15-16 In the table, Bac represents Firmicutes; Cya represents Blue Photosynthetic Bacteria; Pse represents Proteobacteria; Myc represents Mycoplasma; Act represents Actinobacteria; The represents Thermodesulfurobacteria; and Bact represents Bacteroidetes. DETAILED DESCRIPTION

[0056] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are all repeated at least three times, and the results are averaged.

[0057] Example 1. Isolation and identification of strain 6H2

[0058] 1. Isolation and identification of strain 6H2

[0059] The inventor isolated strain 6H2 from forage silage obtained by mixing silage of peas and triticale (1:1), and the strain has good low temperature resistance and growth ability. The 16SrDNA sequence number of strain 6H2 is as follows:

[0060]

[0061] Physiological and biochemical identification and 16SrDNA homology sequencing analysis of strain 6H2 showed that strain 6H2 belongs to Lactobacillus plantarum, hereinafter referred to as Lactobacillus plantarum 6H2. Lactobacillus plantarum 6H2 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on July 9, 2024, with the deposit number: CGMCC No.31240.

[0062] II. Identification of the low temperature tolerance of strain 6H2

[0063] The test strain was Lactobacillus plantarum 6H2; the control group was a commercial strain Lactobacillus plantarum 21804, which was from the China Industrial Microbiological Culture Collection Center and the commercial strain number was CICC 21804.

[0064] Experimental design: The test strain was activated twice in liquid culture medium, inoculated in MRS liquid culture medium with a relative volume inoculation of 2% and cultured for 24 h at a temperature gradient of 5°C, 15°C, 25°C and 35°C, and the cell membrane integrity, cell membrane fatty acid composition and cell membrane fatty acid content of the strain were determined.

[0065] Measurement content and methods:

[0066] β-galactosidase is an intracellular enzyme of microorganisms. When the cell membrane is damaged, β-galactosidase diffuses through the cell membrane into the extracellular environment. The activity of extracellular β-galactosidase reflects the integrity of the cell membrane. The higher the activity of extracellular β-galactosidase, the worse the integrity of the cell membrane. Take the bacterial solution of the test strain after culturing for 24 hours under 4 temperature gradients. Treat 3 replicates in each group at 4℃, 4000r·min -1 Centrifuge for 8 min to take 0.2 g of wet bacteria (the number of viable bacteria is 1×10 5 CFU·g -1 ) was reconstituted with an equal volume of physiological saline, and the supernatant was extracted after centrifugation for 8 min under the above centrifugation conditions. The β-galactosidase activity was determined using a β-galactosidase assay kit (β-GAL, Beijing Solebow Technology Co., Ltd.). Each treated sample was assayed according to the instructions of the β-galactosidase assay kit, and the β-galactosidase activity was calculated after drawing a standard curve.

[0067] Take the test strain cultured at 4 temperature gradients for 24 hours and heat it at 4℃ and 8000r·min. -1 , centrifuged under the condition of 8min and washed 3 times with physiological saline before collecting the cells. According to the Bligh & Dyer method, the cell membrane fatty acids of the cells were extracted, and an equal volume of physiological saline was added to the re-dissolved bacterial solution, and the solution was broken in an ultrasonic disruptor. After breaking for 10min, 0.3g of sodium hydroxide and 1ml of methanol solution (V (methanol): V (deionized water) = 1:1) were added and heated in a 100℃ water bath for saponification. After 30min, the solution was taken out and quickly shaken and cooled. After cooling, 2ml of a mixed solution of hydrochloric acid and methanol (V (hydrochloric acid): V (methanol) = 1:1.2) was added and heated in a 80℃ water bath for methylation. After 0min, take it out and shake it quickly to cool it. After cooling, add 2ml of n-hexane and methyl butyl tert-ether mixed solution (V (n-hexane): V (methyl butyl tert-ether) = 1:1) and shake it slowly for 10min for extraction. After shaking and mixing, let it stand until the liquid is clearly separated. After extracting the upper liquid, use a nitrogen blower to blow dry to obtain fatty acids. Add 3ml of sodium hydroxide solution (solution concentration is 1.2mol / L) to the sample and shake it slowly until the liquid is clearly separated. Take the upper liquid phase and pass it through an organic filter membrane and save it in a gas phase injection bottle for GC / MS analysis. The sample GC / MS detection was completed by Shanghai Meiji Biotechnology Co., Ltd.

[0068] Data statistics and analysis: Excel 2017 was used to organize the data, IBM SPSS Statistics 26 software was used to perform data difference analysis, and Origin 2022 was used to draw the bar chart.

[0069] Results and analysis:

[0070] Integrity of lactic acid bacteria cell membrane at different temperatures: β-galactosidase is an essential enzyme for organisms to degrade lactose. It is found in microorganisms, animals and plants and is an endogenous enzyme of lactic acid bacteria. β-galactosidase, as a macromolecular protein, will only cross the cell membrane and enter the extracellular environment when the cell membrane is damaged. The activity of β-galactosidase can be used as a detection indicator of the integrity of microbial cell membranes. Under the temperature condition of 5°C, the extracellular β-galactosidase activity of commercial strain D (enzyme activity is 0.90U / g) is significantly higher than that of Lactobacillus plantarum 6H2 (enzyme activity is 0.48U / g) (P<0.05), indicating that the cell membrane integrity of commercial strain D is poor and the cell membrane is seriously damaged. At 15°C, the extracellular β-galactosidase activity of commercial strain D was 0.65U / g, which was significantly higher than that of Lactobacillus plantarum 6H2 (enzyme activity was 0.33U / g) (P<0.05), and the cell membrane integrity was lower than that of Lactobacillus plantarum 6H2. At 25°C, the extracellular β-galactosidase activity of commercial strain D was 0.47U / g, which was significantly higher than that of Lactobacillus plantarum 6H2 (enzyme activity was 0.28U / g) (P<0.05), and the cell membrane integrity was lower than that of Lactobacillus plantarum 6H2. At 35°C, the extracellular β-galactosidase activity of commercial strain D was 0.45 U / g, which was significantly higher than that of Lactobacillus plantarum 6H2 (enzyme activity was 0.24 U / g) (P<0.05), and the cell membrane integrity was lower than that of Lactobacillus plantarum 6H2. This indicates that Lactobacillus plantarum 6H2 has a higher cold tolerance than commercial strain D.

[0071] Fatty acid composition of lactic acid bacteria cell membrane at different temperatures: The saturated fatty acid content of lactic acid bacteria at different temperatures is shown in Table 1. The types of saturated fatty acids in the cell membrane of Lactobacillus plantarum 6H2 and commercial strain D are mainly myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0). The myristic acid content in the cell membrane of Lactobacillus plantarum 6H2 and commercial strain D increased with decreasing temperature; the palmitic acid content and stearic acid content in the cell membrane of Lactobacillus plantarum 6H2 and commercial strain D showed a decreasing trend with decreasing temperature.

[0072] Table 1. Saturated fatty acid content of lactic acid bacteria cell membrane at different temperatures

[0073]

[0074] Note: ND, not detected.

