A compound probiotic silage agent suitable for the cold season of the Qinghai-Tibet Plateau and an application method thereof

Abstract

The application provides a compound probiotic silage agent suitable for Qinghai-Tibet Plateau cold season silage and an application method thereof, the compound probiotic silage agent comprises an additive A and an additive B, the additive A comprises lactobacillus plantarum, bacillus subtilis, cellulase, xylanase, seaweed extract and acetic acid, the additive B comprises lactobacillus curvatus, pectinase, beta-mannanase and seaweed extract, the additive A is added before fermentation, and the additive B is added at 7-10 days after fermentation; under the preparation process of the application, oat straw-oat straw mixed silage can realize efficient fermentation under high-cold conditions, the prepared silage feed products all reach the I level standard, the process is beneficial to protein and starch reservation, can effectively degrade fiber substances, improves palatability and digestibility, and can effectively improve the comprehensive quality of oat straw-oat straw mixed silage in high-cold regions, and the sensory quality is excellent.

Description

Technical Field

[0001] This invention relates to the field of feed fermentation technology, specifically to a compound probiotic silage agent adapted for cold-season silage in the Qinghai-Tibet Plateau and its application method. Background Technology

[0002] As an important pastoral area in my country, the Qinghai-Tibet Plateau's high altitude, low temperature, and low oxygen climate results in a long cold season and a shortage of forage. Forage preservation and supply have become the core bottleneck restricting the development of local animal husbandry. Winter feeding of unique livestock such as yaks and Tibetan sheep is highly dependent on high-quality forage. Silage technology, as an efficient forage preservation method, transforms fresh forage into high-quality feed that can be stored for a long time through anaerobic fermentation. It is key to solving the forage shortage in the cold season, reducing livestock weight loss, and ensuring the stable development of animal husbandry in pastoral areas.

[0003] However, the extreme environment of the Qinghai-Tibet Plateau during the cold season presents numerous technical challenges to silage fermentation, and existing silage inoculants and technologies have significant limitations. On the one hand, the low-temperature environment significantly inhibits the activity of beneficial microorganisms, leading to slow fermentation initiation, insufficient lactic acid production, and an inability to quickly lower the pH to a safe range. This easily triggers the proliferation of mold and yeast, causing silage spoilage, nutrient loss, and even the production of harmful substances that endanger livestock health. On the other hand, the local high-yield silage raw materials (oats, crested wheatgrass, barley straw, etc.) have high fiber content and high lignification. Existing inoculants lack targeted enzymatic hydrolysis systems, making it difficult to degrade recalcitrant components. Furthermore, the strains lack sufficient resistance, cannot adapt to the high-altitude, low-oxygen environment, and have low survival and reproduction rates. Moreover, when applied during the cold season on the plateau, the fermentation cycle is long, the success rate is low, and the quality is unstable. Single low-temperature resistant lactic acid bacteria cannot meet the needs of multiple fermentations and cannot fundamentally solve the core problems. In addition, existing application methods often involve adding the inoculant all at once without optimizing the timing of addition based on different fermentation stages, affecting the effectiveness of beneficial bacteria. Therefore, developing compound probiotic silage agents that are adapted to the cold season environment of the Qinghai-Tibet Plateau and can significantly improve the nutritional value of silage is of great practical significance for promoting the upgrading of plateau cold season silage technology, ensuring forage supply, and promoting the high-quality development of animal husbandry. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a compound probiotic silage agent suitable for cold-season silage in the Qinghai-Tibet Plateau and its application. The compound probiotic silage agent includes adjuvant A and adjuvant B. Adjuvant A includes Lactobacillus plantarum, Bacillus subtilis, cellulase, xylanase, seaweed extract, and acetic acid. Adjuvant B includes Lactobacillus curvatureii, pectinase, β-mannanase, and seaweed extract. Adjuvant A is added before fermentation, and adjuvant B is added 7-10 days after fermentation. By adding the compound probiotic silage agent in stages and combining the effective components in adjuvant A and adjuvant B, efficient fermentation of cold-season silage in the high-altitude and cold-climate conditions can be achieved. The silage product prepared can meet the Grade I standard, with high protein and starch content, and can improve palatability and digestibility, effectively improving the overall quality of barley-oat mixed silage in high-altitude and cold-climate regions.

