A biophysical coupling pretreatment method for high fiber raw materials and application thereof

Abstract

The present application relates to the field of feed processing and pretreatment, and discloses a biophysical coupling pretreatment method for high-fiber raw materials and application thereof, which comprises the following steps: mixing high-fiber raw materials, corn powder, soybean meal and molasses into mixed raw materials, conditioning and stacking treatment by connecting with an acidic conditioning liquid, extruding and vaporizing flash evaporation by a double-phase stainless steel double-screw extrusion expander, preparing expanded raw materials, connecting with depolymerase bacterial liquid for shallow tank tumbling aerobic fermentation after cooling, moving into a mixing machine for homogenization and spraying phase change promoting liquid, then forming a material package by high-density silage hydraulic packing machine, and simultaneously connecting with lactobacillus plantarum for multi-layer stretch film sealed fermentation. The present application realizes gradient deconstruction and efficient conversion of lignocellulose structure by coupling of physical modification and biological conversion, so that the product is rich in active substances such as small peptides and organic acids, and the nutritional value and utilization rate of high-fiber raw materials are improved, and the formula cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of feed processing and pretreatment, specifically to a biophysical coupling pretreatment method for high-fiber raw materials and its application. Background Technology

[0002] Currently, international high-fiber feed processing is developing towards refinement, improving the conversion efficiency of fiber raw materials through the synergistic effect of biotechnology and mechanical equipment.

[0003] However, existing processing methods typically involve isolated combinations of physical processing, microbial fermentation, or single enzymatic hydrolysis, lacking deep-level technological coupling. Limited by the stable physicochemical structure of lignocellulose, a single pathway is insufficient to effectively break down the cross-linked network between lignin and cellulose, resulting in limited increases in the specific surface area of ​​the raw materials and hindering enzyme molecules from penetrating the underlying structure. Due to the lack of targeted biochemical regulatory mechanisms, the generation rates of functional substances such as bioactive peptides and organic acids in fermentation products are low, leading to the materials being long considered inefficient fillers, with limited potential for improvement in their substitution ratio and nutritional value in feed formulations. Furthermore, for resources such as distiller's grains and straw, which are scattered and have high moisture content, existing technologies still suffer from low processing efficiency and high overall processing costs, failing to develop end-use solutions that balance deconstruction efficiency and nutritional value enhancement. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a biophysical coupling pretreatment method for high-fiber raw materials and its application. This method solves the problem that existing technologies lack synergy between physical modification and bioconversion, resulting in incomplete degradation of high-fiber raw materials and low nutritional value of the products, making it difficult to achieve a high proportion of substitution for bulk feed ingredients.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a biophysical coupling pretreatment method for high-fiber raw materials, employing the following technical solution:

[0007] A biophysical coupling pretreatment method for high-fiber raw materials includes the following steps:

[0008] By weight percentage, select 70%–85% high-fiber raw materials, 7%–15% corn flour, 7%–12% soybean meal, and 1%–3% molasses, mix them to form a mixed base material, send it into a continuous twin-shaft paddle conditioner, introduce acidic conditioning liquid for conditioning treatment, and then transport it to a buffer silo for heaping treatment.

[0009] The bottom material after the slumping treatment is continuously fed into a duplex stainless steel twin-screw extruder for extrusion. The material is heated and subjected to shear force in the cavity, and is extruded through the die hole to a normal pressure environment to achieve vaporization and flash evaporation, and is collected to form a porous sponge-like extruded bottom material.

[0010] After being cooled, the puffed base material is introduced into the depolymerization enzyme liquid through an atomizing spraying device, and then spread on a shallow trough turning fermentation bed with bottom ventilation for aerobic fermentation to obtain aerobic fermented material.

[0011] The aerobic fermentation material is transferred into a high-shear twin-shaft paddle mixer for homogenization. During the homogenization process, phase change accelerator is continuously sprayed by atomization and the overall moisture content is adjusted. After homogenization, the material is bundled and compacted by a high-density silage hydraulic baler to form bales.