[0075] The contents of unsaturated fatty acids in the cell membrane of lactic acid bacteria at different temperatures are shown in Table 2. The types of unsaturated fatty acids in the cell membrane of each strain are mainly trans-palmitoleic acid (C16:1T), palmitoleic acid (C16:1), elaidic acid (C18:1n9t), oleic acid (C18:1n9c) and vaccenic acid (C18:1n11c). The contents of trans-palmitoleic acid, palmitic acid and vaccenic acid in the cell membrane of Lactobacillus plantarum 6H2 increased with decreasing temperature, while the contents of elaidic acid and oleic acid decreased with decreasing temperature.

[0076] Table 2. Unsaturated fatty acid content of lactic acid bacteria cell membrane at different temperatures

[0077]

[0078] The relative content of fatty acids in the cell membrane of lactic acid bacteria at different temperatures is shown in Table 3. As the temperature decreases, the saturated fatty acid content in the membrane of strain D decreases, the unsaturated fatty acid content increases, and the U / S ratio increases relatively. The saturated fatty acid content in the cell membrane of strain Lactobacillus plantarum 6H2 increases as the temperature decreases, and the unsaturated fatty acid content decreases as the temperature decreases. The U / S ratio of strain D under 5°C is 1.0993, which is 23.50% higher than that of the same strain under 35°C. The U / S ratio of Lactobacillus plantarum 6H2 under 5°C is 0.9205, which is 3.5% lower than that of the same strain under 35°C. U / S is UFA / SFA, i.e. unsaturated fatty acids / saturated fatty acids.

[0079] Table 3. Relative content of fatty acids in lactic acid bacteria cell membrane at different temperatures

[0080]

[0081] The cell membrane is an important structure for the intracellular environment to resist external environmental stress. Chai Jikuan and other studies believe that microorganisms can resist low temperature stress by regulating their own cell membrane fatty acid composition. In the above experiment, the content of saturated fatty acids C16:0 and C18:0 in Lactobacillus plantarum 6H2 and commercial strain D gradually decreased with the decrease of temperature, and the content of unsaturated fatty acids C18:1 and C18:2 increased, indicating that the lactic acid bacteria strains may change the cell membrane components through self-regulation under low temperature conditions to cope with the low temperature stress environment. Chen Chengming and other studies have found that the increase in unsaturated fatty acid content is conducive to maintaining cell membrane fluidity and thus maintaining the activity of microbial cells under low temperature stress. In the above experiment, under low temperature stress conditions, the C16:0 content in the cell membrane of Lactobacillus plantarum 6H2 decreased, and the C16:1T content and C16:1 content increased. The fatty acid composition and fatty acid content in the microbial cell membrane under low temperature conditions changed, which helped to enhance the cell membrane fluidity and was conducive to the maintenance of a relatively ideal growth ability of microorganisms under low temperature stress.

[0082] Example 2: Application of Lactobacillus plantarum 6H2 in oat and pea silage

[0083] 1. Materials and Methods

[0084] 1. Overview of the test site

[0085] The experimental site is located in Xiangla Administrative Village, Kagaman Township, Hezuo City, Gansu Province, and Xinruifeng Plateau Cattle and Sheep Breeding and Planting Technology Development Farmers' Professional Cooperative. The experimental site is located in the north of Hezuo City, with high terrain in the northeast and low terrain in the southwest. The average altitude is 2737m, the average annual temperature is 3.0℃, the growth period is from April to August, the average temperature reaches 10.4℃, the precipitation is 282.8mm, and the sunshine hours are 878.6h.

[0086] 2. Test Materials

[0087] The tested forage varieties were Avena sativa cv. Longyan No. 3 for oats and Lathyrus sativa cv. Ximu 333 for peas. The main chemical components and enzyme activities of fresh forage are shown in Table 4. There were two lactic acid bacteria additives, both of which were Lactobacillus plantarum, with strain number CICC21804 (referred to as CI) with strong acid production ability, from the China Industrial Microbiological Culture Collection Management Center. Low temperature resistant Lactobacillus plantarum 6H2 (referred to as 6H2).

[0088] Table 4. Nutritional composition and enzyme activity of oats and pea mixture before ensiling

[0089] project content Dry matter DM (%) 37.43 Crude protein CP (%) 13.85 Neutral detergent fiber NDF (%) 50.09 Acid detergent fiber ADF (%) 30.40 Soluble carbohydrates WSC (% DM) 13.00 Ash(%) 6.72 Crude fat EE (%) 2.76 Acid xylanase ACX(u / g) 14.17 Acid protease ACP (u / g) 10.36

[0090] 3. Experimental Design

[0091] Oats and peas were sown by broadcasting at a sowing rate of 255 kg / hm2. 2 and 60kg / hm 2 By the end of August of the same year, when oats were in the grain filling stage and peas were in the flowering stage, they were mowed evenly with a disc mower (the mass ratio of oats to peas was 4.25:1), and turned over and dried under natural conditions. The moisture content of the forage was monitored with a moisture meter. When the moisture content reached 65% to 70%, the forage was cut into small pieces of about 1 cm by a shredder, and CI strain suspension (obtained by culturing CICC21804) and 6H2 strain suspension (obtained by culturing Lactobacillus plantarum 6H2) were added respectively, with the addition amount of 1.0×10 6 cfu / g forage, CK means no strain added. Mix evenly and put into 30cm×40cm polyethylene vacuum bags, 500g per bag, evacuate with a vacuum machine and seal. Place in a local hay shed and ferment at natural temperature (-6℃~18℃) for 7d, 14d, 45d and 90d. On each silage day, 3 bags were randomly selected for each treatment. Samples were taken from different positions of each vacuum bag to 300g. After mixing evenly, they were stored at low temperature and brought back to the laboratory to test the fermentation quality and nutritional quality of the silage.

[0092] 4. Determination indicators and methods

[0093] 4.1 Fermentation quality

[0094] Take 20g of silage, add 180mL of distilled water and mix, seal and extract in a refrigerator at 4℃ for 24h, filter with 4 layers of gauze, and use the filtrate to determine the fermentation quality. Take a part of the filtrate to measure the pH value with a pH meter, and use the phenol-sodium hypochlorite colorimetric method to determine the ammonia nitrogen (AN) content; after filtering the filtrate through a 0.22 filter membrane, place it in an injection bottle, and use ultra-high performance liquid chromatography to determine the lactic acid (LA), acetic acid (AA), propionic acid (PA) and butyric acid (BA) content.

[0095] 4.2 Nutritional quality

[0096] The silage was placed in an oven at 65°C and dried to constant weight. The dried sample was crushed and passed through a 0.45 mm sieve to determine its nutritional components. Dry matter (DM) was determined by conventional drying method. Crude protein (CP) was determined by Kjeldahl nitrogen determination; neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using Van der Waals detergent fiber method; soluble carbohydrates (WSC) were determined by anthrone-sulfuric acid colorimetry; crude ash (Ash) was determined by low-temperature carbonization in an electric furnace and then burned in a muffle furnace. Crude fat (EE) was determined by Soxhlet extraction.

