A method for preparing a rumen microorganism enzyme fermented feed for ruminants

Abstract

The present application belongs to the technical field of feed processing, and discloses a preparation method of a ruminant fungus enzyme fermented feed, comprising the following steps: S1. low-pressure steam and organic acid are used for synergistic pretreatment of straws; S2. microcapsule-encapsulated composite microbial agents are added to the pretreated straws together with a nutrient substrate, and then mixed thoroughly, and then fermented at 18-22 DEG C for 3-4 weeks to obtain the ruminant fungus enzyme fermented feed; the microcapsule-encapsulated composite microbial agents are formed by layer-by-layer self-assembly technology using sodium alginate and chitosan to form an inner film after mixing of composite microbial species, trehalose and pregelatinized starch, and then coated by hydroxypropyl methylcellulose phthalate to form an outer film; the nutrient substrate is a composite of multiple functional components; the preparation method of the ruminant fungus enzyme fermented feed can efficiently degrade anti-nutritional factors in straws, significantly improve the palatability, digestibility and nutritional value of the feed, and simultaneously enhance rumen health and production performance of ruminants.

Description

Technical Field

[0001] This invention belongs to the field of feed processing technology, specifically relating to a method for preparing fermented feed for ruminants. Background Technology

[0002] Microbial-enzyme synergistic fermentation of feed organically combines the efficacy of added enzymes and microbial strains. Through the synergistic effect of fermentation processes and enzyme engineering technologies, fermentation time is significantly shortened, thereby effectively improving fermentation production efficiency and obtaining high-quality fermented products. Simultaneously, using microbial-enzyme synergistic fermentation in feed allows for more complete degradation and fermentation of certain nutrients. By enhancing the conversion and utilization rate of specific substances in feed through microbial fermentation, the content of anti-nutritional factors in feed is reduced, the content of specific products is increased, feed flavor and palatability are improved, and the utilization value of feed ingredients is enhanced.

[0003] The invention patent with publication number CN120458186A discloses a fermented wheat straw feed and its preparation method. The method involves crushing wheat straw, mixing it with cornmeal, adding a compound bacterial solution, and then anaerobically fermenting it in a fermentation container for 10-30 days. This method improves the palatability and nutritional value of wheat straw by mixing it with cornmeal and using a unique fermentation compound bacterial solution. However, when fermentation agents or enzymes are directly added, most of the active ingredients are rapidly inactivated or degraded in the highly acidic and microbially competitive environment of the rumen after feeding to ruminants. These ingredients cannot effectively reach the hindgut to exert their intended probiotic or enzymatic functions, resulting in resource waste and limited ability to precisely control rumen fermentation. Furthermore, most of the nutritional additives used (cornmeal) are relatively simple, providing only basic carbon and nitrogen sources. They are not precisely designed to address the metabolic characteristics of low-temperature fermentation bacteria and the nutritional needs of ruminants, and cannot effectively activate and maintain the synergistic degradation activity of the compound bacterial enzymes, especially in terms of the efficiency of treating recalcitrant components such as lignin. Summary of the Invention

[0004] To address the shortcomings mentioned in the background art, the present invention aims to provide a method for preparing fermented feed for ruminants using microbial enzymes. By pretreating straw with low-pressure steam and organic acids, and then mixing it with capsule-encapsulated compound microbial agents and nutrient substrates for fermentation, the method can efficiently degrade anti-nutritional factors in straw, significantly improve the palatability, digestibility, and nutritional value of the feed, and enhance rumen health and production performance of ruminants.

[0005] The objective of this invention can be achieved through the following technical solutions: A method for preparing fermented feed for ruminants using microbial enzymes includes the following steps: S1. After drying the straw, crush it to 2-5 mm. Treat the crushed straw in saturated steam at 0.3-0.5 MPa and 110-120℃ for 15-20 min. Immediately afterward, spray it with a mixed organic acid solution with pH 3.5-4.0 at a rate of 10-15% of the dry weight of the straw. After treatment, adjust the moisture content of the material to 60-65%. S2. Add the microencapsulated compound microbial agent and nutrient matrix to the pretreated straw and mix thoroughly. Pack the straw into a fermentation tank and compact it. Control the fermentation temperature at 18~22℃ and ferment for 3~4 weeks. Intermittent aeration management with turning the pile 1~2 times a week to ensure adequate oxygen supply. After fermentation, the fermented feed for ruminants is obtained. The microcapsule-encapsulated compound bacterial agent is formed by mixing compound bacterial strains with trehalose and pregelatinized starch, then using sodium alginate and chitosan to form an inner membrane through layer-by-layer self-assembly technology, and then encapsulating it with hydroxypropyl methylcellulose phthalate to form an outer membrane. The nutrient matrix includes microcrystalline cellulose, xylooligosaccharides, fermented soybean meal, yeast hydrolysate, isopropyl methionine hydroxy analogue, nicotinic acid, zinc sulfate, manganese sulfate, lanthanum chloride, and modified attapulgite.