[0012] During the sealing process, Lactobacillus plantarum bacterial solution is introduced through a synchronous inoculation device, the feed package is sealed with a stretch film and placed in a temperature-controlled chamber for static fermentation to obtain functional fermented feed, thus completing the pretreatment.

[0013] In this process system, the combined effects of acidic pretreatment and instantaneous flash evaporation initially weaken the density of the substrate cell walls. Subsequent aerobic and anaerobic dual-stage fermentation achieves the directional degradation of lignin and the controlled enrichment of functional peptides, thereby improving the final product in terms of both lignocellulose degradation rate and nutritional composition.

[0014] Preferably, the high-fiber raw material is corn stalks or liquor lees.

[0015] Preferably, the spraying amount of the acidic conditioning liquid is controlled so that the overall moisture content of the mixed base material system is 25% to 35%, and the heaping treatment time is 15 to 20 minutes.

[0016] Preferably, the temperature of the heating zone inside the extrusion chamber of the duplex stainless steel twin-screw extruder is 105–130°C, and the residence time of the material in the chamber is 20–45 seconds.

[0017] Preferably, during the aerobic fermentation stage, the extruded substrate is cooled to 35-37°C and then inoculated with depolymerase bacterial solution; the bottom ventilation volume of the shallow trench turning fermentation bed is 0.5-1.0 vvm, the substrate thickness is controlled at 30-40cm, and the fermentation duration is 24-36h.

[0018] Preferably, during the homogenization process, high-intensity shear homogenization is carried out in a high-shear biaxial paddle mixer for 1-2 minutes, and the overall moisture content of the aerobic fermentation material system after replenishment is stabilized at 50%-55%; the compaction density of the baled and compacted material is controlled at 600-1000 kg / m³. 3 .

[0019] Preferably, the sealed package is placed in a temperature-controlled chamber at 30-32°C and allowed to ferment for 48-72 hours.

[0020] Preferably, the acidic conditioning solution is prepared from an aqueous solution of citric acid with a mass concentration of 1.5% to 2.0% or lactic acid with a mass equivalent concentration of 1.0%.

[0021] Preferably, the depolymerase bacterial solution has an effective viable count of 1.0 × 10⁻⁶ cells. 7 ~5.0×10 8 It is prepared by mixing CFU / mL liquid mycelial fermentation broth of *Amanita muscaria*, 500–1000 U / g of industrial laccase, and 200–500 U / g of xylanase.

[0022] Preferably, the preparation steps of the phase change activating solution include: dissolving 1.0%–2.0% (w / w) of soluble starch and 0.5%–1.0% (w / w) of yeast extract in deionized water, sterilizing and cooling; adding industrial cellulase at a concentration of 1000–2000 U / g (based on the dry weight of the aerobic fermentation material) and neutral protease at a concentration of 100–300 U / g; and inoculating with an effective viable cell count of 1.0 × 10⁻⁶ cells / g at a volume ratio of 1.5:1–2:1. 8 The CFU / mL Bacillus subtilis bacterial suspension had an effective viable count of 5.0 × 10⁻⁶. 7 Saccharomyces cerevisiae culture at CFU / mL.

[0023] The reaction mechanism involved in this invention is as follows:

[0024] This involves a structural activation process involving acid-thermal-mechanical coupling. During the conditioning and heaping stages, weak acid molecules in the acidic conditioning solution gradually penetrate into the plant cell walls. After entering the extrusion chamber, under high temperature and strong shear stress, the acetyl groups of the hemicellulose side chains undergo self-hydrolysis to produce acetic acid. Through the synergistic effect of this process, the linkage between lignin and polysaccharide components is induced to break.

[0025] When the base material is ejected through the die, the sudden change in pressure environment causes the moisture inside the material to flash evaporate instantly. The resulting expansion work destroys the dense network of lignin and carbohydrate complexes at the microscale, forming a porous structure that facilitates the diffusion of enzyme molecules.