[0097] 4.3 Enzyme activity

[0098] Acid xylanase (ACP) was determined using an ACP kit (Suzhou Keming Biotechnology Co., Ltd.), and acid protease (ACX) was determined using an ACX kit (Suzhou Keming Biotechnology Co., Ltd.).

[0099] 5. Data Processing and Analysis

[0100] Excel 2020 was used for preliminary data processing, and SPSS 20.0 software was used for univariate ANOVA model analysis, combined with the Duncan method for multiple comparisons (P<0.05); SPSS 17.0 software was used for multivariate generalized linear GLM model analysis, and the SNK method was used to analyze the significance of the interaction effect (P<0.01).

[0101] 2. Results and Analysis

[0102] 1. Effects of different lactic acid bacteria additives and silage time on fermentation parameters of oat and pea mixed silage

[0103] As shown in Table 5, lactic acid bacteria additives and silage time had extremely significant effects on pH value, ammonia nitrogen and organic acid content in the mixed silage process of oats and samphire peas (P<0.01), and the interaction between the two also reached an extremely significant level (P<0.01).

[0104] Table 5. ANOVA of the interaction between additives and silage time

[0105]

[0106] Note: * indicates that there are significant differences between different treatments of the same item at the 0.01 level (P<0.01).

[0107] As shown in Table 6, the pH value and ammonia nitrogen content of the 6H2 and CI groups were significantly lower than those of the CK group at each ensiling time point (P<0.05), and the lactic acid content of the 6H2 and CI groups was significantly higher than that of the CK group at each ensiling time point (P<0.05). At 45d and 90d of ensiling, the pH value of the 6H2 group was significantly lower than that of the CI group; the lactic acid and acetic acid contents of the 6H2 group were significantly higher than those of the CI group (P<0.05). Propionic acid was only detected at 14d of ensiling. Li Haiping's research shows that lactic acid bacteria with freeze-thaw resistance can effectively reduce the pH value of oat silage, increase the production of lactic acid and acetic acid, and have a positive effect on the preservation of silage nutrition and the extension of the shelf life of silage in the Qinghai-Tibet Plateau. In the above experiment, the acetic acid and lactic acid contents of the CI and 6H2 groups were higher than those of the CK group at each ensiling time point, but the acetic acid and lactic acid contents of the 6H2 group were higher than those of the CI group in the late ensiling period.

[0108] Li Haiping's research shows that lactic acid bacteria with freeze-thaw resistance can effectively reduce the pH value of oat silage, increase the production of lactic acid and acetic acid, and have a positive effect on the preservation of silage nutrition and the extension of silage shelf life in the Qinghai-Tibet Plateau. The acetic acid and lactic acid content of CI and 6H2 groups of the present invention are higher than those of CK group at each silage time point, and the acetic acid and lactic acid content of 6H2 group is higher than that of CI in the late silage period, which is helpful for the preservation of silage nutrition and the extension of silage shelf life.

[0109] Table 6 Effects of additives and silage time on fermentation indexes of mixed silage of oats and peas

[0110]

[0111] Note: Different capital letters indicate that the measured items were significantly different among different additive treatments at the same silage days (P<0.05); Different lowercase letters indicate that the measured items were significantly different among different silage days at the same additive treatment (P<0.05).

[0112] Note: Butyric acid (BA) was not detected in any treatment and therefore not listed.

[0113] 2. Effects of different lactic acid bacteria additives and silage time on the nutritional indexes of oat and pea mixed silage

[0114] As shown in Table 7, lactic acid bacteria additives and silage time had extremely significant effects on various measured indicators during the mixed silage of oats and samphire peas (P<0.01), and the interaction between the two also reached an extremely significant level (P<0.01).

[0115] Table 7. ANOVA of the interaction between additives and silage time

[0116]

[0117] Note: * indicates that there are significant differences between different treatments of the same item at the 0.01 level (P<0.01).

[0118] As shown in Table 8, at 14d, 45d, and 90d of ensiling, the dry matter and crude protein contents of the CK group were significantly lower than those of the 6H2 and CI groups. The soluble carbohydrate and crude fat contents of the 6H2 and CI groups were significantly lower than those of the CK group at each ensiling time point (P<0.05). There was no significant difference in the dry matter and crude protein contents between the 6H2 and CI groups at each ensiling time (P>0.05). At 45d of ensiling, the crude ash content of the CI group was significantly lower than that of the CK and 6H2 groups (P<0.05); at 45d and 90d of ensiling, the soluble carbohydrate and crude fat contents of the 6H2 group were significantly lower than those of the CI group; the acid detergent fiber content of the CK group was significantly lower than that of the CI and 6H2 groups (P<0.05). At 90d of ensiling, the crude ash content of the 6H2 group was significantly lower than that of the CK and CI groups (P<0.05). The dry matter, crude protein and soluble carbohydrate contents of 90d silage were significantly lower than those of 7d and 14d silage (P<0.05); at 90d silage, the dry matter, crude protein and soluble carbohydrate contents of 6H2 group were significantly lower than those of 45d silage (P<0.05); the neutral detergent fiber and acid detergent fiber contents of 90d silage were significantly lower than those of 7d, 14d and 45d silage (P<0.05); while the changes in crude ash content were just the opposite, the crude ash content of 7d silage was significantly lower than that of 14d, 45d and 90d silage (P<0.05); the crude fat content of 45d silage was significantly higher than that of 7d, 14d and 90d silage.

[0119] In the above experiment, the dry matter content of CI and 6H2 groups at each ensiling time point was higher than that of CK group. As the ensiling time prolonged, the dry matter content of CK and CI groups gradually stabilized. At each ensiling time point, the crude protein content of CI and 6H2 groups was significantly higher than that of CK group. Crude fat is also an important indicator for measuring feed quality. At each ensiling time point, the crude fat content of CK group was significantly higher than that of CI and 6H2 groups. Li Feifei et al. found that adding Lactobacillus plantarum would reduce the crude fat content in silage; Huang Yuan et al. also found that with the increase in the amount of Lactobacillus plantarum added, the crude fat content in silage showed a downward trend, indicating that Lactobacillus plantarum can utilize crude fat. This is consistent with the trend of crude fat content changes in CI and 6H2 groups in the above experiment.

[0120] Table 8 Effects of additives and silage time on nutritional indexes of mixed silage of oats and peas

[0121]

[0122] Note: Different capital letters indicate that the measured items were significantly different among different additive treatments at the same silage days (P<0.05); Different lowercase letters indicate that the measured items were significantly different among different silage days at the same additive treatment (P<0.05).

[0123] 3. Effects of different lactic acid bacteria additives and silage time on enzyme activity of mixed oat and pea silage

[0124] As shown in Table 9, lactic acid bacteria additives and silage time had extremely significant effects on the activities of acid xylanase and acid protease in the mixed silage process of oats and sagittaria (P<0.01), and the interaction between the two also reached an extremely significant level (P<0.01).

[0125] Table 9. ANOVA of the interaction between additives and silage time

[0126]

[0127] Note: * indicates that there are significant differences between different treatments of the same item at the 0.01 level (P<0.01).

