Ensilage fermented feed and preparation method thereof

Abstract

The invention provides an ensilage fermented feed and a preparation method thereof, which adopt a staged sequential fermentation strategy: firstly, trichoderma viride secretes cellulase under an aerobic condition to decompose fibers and release soluble sugar, and then lactobacillus rhamnosus quickly produces acid under an anaerobic environment to inhibit putrefying bacteria; the digestibility and the stability of the feed are obviously improved; meanwhile, pH-responsive nutritional microcapsules are also added into the raw materials during preparation of the ensilage fermented feed: pectin oligosaccharide, chitosan and cassava starch are used as wall materials, rosmarinic acid, calcium lactate and bacillus coagulans are embedded, and core materials are released by stages through pH responsiveness, so that the ensilage fermented feed is prepared; the nutritional value of the feed is improved, digestion and absorption are promoted, and the intestinal health of animals is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of feed processing, and particularly relates to silage fermented feed and a preparation method thereof. Background Art

[0002] Silage, an important source of nutrition for ruminants, is directly influenced by its fermentation process, which directly impacts its digestibility, nutritional value, and storage stability. Traditional silage technology, which relies on simultaneous fermentation with either a single or a combination of inoculants dominated by lactic acid bacteria, can inhibit some spoilage bacteria through acid production, but it still suffers from significant technical drawbacks.

[0003] First, in the co-fermentation process where composite microbial agents are added simultaneously, the metabolic competition and adaptability conflicts among different bacterial strains are difficult to reconcile. For example, lactic acid bacteria preferentially consume soluble sugars to rapidly produce acid, resulting in insufficient substrate for cellulolytic bacteria to fully degrade fiber, resulting in high crude fiber residues in the feed and limited digestibility. At the same time, bacterial strains differ significantly in their adaptability to environmental conditions such as temperature and pH. For example, the activity of cellulolytic bacteria is inhibited in an acidic environment (pH < 4.5), while the proliferation of lactic acid bacteria is hindered under high temperature conditions, resulting in low fermentation efficiency and insufficient stability.

[0004] Secondly, in the traditional process, the pH rises to above 5.0 in the late fermentation stage due to the consumption of lactic acid, creating conditions for the reproduction of mold, and the risk of feed moldiness increases significantly. In addition, feeds that rely solely on microbial fermentation have the problem of nutritional monotony, lacking minerals (such as calcium and phosphorus) and antioxidants, and are difficult to meet the growth needs of animals. Existing improved technologies attempt to improve feed quality by exogenously adding cellulase or complex nutrients, but free enzymes are easily inactivated in acidic environments, and directly supplemented minerals (such as phosphates) will neutralize the acid production process of lactic acid bacteria, while antioxidants (such as rosmarinic acid) are easily degraded by microorganisms or become ineffective prematurely in the early stages of fermentation, and cannot exert their antibacterial and antioxidant effects at critical stages.

[0005] Furthermore, while acid-resistant probiotics (such as Bacillus coagulans) can assist in acidifying the environment, they are inactive at high pH levels during the initial fermentation phase, making it difficult to achieve a synergistic effect with lactic acid bacteria. The lack of control over the timing of bacterial addition and the release of functional ingredients in existing technologies leads to problems such as incomplete fiber degradation, limited nutritional enhancement, and a short storage period. Therefore, there is an urgent need for a method for preparing silage fermented feed that can coordinate the timing of bacterial metabolism, precisely control the release of functional ingredients, and simultaneously improve the nutritional value and storage stability of the feed. Summary of the Invention

[0006] Technical problem to be solved: In response to the above technical problems, the purpose of the present invention is to provide an silage fermented feed and a preparation method thereof, which adopts a staged sequential fermentation strategy: first, Trichoderma viride is used to secrete cellulase under aerobic conditions to decompose fiber and release soluble sugars, and then Lactobacillus rhamnosus is used to quickly produce acid under an anaerobic environment to inhibit putrefactive bacteria, avoiding carbon source competition between bacterial species and low pH inhibition of fiber degradation, thereby significantly improving feed digestibility and stability; at the same time, the present invention also adds pH-responsive nutritional microcapsules to the raw materials when preparing the silage fermented feed: pectin oligosaccharides, chitosan and cassava starch are used as wall materials, rosmarinic acid, calcium lactate and Bacillus coagulans are embedded, and the core material is released in a pH-responsive staged manner - pectin oligosaccharides are released to supplement the carbon source during the Trichoderma viride fermentation stage (pH 5.0-6.5), and minerals, antioxidants and acid-resistant Bacillus are released during the Lactobacillus rhamnosus-dominated stage (pH 4.0-4.5), synergistically enhancing acidification to inhibit mildew, enhance the nutritional value of feed, promote digestion and absorption, and improve animal intestinal health.