[0026] The mechanism involves a directional and targeted degradation process during the aerobic stage. Laccase in the depolymerizing bacterial solution and *Acer velutipes* function under aerobic conditions. Laccase uses phenolic substances released in situ from the substrate as a mediator to attack chemical bonds such as β−O−4 in lignin molecules through a single-electron oxidation pathway, causing the macromolecular lignin to depolymerize.

[0027] Based on this, xylanase further hydrolyzes the remaining hemicellulose components, eliminating the physical barrier that originally covered the cellulose surface and providing a usable substrate exposure surface for subsequent biochemical transformation.

[0028] This involves the controlled degradation of proteins under mechanically induced phase transitions. Through high-density bundling and compaction, the material environment is transformed from an aerobic to an anaerobic state. During this process, the protease system in the phase transition facilitator begins to function. As *Lactobacillus plantarum* metabolizes and produces lactic acid, causing a decrease in the system's pH, the acidic environment inhibits other bacteria while activating specific acidic proteases. Under the action of enzymes activated at different pH levels, large protein molecules are gradually converted into acid-soluble small peptides.

[0029] Compaction not only restricts oxygen mass transfer but also shortens the diffusion distance between enzyme molecules and substrates, thereby increasing the rate and total amount of protein conversion into nutrient peptides.

[0030] Secondly, this invention provides the application of a biophysical coupling pretreatment method for high-fiber raw materials in the preparation of functional fermented feeds made from high-fiber raw materials.

[0031] Based on the above applications, the method of the present invention can be applied to various high-fiber biomass such as corn stalks and liquor lees. The degradation rate of lignocellulose components in the products obtained after treatment is improved, and the content of acid-soluble small peptides is increased, which is beneficial to improving the digestion and absorption of feed components by animals.

[0032] This invention provides a biophysical coupling pretreatment method for high-fiber raw materials and its application. It has the following beneficial effects:

[0033] 1. This invention overcomes the limitations of single physical or biological treatments in deconstructing high-fiber raw materials by constructing an integrated process of acid-thermal-mechanical coupled pretreatment and multi-stage microbial fermentation. Utilizing the thermomechanical work of a duplex stainless steel twin-screw extruder during extrusion and the chemical induction effect of the acidic conditioning solution, the lignin and cellulose barriers are effectively destroyed, increasing the specific surface area of ​​the material and achieving a tiered deconstruction of the fiber structure. The integrated system provides a good working surface for the complex enzyme system in the subsequent depolymerization enzyme solution and phase change accelerator, improving the conversion efficiency of fiber components.

[0034] 2. This invention, through targeted regulation of the fermentation process, enriches the final product with bioactive substances such as small peptides, organic acids, and antimicrobial peptides. The process is not limited to simple fiber degradation, but rather transforms the originally low-digestibility fiber raw materials into functional sources with nutritional value through multi-strain combined fermentation and efficient enzymatic hydrolysis of compound enzymes. This transformation process enhances the product's effectiveness in improving animal intestinal health and reducing diarrhea rates, realizing the transformation of raw materials from inefficient fillers to high-value functional feeds.

[0035] 3. This invention transforms conventionally difficult-to-utilize high-fiber raw materials into efficient nutrient sources through biological and physical coupling, thereby increasing the proportion and nutritional value of high-fiber raw materials in feed formulations. Based on the nutritional characteristics and nutritional model optimization of pretreated raw materials, it achieves the substitution of bulk raw materials such as corn and soybean meal, effectively reducing the formulation cost of finished feed. Attached Figure Description

[0036] Figure 1 This is a comparison of high-performance liquid chromatography (HPLC) images of acetic acid release from materials in the embodiments and comparative examples of the present invention.

[0037] Figure 2 This is a comparison of high-performance liquid chromatography (HPLC) images of ferulic acid release in the materials of the embodiments and comparative examples of the present invention.