[0128] As shown in Table 10, at 14d, 45d, and 90d of ensiling, the acid xylanase activity of the CK and 6H2 groups was significantly lower than that of the CI group (P<0.05); at 14d of ensiling, the acid protease activity of the CK group was significantly lower than that of the 6H2 group (P<0.05); at 45d and 90d of ensiling, the acid xylanase activity of the CK and CI groups was significantly lower than that of the 6H2 group (P<0.05); at 90d of ensiling, the acid protease activity was the highest under the 6H2 treatment, and the acid xylanase activity was the highest under the CI treatment. The acid xylanase and acid protease activities at 90d of ensiling were significantly lower than those at 7d, 14d, and 45d of ensiling (P<0.05).

[0129] Table 10. Effects of additives and silage time on enzyme activity of mixed oat and pea silage

[0130]

[0131] Note: Different capital letters indicate that the measured items were significantly different among different additive treatments at the same silage days (P<0.05); Different lowercase letters indicate that the measured items were significantly different among different silage days at the same additive treatment (P<0.05).

[0132] Acid xylanase plays an important role in the degradation of hemicellulose in plants. It can degrade xylan into a mixture of oligoxylose and glucose. In the above experiment, the acid xylanase content in the CI and 6H2 treatment groups was significantly higher than that in the control group at each ensiling time point. Acid protease decomposes the protein in the raw material through the deamination action of yeast intracellular enzymes to produce small molecular peptides and amino acids. These components not only provide nutrition for the growth and reproduction of microorganisms, but also are precursors for the production of higher alcohols and organic acids. In the above experiment, the acid protease was still active after 90 days of ensiling. Acid protease has the highest activity under 6H2 treatment at any ensiling time.

[0133] The above experimental results show that lactic acid bacteria additives have improved the fermentation quality, nutritional content and enzyme activity of mixed oat and pea silage to a certain extent, and reduced the loss of dry matter and the generation of ammonia nitrogen during silage. From the perspective of indicators such as lactic acid, pH value and acid protease, Lactobacillus plantarum 6H2, which has a low temperature resistance effect, has a better effect.

[0134] Example 3: Application of Lactobacillus plantarum 6H2 in oat silage

[0135] 1. Materials and Methods

[0136] 1. Overview of the test site

[0137] Huajialing Township is located in the northwest of Tongwei County, Dingxi City, Gansu Province, China. It is located in the northwest inland Loess Plateau, 105°12' east longitude, 34°55' north latitude. It has a very strong continental temperate monsoon climate, which is characterized by cold and damp, strong winds, heavy rain and fog. The territory is crisscrossed with valleys and ridges. It belongs to the two yin and warm mountainous areas. The terrain is relatively flat, with an average altitude of more than 2200m, an average annual temperature of 3.4℃, an average annual sunshine of 2428.4h, an average annual growing season of 190d, a frost-free period of 80d, and an average annual rainfall of 451.1mm.

[0138] 2. Test materials

[0139] Test materials: 'Dingyan No. 2' oats (Avena sativa L.'Dingyan No. 2'), provided by the College of Grassland, Gansu Agricultural University.

[0140] Test strain: Lactobacillus plantarum 6H2 (Lactobacillus plantarum 6H2) (abbreviated as H).

[0141] Commercial composite bacteria Zhuanglemei type IV (abbreviated as F), a product of Sichuan Gaofuji Biotechnology Co., Ltd., the main ingredient is Lactobacillus plantarum 550 (≥2.0×10 10 CFU / g), Lactobacillus plantarum 360 (≥2.0×10 10 CFU / g), Enterococcus faecium (≥1.0×10 8 CFU / g), maltodextrin.

[0142] Cellulase: Beijing Coolable Technology Co., Ltd.

[0143] 3. Experimental Design

[0144] Oat fertilization method: Apply basal fertilizer (urea 74.96kg·hm -2 , diammonium 299.85kg·hm -2 ), urea 24.89 kg hm-1 was applied after rain at the jointing stage -2 , 50.07kg·hm2 urea was applied after rain at heading stage -2 . 3 replicates, plot area 111m 2 .

[0145] The oat straw was harvested at the milky stage, dried to a moisture content of about 65%, chopped with a kneading and tearing machine, mixed evenly, and packed into an aluminum foil vacuum bag. The bag was evacuated and sealed with a vacuum machine, and the silage agent Lactobacillus plantarum 6H2 (added at 1×10 6cfu / g forage), cellulase (added at 0.15 g / kg forage), commercial compound bacteria Zhuanglemei IV (added at 1×10 6 cfu / g forage), Lactobacillus plantarum 6H2 + cellulase (added amount is Lactobacillus plantarum 6H2 1×10 6 cfu / g forage, cellulase 0.15g / kg forage), commercial compound bacteria Zhuanglemei type IV + cellulase (the addition amount is commercial compound bacteria Zhuanglemei type IV 1×10 6 cfu / g forage, cellulase 0.15g / kg forage), and control (no silage agent added), which were recorded as H group, X group, F group, H+X group, F+X group, and CK group, with 6 treatments in total and 3 replicates for each treatment; after natural fermentation at 1℃-12℃ in Huajialing, Tongwei County for 0d, 7d, 14d, 30d, 60d, and 90d, the silage bags were opened for sampling.

[0146] Weigh 20g of silage from each bag in a clean bench, put it into sterile water and shake it for 30min. The supernatant after shaking is filtered through a 0.22μm filter membrane, and the filter membrane is stored in a -80℃ refrigerator for total DNA extraction. Take 20g of silage from the bag and put it into a conical flask, add deionized water for the determination of fermentation indicators, and place the remaining silage oats in a 105℃ oven, dry them, crush them with a grinder, pass them through a 22-mesh sieve, and determine the nutritional indicators.

[0147] 4. Measurement indicators and methods

[0148] 4.1 Determination of nutritional indexes of silage oats

[0149] The crude protein (CP), dry matter (DM) and crude ash (Ash) contents were determined according to the AOAC conventional method, the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined by the Van Soest detergent fiber determination method, and the soluble sugar (WSC) content was determined by the anthrone colorimetric method.

[0150] 4.2 Determination of fermentation indexes of oat silage

[0151] The pH value of the oat silage extract was directly determined using a pH meter; the ammonia nitrogen content was determined using the phenol-hypochlorite colorimetric method; and the content of organic acids (lactic acid, acetic acid, propionic acid, and butyric acid) was determined using liquid chromatography.

[0152] 4.3 Total DNA extraction from oat silage

[0153] The extraction of nucleic acid was successfully completed using the TGuide S96 magnetic bead soil / feces genomic DNA extraction kit, and the nucleic acid concentration was accurately detected using the synergy HTX microplate reader. Using a specific concentration of DNA as a template, PCR amplification was performed using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for the V3V4 region of bacterial 16Sr DNA. The PCR product was detected by electrophoresis technology, and the corresponding detection library was established accordingly.