[0007] Technical solution: A method for preparing silage fermented feed, comprising the following steps: S1 raw material mixing: 100-120 parts of corn stalks, 80-90 parts of sweet potato vines and 70-80 parts of alfalfa crushed to a length of 4-5cm, control the moisture at 60-70%, then add 10-15 parts of pH-responsive nutrient microcapsules, mix well to obtain a mixed raw material; S2 first fermentation: spraying the mixed raw material with a spore suspension of Trichoderma viride, fermenting for 3-5 days at 28-32 ° C, inactivating the fermentation raw material; S3. Secondary fermentation: Compact and seal the fermentation raw materials, spray Lactobacillus rhamnosus solution again, and ferment at 30-35°C for 15-20 days to produce silage fermented feed.

[0008] Furthermore, the pH-responsive nutrient microcapsules in step S1 are composed of a wall material and a core material, and the specific preparation steps are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a solution with a concentration of 4-5 wt%, then add pectinase, react in a 40-50°C water bath for 2-4 hours, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides; Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 2-3 wt%; Step 3. Dissolve cassava starch in water and heat to 60-70° C. to prepare a cassava starch solution with a concentration of 5-8 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 2-4 wt%, chitosan solution and cassava starch solution are added and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water, add rosmarinic acid, stir and dissolve at 60°C, adjust the pH to 3.5-4.5 to obtain a mixed solution, cool it, add Bacillus coagulans solution, mix well, and obtain a core material solution; Step 6. Slowly add the core material solution to the wall material solution, stir at 50°C for 0.5-1h, homogenize at 3000-5000rpm for 5-10min, and freeze-dry to obtain pH-responsive nutrient microcapsules.

[0009] Furthermore, the pectinase in step 1 includes pectin methylesterase and polygalacturonase; the added amount of the pectinase is 0.5-0.8% of the mass of citrus pectin.

[0010] Furthermore, in step 4, the volume ratio of the pectin oligosaccharide solution, the chitosan solution and the cassava starch solution is (4-6):2:(3-4).

[0011] Furthermore, in step 5, the mass volume ratio of calcium lactate, rosmarinic acid and water is (1-1.5) g: (0.05-0.07) g: 100 mL; the volume ratio of the mixed solution to the Bacillus coagulans liquid is (5-6): 1; the concentration of the Bacillus coagulans liquid is 1×10 8 -1×10 9 CFU / mL.

[0012] Furthermore, in step 6, the volume ratio of the core material solution to the wall material solution is 1:(3-4).

[0013] Furthermore, the concentration of the Trichoderma viride spore suspension in step S2 is 1×10 6 -1×10 7 CFU / mL, with an inoculum size of 10-15%.

[0014] Furthermore, the concentration of the Lactobacillus rhamnosus solution in step S3 is 1×10 8 -1×10 9 CFU / mL, the inoculum size is 3-5%.

[0015] Silage fermented feed prepared by the above preparation method. Beneficial effects