[0005] To achieve the above technical effects, the following technical solution is adopted: A compound probiotic silage agent adapted for cold-season silage on the Qinghai-Tibet Plateau includes adjuvant A and adjuvant B. Adjuvant A includes Lactobacillus plantarum, Bacillus subtilis, cellulase, xylanase, seaweed extract, and acetic acid. Adjuvant B includes Lactobacillus curvaturei, pectinase, β-mannanase, and seaweed extract. Furthermore, the weight ratio of each component in adjuvant A is as follows: Lactobacillus plantarum: Bacillus subtilis: cellulase: xylanase: seaweed extract: acetic acid = 2.5~4.5: 1.5~3.0: 0.8~1.8: 0.5~1.2: 1.0~2.0: 0.3~0.8, and the volume concentration of acetic acid is 30%~50%. Furthermore, the weight ratio of each component in adjuvant B is: Lactobacillus curvature: pectinase: β-mannanase: seaweed extract = 3.0~5.0: 0.6~1.5: 0.4~1.0: 1.2~2.2; Furthermore, the viable count of *Lactobacillus plantarum* is ≥1.0 × 10⁻⁶. 10 CFU / g, Bacillus subtilis viable count ≥8×10 9 CFU / g, viable count of Curvular Lactobacillus ≥1.2×10⁻⁶ 10 CFU / g; Furthermore, the weight ratio of additive A to additive B is 1.5~3:1; This invention also provides the application of a compound probiotic silage agent adapted to the cold season silage of the Qinghai-Tibet Plateau in the fermentation of cold season silage on the Qinghai-Tibet Plateau; Furthermore, the application method includes the following steps: Step S1: After harvesting the barley and oat straw, immediately crush them to 2-5 cm and mix them together. The weight ratio of the two mixtures should be 0.5-2:1. Step S2: Spray the 0.5%~1.5% (w / w) solution of adjuvant A evenly onto the surface of the mixed silage raw material. The spraying amount is 0.6~1.5 L per 100 kg of silage raw material. Stir while spraying to ensure that the diluted adjuvant A solution is evenly mixed with the silage raw material. Then, fill, compact, and seal the mixed silage in layers and ferment it at 0~15℃. Step S3: On the 7th to 10th day after fermentation, spray the 0.5% to 1.5% (w / w) solution of adjuvant B evenly onto the surface of the silage raw material. The spraying amount is 0.4 to 1 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material 5 to 10 cm to ensure that the diluted adjuvant B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. Then reseal and compact the insulation layer. Step S4: Continue anaerobic fermentation, and obtain silage after fermentation is completed; Furthermore, in step S1, after the silage raw materials are crushed and mixed, the moisture content of the raw materials is adjusted to 60%~70%, and the total fermentation cycle in steps S2~S4 is 30~45 days.

[0006] Furthermore, in step S3, the addition of adjuvant B is carried out on the 8th to 9th day after the start of fermentation, and the spraying and turning process is completed within 30 minutes.

[0007] Furthermore, in steps S2 and S3, mechanical compaction is used for compaction; the insulation layer is either straw or insulation cotton.

[0008] The beneficial effects of this invention are as follows: This invention, through the two-stage addition of a compound probiotic silage agent and the combination of the effective components in adjuvants A and B, enables efficient fermentation of cold-season silage on the Qinghai-Tibet Plateau under high-altitude and cold conditions. This effectively improves the overall quality of mixed silage from oat straw and barley straw in high-altitude and cold regions. The prepared mixed silage products all meet Grade I standards, exhibiting excellent performance in various indicators: pH close to 4.2, ammonia nitrogen / total nitrogen ≤9.8%, lactic acid / total organic acid ≥61.2%. Its sensory quality is yellow-green, with a rich sour aroma, loose and pliable texture, free from mold and stickiness. The process is beneficial for protein and starch retention while effectively degrading fibrous substances, improving palatability and digestibility, significantly enhancing the nutritional value and feed conversion rate of silage, and improving feed taste. Its effects are as follows: By adding adjuvant B 7-10 days after fermentation, this invention strengthens the fermentation effect and optimizes the fermentation quality, thereby compensating for insufficient fermentation caused by low temperatures in high-altitude and cold regions; plant milk... Bacillus subtilis and Bacillus thuringiensis, as probiotics, can rapidly colonize and inhibit the growth of putrefactive bacteria, lowering the pH value of silage. Cellulase and xylanase can degrade fibrous materials, improving palatability and nutrient utilization. Lactobacillus curvature further consolidates the fermentation environment, while pectinase and β-mannanase assist in the degradation of unusable substances, improving nutrient conversion efficiency. Seaweed extract can enhance the activity of probiotics under high and low temperatures, alleviating the inhibition of fermentation by low temperatures, while improving the water retention and nutritional stability of silage, and improving sensory quality. Acetic acid can rapidly lower the initial pH value of silage, inhibit the reproduction of harmful bacteria, extend the shelf life of silage, and prevent mold growth caused by slow fermentation in high and cold environments. The various components of the compound probiotic silage agent have a synergistic effect, not only rapidly lowering the pH value in the early stage of fermentation, but also effectively blocking the consumption of substrate by harmful microorganisms, thereby effectively optimizing the nutritional indicators of fermentation products and improving the overall quality of barley straw-oat straw silage in high and cold regions. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0011] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0012] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used are conventional methods and techniques in the art.