[0154] After the library was strictly inspected by QseP-400 to ensure its quality, it was sequenced using the Illumina Novasek6000 sequencing platform. In order to ensure the accuracy and reliability of the data, the sequencing results were further processed using UCHIMEv4.2 software to remove the chimera sequences, and finally obtain effective and high-quality sequence data. These sequence data will provide a solid foundation for subsequent bacterial community structure analysis and functional research.

[0155] 5. Data Analysis

[0156] Data were collated using Excel 2016, data were analyzed using SPSS 20.00, and correlation heat maps were drawn using origin 2021. Fuzzy membership functions were used to comprehensively evaluate the silage treatments according to the method of Sun Donglei et al.

[0157] 2. Results and Analysis

[0158] 1. Effect of additives on the nutritional quality of oats

[0159] 1.1 Effect of additives on dry matter of oat silage

[0160] Effects of different additives on dry matter of oat silage Figure 1 As shown: the dry matter content decreased slowly throughout the ensilage process. The control treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment showed a significant decrease on the 7th day, while the Lactobacillus plantarum 6H2 treatment showed a significant decrease on the 14th day (P<0.05).

[0161] At 0 days of ensiling, there was no significant difference between the treatments; at the 7th day of ensiling, among the treatment groups, the group with added Lactobacillus plantarum 6H2 had a significantly higher effect than all other treatment groups. In contrast, the group with added cellulase was significantly inferior to the Lactobacillus plantarum 6H2 treatment group and the control treatment group. This result shows that Lactobacillus plantarum 6H2 has an advantage in improving relevant indicators or effects, while the treatment effect of cellulase is relatively weak (P<0.05); at the 14th day of ensiling, the effects of the control treatment and Lactobacillus plantarum 6H2 treatment were statistically significantly better than those of other treatment groups (P<0.05), which shows that these two treatments are outstanding in improving relevant indicators or effects. However, the effects of cellulase treatment and combined treatment of Zhuanglemei and cellulase were significantly lower than those of combined treatment of Lactobacillus plantarum 6H2 and cellulase (P<0.05), which showed the advantage of the synergistic effect of Lactobacillus plantarum 6H2 and cellulase in improving the treatment effect; on the 30th day of ensiling, the control treatment was significantly higher than other treatments (P<0.05), and the Lactobacillus plantarum 6H2 treatment was significantly higher than the cellulase treatment and Zhuanglemei + cellulase treatment; on the 60th day of ensiling, the control treatment was significantly higher than other treatments, and the Lactobacillus plantarum 6H2 treatment, Lactobacillus plantarum 6H2 + cellulase treatment, and Zhuanglemei + cellulase treatment were significantly higher than the cellulase treatment (P<0.05); on the 90th day of ensiling, the control treatment was significantly higher than other treatments (P<0.05).

[0162] After ensiling was completed, the Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were reduced by 1.03%, 2.12%, 1.66%, 1.11% and 1.06% respectively compared with before ensiling, and the Lactobacillus 6H2 treatment had the least decrease.

[0163] 1.2 Effect of additives on soluble sugar in oat silage

[0164] Effects of different additives on soluble sugar content of oat silage Figure 2 As shown: During the entire ensiling process, the soluble sugar content in each treatment showed a significant downward trend at 7 days.

[0165] On the 0th and 7th days, there were no significant differences among the treatments; on the 14th day, the Lactobacillus plantarum 6H2 treatment was significantly higher than the other treatment groups (P<0.05), and the cellulase treatment was significantly lower than the Lactobacillus plantarum 6H2+cellulase treatment (P<0.05); on the 30th and 60th days, the control treatment was significantly lower than the other additive treatments (P<0.05); on the 90th day of ensiling, the Lactobacillus plantarum 6H2 treatment was significantly higher than the other treatment groups (P<0.05), and the control treatment was significantly lower than the other treatments (P<0.05).

[0166] After ensiling was completed, the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were reduced by 6.91%, 5.17%, 5.75%, 5.70%, 5.69% and 5.84% respectively compared with before ensiling, and the Lactobacillus plantarum 6H2 treatment had the least decrease.

[0167] 1.3 Effect of additives on crude protein content of oat silage

[0168] Effects of different additives on crude protein content of oat silage Figure 3 As shown: the crude protein content showed a downward trend throughout the ensiling process. The control treatment group, Lactobacillus plantarum 6H2 treatment, Zhuanglemei treatment and Zhuanglemei + cellulase treatment showed a significant decrease on the 7th day of ensiling; the treatment with cellulase alone showed a significant decrease on the 14th day; the Lactobacillus plantarum 6H2 + cellulase treatment slowly decreased and became significant on the 60th day.

[0169] On the 0th day of ensiling, there was no significant difference among the treatments. On the 7th day of ensiling, the Lactobacillus plantarum 6H2 treatment was significantly higher than the control treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment, and the two bacterial enzyme mixed additive treatments were significantly lower than the other treatment groups (P<0.05). On the 14th day of ensiling, the Lactobacillus plantarum 6H2 treatment was significantly higher than the cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment. On the 30th, 60th and 90th days of ensiling, the Lactobacillus plantarum 6H2 treatment was significantly higher than the other treatments (P<0.05).

[0170] After ensiling was completed, the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were reduced by 2.67%, 0.93%, 3.25%, 2.61%, 3.29% and 3.53% respectively compared with before ensiling, and the Lactobacillus plantarum 6H2 treatment had the least decrease.

[0171] 1.4 Effect of additives on crude fat content of oat silage

[0172] Effects of different additives on crude fat content of oat silage Figure 4 As shown: the crude fat content showed an irregular upward trend during the whole silage process. However, only the treatment of Zhuanglemei + cellulase showed significant differences. The treatment of adding Zhuanglemei alone and the treatment of Lactobacillus plantarum 6H2 + cellulase showed a trend of first decreasing and then increasing. The control treatment, Lactobacillus plantarum 6H2 treatment, and cellulase treatment showed a significant increase on the 30th day.

[0173] There was no significant difference on the 0th day; on the 7th day of ensiling, the Lactobacillus plantarum 6H2 + cellulase treatment was significantly higher than the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, and Zhuanglemei treatment; on the 14th day of ensiling, there was no significant difference among the treatments; on the 30th day of ensiling, the Zhuanglemei treatment and Zhuanglemei + cellulase treatment were significantly lower than the Lactobacillus plantarum 6H2 treatment and cellulase treatment (P<0.05); on the 60th day of ensiling, the Lactobacillus plantarum 6H2 treatment was significantly higher than the other treatment groups (P<0.05); on the 90th day of ensiling, the Lactobacillus plantarum 6H2 + cellulase treatment was significantly higher than the Lactobacillus plantarum 6H2 treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment, and Zhuanglemei + cellulase treatment (P<0.05).

[0174] After ensiling was completed, the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were increased by 0.69%, 0.64%, 0.91%, 0.68%, 1.08% and 0.62% respectively compared with before ensiling.

[0175] 1.5 Effect of additives on the neutral detergent fiber content of oat silage

[0176] Effects of different additives on the neutral detergent fiber content of oat silage Figure 5 As shown in the figure, the neutral detergent fiber content gradually decreased during the whole silage process. The neutral detergent fiber content of cellulase treatment decreased significantly on the 7th day, Lactobacillus plantarum 6H2 treatment decreased significantly on the 14th day, the control treatment group decreased significantly on the 30th day, and the treatment of single addition of Zhuanglemei, Lactobacillus plantarum 6H2 + cellulase treatment group and Zhuanglemei + cellulase treatment decreased significantly on the 60th day.