[0006] Preferably, the straw is one or a mixture of corn straw, wheat straw and rice straw, and the mixed organic acid solution is a compound of citric acid, malic acid and lactic acid.

[0007] Preferably, the microcapsule-encapsulated complex bacterial strains are calculated at 1×10⁻⁶ effective viable bacteria count. 6 ~5×10 6 The ratio of CFU / g dry weight of straw is added to the pretreated straw.

[0008] Preferably, the compound microbial strain includes Trichoderma reesei, Bacillus subtilis, and white-rot fungi.

[0009] Preferably, the preparation method of microcapsule-encapsulated compound microbial agent includes the following steps: (1) Trichoderma reesei was cultured to obtain spore suspension and fermentation supernatant rich in extracellular enzymes. Bacillus subtilis was cultured and centrifuged to collect the cells and resuspended in physiological saline. White rot fungi were cultured to obtain white rot fungal hyphae. (2) Mix Trichoderma reesei spore suspension, Bacillus subtilis suspension and white rot fungal hyphae, then add 20% trehalose and 15% pregelatinized corn starch of the total weight of the compound strain, and homogenize at 4℃ to form a viscous and uniform mixed slurry. (3) Mix the mixed slurry and sodium alginate solution at a volume ratio of 1:3. Drop the mixture into a 1.5wt% calcium chloride solution to form gel microspheres. Stir and solidify for 15-25 minutes. After filtration, wash with deionized water to obtain composite bacterial agent sodium alginate microspheres. (4) Immerse the composite bacterial agent sodium alginate microspheres in a 0.5wt% chitosan acetate solution, adjust the pH to 5~5.5, stir for 10~20min, then transfer the microspheres into a 0.5wt% sodium alginate solution and stir for 10~20min. Repeat this process 2~4 times to deposit and form a multilayer chitosan sodium alginate composite inner membrane. (5) Dissolve 6 wt% hydroxypropyl methylcellulose phthalate in 80 vol% ethanol aqueous solution, stir thoroughly until completely dissolved and clear, cool to 4°C and add cellulase and xylanase. Place the microspheres with protective film obtained in step (4) into a fluidized bed coating machine, and spray the enzyme-containing hydroxypropyl methylcellulose phthalate solution evenly onto the surface of the microspheres in the form of atomization through a micro sprayer. The hydroxypropyl methylcellulose phthalate forms an outer film on the surface of the microspheres. Dry at room temperature for 24 h to obtain microcapsule-encapsulated composite bacterial agent.

[0010] Preferably, in step (1), Trichoderma reesei is activated at 28°C using potato dextrose agar plates, then inoculated into a liquid fermentation medium containing microcrystalline cellulose, and cultured at 28°C and 180 rpm for 72-96 h with shaking. The spore suspension and fermentation supernatant rich in extracellular enzymes are harvested, and the supernatant is concentrated 10 times by centrifugation to obtain enzyme solution. Bacillus subtilis was activated on LB plates at 30°C, inoculated into LB liquid medium, and cultured at 30°C with shaking at 200 rpm until the late logarithmic growth stage. The cells were collected by centrifugation and resuspended in physiological saline. White-rot fungi were slowly activated on malt extract agar plates at 25°C for 7-10 hours. Mycelial blocks were then inoculated onto nitrogen-limited liquid culture medium and incubated statically at 25°C for 10-14 days. Mycelial balls were then collected and appropriately broken up.

[0011] Preferably, in step (2), the Trichoderma reesei spore suspension, Bacillus subtilis suspension and white rot fungal hyphae are mixed in a spore-cell ratio of 5:3:2.