[0016] The present invention adopts a staged sequential fermentation strategy when preparing silage fermented feed. First, green Trichoderma fermentation is carried out under aerobic conditions, which can secrete cellulase and hemicellulase, efficiently decompose cellulose, hemicellulose and lignin in the raw materials, break the fiber composite structure, and improve the feed digestibility. At the same time, it can also release fermentable sugars (such as glucose and xylose) to provide substrates for subsequent lactic acid bacteria fermentation; secondly, rhamnosus lactobacillus fermentation is carried out under anaerobic conditions, which can quickly utilize soluble sugars to generate lactic acid, lower the pH, inhibit the formation of putrefactive bacteria and mycotoxins, reduce the ammonia nitrogen content, and improve the protein preservation rate, thereby effectively improving the quality of silage feed; if the fermentation is reversed, that is, rhamnosus lactobacillus fermentation is carried out first, the low pH environment may inhibit the enzyme activity of green Trichoderma, resulting in insufficient fiber degradation and affecting the feed digestibility; furthermore, rhamnosus lactobacillus has a high utilization rate of soluble sugars (glucose and fructose), and its rapid metabolism will deplete the free sugars in the raw materials. The subsequent addition of green Trichoderma will cause growth retardation due to lack of carbon source, affecting the overall fermentation efficiency; The present invention further adds pH-responsive nutritional microcapsules to the raw materials when preparing silage fermented feed. The microcapsules use pectin oligosaccharides, chitosan, and cassava starch as wall materials and encapsulate rosmarinic acid, calcium lactate, and Bacillus coagulans. During the Trichoderma viride fermentation stage (pH 5.0-6.5), the pectin oligosaccharides are used as a carbon source supplement, allowing the Trichoderma viride to utilize and promote fermentation. The overall structure of the microcapsule remains intact, only a portion of the pectin oligosaccharides is degraded, and the core material remains completely coated. During the Lactobacillus rhamnosus fermentation stage (pH 4.0-4.5), the Lactobacillus rhamnosus metabolizes to produce a large amount of lactic acid, rapidly lowering the environmental pH and triggering the acidic response mechanism of the microcapsule wall material: under acidic conditions, the amino groups (-NH2) in the chitosan molecular chain are protonated (-NH3+), resulting in the breaking of intermolecular hydrogen bonds. The glycosidic bonds of the pectin oligosaccharides are also further broken, accelerating the loosening of the wall material structure, thereby causing the wall material structure of the microcapsule to disintegrate and slowly releasing rosmarinic acid, calcium lactate, and Bacillus coagulans.

[0017] The microcapsules prepared by the present invention release rosmarinic acid, calcium lactate and Bacillus coagulans in response to acidic conditions. Rosmarinic acid can play an antioxidant role, protect lipids and microorganisms in feed, and prolong the storage period; calcium lactate can supplement minerals and improve the nutritional value of feed; Bacillus coagulans is an acid-resistant Bacillus that can germinate and metabolize to produce lactic acid in a low pH environment, assisting Lactobacillus rhamnosus to further acidify the environment, inhibit mold, improve intestinal health, and promote animal digestion and absorption; if the core material is not microencapsulated and protected, but rosmarinic acid is directly added, it is easy to be metabolized or oxidized by microorganisms (such as mold) If Bacillus coagulans is directly added, it can utilize soluble sugars to grow under aerobic conditions and compete with Trichoderma viride for carbon sources (such as glucose), which may lead to a decrease in the enzyme production efficiency of Trichoderma due to insufficient substrate. At the same time, the activity of Bacillus coagulans may decline due to metabolic consumption in the early stage, resulting in the inability to continuously produce acid and maintain a low pH environment in the later stage of fermentation. The mold inhibition effect is weakened, affecting the acidification process of silage fermentation. Therefore, the present invention chooses to adopt microencapsulation technology for rosmarinic acid, calcium lactate and Bacillus coagulans, which is more conducive to improving the nutritional value of silage. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples: Example 1

[0019] The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 4:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1g:0.05g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:3, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules.

[0020] Example 2 The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 4:2:4 (i.e., 2:1:2), and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1g:0.05g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:3, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Example 3

[0021] The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1g:0.05g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:3, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Example 4

[0022] The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add polygalacturonase at a concentration of 0.8% by weight of the pectin. React in a 50°C water bath for 3 hours, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 6:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1g:0.05g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:3, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Example 5

[0023] The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1.5g:0.07g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:3, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Example 6

[0024] The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1g:0.07g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:3, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Example 7

[0025] The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1.5g:0.07g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:4, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules.