[0038] Figure 3 The following are comparison charts showing the dynamic monitoring characteristics of the fermentation systems of the embodiments and comparative examples of the present invention; wherein, (a) is a comparison chart showing the dynamic changes in carbon dioxide release rate of Example 1 and Comparative Example 5, and (b) is a comparison chart showing the dynamic changes in pH value of the systems of Example 1 and Comparative Example 5. Detailed Implementation

[0039] The technical solutions in the preparation examples and embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, comparative examples, and test examples. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Preparation Examples 1-9:

[0041] Preparation Example 1:

[0042] This preparation example provides a method for preparing an acidic conditioning solution, including the following steps:

[0043] At room temperature, solid citric acid was slowly added to deionized water at a mass concentration of 1.5%, and mechanically stirred for 10 minutes until completely dissolved to obtain an acidic conditioning solution.

[0044] Preparation Example 2:

[0045] This preparation example provides a method for preparing an acidic conditioning solution, including the following steps:

[0046] At room temperature, an 80% lactic acid aqueous solution is slowly added to deionized water at a ratio of 1.0% (converted to a mass concentration of 1.0%), and mechanically stirred for 10 minutes until the mixture is homogeneous to obtain an acidic conditioning solution.

[0047] Preparation Example 3:

[0048] This preparation example provides a method for preparing an acidic conditioning solution, including the following steps:

[0049] At room temperature, solid citric acid was slowly added to deionized water at a mass concentration of 2.0%, and mechanically stirred for 15 minutes until completely dissolved to obtain an acidic conditioning solution.

[0050] Preparation Example 4:

[0051] This preparation example provides a method for preparing depolymerase bacterial solution, including the following steps:

[0052] Under aseptic conditions, the liquid mycelium fermentation broth of *Amanita muscaria* in the logarithmic growth phase (effective viable count of 1.0 × 10⁻⁶) was used. 8 Using CFU / mL as the base solution, industrial laccase (added at a rate of 750 U / g based on the dry weight of the puffed base material to be treated) and xylanase (added at a rate of 350 U / g based on the dry weight of the puffed base material to be treated) were added to it. The mixture was stirred at a low speed of 65 rpm at 28°C to obtain the depolymerization enzyme solution.

[0053] Preparation Example 5:

[0054] This preparation example provides a method for preparing depolymerase bacterial solution, including the following steps:

[0055] Under aseptic conditions, the liquid mycelium fermentation broth of *Amanita muscaria* in the logarithmic growth phase (effective viable count of 1.0 × 10⁻⁶) was used. 7 Using CFU / mL as the base solution, industrial laccase (added at a rate of 500 U / g based on the dry weight of the puffed base material to be treated) and xylanase (added at a rate of 200 U / g based on the dry weight of the puffed base material to be treated) were added to it. The mixture was stirred at a low speed of 50 rpm at 25°C to obtain the depolymerization enzyme solution.

[0056] Preparation Example 6:

[0057] This preparation example provides a method for preparing depolymerase bacterial solution, including the following steps:

[0058] Under aseptic conditions, the liquid mycelium fermentation broth of *Amanita muscaria* in the logarithmic growth phase (effective viable count of 5.0 × 10⁻⁶) was used. 8 Using CFU / mL as the base solution, industrial laccase (added at a rate of 1000 U / g based on the dry weight of the puffed base material to be treated) and xylanase (added at a rate of 500 U / g based on the dry weight of the puffed base material to be treated) were added to it. The mixture was stirred at 80 rpm at 30°C to obtain the depolymerization enzyme solution.

[0059] Preparation Example 7:

[0060] This preparation example provides a method for preparing a phase change activating liquid, including the following steps:

[0061] Dissolve 1.5% (w / w) of soluble starch and 0.8% (w / w) of yeast extract in deionized water, sterilize at 121°C for 20 min, then cool to 35°C. Add industrial cellulase (at a rate of 1500 U / g based on the dry weight of the subsequent aerobic fermentation material) and neutral protease (at a rate of 200 U / g based on the dry weight of the subsequent aerobic fermentation material) sequentially and stir to dissolve. Then inoculate with Bacillus subtilis culture (effective viable count 1.0 × 10⁻⁶). 8 CFU / mL) and Saccharomyces cerevisiae culture (effective viable count 5.0 × 10⁻⁶) 7 The two solutions (CFU / mL) were mixed in a 1:1 volume ratio to obtain a phase change activating solution.

