Method for ensiling oats and common vetches

Abstract

The invention discloses an ensiling method of oats and common vetches. The method for ensiling the oats and the vicia sativa, disclosed by the invention, comprises the following steps: adding lactobacillus plantarum 6H2 into mixed forage grass consisting of the oats and the vicia sativa for ensiling, so as to complete ensiling of the oats and the vicia sativa, the preservation number of the lactobacillus plantarum 6H2 (Lactobacillus plantarum 6H2) in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC (China General Microbiological Culture Collection Center). 31240. Experiments prove that the low temperature resistant lactic acid bacteria lactobacillus plantarum 6H2 (Lactobacillus plantarum 6H2) shows more excellent fermentation quality and nutritional ingredient optimization effect in the mixed silage of the oats and the vicia sativa at a low temperature.

Description

Technical Field

[0001] The invention relates to an silage method for oats and peas in the field of biological agriculture. Background Art

[0002] In recent years, overloading and overgrazing of natural grasslands have seriously affected grassland ecological restoration and herders' income. In order to resolve the contradiction between grassland ecological protection and economic development in pastoral areas, traditional grassland animal husbandry has gradually transformed and upgraded to modern grassland animal husbandry. The transformation and development of animal husbandry faces new opportunities and challenges, and the demand for high-quality and safe forage has risen rapidly. The development and utilization of high-quality silage can provide high-quality forage throughout the year and promote the green development of modern animal husbandry.

[0003] Oats (Avena sativa) are annual grasses that prefer cool weather. Its soluble carbohydrate content is higher than that of leguminous forages, but its crude protein content is lower, which is not enough to meet the high nutritional needs of livestock. Vicia sativa is a leguminous forage with a high crude protein content, but its high buffering energy value and water content make it difficult to successfully ensilage it alone. Therefore, mixing oats and vicia sativa and then silaging them can not only solve the problem of poor silage quality of a single forage. It also increases yield, improves nutritional quality and feeding value. In terms of mixed silage of oats and vicia sativa, research by Liu Wen and others showed that the crude protein content increased after oats and peas were mixed for silage, and the mixture of the two significantly improved the nutritional value of silage. Although oat and pea mixed silage is rich in nutritional value, most forage grasses in the Qinghai-Tibet Plateau are harvested from the end of August to September, when the highest temperature has dropped to around 15-18°C and the lowest temperature is about -6°C. There is also a huge temperature difference between day and night. Too high or too low temperature has a great impact on the length of silage fermentation time and silage quality. Therefore, it is very important to select appropriate lactic acid bacteria additives and clarify the time for silage fermentation to be completed.

[0004] In recent years, the mixed planting model of oats and peas has been promoted in the Qinghai-Tibet Plateau, and the promotion area as a high-quality forage has increased year by year. However, the best lactic acid bacteria additive and fermentation completion time for mixed silage of oats and peas in this region are still unclear. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to add a low-temperature resistant lactic acid bacteria agent to perform silage of oats and peas at low temperature.

[0006] In order to solve the above technical problems, the present invention first provides an oat and sagittaria ensiling method, the method comprising: adding Lactobacillus plantarum 6H2 to a mixed forage consisting of oats and sagittaria ensiling to complete the ensiling of oats and sagittaria ensiling;

[0007] The Lactobacillus plantarum 6H2 has a deposit number of CGMCC No. 31240 in the General Microbiological Center of China National Committee for the Preservation of Microorganisms.

[0008] In the above method, the amount of Lactobacillus plantarum 6H2 added can be 1×10 6 cfu / g forage.

[0009] In the above method, the mass ratio of oats to peas in the mixed forage can be (3-5):1, such as 4.25:1.

[0010] In the above method, the ensiling can be carried out at -6°C to 18°C ​​(such as -6°C to 15°C, etc.).

[0011] In the above method, the ensiling time can be 7-90 days (such as 14 days, 30 days, 60 days, etc.).

[0012] The present invention also provides the use of Lactobacillus plantarum 6H2 in improving the lactic acid and / or acetic acid content of oat and pea silage, or in preparing a product for improving the lactic acid and / or acetic acid content of oat and pea silage.

[0013] The present invention also provides the use of Lactobacillus plantarum 6H2 in improving the dry matter content and / or crude protein content of oat and pea silage, or in preparing a product with improved dry matter content and / or crude protein content of oat and pea silage.