[0012] Preferably, the method for preparing the nutrient substrate includes the following steps: A. Crush natural attapulgite, calcine it at 500℃ for 1.5~2.5h, cool it and mix it with citric acid solution. Stir and react in a 70℃ water bath for 3~5h. After the reaction is completed, wash it repeatedly with deionized water until the filtrate is neutral. Dry the filter cake to constant weight and crush it through a 200-mesh sieve to obtain modified attapulgite. B. Add zinc sulfate, manganese sulfate and lanthanum chloride to warm water at 40~50℃ and stir until completely dissolved to obtain a trace element concentrate. Dissolve methionine hydroxy analogue isopropyl ester and nicotinic acid in warm water to obtain a nutrient element precursor solution. C. Add the modified attapulgite to the mixer, and while stirring, spray the concentrated trace element solution onto the attapulgite using a spraying device. After spraying, continue mixing for 5-15 minutes. Then, evenly spray the nutrient element precursor solution onto the modified attapulgite carrier loaded with trace elements, and continue mixing for 10-20 minutes to allow the liquid components to be fully adsorbed by the carrier. D. Add microcrystalline cellulose, xylooligosaccharides, fermented soybean meal and yeast hydrolysate to the mixer in sequence, mix for 20-30 minutes, dry with low-temperature airflow until the moisture content is ≤10%, and pass through a 60-mesh sieve to obtain the nutrient matrix.

[0013] Preferably, attapulgite clay is mixed with 1 mol / L citric acid solution at a solid-liquid ratio of 1:5.

[0014] Preferably, the content of each raw material in the nutrient matrix is ​​as follows: 2-3 parts microcrystalline cellulose, 0.6-1.0 parts xylooligosaccharide, 1.0-1.4 parts fermented soybean meal, 0.6-1.0 parts yeast hydrolysate, 0.04-0.06 parts isopropyl methionine hydroxy analogue, 0.01-0.03 parts nicotinic acid, 0.01-0.02 parts zinc sulfate, 0.005-0.015 parts manganese sulfate, 0.001-0.003 parts lanthanum chloride, and 1.0-2 parts modified attapulgite.

[0015] The beneficial effects of this invention are: This invention utilizes low-pressure steam and organic acids to synergistically pretreat straw, followed by fermentation with encapsulated compound microbial agents and a nutrient substrate. This process efficiently degrades anti-nutritional factors in straw, significantly improving feed palatability, digestibility, and nutritional value, while also enhancing rumen health and production performance in ruminants. The nutrient substrate provides an optimal growth environment for the microorganisms within the microcapsules after exocapsulation, while the microcapsules protect and efficiently deliver the nutrient-optimized microbial community. Together, they construct a stable, efficient, and controllable solid-state fermentation system, ultimately producing fermented feed for ruminants with significantly improved nutritional value and feeding efficacy.

[0016] This invention utilizes a microcapsule-encapsulated compound microbial agent. The compound microbial strain is mixed with trehalose and pregelatinized starch, and an inner membrane is formed using sodium alginate and chitosan through a layer-by-layer self-assembly technique. An outer membrane is then formed by encapsulation with hydroxypropyl methylcellulose phthalate. The sodium alginate-chitosan inner membrane provides a physical barrier for the microorganisms, resisting environmental stresses during fermentation and enabling the slow release of active substances. The hydroxypropyl methylcellulose phthalate outer membrane imparts pH-responsive properties to the microcapsules, allowing them to pass through the rumen and release into the intestine. This alters the sites of action and rhythms of functional microorganisms and enzymes in the digestive tract of ruminants, achieving precise nutritional regulation, improving feed utilization, and promoting hindgut health.

[0017] The nutrient matrix of this invention is a composite of various functional components. Xylooligosaccharides and other readily available carbon sources, along with yeast hydrolysate and other organic nitrogen sources, provide the microorganisms with the energy to initiate fermentation, ensuring the rapid establishment of the microbial community in the early stages. Microcrystalline cellulose, as a slow-release carbon source, continuously induces cellulase secretion. Methionine hydroxy analogue isopropyl ester and nicotinic acid precisely supplement limiting nutrients, optimizing the microbial metabolic flux. In particular, extremely low concentrations of lanthanum chloride can significantly improve the efficiency and stability of key enzymes at low temperatures by stabilizing enzyme protein conformation or participating in electron transfer at the enzyme's active site. The porous structure of modified attapulgite adsorbs and slowly releases nutrients, making the nutrient supply more matched to microbial consumption, maintaining the continuity of metabolism in the later stages of fermentation, and preventing premature nutrient depletion.