[0062] Preparation Example 8:

[0063] This preparation example provides a method for preparing a phase change activating liquid, including the following steps:

[0064] Dissolve 1.0% (w / w) of soluble starch and 0.5% (w / w) of yeast extract in deionized water, sterilize at 121℃ for 20 min, and then cool to 35℃. Add industrial cellulase (at a rate of 1000 U / g based on the dry weight of the subsequent aerobic fermentation material) and neutral protease (at a rate of 100 U / g based on the dry weight of the subsequent aerobic fermentation material) sequentially and stir to dissolve. Then inoculate with Bacillus subtilis culture (effective viable count 1.0 × 10⁻⁶). 8 CFU / mL) and Saccharomyces cerevisiae culture (effective viable count 5.0 × 10⁻⁶) 7 The two solutions (CFU / mL) were mixed in a volume ratio of 1.5:1 to obtain a phase change activating solution.

[0065] Preparation Example 9:

[0066] This preparation example provides a method for preparing a phase change activating liquid, including the following steps:

[0067] Dissolve 2.0% (w / w) of soluble starch and 1.0% (w / w) of yeast extract in deionized water, sterilize at 121℃ for 20 min, then cool to 35℃. Add industrial cellulase (2000 U / g based on the dry weight of the subsequent aerobic fermentation material) and neutral protease (300 U / g based on the dry weight of the subsequent aerobic fermentation material) sequentially and stir to dissolve. Then inoculate with Bacillus subtilis culture (effective viable count 1.0 × 10⁻⁶). 8 CFU / mL) and Saccharomyces cerevisiae culture (effective viable count 5.0 × 10⁻⁶) 7The two solutions (CFU / mL) were mixed in a volume ratio of 2:1 to obtain a phase change activating solution.

[0068] Examples 1-4:

[0069] Example 1:

[0070] This embodiment provides a biophysical coupling pretreatment method for high-fiber raw materials, including the following steps:

[0071] By mass percentage, 80% corn stalks, 10% corn flour, 8% soybean meal, and 2% molasses were selected as the mixed base material and fed into a continuous twin-shaft paddle conditioner. The acidic conditioning liquid prepared in Preparation Example 1 was introduced through an atomizing spraying device. By adjusting the spraying amount of the acidic conditioning liquid, the overall moisture content of the mixed base material system was controlled at 30%. After the conditioning and mixing were uniform, the mixture was transported to a buffer chamber and left to stand for 18 minutes to allow acid molecules to penetrate into the micropores of the plant cell walls.

[0072] The bottom material after being piled up is continuously fed into a duplex stainless steel twin-screw extruder. The temperature of the heating zone in the extrusion chamber is set to 110℃. By adjusting the screw speed, the residence time of the bottom material after being piled up in the chamber is maintained at 30s. The bottom material after being piled up is extruded through the die hole to a normal pressure environment to achieve instantaneous vaporization and flash evaporation, and is collected to form a porous sponge-like extruded bottom material.

[0073] When the puffed base material is cooled to 36°C by cold air convection, it is introduced into the depolymerase bacterial solution prepared in Preparation Example 4 through an atomizing spraying device. The inoculated material is spread evenly in a shallow trough turning fermentation bed equipped with bottom forced ventilation (ventilation volume of 0.8 vvm). The thickness of the material is controlled at 35 cm. The aerobic fermentation at this stage is maintained at the ambient temperature for 30 hours to obtain aerobic fermented material.

[0074] After aerobic fermentation, the aerobic fermented material was quickly transferred to a high-shear biaxial paddle mixer. During the 1.5-minute high-intensity shear homogenization process, the phase change accelerator prepared in Example 7 was continuously atomized and sprayed. By controlling the amount of liquid added, the overall moisture content of the aerobic fermented material system was adjusted and stabilized at 53% based on the measured moisture content before homogenization. After homogenization, the material was seamlessly connected to a high-density silage hydraulic baler, where hydraulic mechanical force was applied for baling and compaction, with the compaction density controlled at 800 kg / m³. 3 .