[0013] The following examples illustrate the multifunctional flame retardant and its preparation method provided by the present invention. The scope of protection of the present invention is not limited by the following examples.

[0014] In the following examples and comparative examples, *Lactobacillus plantarum* was purchased from Shandong Pingao Pharmaceutical Co., Ltd., catalog number PG-LPZ131; *Bacillus subtilis* was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, catalog number BNCC109047; *Lactobacillus curvatureii* was purchased from Shanghai Xuanya Biotechnology Co., Ltd., catalog number XY-WSW-1556; cellulase, xylanase, and pectinase were all purchased from Taian Xindeli Bioengineering Co., Ltd.; β-mannanase was purchased from Weifang Ruichen Biotechnology Co., Ltd.; and the seaweed extract was obtained from Fufeng Sinote Biotechnology Co., Ltd., catalog number SNT1962.

[0015] Example 1 Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 weight ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9 The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material. The mixture was stirred continuously during spraying to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, and compacted mechanically to a density ≥600 kg / m³ for each layer.3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0016] Example 2 Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.3 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 2.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 3 samples of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9 The mixture of 1.8 parts cellulase (activity 1600 U / g), 1.2 parts xylanase (activity 1300 U / g), and 2 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material. 0.6 L of additive solution A was applied per 100 kg of silage material, with continuous stirring to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, and compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 3.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10Add 1.5 parts of pectinase (activity 1100 U / g), 1.0 part of β-mannanase (activity 900 U / g), and 2.2 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 7 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.5 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0017] Comparative Example 1 In step S3, both auxiliary agent B solution and auxiliary agent A solution are sprayed onto the surface of the mixed oat straw and highland barley straw silage raw materials before fermentation. The specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9 The following ingredients were mixed thoroughly: 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract to prepare auxiliary agent A solution; 4.0 parts of Lactobacillus curvatureis (activity 1.3 × 10⁻⁶) were added. 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 parts of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix thoroughly to obtain auxiliary agent B solution. Spray auxiliary agent A and auxiliary agent B sequentially onto the surface of the mixed oat straw and highland barley straw silage raw material, applying 1 L of auxiliary agent A solution and 0.7 L of auxiliary agent B solution per 100 kg of silage raw material, stirring continuously while spraying to ensure uniform mixing. Fill the silage pit in layers and compact using mechanical compaction, with each layer compacted to a density ≥600 kg / m³. 3The silage pit is covered with a straw insulation layer, sealed, and fermented at 8°C. The total fermentation period is 38 days, and silage is obtained after fermentation.