[0018] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: A microcapsule-encapsulated composite bacterial agent. The microcapsule-encapsulated composite bacterial agent is prepared by mixing a composite bacterial strain with trehalose and pregelatinized starch, then using sodium alginate and chitosan to form an inner membrane through a layer-by-layer self-assembly technique, and finally encapsulating it with hydroxypropyl methylcellulose phthalate to form an outer membrane. The preparation method includes the following steps: (1) Trichoderma reesei was activated at 28°C using potato dextrose agar plates and then inoculated into a liquid fermentation medium containing microcrystalline cellulose. The medium was cultured at 28°C and 180 rpm for 84 h with shaking. The spore suspension and the fermentation supernatant rich in extracellular enzymes were harvested. The supernatant was concentrated 10 times by centrifugation to obtain the enzyme solution. Bacillus subtilis was activated on LB plates at 30°C, inoculated into LB liquid medium, and cultured at 30°C and 200 rpm with shaking until the late logarithmic growth phase. The cells were collected by centrifugation and resuspended in physiological saline. The white-rot fungus was slowly activated at 25°C for 8.5 hours using malt extract agar plates. The mycelial blocks were then inoculated into nitrogen-limited liquid medium and incubated at 25°C for 12 days. The mycelial balls were then collected and appropriately broken up. (2) Mix Trichoderma reesei spore suspension, Bacillus subtilis suspension and white rot fungal hyphae in a spore-to-cell ratio of 5:3:2, then add 20% trehalose and 15% pregelatinized corn starch of the total weight of the compound strain, and homogenize at 4°C to form a viscous and uniform mixed slurry. (3) Mix the mixed slurry and sodium alginate solution at a volume ratio of 1:3. Drop the mixture into a 1.5wt% calcium chloride solution to form gel microspheres. Stir and solidify for 20 minutes. After filtration, wash with deionized water to obtain composite bacterial agent sodium alginate microspheres. (4) Immerse the composite bacterial agent sodium alginate microspheres in a 0.5wt% chitosan acetate solution, adjust the pH to 5~5.5, stir for 15min, then transfer the microspheres into a 0.5wt% sodium alginate solution and stir for 15min. Repeat this process 2~4 times to deposit and form a multilayer chitosan sodium alginate composite inner membrane. (5) Dissolve 6 wt% hydroxypropyl methylcellulose phthalate in 80 vol% ethanol aqueous solution, stir thoroughly until completely dissolved and clear, cool to 4°C and add cellulase and xylanase. Place the microspheres with protective film obtained in step (4) into a fluidized bed coating machine, and spray the enzyme-containing hydroxypropyl methylcellulose phthalate solution evenly onto the surface of the microspheres in the form of atomization through a micro sprayer. The hydroxypropyl methylcellulose phthalate forms an outer film on the surface of the microspheres. Dry at room temperature for 24 h to obtain microcapsule-encapsulated composite bacterial agent.

[0021] Example 2: A nutrient matrix comprises 2.5 parts microcrystalline cellulose, 0.8 parts xylooligosaccharides, 1.0-1.4 parts fermented soybean meal, 0.8 parts yeast hydrolysate, 0.05 parts isopropyl methionine hydroxy analogue, 0.02 parts nicotinic acid, 0.015 parts zinc sulfate, 0.01 parts manganese sulfate, 0.002 parts lanthanum chloride, and 1.5 parts modified attapulgite. The preparation method of the nutrient matrix includes the following steps: A. Crush natural attapulgite, calcine at 500℃ for 2 hours, cool and mix attapulgite with 1 mol / L citric acid solution at a solid-liquid ratio of 1:5, stir and react in a 70℃ water bath for 4 hours, after the reaction is completed, wash repeatedly with deionized water until the filtrate is neutral, dry the filter cake to constant weight and crush it through a 200-mesh sieve to obtain modified attapulgite. B. Add zinc sulfate, manganese sulfate and lanthanum chloride to warm water at 45°C and stir until completely dissolved to obtain a concentrated trace element solution. Dissolve isopropyl methionine hydroxy analogue and nicotinic acid in warm water to obtain a nutrient element precursor solution. C. Add the modified attapulgite to the mixer, and spray the concentrated trace element solution onto the attapulgite using a spraying device while stirring. After spraying, continue mixing for 10 minutes. Then, evenly spray the nutrient element precursor solution onto the modified attapulgite carrier loaded with trace elements and continue mixing for 15 minutes to allow the liquid components to be fully adsorbed by the carrier. D. Add microcrystalline cellulose, xylooligosaccharides, fermented soybean meal and yeast hydrolysate to a mixer in sequence, mix for 25 minutes, dry with low-temperature airflow until the moisture content is ≤10%, and pass through a 60-mesh sieve to obtain the nutrient matrix.