[0075] During the sealing process, *Lactobacillus plantarum* bacterial suspension (with an effective viable count of 1.0 × 10⁻⁶) was introduced via a synchronous inoculation device. 8 (CFU / mL, inoculation amount is 1.0% based on the wet weight of the material) The feed bag is sealed in multiple layers using stretch film. The sealed feed bag is placed in a temperature-controlled chamber at 31℃ and allowed to ferment for 60 hours. After fermentation, the pH value of the system drops to 4.0, and functional fermented feed is obtained, thus completing the pretreatment.

[0076] Example 2:

[0077] This embodiment provides a biophysical coupling pretreatment method for high-fiber raw materials, including the following steps:

[0078] By mass percentage, 70% corn stalks, 15% corn flour, 12% soybean meal, and 3% molasses were selected as the mixed base material and fed into a continuous twin-shaft paddle conditioner. The acidic conditioning solution prepared in Example 2 was atomized and sprayed. By adjusting the spraying amount of the acidic conditioning solution, the overall moisture content of the mixed base material system was controlled at 25%. After mixing, the mixture was piled in a buffer chamber for 15 minutes to induce the initial penetration of weak acid molecules.

[0079] The bottom material after being piled up is fed into a duplex stainless steel twin-screw extruder. The extrusion chamber temperature is set to 105℃. The residence time of the piled bottom material in the chamber is controlled to 20s by adjusting the screw speed. After being extruded through the die, the bottom material is subjected to a sudden pressure drop to achieve flash evaporation of moisture and is collected to obtain the puffed bottom material.

[0080] When the expanded base material is cooled down to 35°C by cold air, the depolymerase bacterial solution prepared in Preparation Example 5 is introduced. The inoculated material is spread evenly in a shallow trough turning fermentation bed with bottom forced ventilation (ventilation volume of 0.5 vvm). The thickness of the material is maintained at 30 cm. The aerobic fermentation stage is maintained at the ambient temperature for 24 hours to obtain aerobic fermented material.

[0081] The aerobic fermentation material was transferred to a high-shear biaxial paddle mixer, and the phase change accelerator prepared in Example 8 was sprayed during a 1-minute high-intensity shear homogenization process. By controlling the amount of liquid added, the overall moisture content of the aerobic fermentation material system was adjusted and stabilized at 50% based on the measured moisture content before homogenization. Subsequently, it was baleed in a high-density silage hydraulic baler, and the compaction density was controlled at 600 kg / m³. 3 And use stretch film for sealing and wrapping.

[0082] During the sealing process, *Lactobacillus plantarum* bacterial suspension (with an effective viable count of 5.0 × 10⁻⁶) was simultaneously introduced. 7 (CFU / mL, inoculum amount is 0.5% based on wet weight of material), the sealed package is placed in a 30℃ environment for static fermentation for 48h. After fermentation, the pH value reaches 4.5, realizing the bioconversion of high-fiber raw materials and completing the pretreatment.

[0083] Example 3:

[0084] This embodiment provides a biophysical coupling pretreatment method for high-fiber raw materials, including the following steps:

[0085] By mass percentage, 85% corn stalks, 7% corn flour, 7% soybean meal and 1% molasses were selected as the mixed base material and fed into a continuous twin-shaft paddle conditioner. The acidic conditioning solution prepared in Preparation Example 3 was sprayed on the mixture. By adjusting the spraying amount of the acidic conditioning solution, the overall moisture content of the mixed base material system was controlled at 35%. After conditioning, the mixture was piled in a buffer chamber for 20 minutes.

[0086] After the bottom material is piled up, it enters the duplex stainless steel twin-screw extruder. The heating zone temperature is set to 130℃, and the residence time of the piled bottom material in the cavity is controlled to be 45s. After the piled bottom material is extruded through the die, it undergoes a violent flash phase change to form an extruded bottom material with a high specific surface area.