[0177] There were no significant differences among the treatments on the 0th and 7th days of ensiling; on the 14th day of ensiling, the Lactobacillus plantarum 6H2 treatment was significantly lower than the control treatment and the Zhuanglemei + cellulase treatment (P<0.05); on the 30th and 60th days of ensiling, the control treatment was significantly lower than the other treatments (P<0.05); on the 90th day of ensiling, the cellulase treatment was significantly lower than the control treatment, Lactobacillus plantarum 6H2 treatment, and Zhuanglemei treatment.

[0178] After ensiling was completed, the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were reduced by 14.59%, 13.84%, 17.47%, 15.91%, 16.76% and 16.53% respectively compared with before ensiling.

[0179] 1.6 Effect of additives on the acid detergent fiber content of oat silage

[0180] Effects of different additives on the acid detergent fiber content of oat silage Figure 6 The results showed that the acid detergent fiber content gradually decreased during the whole silage process (P<0.05). The treatments that showed a significant decrease on the 60th day included the Lactobacillus plantarum 6H2 treatment group, the Zhuanglemei treatment group, and the Zhuanglemei + cellulase treatment group; the cellulase treatment group showed a trend of first increasing and then decreasing, reaching the lowest value on the 90th day.

[0181] There were no significant differences among the treatments on the 0th and 7th days of ensiling; on the 30th and 60th days of ensiling, the cellulase treatment was significantly higher than the other treatment groups (P<0.05); on the 90th day of ensiling, the Lactobacillus plantarum 6H2 treatment and the cellulase treatment were significantly lower than the other treatment groups (P<0.05).

[0182] After ensiling was completed, the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were reduced by 5.81%, 9.90%, 8.92%, 7.39%, 7.91% and 7.73% respectively compared with before ensiling.

[0183] 1.7 Effect of additives on crude ash content of oat silage

[0184] Effects of different additives on the crude ash content of oat silage Figure 7 As shown: the crude ash content did not change regularly during the ensiling process. The crude ash content of the control treatment group, Lactobacillus plantarum 6H2 treatment group, cellulase treatment group and Zhuanglemei treatment group after 90 days of ensiling was significantly higher than that on the 1st day and 60th day of ensiling.

[0185] There were no significant differences among the treatments at 0d, 14d, and 30d of ensiling; on the 7th day of ensiling, the Lactobacillus plantarum 6H2+cellulase treatment and the Zhuanglemei+cellulase treatment were significantly higher than the other treatments (P<0.05); on the 60th day, the Zhuanglemei treatment was significantly lower than the Lactobacillus plantarum 6H2 and Zhuanglemei+cellulase treatments (P<0.05); on the 90th day of ensiling, the control treatment was significantly higher than the other treatment groups (P<0.05).

[0186] After ensiling was completed, the control treatment, Lactobacillus plantarum 6H2 treatment, cellulase treatment, Zhuanglemei treatment, Lactobacillus plantarum 6H2 + cellulase treatment and Zhuanglemei + cellulase treatment were increased by 1.43%, 1.24%, 1.16%, 0.91%, 1.18% and 1.04% respectively compared with before ensiling.

[0187] 2. Effect of additives on oat fermentation quality

[0188] 2.1 Effect of additives on pH of oat fermentation

[0189] Effects of different additives on pH changes during silage of oats Figure 8 As shown: During the ensiling process, the pH showed a downward trend as the ensiling time prolonged. On the 7th day of ensiling, the pH in the Lactobacillus plantarum 6H2-treated group decreased significantly, followed by Lactobacillus plantarum 6H2+cellulase, and the control treatment group had the least decrease.

[0190] The pH of the control treatment decreased rapidly from the 0th day to the 30th day of ensiling, and tended to be stable after 30 days; the pH of the Lactobacillus plantarum 6H2 treatment reached 4.14 on the 7th day, and reached a stable state after the 7th day, with the final pH being 3.83; the pH of the cellulase treatment decreased rapidly before 30 days of ensiling, tended to be stable after 30 days, and the final pH was 3.93; the pH of the Zhuanglemei treatment continued to decrease slowly, and finally reached 3.93; the pH of the Lactobacillus plantarum 6H2 + cellulase treatment decreased rapidly on the 7th day of ensiling, and then decreased slowly and tended to be stable, with the final pH being 3.85; the pH of the Zhuanglemei + cellulase treatment continued to decrease slowly, and finally reached 3.89.

[0191] 2.2 Effect of additives on lactic acid during oat fermentation

[0192] Effects of different additives on the lactic acid content of oat silage during silage Fig. 9 As shown in the figure, the lactic acid content showed an upward trend with the extension of ensiling time, and all treatments increased significantly on the 7th day.

[0193] On the 0th day, the lactic acid content of Lactobacillus plantarum 6H2 treatment was significantly higher than that of other treatment groups (P<0.05), and the control treatment group was the lowest; on the 7th day of ensiling, the lactic acid content of Zhuanglemei + cellulase treatment was the highest, followed by Zhuanglemei treatment, and the lactic acid content in the control group was the lowest; on the 90th day of ensiling, the lactic acid content of Lactobacillus plantarum 6H2 + cellulase treatment was the highest (P<0.05), followed by Zhuanglemei + cellulase treatment, and the lactic acid content in the control treatment was the lowest.

[0194] 2.3 Effect of additives on acetic acid during oat fermentation

[0195] Effects of different additives on the changes of acetic acid content in oat silage Fig.10 As shown in the data, the acetic acid content increased continuously with the extension of time during the ensiling process. On the 30th day, the acetic acid content in the control treatment increased significantly (P<0.05) and became the highest value among all treatment groups. On the 90th day of ensiling, the acetic acid content in the Zhuanglemei + cellulase treatment was significantly higher than that in other treatment groups (P<0.05).

[0196] 2.4 Effect of additives on propionic acid during oat fermentation

[0197] Effects of different additives on the changes of propionic acid content in oat silage Fig.11 As shown: the propionic acid content continued to rise during the ensiling process. On the 0th day, the propionic acid content of Lactobacillus plantarum 6H2 treatment and Lactobacillus plantarum 6H2 + cellulase treatment was undetectable. From the 7th day of ensiling to the completion of ensiling, the propionic acid content in the control treatment was significantly higher than that in other treatment groups at all times. On the 7th day, the Lactobacillus plantarum 6H2 treatment was significantly lower than that in other treatment groups; on the 30th and 60th days, the propionic acid content of the Zhuanglemei treatment was significantly lower than that in other treatment groups; on the 90th day of ensiling, the Lactobacillus plantarum 6H2 + cellulase treatment and the Zhuanglemei + cellulase treatment were significantly lower than those in other treatments.