[0018] Comparative Example 2 The seaweed extract component of auxiliary agent B is added to auxiliary agent A, and the specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9 The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 3.2 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material, stirring continuously to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g) and 0.7 parts of β-mannanase (activity 900 U / g) to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0019] Comparative Example 3 Neither A nor B contains seaweed extract. The specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9 The mixture of 1.3 parts cellulase (activity 1600 U / g) and 0.8 parts xylanase (activity 1300 U / g) was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material. The mixture was stirred continuously during spraying to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, and compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g) and 0.7 parts of β-mannanase (activity 900 U / g) to 700 parts of deionized water, mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0020] Comparative Example 4 Additive A does not contain acetic acid. The specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: By weight, add 3.5 parts of Lactobacillus plantarum (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9 Add 1.3 parts of cellulase (activity 1600 U / g), 0.8 parts of xylanase (activity 1300 U / g), and 1.5 parts of seaweed extract to 900 parts of deionized water, mix thoroughly, and then spray the prepared adjuvant A solution evenly onto the surface of the mixed oat straw and highland barley straw silage raw material. Apply 1 L of adjuvant A solution per 100 kg of silage raw material, stirring continuously while spraying to ensure uniform mixing. Fill the silage pit in layers and compact using mechanical compaction, with each layer having a compaction density ≥600 kg / m³. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0021] Comparative Example 5 Additive A does not contain Lactobacillus plantarum, but Bacillus subtilis is added in equal amounts. The specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 5.7 parts of Bacillus subtilis (active 9.0 × 10⁻⁶). 9The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material, stirring continuously to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0022] Comparative Example 6 Additive A does not contain Bacillus subtilis, but contains an equal amount of Lactobacillus plantarum. The specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 5.7 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material, stirring continuously to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0023] Comparative Example 7 The Lactobacillus plantarum is used to replace Lactobacillus curvilinearis in adjuvant B in an equal amount. The specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material. The mixture was stirred continuously during spraying to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, and compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus plantarum (active 1.2 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0024] Comparative Example 8 The xylanase in adjuvant A is replaced with an equal amount of cellulase, and the specific process is as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9The mixture of 2.1 parts cellulase (activity 1600 U / g) and 1.5 parts seaweed extract was thoroughly mixed. The resulting adjuvant solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of adjuvant solution A per 100 kg of silage material, stirring continuously to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0025] Comparative Example 9 Use cellulase to replace pectinase in adjuvant B in an equal amount. Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material. The mixture was stirred continuously during spraying to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, and compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of cellulase (activity 1600 U / g), 0.7 part of β-mannanase (activity 900 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0026] Comparative Example 10 The process involves replacing β-mannanase in adjuvant B with an equal amount of cellulase, as follows: Step S1: Select oat straw and barley straw harvested in the cold season (late October, average daily temperature 8℃) in Shigatse, Tibet. Remove weeds, rotten leaves and soil impurities. Crush them to 3 cm using a silage crusher. Mix the crushed oat straw and barley straw in a 1:1 ratio. Adjust the moisture content to 65% with sterile water and stir well for later use. Step S2: Dissolve 0.5 parts by weight of 40% acetic acid in 900 parts of deionized water, then add 3.5 parts of *Lactobacillus plantarum* (active 1.2 × 10⁻⁶). 10 CFU / g), 2.2 portions of Bacillus subtilis (activity 9.0 × 10⁻⁶). 9The mixture of 1.3 parts cellulase (activity 1600 U / g), 0.8 parts xylanase (activity 1300 U / g), and 1.5 parts seaweed extract was thoroughly mixed. The resulting additive solution A was then evenly sprayed onto the surface of the mixed oat straw and barley straw silage material, using 1 L of additive solution A per 100 kg of silage material. The mixture was stirred continuously during spraying to ensure uniform mixing. The mixed material was then layered and filled into the silage pit, and compacted mechanically to a density ≥600 kg / m³ for each layer. 3 Cover with a straw insulation layer, seal the silage pit, and begin fermentation at 8℃; Step S3: Take 4.0 parts by weight of Lactobacillus curvature (active 1.3 × 10⁻⁶). 10 Add 1.0 part of pectinase (activity 1100 U / g), 0.7 parts of cellulase (activity 1600 U / g), and 1.7 parts of seaweed extract to 700 parts of deionized water and mix well to obtain auxiliary agent B solution; after fermentation for 8 days in step S2, spray the auxiliary agent B solution evenly on the surface of the silage raw material at a rate of 0.7 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material by 8 cm to ensure that the diluted auxiliary agent B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. The entire spraying and turning process should be completed within 20 minutes to reduce air entry. Then, re-compact the material mechanically, cover it with a straw insulation layer, reseal the silage pit, check the integrity of the seal, and repair any damaged areas. Step S4: Continue anaerobic fermentation for a total fermentation period of 38 days. After fermentation, silage is obtained.