[0087] After the puffed base material is cooled to 37°C by cold air, the depolymerase bacterial solution prepared in Preparation Example 6 is introduced. The material is spread evenly in a shallow trough turning fermentation bed equipped with bottom forced ventilation (ventilation volume of 1.0 vvm), with a material layer thickness of 40 cm. This aerobic fermentation stage is maintained for 36 hours to obtain aerobic fermented material.

[0088] The aerobic fermentation material was fed into a high-shear biaxial paddle mixer, where the phase change accelerator prepared in Example 9 was sprayed during a 2-minute high-intensity shear homogenization process. By controlling the amount of liquid added, the overall moisture content of the aerobic fermentation material system was adjusted and stabilized at 55% based on the measured moisture content before homogenization. Subsequently, it was compacted and baled by a high-density silage hydraulic baler, achieving a compaction density of 1000 kg / m³. 3 Use stretch film for sealing and wrapping.

[0089] During the sealing process, *Lactobacillus plantarum* bacterial suspension (with an effective viable count of 2.0 × 10⁻⁶) was simultaneously introduced. 8 (CFU / mL, inoculation amount is 1.5% based on the wet weight of the material), the sealed package is placed in a 32℃ environment for static fermentation for 72h. After the fermentation is completed, the pH value of the system naturally drops to 3.5, and the pretreatment is completed.

[0090] Example 4:

[0091] This embodiment provides a biophysical coupling pretreatment method for high-fiber raw materials, including the following steps:

[0092] By mass percentage, 80% of liquor lees, 10% of corn flour, 8% of soybean meal and 2% of molasses were selected as the mixed base material and fed into a continuous twin-shaft paddle conditioner. The acidic conditioning liquid prepared in Example 1 was atomized and sprayed. By adjusting the spraying amount of the acidic conditioning liquid, the overall moisture content of the mixed base material system was controlled at 30%, and the mixture was left to stand in the buffer chamber for 18 minutes.

[0093] After being piled up, the bottom material enters a duplex stainless steel twin-screw extruder. The extrusion chamber temperature is set to 110℃ and the residence time is controlled at 30s. After being extruded, the piled bottom material is collected to form a loose and porous puffed bottom material.

[0094] After the puffed base material was cooled to 36°C, the depolymerase bacterial solution prepared in Preparation Example 4 was introduced. Aerobic fermentation was maintained for 30 hours in a shallow trough turning fermentation bed (material thickness 35cm, bottom ventilation 0.8vvm) to obtain aerobic fermented material.

[0095] The aerobic fermentation material was fed into a high-shear biaxial paddle mixer. During the 1.5-minute high-intensity shear homogenization process, the phase change accelerator prepared in Example 7 was sprayed on it. By controlling the amount of liquid added, the overall moisture content of the aerobic fermentation material system was adjusted and stabilized at 53% based on the measured moisture content before homogenization. Subsequently, it was baled using a high-density silage hydraulic baler, and the compaction density was 800 kg / m³. 3 Multiple layers of stretch membrane are used for sealing.

[0096] During the sealing process, *Lactobacillus plantarum* bacterial solution was simultaneously introduced (the effective viable count of *Lactobacillus plantarum* bacterial solution was 1.0 × 10⁻⁶). 8 (CFU / mL, inoculation amount is 1.0% based on the wet weight of the material), the feed package is placed in a static fermentation environment at 31℃ for 60h. After fermentation, the pH value drops to 4.0, and fermented feed with liquor lees as the base material is obtained, thus completing the pretreatment.

[0097] Comparative Examples 1-5:

[0098] Comparative Example 1:

[0099] Compared with Example 1, the difference is that the mixed base material is directly subjected to conventional mechanical crushing, without acid conditioning, extrusion puffing, inoculation of depolymerization enzyme liquid, shallow tank aerobic fermentation, homogenization and replenishment of phase change promoting liquid, and hydraulic bundling and compaction. Water is directly added to the total moisture content of 53%, and after inoculation with Lactobacillus plantarum, it is sealed for fermentation. The sealing method, fermentation temperature and fermentation time are the same as in Example 1.