[0198] 2.5 Effect of additives on ammonia nitrogen during oat fermentation

[0199] Effects of different additives on the changes of ammonia nitrogen content in oat silage Fig.12 As shown: the ammonia nitrogen content continued to rise during the ensiling process. On the 0th day of ensiling, the ammonia nitrogen content of the control treatment was significantly higher than that of the cellulase treatment, the Zhuanglemei treatment, the Lactobacillus plantarum 6H2+cellulase treatment, and the Zhuanglemei+cellulase treatment; on the 7th day of ensiling, the control treatment, the Lactobacillus plantarum 6H2+cellulase treatment, and the Zhuanglemei+cellulase treatment were significantly higher than the other treatment groups; on the 14th day of ensiling, the Zhuanglemei treatment was significantly higher than the cellulase treatment, and there was no significance between the other treatment groups; after 30 days of ensiling, the ammonia nitrogen content of the control group, the cellulase treatment group, and the Zhuanglemei treatment group showed a significant increase compared with the other treatment groups (P<0.05). When the ensiling was carried out for 60 days, the ammonia nitrogen content of the Zhuanglemei treatment group significantly exceeded that of the other treatment groups. By the 90th day of ensiling, the ammonia nitrogen content of the control group once again significantly exceeded that of the other treatment groups (P<0.05).

[0200] 3. Comprehensive evaluation of nutritional quality and fermentation quality and screening of additives

[0201] The quality of oat silage was comprehensively evaluated by membership function, as shown in Table 11: the scores were ranked from high to low as Lactobacillus plantarum 6H2 treatment > Lactobacillus plantarum 6H2 + cellulase treatment > Zhuanglemei + cellulase treatment > cellulase treatment > Zhuanglemei treatment > control treatment.

[0202] Table 11. Comprehensive evaluation of oat silage quality under different additive treatments

[0203]

[0204] 4. Characteristics of oat silage microbial community

[0205] 4.1 Characteristics of oat silage microbial communities

[0206] The characteristics of the microbial community at the phylum level after ensiling with the additive treatment of Lactobacillus plantarum 6H2 are as follows: the top ten dominant bacterial phyla at the phylum level with a relative abundance of >1% are Firmicutes (86.1%), Blue Photosynthetic Bacteria (5.2%), Proteobacteria (8.1%), Fig.13 .

[0207] The characteristics of the microbial community at the genus level after ensiling with the additive treatment of Lactobacillus plantarum 6H2 are as follows: the top ten dominant genera at the genus level with a relative abundance of >1% are Lactobacillus (83.4%), Neurospora (5.2%), Anaerobic Bacillus (2.2%), Pantoea (2.7%), Serratia (3.1%), Hafnia (1.5%), Fig.14 .

[0208] 5. Correlation between microbial communities and silage quality

[0209] 5.1 Correlation between microbial communities and nutritional quality

[0210] Correlation between bacterial community and nutritional quality of silage Fig.15As shown in the figure, there is a significant correlation between the distribution of microbial communities and various material components during the fermentation of silage oats. Specifically, the microorganisms of the Firmicutes phylum showed a very strong negative correlation with the content of dry matter and acid detergent fiber, and also showed a significant negative correlation with soluble sugars and neutral detergent fiber. However, it was positively correlated with the content of crude fat and crude ash, and negatively correlated with crude protein. The Blue Photosynthetic Bacteria phylum had a significant positive correlation with the content of dry matter, and also showed a positive correlation with soluble sugars, crude protein, neutral detergent fiber and acid detergent fiber. However, it was negatively correlated with the content of crude fat and crude ash. There was a significant positive correlation between the content of Proteobacteria and neutral detergent fiber. At the same time, it also showed a positive correlation with the content of dry matter, soluble sugars, crude protein and acid detergent fiber. However, it is worth noting that there is a very significant negative correlation between the content of Proteobacteria and the content of crude fat, and it also showed a negative correlation with crude ash. Mycoplasma phylum was positively correlated with the contents of soluble sugar, dry matter, crude protein, neutral detergent fiber and acid detergent fiber, but negatively correlated with crude fat and crude ash. Actinobacteria phylum was extremely significantly negatively correlated with the content of acid detergent fiber, and was also significantly negatively correlated with dry matter, soluble sugar and neutral detergent fiber. However, it showed a significant positive correlation with crude ash and a positive correlation with crude fat. Thermodesulfobacterium phylum was positively correlated with soluble sugar, dry matter, crude protein, neutral detergent fiber and acid detergent fiber, but negatively correlated with crude fat and crude ash. Bacteroidetes phylum was negatively correlated with soluble sugar and crude protein, positively correlated with crude ash, negatively correlated with dry matter and neutral detergent fiber, and positively correlated with crude fat.

[0211] 5.2 Correlation between microbial community and fermentation quality

[0212] Correlation between bacterial community and silage fermentation quality Fig.16 As shown in the figure, the number of Firmicutes is significantly negatively correlated with pH value, and significantly positively correlated with the content of lactic acid and ammonia nitrogen, while the relationship with acetic acid and propionic acid is positively correlated. The number of Blue Photosynthetic Bacteria, Mycoplasma and Thermodesulfurobacterium is positively correlated with the pH value of the environment, but negatively correlated with the content of lactic acid, acetic acid, propionic acid and ammonia nitrogen. The number of Proteobacteria is positively correlated with pH value, but has a very significant negative correlation with the content of acetic acid, a significant negative correlation with the content of lactic acid and ammonia nitrogen, and a negative correlation with propionic acid. The number of Actinobacteria is significantly negatively correlated with pH value, but has a significant positive correlation with the content of lactic acid and ammonia nitrogen, and is also positively correlated with acetic acid and propionic acid. Finally, the number of Bacteroidetes is extremely significantly negatively correlated with pH value, significantly positively correlated with the content of propionic acid and ammonia nitrogen, and is also positively correlated with lactic acid and acetic acid.

[0213] 6. Effects of different additives on oat silage quality

[0214] The application of silage additives is to create an ideal environment conducive to lactic acid fermentation, thereby promoting the production of lactic acid, achieving feed acidification, thereby accelerating the silage process, reducing nutrient loss, enhancing oxygen stability, and ultimately improving the overall quality of silage. Through scientific and reasonable selection of additives, the fermentation effect of silage can be effectively optimized to achieve the purpose of improving crop quality.