[0027] Analysis of experimental results: 1. Comprehensive evaluation of mixed silage quality This experiment comprehensively evaluated the quality of the mixed silage products prepared by each experimental group. The method referred to "NY / T4469-2025 Comprehensive Index Method for Comprehensive Evaluation of Whole Plant Maize Silage Quality", and the specific scoring criteria are shown in Table 1. An Agilent 1260 high-performance liquid chromatography (HPLC) system was used with a Shodex KC-811 column, a column temperature of 50℃, a flow rate of 1.0 mL / min, a detection wavelength of 210 nm, and an injection volume of 5 μL. The organic acid content was determined according to NY / T 2796-2015, the ammonia nitrogen content was determined by the phenol-sodium hypochlorite colorimetric method, and the total nitrogen content was determined by the Kjeldahl method according to GB / T 6432-2018.

[0028] Table 1: Comprehensive Quality Scoring Criteria for Mixed Silage

[0029] The comprehensive quality evaluation results of fermented mixed silage under different treatment groups are shown in Table 2: Table 2. Overall quality evaluation results of fermented mixed silage under different treatment groups

[0030] As can be seen from the experimental results in Table 2, Examples 1-2, which used a step-by-step addition of compound silage components, including adjuvant A and adjuvant B, achieved comprehensive quality evaluation scores of 99 and 97 points respectively, both reaching Grade I (excellent) standards. All indicators showed excellent performance: pH was close to 4.2, ammonia nitrogen / total nitrogen ≤9.8%, lactic acid / total organic acid ≥61.2%, and the sensory quality was yellow-green, with a rich sour aroma, loose and pliable texture, and no mold or stickiness. This indicates that under the aforementioned preparation process, oat straw-barley straw mixed silage can achieve efficient fermentation under cold conditions, resulting in better sensory quality. In contrast, Comparative Examples 1-10, when their preparation process was changed or one of the silage components was missing, showed significantly worse comprehensive quality evaluation results than Examples 1-2, with lower overall scores. Their processes are not suitable for silage fermentation under cold conditions.

[0031] 2. Determination of the nutritional quality of silage Take 300 g of mixed silage samples after fermentation from each experimental group, inactivate enzymes at 105℃ for 15 min, then dry in an oven at 65℃ until constant weight. After pulverizing, pass through a 40-mesh sieve and pack into self-sealing bags for the determination of nutritional quality of dry matter (DM), crude protein (CP), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), crude fat (EE), crude ash (Ash), starch (ST), and watersolule arohyrates (WSC). The determination of DM, CP, EE, and Ash follows the standards GB / T 6435-1986, GB / T 6432-1994, and GB / T 6435-1986, GB / T 6432-1994, and GB / T 6435-1986, respectively. The determination methods were based on GB / T 6433-1994 and GB / T 6438-1992. Crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined using the Van Soest method. Starch (ST) and soluble carbohydrates (WSC) were determined using Nanjing Jiancheng reagent kits (catalog numbers A148-1-1 and A145-1-1, respectively). The results are shown in Table 3.

[0032] Table 3. Effects of different treatment groups on the nutritional quality of fermented mixed silage

[0033] As shown in Table 3, the nutritional quality of the mixed fermented silage prepared in Examples 1-2 is significantly better than that of the comparative examples. Specifically, the dry matter content, as well as the contents of crude protein, starch, and soluble carbohydrates, are significantly higher than those in Comparative Examples 1-10, while the contents of crude fiber, neutral detergent fiber, acid detergent fiber, and acid detergent lignin are significantly lower than those in Comparative Examples 1-10. This indicates that the mixed silage prepared in Examples 1-2 has more reasonable moisture control, which is conducive to silage preservation. Its process is more conducive to the retention of protein and starch, while effectively degrading fibrous substances, improving palatability and digestibility, effectively improving the conversion rate of silage, improving feed taste, and thus helping to increase animal milk production and daily weight gain. Examples 1-2 of this invention can not only rapidly reduce the pH value in the early stage of fermentation, but also effectively block the consumption of substrate by harmful microorganisms, thereby effectively optimizing the nutritional indicators of fermentation products, improving the nutritional value, palatability, and digestibility of silage. In contrast, when the preparation process of Comparative Examples 1-10 is changed or one of the silage agent components is missing, the crude protein and starch content of the silage decreases, as does its palatability and digestibility.