[0026] Comparative Example 1 The difference between this comparative example and Example 7 is that pectin oligosaccharide is replaced with ordinary pectin, specifically as follows: The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 2. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 3. Dissolve citrus pectin in water and heat to 70°C to dissolve to prepare a 3 wt% pectin solution. Then, add the chitosan solution and the cassava starch solution in a volume ratio of 5:2:3 and stir evenly to obtain a wall material solution. Step 4. Dissolve calcium lactate in water and add rosmarinic acid. The mass volume ratio of calcium lactate, rosmarinic acid and water is 1.5g:0.07g:100mL. Stir and dissolve at 60℃. Adjust the pH to 4.0 to obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 5. Slowly add the core material solution to the wall material solution at a volume ratio of 1:4, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Comparative Example 2

[0027] The difference between this comparative example and Example 7 is that rosmarinic acid is not added, specifically as follows: The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water at a mass volume ratio of 1.5 g to 100 mL, stir and dissolve at 60 °C, adjust the pH to 4.0, and obtain a mixed solution. After cooling, add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:4, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Comparative Example 3

[0028] The difference between this comparative example and Example 7 is that calcium lactate is not added, specifically as follows: The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve rosmarinic acid in water with a mass volume ratio of rosmarinic acid to water of 0.07 g:100 mL to obtain a rosmarinic acid solution, and then add 1×10 9 CFU / mL of Bacillus coagulans bacterial solution, with a volume ratio of 5:1, were mixed evenly to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:4, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules. Comparative Example 4

[0029] The difference between this comparative example and Example 7 is that Bacillus coagulans was not added, specifically as follows: The specific preparation steps of pH-responsive nutrient microcapsules are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a 4 wt% solution, then add pectin methylesterase at 0.8% of the pectin mass, react in a 50°C water bath for 3 h, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides. Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%; Step 3. Dissolve cassava starch in water and heat to 70° C. to prepare a cassava starch solution with a concentration of 6 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 3 wt%, chitosan solution and cassava starch solution were added at a volume ratio of 5:2:3, and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water, then add rosmarinic acid in a mass volume ratio of calcium lactate, rosmarinic acid, and water of 1.5 g:0.07 g:100 mL, stir and dissolve at 60°C, and adjust the pH to 4.0 to prepare a core material solution; Step 6. Slowly add the core material solution to the wall material solution at a volume ratio of 1:4, stir at 50°C for 1 hour, homogenize at 4000 rpm for 10 minutes, and freeze-dry to obtain pH-responsive nutrient microcapsules.

[0030] Performance testing: (1) Microcapsule embedding rate The microcapsule structure was destroyed by high-speed centrifugation, and the entrapment rate of rosmarinic acid was determined by HPLC, the entrapment rate of calcium lactate was determined by atomic absorption spectrometry, and the entrapment rate of Bacillus coagulans was determined by plate colony counting.

[0031] Table 1 Encapsulation efficiency of rosmarinic acid, calcium lactate and Bacillus coagulans in microcapsules

[0032] As can be seen from Table 1, the encapsulation efficiency of rosmarinic acid in the microcapsules prepared in Examples 1-7 was 86.4-89.3%, the encapsulation efficiency of calcium lactate was 80.8-82.6%, and the encapsulation efficiency of Bacillus coagulans was 91.5-92.4%. In Comparative Example 1, pectin oligosaccharides were replaced with ordinary pectin. Ordinary pectin has a long molecular chain and high rigidity, which increases the porosity of the microcapsules and causes the loss of core material, thereby reducing the encapsulation efficiency.

[0033] (2) Microcapsule release rate The microcapsules were placed in a pH 4.0 buffer solution to simulate the fermentation stage of Lactobacillus rhamnosus in the microcapsules, and the release rates of rosmarinic acid, calcium lactate and Bacillus coagulans in the microcapsules were measured.

[0034] Table 2 Release rates of rosmarinic acid, calcium lactate and Bacillus coagulans from microcapsules

[0035] As can be seen from Table 2, when the microcapsules are in the rhamnose fermentation stage, the release rates of rosmarinic acid, calcium lactate and Bacillus coagulans are all high. Among them, the release rate of rosmarinic acid in the microcapsules prepared in Example 7 is 92.4%, the release rate of calcium lactate is 93.7%, and the release rate of Bacillus coagulans is 96.4%. Therefore, the microcapsules prepared in Example 7 were selected for the subsequent preparation of silage fermentation feed.