[0022] Example 3 A method for preparing fermented feed for ruminants using microbial enzymes, comprising the following steps: S1. After drying corn stalks and wheat stalks, crush them to 2-5 mm. Treat the crushed stalks in saturated steam at 0.3 MPa and 120℃ for 15 min. Immediately afterward, spray with an organic acid solution composed of citric acid, malic acid and lactic acid at pH 4.0. The spraying amount is 10% of the dry weight of the stalks. After treatment, adjust the moisture content of the material to 65%. S2. Add the microcapsule-encapsulated compound microbial agent prepared in Example 1 and the nutrient matrix prepared in Example 2 to the pretreated straw and mix thoroughly. The microcapsule-encapsulated compound microbial strain is calculated at 1×10⁻⁶ effective viable bacteria count. 6 Add CFU / g of straw dry weight, pack into fermentation tank and compact, control fermentation temperature at 22℃ for 4 weeks, and manage intermittent aeration management with weekly turning to ensure adequate oxygen supply. After fermentation, ruminant fermented feed is obtained, which is yellowish-brown and has an alcoholic or slightly acidic aroma.

[0023] Example 4 A method for preparing fermented feed for ruminants using microbial enzymes, comprising the following steps: S1. After drying corn stalks and rice stalks, crush them to 2-5 mm. Treat the crushed stalks in saturated steam at 0.5 MPa and 110℃ for 20 min. Immediately afterward, spray with an organic acid solution composed of citric acid, malic acid and lactic acid at pH 3.5. The spraying amount is 15% of the dry weight of the stalks. After treatment, adjust the moisture content of the material to 60%. S2. Add the microcapsule-encapsulated compound microbial agent prepared in Example 1 and the nutrient matrix prepared in Example 2 to the pretreated straw and mix thoroughly. The microcapsule-encapsulated compound microbial strain is calculated at 5 × 10⁻⁶ effective viable bacteria. 6 Add CFU / g of straw dry weight, pack into fermentation tank and compact, control fermentation temperature at 18℃ for 3 weeks, intermittent aeration management with turning twice a week to ensure adequate oxygen supply, after fermentation, obtain fermented feed for ruminants, the feed is yellowish-brown and has an alcoholic or slightly acidic aroma.

[0024] Example 5 A method for preparing fermented feed for ruminants using microbial enzymes, comprising the following steps: S1. Dry wheat straw and rice straw and then crush them to 2-5 mm. Treat the crushed straw in saturated steam at 0.4 MPa and 115℃ for 18 min. Immediately afterward, spray with an organic acid solution composed of citric acid, malic acid and lactic acid at pH 3.8. The spraying amount is 12% of the dry weight of the straw. After treatment, adjust the moisture content of the material to 62.5%. S2. Add the microcapsule-encapsulated compound microbial agent prepared in Example 1 and the nutrient matrix prepared in Example 2 to the pretreated straw and mix thoroughly. The microcapsule-encapsulated compound microbial strain is calculated based on an effective viable count of 2.5 × 10⁻⁶. 6 Add CFU / g of straw dry weight, pack into fermentation tank and compact, control fermentation temperature at 20℃ for 4 weeks, and manage intermittent aeration management with weekly turning to ensure adequate oxygen supply. After fermentation, ruminant fermented feed is obtained, which is yellowish-brown and has an alcoholic or slightly acidic aroma.

[0025] Comparative Example 1: A method for preparing fermented feed for ruminants using microbial enzymes, comprising the following steps: S1. Dry wheat straw and rice straw and then crush them to 2-5 mm. Treat the crushed straw in saturated steam at 0.4 MPa and 115℃ for 18 min. Immediately afterward, spray with an organic acid solution composed of citric acid, malic acid and lactic acid at pH 3.8. The spraying amount is 12% of the dry weight of the straw. After treatment, adjust the moisture content of the material to 62.5%. S2. Add the compound microbial strain and the nutrient substrate prepared in Example 2 to the pretreated straw and mix thoroughly. The compound microbial strain is a mixture of Trichoderma reesei, Bacillus subtilis, and white-rot fungi in a spore-to-cell ratio of 5:3:2. The compound microbial strain is calculated based on an effective viable count of 2.5 × 10⁻⁶ cells / year. 6 Add CFU / g of straw dry weight, pack into fermentation tank and compact, control fermentation temperature at 20℃ for 4 weeks, and manage intermittent aeration management with weekly turning to ensure adequate oxygen supply. After fermentation, ruminant fermented feed is obtained, which is yellowish-brown and has an alcoholic or slightly acidic aroma.