[0034] In summary, this invention, by adding a compound probiotic silage agent in two stages and combining the effective components of adjuvants A and B, enables efficient fermentation under high-altitude and cold conditions, significantly improving the nutritional value, palatability, and digestibility of silage. The resulting mixed silage products all meet Grade I (superior) standards, exhibiting excellent performance across various indicators: pH close to 4.2, ammonia nitrogen / total nitrogen ≤9.8%, lactic acid / total organic acid ≥61.2%, and sensory quality characterized by a yellow-green color, rich sour aroma, loose and pliable texture, and absence of mold and stickiness. The process described in this invention facilitates the retention of protein and starch while effectively degrading fibrous substances, improving palatability and digestibility, thereby effectively increasing the conversion rate of silage and improving feed taste.

[0035] Therefore, those skilled in the art will recognize that although embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. A compound probiotic silage agent suitable for cold-season silage on the Qinghai-Tibet Plateau, characterized in that, It includes components A and B, wherein component A includes Lactobacillus plantarum, Bacillus subtilis, cellulase, xylanase, seaweed extract, and acetic acid, and component B includes Lactobacillus curvatureii, pectinase, β-mannanase, and seaweed extract.

2. The compound probiotic silage agent adapted for cold-season silage on the Qinghai-Tibet Plateau as described in claim 1, characterized in that, The weight ratio of each component in adjuvant A is as follows: Lactobacillus plantarum: Bacillus subtilis: Cellulase: Xylanase: Seaweed extract: Acetic acid = 2.5~4.5: 1.5~3.0: 0.8~1.8: 0.5~1.2: 1.0~2.0: 0.3~0.8, and the volume concentration of acetic acid is 30%~50%.

3. The compound probiotic silage agent adapted for cold-season silage on the Qinghai-Tibet Plateau as described in claim 1, characterized in that, The weight ratio of each component in adjuvant B is: Lactobacillus curvature: pectinase: β-mannanase: seaweed extract = 3.0~5.0: 0.6~1.5: 0.4~1.0: 1.2~2.

2.

4. A compound probiotic silage agent adapted for cold-season silage on the Qinghai-Tibet Plateau as described in claims 1-3, characterized in that, Lactobacillus plantarum viable count ≥1.0×10⁻⁶ 10 CFU / g, Bacillus subtilis viable count ≥8×10 9 CFU / g, viable count of Curvular Lactobacillus ≥1.2×10⁻⁶ 10 CFU / g.

5. The compound probiotic silage agent adapted for cold-season silage on the Qinghai-Tibet Plateau as described in claim 1, characterized in that, The weight ratio of additive A to additive B is 1.5~3:

1.

6. The application of the compound probiotic silage agent adapted to the cold season silage of the Qinghai-Tibet Plateau as described in claims 1 to 5 in the fermentation of cold season silage in the Qinghai-Tibet Plateau.

7. The application method as described in claim 6, characterized in that, Includes the following steps: Step S1: After harvesting the barley and oat straw, immediately crush them to 2-5 cm and mix them together. The weight ratio of the two mixtures should be 0.5-2:

1. Step S2: Spray the 0.5%~1.5% (w / w) solution of adjuvant A evenly onto the surface of the mixed silage raw material. The spraying amount is 0.6~1.5 L per 100 kg of silage raw material. Stir while spraying to ensure that the diluted adjuvant A solution is evenly mixed with the silage raw material. Then, fill, compact, and seal the mixed silage in layers and ferment it at 0~15℃. Step S3: On the 7th to 10th day after fermentation, spray the 0.5% to 1.5% (w / w) solution of adjuvant B evenly onto the surface of the silage raw material. The spraying amount is 0.4 to 1 L per 100 kg of silage raw material. After spraying, quickly turn over the surface layer of the raw material 5 to 10 cm to ensure that the diluted adjuvant B solution is fully mixed with the surface layer and the shallow inner layer of the raw material. Then reseal and compact the insulation layer. Step S4: Continue anaerobic fermentation, and silage is obtained after fermentation.

8. The application method as described in claim 7, characterized in that, In step S1, after the silage raw materials are crushed and mixed, the moisture content of the raw materials is adjusted to 60%~70%. The total fermentation cycle in steps S2~S4 is 30~45 days.

9. The application method as described in claim 7, characterized in that, In step S3, the addition of adjuvant B is performed on the 8th to 9th day after fermentation starts, and the spraying and turning process is completed within 30 minutes.

10. The application method as described in claim 7, characterized in that, In steps S2 and S3, mechanical compaction is used for compaction; the insulation layer is either straw or insulation cotton.

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