[0014] The present invention also provides the use of Lactobacillus plantarum 6H2 in improving the acid protease activity and / or acid xylanase activity of oat and pea silage, or in preparing a product for improving the acid protease activity and / or acid xylanase activity of oat and pea silage.

[0015] The present invention also provides the application of Lactobacillus plantarum 6H2 in reducing the pH of oat and pea silage; or the application of Lactobacillus plantarum 6H2 in preparing a product for reducing the pH of oat and pea silage.

[0016] The present invention studies the dynamic change law of the enzyme activity and silage quality of mixed silage of oats and saguaro peas under low temperature by low temperature resistant Lactobacillus plantarum 6H2. Oats and saguaro peas were mixed for silage, and two lactic acid bacteria additives, Lactobacillus plantarum (CI) and Lactobacillus plantarum (6H2), were used. CK was not added, and the quality and enzyme activity were measured at 7d, 14d, 45d and 90d of silage. The results show that the silage time and lactic acid bacteria additives have a significant effect on the quality and enzyme activity of mixed silage of oats and saguaro peas. In terms of fermentation quality, the pH value and ammonia nitrogen content of the 6H2 and CI groups were significantly lower than those of the CK group at each silage time point, while the lactic acid content was significantly higher than that of the CK group (P<0.05). In particular, at 45d and 90d of silage, the lactic acid and acetic acid contents of the 6H2 group were significantly higher than those of the CI group, which indicates that the fermentation effect of low temperature resistant lactic acid bacteria 6H2 is better under low temperature environment. In terms of nutritional quality, compared with the CK group, the 6H2 and CI groups were able to significantly increase the dry matter and crude protein content and reduce the crude fat and soluble carbohydrate content (P<0.05). This shows that the addition of lactic acid bacteria helps to reduce dry matter loss and improve the nutritional value of feed. In terms of enzyme activity, the acid protease activity of the 6H2 group was significantly higher than that of the CI and CK groups at 45 and 90 days of silage (P<0.05). This shows that 6H2 can decompose the protein in the silage through deamination under low temperature conditions, provide the nutrients required for the growth and reproduction of microorganisms, and promote the fermentation of silage. In summary, lactic acid bacteria 6H2 and CI improved the nutritional content, fermentation quality and enzyme activity of mixed silage of oats and sagittaria peas to a certain extent, and the low-temperature-resistant lactic acid bacteria Lactobacillus plantarum 6H2 showed superior fermentation quality and nutrient optimization effect in mixed silage of oats and sagittaria peas at low temperatures. With the maturity of silage technology, the role of plant enzymes in silage fermentation is clarified, providing a scientific basis for the regulation of silage fermentation quality, the combination of bacteria and enzymes, and the establishment of enzyme preparation technology systems to improve the overall quality and nutritional value of silage.

[0017] 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.

[0018] Description of biological material deposit

[0019] Abbreviation of depository unit: CGMCC

[0020] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0021] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0022] Deposit date: July 9, 2024

[0023] CGMCC Registration Number: CGMCC No.31240

[0024] Classification name: Lactobacillus plantarum

[0025] Strain ID: 6H2 DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1. Isolation and identification of strain 6H2

[0028] 1. Isolation and identification of strain 6H2

[0029] 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:

[0030]

[0031] 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.

[0032] II. Identification of the low temperature tolerance of strain 6H2

[0033] 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.

[0034] 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 at a temperature gradient of 5°C, 15°C, 25°C and 35°C for 24 h to determine the cell membrane integrity, cell membrane fatty acid composition and cell membrane fatty acid content of the strain.

[0035] Measurement content and methods:

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Results and analysis:

[0040] 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.

[0041] 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.

[0042] Table 1. Saturated fatty acid content of lactic acid bacteria cell membrane at different temperatures

[0043]

[0044] Note: ND, not detected.

[0045] 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.

[0046] Table 2 Unsaturated fatty acid content of lactic acid bacteria cell membrane at different temperatures

[0047]

[0048] 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.

[0049] Table 3. Relative content of fatty acids in lactic acid bacteria cell membrane at different temperatures

[0050]

[0051] 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 the fatty acid composition of their own cell membranes. 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 in 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.

[0052] Example 2: Application of Lactobacillus plantarum 6H2 in oat and pea silage

[0053] 1. Materials and Methods

[0054] 1. Overview of the test site

[0055] 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.