[0215] In the experiment of the present embodiment, it is observed that as the silage fermentation time increases, the dry matter content of each group of silage gradually decreases. This is because during the silage process, the respiration of plant cells and the activity of aerobic microorganisms will cause nutrients to be continuously consumed, produce water, carbon dioxide and free ammonia, and ultimately lead to a reduction in the dry matter content in the silage. The lactic acid bacteria additive used is the plant lactobacillus 6H2 screened by the research group in the early stage. The results of the present embodiment show that the addition of plant lactobacillus 6H2 significantly increases the crude fat content, and also correspondingly reduces the neutral detergent fiber and acid detergent fiber content. When the lactic acid bacteria additive is used to treat silage for 90d, it is 1.74% higher than the control group. Adding lactic acid bacteria can effectively increase the lactic acid content during silage. After the silage is completed, the lactic acid content of the lactic acid bacteria treatment group is significantly higher than (P<0.05) the lactic acid content of the lactic acid bacteria treatment group that is not added. Adding lactic acid bacteria to the silage modulation process can accelerate the pH value to decline, prevent protein degradation and loss, and enhance the digestion efficiency of feed, thereby improving the overall quality of feed. Lactic acid bacteria additives can ensure that silage is fermented quickly in the early stage of silage, lactic acid is accumulated quickly, pH value is rapidly reduced, it is conducive to reducing the number of harmful microorganisms, reducing nutrient loss, in the present embodiment, after 90d fermentation, adding lactic acid bacteria treatment in dry matter, soluble sugar, crude protein content is significantly higher than other treatments, and lactic acid content is also higher than the control group, pH value and ammonia nitrogen content are also significantly lower than the control group, these results all show that adding lactic acid bacteria can effectively improve the silage quality of feeding oats. But there are also studies showing that adding lactic acid bacteria does not effectively improve the fermentation quality of forage, which may be caused by being superior to the soluble carbohydrates in forage, but in the present embodiment, oats contain enough soluble carbohydrates, which is conducive to providing an excellent environment for lactic acid bacteria to compete with other undesirable microorganisms.

[0216] Cellulase can hydrolyze the structural polysaccharides in the plant cell wall into monosaccharides, provide sufficient fermentation substrates for lactic acid bacteria, generate a large amount of lactic acid and a small amount of acetic acid, accelerate the fermentation rate, and inhibit the growth of clostridium and other harmful bacteria. Cellulose-degrading bacteria secrete efficient cellulase, which can effectively degrade the structural carbohydrates in silage materials, thereby decomposing the polysaccharides in the cell wall and releasing soluble sugars. In addition, enterococci have dual functions, which can degrade cellulose and perform lactic acid fermentation. It can use the soluble sugars produced by cellulose degradation for lactic acid fermentation, increase the content of lactic acid, and reduce the pH value. In the later stage of silage, the structural carbohydrates in the silage material are further decomposed by enzymatic hydrolysis. The research of Li Xuye et al. showed that the addition of cellulase to corn can better improve the silage quality of corn. The results of He Guoqiong et al.'s study on the effect of adding different concentrations of cellulase on the quality of tall wheatgrass silage showed that the treatment effect of adding cellulase at a concentration of 0.15g / kg was the best. In this example, the neutral detergent fiber and acid detergent fiber of the cellulase-treated group were significantly lower than those of the control group (P<0.05).

[0217] The five additive treatment groups in this example can all improve the silage quality, among which the treatment group with Lactobacillus plantarum 6H2 and the treatment group with Lactobacillus plantarum 6H2+cellulase have the most significant effects.

[0218] 7. Effect of microbial communities on silage quality

[0219] In this embodiment, the addition of Lactobacillus plantarum 6H2 significantly increased the relative abundance of Lactobacillus plantarum, thereby improving the silage quality. Lactobacillus is the dominant genus in the silage microbial community treated with many bacterial agents. The increase in the abundance of Lactobacillus is one of the main reasons for the good quality and high lactic acid content of oat silage treated with Lactobacillus plantarum 6H2 and Lactobacillus plantarum 6H2 + cellulase.

[0220] In the experiment of this embodiment, Firmicutes and Proteobacteria are absolutely dominant before and after ensiling. Most bacteria of Firmicutes have the function of degrading macromolecular compounds such as cellulose, starch, and protein. In this embodiment, as the ensiling time increases, Firmicutes also increases accordingly. Firmicutes increases by 22.04% on the 90th day of ensiling compared with the first day. In this embodiment, as the ensiling time increases, the relative abundance of Proteobacteria decreases accordingly. Proteobacteria decreases by 5.53% on the 90th day of ensiling compared with the first day. Proteobacteria is the largest phylum among bacteria. Escherichia coli, Salmonella, etc. in Proteobacteria will compete with lactic acid bacteria for substrates, affecting the fermentation quality of silage.

[0221] In this embodiment, the community structure at the bacterial genus level was analyzed, and it was found that Lactobacillus always occupied the dominant genus position throughout the anaerobic process. Lactobacillus is a common microorganism in silage. Lactobacillus inhibits the growth of other harmful bacteria by producing lactic acid during anaerobic fermentation. On the first day, the relative proportion of Lactobacillus had reached 47.53%. Oats provided sufficient fermentation substrate for Lactobacillus, allowing oats to ferment rapidly in the early stage of silage, significantly reducing the pH and stabilizing in the later stage.

[0222] In this example, by analyzing the effects of different additives on the quality of oat silage, the treatment with the addition of low-temperature-resistant Lactobacillus plantarum 6H2 had the highest soluble sugar content and crude protein content, which were 3.94% and 9.78%, respectively; the acid detergent fiber was the lowest value among the treatments; the pH was the lowest value among the treatments, 3.83; through comprehensive analysis of the membership function, the comprehensive scores were as follows: Lactobacillus plantarum 6H2 treatment > Lactobacillus plantarum 6H2 + cellulase treatment > Zhuanglemei + cellulase treatment > cellulase treatment > Zhuanglemei treatment > control treatment.

[0223] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Lactobacillus plantarum 6H2 (Lactobacillus plantarum 6H2), whose deposit number in the General Microbiological Center of China Microorganism Culture Collection Administration is CGMCC No.31240.

2. A bacterial agent, the active ingredient of which is the Lactobacillus plantarum 6H2 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is used for silage of forage.

4. The bacterial agent according to claim 3, characterized in that: The forage grass is grass of the gramineous family and / or grass of the leguminous family.

5. Any of the following uses of the Lactobacillus plantarum 6H2 according to claim 1 or the bacterial agent according to claim 2 or 3: X1. Application in forage silage; X2. Application in the preparation of forage silage products; X3. Application in increasing the lactic acid and / or acetic acid content of forage silage; X4. Application in the preparation of products for increasing the lactic acid and / or acetic acid content of forage silage; X5. Application in reducing pH of forage silage; X6. Application in the preparation of products for lowering the pH of forage silage; X7. Application in increasing the dry matter content and / or crude protein content of forage silage; X8. Application in the preparation of products for increasing the dry matter content and / or crude protein content of forage silage; X9. Application in improving the acid protease activity and / or acid xylanase activity of forage silage; X10. Application in the preparation of products for improving the acid protease activity and / or acid xylanase activity of forage silage.

6. The use according to claim 5, characterized in that: The forage grass is grass of the gramineous family and / or grass of the leguminous family.

7. Methods of silage of forage grass, including: The Lactobacillus plantarum 6H2 described in claim 1 is added to the forage for ensiling to complete the ensiling of the forage.

8. The method according to claim 7, characterized in that: The amount of Lactobacillus plantarum 6H2 added in the method is 1×10 6 cfu / g forage.

9. The method according to claim 7 or 8, characterized in that: The silage is carried out at -6°C to 18°C; And / or, the ensiling time is 7-90 days.

10. The method according to any one of claims 7 to 9, characterized in that: The forage grass is grass of the gramineous family and / or grass of the leguminous family.

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