[0026] Comparative Example 2: A method for preparing fermented feed for ruminants using microbial enzymes, comprising the following steps: S1. Dry wheat straw and rice straw and then crush them to 2-5 mm. Treat the crushed straw in saturated steam at 0.4 MPa and 115℃ for 18 min. Immediately afterward, spray with an organic acid solution composed of citric acid, malic acid and lactic acid at pH 3.8. The spraying amount is 12% of the dry weight of the straw. After treatment, adjust the moisture content of the material to 52.5%. S2. Add the microcapsule-encapsulated composite microbial agent prepared in Example 1 to the pretreated straw and mix thoroughly. The microcapsule-encapsulated composite microbial strain is calculated based on an effective viable count of 2.5 × 10⁻⁶. 6 Add CFU / g of straw dry weight, pack into fermentation tank and compact, control fermentation temperature at 20℃ for 4 weeks, and manage intermittent aeration management with weekly turning to ensure adequate oxygen supply. After fermentation, ruminant fermented feed is obtained, which is yellowish-brown and has an alcoholic or slightly acidic aroma.

[0027] Performance testing The preparation process and fermentation products of ruminant microbial enzyme fermented feed in Example 5, Comparative Example 1 and Comparative Example 2 were tested, and straw that had only undergone physicochemical pretreatment (without any added nutrient substrate or microbial agent) was set up as Comparative Example 3.

[0028] (1) Dynamic monitoring of the fermentation process Samples were taken on days 0, 7, 14, 21, and 28 of fermentation to determine the pH value, lactic acid, and volatile fatty acid (VFA, mainly including acetic acid, propionic acid, and butyric acid) content (using high performance liquid chromatography), and the total number of viable bacteria was determined (using plate count method; the microcapsule samples of Example 5 and Comparative Example 2 required capsule rupture). The data are shown in Table 1 below.

[0029] Table 1. Dynamic monitoring results of the fermentation process As shown in Table 1, Comparative Example 1 exhibited a faster acidification rate and higher viable cell count than the experimental group in the early stages of fermentation (days 7 and 14), possibly due to direct exposure of the cells, which rapidly initiated metabolism. However, in the later stages of fermentation (days 21 and 28), its viable cell count decreased significantly, while Example 5 maintained a high viable cell count through the sustained release and protective effect of the microcapsules, indicating that the microcapsules provided long-term protection. Comparative Example 2 showed a much lower rate and total accumulation of pH, lactic acid, and VFA compared to Example 5, and its viable cell proliferation was also slower. This indicates that the lack of a nutrient substrate severely limited the initial colonization and metabolic activity of the microorganisms.

[0030] (2) Detection of feed nutritional quality and degradation efficiency at the fermentation endpoint Samples were taken on day 28 of fermentation to determine the conventional nutrient components (crude protein CP, neutral detergent fiber NDF, acid detergent fiber ADF), cellulase and xylanase activities (using the DNS method), and lignin degradation rate (calculated using the ADL method for acid detergent lignin). The data are shown in Table 2 below.

[0031] Table 2 Results of feed nutrient quality and degradation efficiency at the fermentation endpoint As shown in Table 2, although Comparative Example 1 exhibited active metabolism in the initial stage, the end-product enzyme activities (cellulase and xylanase) and lignin degradation rate of Example 5 were significantly higher than those of Comparative Example 1. This indicates that the sustained-release properties of the microcapsules allow the microorganisms and enzymes to function continuously and stably throughout the fermentation cycle, avoiding the problem of explosive substrate consumption in the early stage followed by a decline in activity, thus achieving more thorough degradation. Comparative Example 2, lacking a nutrient substrate, showed significantly lower performance than Example 5 and Comparative Example 1 in all key indicators, including increased CP, decreased NDF / ADF, enzyme activity, and lignin degradation rate. This demonstrates that the composite nutrient substrate serves as both fuel and catalyst for efficient biotransformation.

[0032] (3) In vitro rumen fermentation simulation test A continuous rumen fermentation system (Rusitec) was used, with a basal TMR diet (50:50 concentrate:roughage ratio) as the fermentation substrate. In each experimental group, 50% of the roughage portion was replaced with the same amount of the basal diet. Each group had three replicates, and fermentation lasted for 48 hours. Key parameters were measured: cumulative gas production, ammonia nitrogen concentration, and microbial protein synthesis. The results are shown in Table 3 below.