[0056] 2. Test Materials

[0057] The tested forage varieties were Avena sativa cv. Longyan No.3 for oats and Lathyrus sativa cv. Ximu 333 for arrow peas. The main chemical components and enzyme activities of fresh forage are shown in Table 4. There were two types of 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).

[0058] Table 4. Nutritional composition and enzyme activity of oats and pea mixture before ensiling

[0059] 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

[0060] 3. Experimental Design

[0061] 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. CI strain suspension (obtained by culturing CICC21804) and 6H2 strain suspension (obtained by culturing Lactobacillus plantarum 6H2) were added respectively, and the addition amount was 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.

[0062] 4. Determination indicators and methods

[0063] 4.1 Fermentation quality

[0064] 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 the filtrate is used to determine the fermentation quality. Take a part of the filtrate to measure the pH value with a pH meter, and determine the ammonia nitrogen (AN) content with phenol-sodium hypochlorite colorimetry; filter 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 (LLA), acetic acid (AA), propionic acid (PA) and butyric acid (BA) content.

[0065] 4.2 Nutritional quality

[0066] 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.

[0067] 4.3 Enzyme activity

[0068] 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.).

[0069] 5. Data Processing and Analysis

[0070] 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).

[0071] 2. Results and Analysis

[0072] 1. Effects of different lactic acid bacteria additives and silage time on fermentation parameters of oat and pea mixed silage

[0073] 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).

[0074] Table 5. ANOVA of the interaction between additives and silage time

[0075]

[0076] Note: * indicates that there are significant differences between different treatments of the same item at the 0.01 level (P<0.01).

[0077] 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.

[0078] 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.

[0079] Table 6 Effects of additives and silage time on fermentation indexes of mixed silage of oats and peas

[0080]

[0081] 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).

[0082] Note: Butyric acid (BA) was not detected in any treatment and therefore not listed.

[0083] 2. Effects of different lactic acid bacteria additives and silage time on the nutritional indexes of oat and pea mixed silage

[0084] 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).

[0085] Table 7. ANOVA of the interaction between additives and silage time

[0086]

[0087] Note: * indicates that there are significant differences between different treatments of the same item at the 0.01 level (P<0.01).

[0088] 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.

[0089] 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.

[0090] Table 8 Effects of additives and silage time on nutritional indexes of mixed silage of oats and peas

[0091]

[0092] 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).

[0093] 3. Effects of different lactic acid bacteria additives and silage time on enzyme activity of mixed oat and pea silage

[0094] 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).

[0095] Table 9. ANOVA of the interaction between additives and silage time

[0096]

[0097] Note: * indicates that there are significant differences between different treatments of the same item at the 0.01 level (P<0.01).

[0098] 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).

[0099] Table 10. Effects of additives and silage time on enzyme activity of mixed oat and pea silage

[0100]

[0101] 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).

[0102] 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.

[0103] 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.

[0104] 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. A method for silage of oats and peas, comprising: Adding Lactobacillus plantarum 6H2 to the mixed forage consisting of oats and sagittaria peas for silage, thereby completing the silage of oats and sagittaria peas; The Lactobacillus plantarum 6H2 has a deposit number of CGMCC No. 31240 in the General Microbiological Center of China National Committee for the Preservation of Microorganisms.

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

3. The method according to claim 1 or 2, characterized in that: The mass ratio of oats to peas in the mixed forage is (3-5):

1.

4. The method according to any one of claims 1 to 3, characterized in that: The silage is carried out at -6°C to 18°C.

5. The method according to any one of claims 1 to 4, characterized in that: The silage time is 7-90 days.

6. Use of Lactobacillus plantarum 6H2 as claimed in claim 1 in increasing the lactic acid and / or acetic acid content of oat and pea silage, or in preparing a product having increased lactic acid and / or acetic acid content of oat and pea silage.

7. Use of Lactobacillus plantarum 6H2 as claimed in claim 1 in increasing the dry matter content and / or crude protein content of oat and pea silage, or in preparing a product having increased dry matter content and / or crude protein content of oat and pea silage.

8. Use of Lactobacillus plantarum 6H2 as claimed in claim 1 in improving the acid protease activity and / or acid xylanase activity of oat and pea silage, or in preparing a product having improved acid protease activity and / or acid xylanase activity of oat and pea silage.

9. Use of Lactobacillus plantarum 6H2 as claimed in claim 1 in reducing the pH of oat and pea silage; or in preparing a product for reducing the pH of oat and pea silage.

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