[0036] Example 8 A method for preparing silage fermented feed comprises the following steps: S1 raw material mixing: 100kg corn stalks, 90kg sweet potato vines and 80kg alfalfa were crushed to a length of 4cm, controlling the moisture content at 60%, and then 15kg of the microcapsules prepared in Example 7 were added and mixed to obtain a mixed raw material; S2. First fermentation: Spray the mixed raw materials with a concentration of 1×10 7 CFU / mL of Trichoderma viride spore suspension, with an inoculation amount of 15%, was fermented at 30°C for 5 days and inactivated to obtain the fermentation raw material; S3. Secondary fermentation: Compact and seal the fermentation raw materials and spray again with a concentration of 1×10 9 The rhamnosus Lactobacillus liquid with a CFU / mL and an inoculation amount of 5% was fermented at 35°C for 15 days to prepare silage fermented feed.

[0037] Comparative Example 5 The difference between this comparative example and Example 8 is that no microcapsules were added, as follows: A method for preparing silage fermented feed comprises the following steps: S1 raw material mixing: 100kg corn stalks, 90kg sweet potato vines and 80kg alfalfa crushed to a length of 4cm, control the moisture at 60%, mix well to obtain a mixed raw material; S2. First fermentation: Spray the mixed raw materials with a concentration of 1×10 7 CFU / mL of Trichoderma viride spore suspension, with an inoculation amount of 15%, was fermented at 30°C for 5 days and inactivated to obtain the fermentation raw material; S3. Secondary fermentation: Compact and seal the fermentation raw materials and spray again with a concentration of 1×10 9 The rhamnosus Lactobacillus liquid with a CFU / mL and an inoculation amount of 5% was fermented at 35°C for 15 days to prepare silage fermented feed.

[0038] Comparative Example 6 The difference between this comparative example and Example 8 is that rosmarinic acid, calcium lactate and Bacillus coagulans are directly added, specifically as follows: A method for preparing silage fermented feed comprises the following steps: S1. Raw material mixing: 100 kg corn stalks, 90 kg sweet potato vines and 80 kg alfalfa were crushed to a length of 4 cm, and the moisture content was controlled at 60%. Then 1.1 kg calcium lactate, 51.7 g rosmarinic acid and 1.48 × 10 10 CFU coagulans bacterial solution, mixed well to obtain a mixed raw material; S2. First fermentation: Spray the mixed raw materials with a concentration of 1×10 7 CFU / mL of Trichoderma viride spore suspension, with an inoculation amount of 15%, was fermented at 30°C for 5 days and inactivated to obtain the fermentation raw material; S3. Secondary fermentation: Compact and seal the fermentation raw materials and spray again with a concentration of 1×10 9 The rhamnosus Lactobacillus liquid with a CFU / mL and an inoculation amount of 5% was fermented at 35°C for 15 days to prepare silage fermented feed.

[0039] Comparative Example 7 The difference between this comparative example and Example 8 is that only Trichoderma viride was inoculated, specifically as follows: A method for preparing silage fermented feed comprises the following steps: S1 raw material mixing: 100kg corn stalks, 90kg sweet potato vines and 80kg alfalfa were crushed to a length of 4cm, controlling the moisture content at 60%, and then 15kg of the microcapsules prepared in Example 7 were added and mixed to obtain a mixed raw material; S2. Fermentation: Spray the mixed raw materials with a concentration of 1×10 7 The spore suspension of Trichoderma viride with a CFU / mL and an inoculation amount of 20% was fermented at 30°C for 20 days to prepare silage fermented feed.

[0040] Comparative Example 8 The difference between this comparative example and Example 8 is that only Lactobacillus rhamnosus was inoculated, specifically as follows: A method for preparing silage fermented feed comprises the following steps: S1 raw material mixing: 100kg corn stalks, 90kg sweet potato vines and 80kg alfalfa were crushed to a length of 4cm, controlling the moisture content at 60%, and then 15kg of the microcapsules prepared in Example 7 were added and mixed to obtain a mixed raw material; S2. Fermentation: Compact and seal the mixed raw materials and spray with a concentration of 1×10 9 The Lactobacillus rhamnosus liquid with a CFU / mL and an inoculation amount of 20% was fermented at 35°C for 20 days to prepare silage fermented feed.