[0033] Table 3 Results of in vitro rumen fermentation simulation test As shown in Table 3, Example 5 exhibited higher in vitro rumen fermentation gas production, lower NH3-N concentration, and higher MCP synthesis, indicating that the feed from Example 5 produced more readily fermentable carbohydrates in the rumen (due to more thorough fiber degradation), while also demonstrating higher nitrogen utilization efficiency (less nitrogen was lost as ammonia, and more was converted into microbial protein). This is directly related to the pH-sensitive outer layer design of the microcapsules. The outer layer is stable in the acidic environment of the rumen, reducing premature consumption of bacteria and enzymes in the rumen, allowing them to enter the hindgut or exert their effects in the small intestine, thereby more effectively releasing fermentable carbon and nitrogen sources simultaneously and optimizing the fermentation balance of rumen microorganisms. Comparative Example 2 showed the lowest gas production and MCP synthesis, but the highest NH3-N concentration, indicating poor quality of the fermentable substrate and uncoordinated nitrogen source utilization.

[0034] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0035] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing fermented feed for ruminants, characterized in that, Includes the following steps: S1. After drying the straw, crush it to 2-5 mm. Treat the crushed straw in saturated steam at 0.3-0.5 MPa and 110-120℃ for 15-20 min. Immediately afterward, spray it with a mixed organic acid solution with pH 3.5-4.0 at a rate of 10-15% of the dry weight of the straw. After treatment, adjust the moisture content of the material to 60-65%. S2. Add the microencapsulated compound microbial agent and nutrient matrix to the pretreated straw and mix thoroughly. Pack the straw into a fermentation tank and compact it. Control the fermentation temperature at 18~22℃ and ferment for 3~4 weeks. Intermittent aeration management with turning the pile 1~2 times a week to ensure adequate oxygen supply. After fermentation, the fermented feed for ruminants is obtained. The microcapsule-encapsulated compound bacterial agent is formed by mixing compound bacterial strains with trehalose and pregelatinized starch, then using sodium alginate and chitosan to form an inner membrane through layer-by-layer self-assembly technology, and then encapsulating it with hydroxypropyl methylcellulose phthalate to form an outer membrane. The nutrient matrix includes microcrystalline cellulose, xylooligosaccharides, fermented soybean meal, yeast hydrolysate, isopropyl methionine hydroxy analogue, nicotinic acid, zinc sulfate, manganese sulfate, lanthanum chloride, and modified attapulgite.

2. The method for preparing fermented feed for ruminants according to claim 1, characterized in that, The straw is one or a mixture of corn straw, wheat straw and rice straw, and the mixed organic acid solution is a compound of citric acid, malic acid and lactic acid.

3. The method for preparing fermented feed for ruminants according to claim 1, characterized in that, The microcapsule-encapsulated complex microbial strains were calculated at 1×10⁻⁶ effective viable bacteria count. 6 ~5×10 6 The ratio of CFU / g dry weight of straw is added to the pretreated straw.

4. The method for preparing fermented feed for ruminants according to claim 1, characterized in that, The composite microbial strain includes Trichoderma reesei, Bacillus subtilis, and white-rot fungi.

5. The method for preparing fermented feed for ruminants according to claim 1, characterized in that, The preparation method of the microcapsule-encapsulated compound bacterial agent includes the following steps: (1) Trichoderma reesei was cultured to obtain spore suspension and fermentation supernatant rich in extracellular enzymes. Bacillus subtilis was cultured and centrifuged to collect the cells and resuspended in physiological saline. White rot fungi were cultured to obtain white rot fungal hyphae. (2) Mix Trichoderma reesei spore suspension, Bacillus subtilis suspension and white rot fungal hyphae, then add 20% trehalose and 15% pregelatinized corn starch of the total weight of the compound strain, and homogenize at 4℃ to form a viscous and uniform mixed slurry. (3) Mix the mixed slurry and sodium alginate solution at a volume ratio of 1:

3. Drop the mixture into a 1.5wt% calcium chloride solution to form gel microspheres. Stir and solidify for 15-25 minutes. After filtration, wash with deionized water to obtain composite bacterial agent sodium alginate microspheres. (4) Immerse the composite bacterial agent sodium alginate microspheres in a 0.5wt% chitosan acetate solution, adjust the pH to 5~5.5, stir for 10~20min, then transfer the microspheres into a 0.5wt% sodium alginate solution and stir for 10~20min. Repeat this process 2~4 times to deposit and form a multilayer chitosan sodium alginate composite inner membrane. (5) Dissolve 6 wt% hydroxypropyl methylcellulose phthalate in 80 vol% ethanol aqueous solution, stir thoroughly until completely dissolved and clear, cool to 4°C and add cellulase and xylanase. Place the microspheres with protective film obtained in step (4) into a fluidized bed coating machine, and spray the enzyme-containing hydroxypropyl methylcellulose phthalate solution evenly onto the surface of the microspheres in the form of atomization through a micro sprayer. The hydroxypropyl methylcellulose phthalate forms an outer film on the surface of the microspheres. Dry at room temperature for 24 h to obtain the microcapsule-encapsulated composite bacterial agent.

6. The method for preparing fermented feed for ruminants according to claim 5, characterized in that, In step (1), Trichoderma reesei was activated at 28°C using potato dextrose agar plates and then inoculated into a liquid fermentation medium containing microcrystalline cellulose. The medium was cultured at 28°C and 180 rpm for 72-96 hours with shaking. The spore suspension and the fermentation supernatant rich in extracellular enzymes were harvested. The supernatant was concentrated 10 times by centrifugation to obtain the enzyme solution. Bacillus subtilis was activated on LB plates at 30°C, inoculated into LB liquid medium, and cultured at 30°C with shaking at 200 rpm until the late logarithmic growth stage. The cells were collected by centrifugation and resuspended in physiological saline. White-rot fungi were slowly activated on malt extract agar plates at 25°C for 7-10 hours. Mycelial blocks were then inoculated onto nitrogen-limited liquid culture medium and incubated statically at 25°C for 10-14 days. Mycelial balls were then collected and appropriately broken up.

7. The method for preparing fermented feed for ruminants according to claim 5, characterized in that, In step (2), Trichoderma reesei spore suspension, Bacillus subtilis suspension and white rot fungal hyphae are mixed in a spore-cell ratio of 5:3:

2.

8. The method for preparing fermented feed for ruminants according to claim 1, characterized in that, The method for preparing the nutrient substrate includes the following steps: A. Crush natural attapulgite, calcine it at 500℃ for 1.5~2.5h, cool it and mix it with citric acid solution. Stir and react in a 70℃ water bath for 3~5h. After the reaction is completed, wash it repeatedly with deionized water until the filtrate is neutral. Dry the filter cake to constant weight and crush it through a 200-mesh sieve to obtain modified attapulgite. B. Add zinc sulfate, manganese sulfate and lanthanum chloride to warm water at 40~50℃ and stir until completely dissolved to obtain a trace element concentrate. Dissolve methionine hydroxy analogue isopropyl ester and nicotinic acid in warm water to obtain a nutrient element precursor solution. C. Add the modified attapulgite to the mixer, and while stirring, spray the concentrated trace element solution onto the attapulgite using a spraying device. After spraying, continue mixing for 5-15 minutes. Then, evenly spray the nutrient element precursor solution onto the modified attapulgite carrier loaded with trace elements, and continue mixing for 10-20 minutes to allow the liquid components to be fully adsorbed by the carrier. D. Add microcrystalline cellulose, xylooligosaccharides, fermented soybean meal and yeast hydrolysate to a mixer in sequence, mix for 20-30 minutes, dry with low-temperature airflow until the moisture content is ≤10%, and pass through a 60-mesh sieve to obtain the nutrient matrix.

9. The method for preparing fermented feed for ruminants according to claim 8, characterized in that, In step A, attapulgite clay and 1 mol / L citric acid solution are mixed at a solid-liquid ratio of 1:

5.

10. The method for preparing fermented feed for ruminants according to claim 8, characterized in that, The content of each raw material in the nutrient matrix is ​​as follows: 2-3 parts microcrystalline cellulose, 0.6-1.0 parts xylooligosaccharide, 1.0-1.4 parts fermented soybean meal, 0.6-1.0 parts yeast hydrolysate, 0.04-0.06 parts isopropyl methionine hydroxy analogue, 0.01-0.03 parts nicotinic acid, 0.01-0.02 parts zinc sulfate, 0.005-0.015 parts manganese sulfate, 0.001-0.003 parts lanthanum chloride, and 1.0-2 parts modified attapulgite.

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