[0041] Comparative Example 9 The difference between this comparative example and Example 8 is that synchronous fermentation is adopted, that is, Trichoderma viride and Lactobacillus rhamnosus are added simultaneously for fermentation, specifically as follows: A method for preparing silage fermented feed comprises the following steps: S1 raw material mixing: 100kg corn stalks, 90kg sweet potato vines and 80kg alfalfa were crushed to a length of 4cm, controlling the moisture content at 60%, and then 15kg of the microcapsules prepared in Example 7 were added and mixed to obtain a mixed raw material; S2. Fermentation: Spray the mixed raw materials with a concentration of 1×10 7 CFU / mL of Trichoderma viride spore suspension, the inoculation amount was 15%, and the spraying concentration was 1×10 9The Lactobacillus rhamnosus liquid with a CFU / mL and an inoculation amount of 5% was fermented at 30°C for 20 days to prepare silage fermented feed.

[0042] Comparative Example 10 The difference between this comparative example and Example 8 is that the reverse fermentation is adopted, that is, the Lactobacillus rhamnosus fermentation is carried out first, and then the Trichoderma viride fermentation is carried out, as follows: A method for preparing silage fermented feed comprises the following steps: S1 raw material mixing: 100kg corn stalks, 90kg sweet potato vines and 80kg alfalfa were crushed to a length of 4cm, controlling the moisture content at 60%, and then 15kg of the microcapsules prepared in Example 7 were added and mixed to obtain a mixed raw material; S2. First fermentation: Compact and seal the mixed raw materials and spray with a concentration of 1×10 9 CFU / mL of Lactobacillus rhamnosus liquid, with an inoculation amount of 5%, was fermented at 35°C for 15 days and inactivated to obtain a fermentation raw material; S3. Secondary fermentation: Spray the fermentation raw materials again with a concentration of 1×10 7 The silage fermented feed was prepared by fermenting a suspension of Trichoderma viride spores with an inoculation rate of 15% at 30°C for 5 days.

[0043] Performance testing: (1) Analysis of feed nutritional components Organic acids (lactic acid, acetic acid) were determined by HPLC; Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using the Van der Waals method; The dry matter (DM) content was determined by drying in a vacuum drying oven at 105 °C to constant weight; Crude protein was determined using the national standard micro-Kjeldahl nitrogen method; Ammoniacal nitrogen content was determined by distillation-titration method; The mineral content was determined by EDTA complexometric titration.

[0044] Table 3 Determination of nutrients in silage fermented feed prepared in Example 8 and Comparative Examples 5-10

[0045] As can be seen from Table 3, the present invention adopts a specific fermentation sequence to prepare silage fermented feed, firstly fermenting with Trichoderma viride and then fermenting with Lactobacillus rhamnosus, wherein Lactobacillus rhamnosus can efficiently produce lactic acid after fermentation, and the acetic acid content is low; Trichoderma viride can efficiently decompose cellulose, while if only relying on Lactobacillus rhamnosus fermentation (Comparative Example 8), the fiber in the raw material cannot be fully decomposed, resulting in high residual neutral detergent fiber and acid detergent fiber; compared with other comparative examples, the feed prepared by the fermentation method of the present invention has a lower ammonia nitrogen content and an improved protein preservation rate, while the other comparative examples have insufficient acidification or a single strain, and the putrefactive bacteria produced decompose the protein, resulting in a lower crude protein content and a higher ammonia nitrogen content; and the calcium lactate released in the microcapsules supplements the mineral calcium content, which is beneficial to improving the nutritional value of the feed and effectively improving the quality of the silage feed.

[0046] (2) Determination of total mold count and mycotoxins The total number of molds was determined according to GB / T 13092-2006 “Determination of the total number of molds in feeds”; The determination of mycotoxins such as aflatoxin B1 adopts T / SDAA 0049-2021 "Rapid Determination of Aflatoxin B1, Zearalenone and Doxynivalenol in Feed by Upconversion Luminescence Method".

[0047] Table 4 Determination of total mold count and aflatoxin B1 in silage fermented feed prepared in Example 8 and Comparative Examples 5-10

[0048] As can be seen from Table 4, the staged fermentation method adopted by the present invention, combined with the sustained release of rosmarinic acid and Bacillus coagulans by microcapsules, can effectively inhibit the growth of putrefactive bacteria and molds. Comparative Example 5 did not add microcapsules and lacked sustained release protection; Comparative Example 6 directly added rosmarinic acid, calcium lactate and Bacillus coagulans, and the content of mold and putrefactive bacteria was relatively high; Comparative Example 7 was fermented only by Trichoderma viride, and although the fiber degradation was sufficient, the acidification was lacking to inhibit putrefactive bacteria; Comparative Example 8 was fermented only by Lactobacillus rhamnosus, and some putrefactive bacteria and mycotoxins remained; and Comparative Example 9 was fermented simultaneously and Comparative Example 10 was fermented in reverse order, and the total mold count and aflatoxin AFB1 content were both higher than those in Example 9.

[0049] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing silage fermented feed, characterized in that: The following steps are involved: S1 raw material mixing: 100-120 parts of corn stalks, 80-90 parts of sweet potato vines and 70-80 parts of alfalfa crushed to a length of 4-5cm, control the moisture at 60-70%, then add 10-15 parts of pH-responsive nutrient microcapsules, mix well to obtain a mixed raw material; S2 first fermentation: spraying the mixed raw material with a spore suspension of Trichoderma viride, fermenting for 3-5 days at 28-32 ° C, inactivating the fermentation raw material; S3. Secondary fermentation: Compact and seal the fermentation raw materials, spray Lactobacillus rhamnosus solution again, and ferment at 30-35°C for 15-20 days to produce silage fermented feed.

2. The method for preparing silage fermented feed according to claim 1, characterized in that: The pH-responsive nutrient microcapsules in step S1 are composed of a wall material and a core material, and the specific preparation steps are as follows: Step 1. Dissolve citrus pectin in 0.1M citric acid-disodium hydrogen phosphate buffer at pH 4.5 to prepare a solution with a concentration of 4-5 wt%, then add pectinase, react in a 40-50°C water bath for 2-4 hours, inactivate the enzyme, centrifuge, concentrate, and spray-dry the supernatant to obtain pectin oligosaccharides; Step 2. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 2-3 wt%; Step 3. Dissolve cassava starch in water and heat to 60-70° C. to prepare a cassava starch solution with a concentration of 5-8 wt %; Step 4. After dissolving pectin oligosaccharides in water to prepare a pectin oligosaccharide solution with a concentration of 2-4 wt%, chitosan solution and cassava starch solution are added and stirred evenly to obtain a wall material solution; Step 5. Dissolve calcium lactate in water, add rosmarinic acid, stir and dissolve at 60°C, adjust the pH to 3.5-4.5 to obtain a mixed solution, cool it, add Bacillus coagulans solution, mix well, and obtain a core material solution; Step 6. Slowly add the core material solution to the wall material solution, stir at 50°C for 0.5-1h, homogenize at 3000-5000rpm for 5-10min, and freeze-dry to obtain pH-responsive nutrient microcapsules.

3. The method for preparing silage fermented feed according to claim 2, wherein: The pectinase in step 1 includes pectin methylesterase and polygalacturonase; the added amount of the pectinase is 0.5-0.8% of the mass of citrus pectin.

4. The method for preparing silage fermented feed according to claim 2, wherein: In step 4, the volume ratio of the pectin oligosaccharide solution, the chitosan solution and the cassava starch solution is (4-6):2:(3-4).

5. The method for preparing silage fermented feed according to claim 2, characterized in that: In step 5, the mass volume ratio of calcium lactate, rosmarinic acid and water is (1-1.5) g: (0.05-0.07) g: 100 mL; the volume ratio of the mixed solution to the Bacillus coagulans liquid is (5-6): 1; the concentration of the Bacillus coagulans liquid is 1×10 8 -1×10 9 CFU / mL.

6. The method for preparing silage fermented feed according to claim 2, characterized in that: In step 6, the volume ratio of the core material solution to the wall material solution is 1:(3-4).

7. The method for preparing silage fermented feed according to claim 1, wherein: The concentration of the Trichoderma viride spore suspension in step S2 is 1×10 6 -1×10 7 CFU / mL, with an inoculum size of 10-15%.

8. The method for preparing silage fermented feed according to claim 1, wherein: The concentration of the Lactobacillus rhamnosus solution in step S3 is 1×10 8 -1×10 9 CFU / mL, the inoculum size is 3-5%.

9. Silage fermented feed prepared by the preparation method according to any one of claims 1 to 8.

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