[0100] Comparative Example 2:

[0101] Compared with Example 1, the difference is that the atomizing spraying device is connected to an equal amount of deionized water instead of an acidic conditioning solution; all other aspects are the same.

[0102] Comparative Example 3:

[0103] Compared with Example 1, the difference is that the extrusion and flash steaming treatment of the duplex stainless steel twin-screw extruder is cancelled. Instead, the treatment process is replaced by steaming the bottom material after it has been piled up at 100°C and atmospheric pressure for 30 seconds and then crushing it with ordinary mechanical means. All other processes are the same.

[0104] Comparative Example 4:

[0105] Compared with Example 1, the difference is that the stages of introducing depolymerase bacterial solution and aerobic fermentation in shallow trough turning fermentation bed are cancelled. After cooling, the puffed base material is directly transferred into a high-shear twin-shaft paddle mixer for homogenization, liquid replenishment and subsequent compaction and packaging operations. All other operations are the same.

[0106] Comparative Example 5:

[0107] Compared with Example 1, the difference is that after homogenization, the material is not bundled and compacted by a high-density silage hydraulic baler and sealed with multiple layers of stretch film. Instead, after homogenization, Lactobacillus plantarum is inoculated at the same amount as in Example 1, and fermentation is carried out directly using conventional aerated stacking method. All other conditions are the same as in Example 1.

[0108] Test Examples 1-4:

[0109] Test Example 1: Determination of Acid-Thermomechanical Coupling-Induced Endogenous Mediator Release

[0110] (1) Collect experimental samples. Take 100g of each of the following materials: the extruded material from Example 1 after being extruded by a duplex stainless steel twin-screw extruder, the material from Comparative Example 2 after undergoing the same extrusion process, and the material from Comparative Example 3 after being subjected to atmospheric pressure cooking and ordinary mechanical crushing. Place them in an ultra-low temperature freezer at -80℃ and freeze-dry them under vacuum for 48h. Grind them through a 60-mesh sieve to obtain the dry basis powder of each group of materials for testing.

[0111] (2) Preparation of the detection extract. Accurately weigh 2.00 g of the dry powder of each of the above groups and place them in 50 mL centrifuge tubes. Add 70% methanol aqueous solution at a material-to-liquid ratio of 1:15 (g / mL). After sealing, place the tubes in a CNC ultrasonic cleaner and extract ultrasonically at 45℃ and 250W for 40 min. After extraction, centrifuge at 8000 r / min for 15 min and collect the supernatant. Take 2 mL of the supernatant and filter it through a 0.22 μm organic microporous membrane. Collect the filtrate and transfer it to a chromatographic sample bottle for later use.

[0112] (3) High-performance liquid chromatography (HPLC) determination. Acetic acid and ferulic acid in the filtrate were quantitatively analyzed using an HPLC system equipped with a diode array detector. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was selected.

[0113] For the detection of acetic acid, the mobile phase was 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid), and the detection wavelength was set to 210 nm. For the detection of ferulic acid, the mobile phase was methanol-0.1% formic acid aqueous solution (volume ratio 45:55), and the detection wavelength was set to 320 nm. The column temperature was maintained at 30℃, the injection volume was 10 μL, and the flow rate was 1.0 mL / min. The release amount of the target analyte in each group was calculated using the internal standard method combined with the standard curve of the analyte.

[0114] Table 1. Results of endogenous mediator release from each group of materials

[0115] Materials corresponding to the process in Example 1 17.82 132.4 Comparative Example 2 Corresponding Process Materials 2.65 48.7 Comparative Example 3 Corresponding Process Materials 9.43 35.2

[0116] See appendix Figure 1 , Figure 1 The data reflects the acetic acid chromatographic peak distribution of the extruded base material from Example 1 via a duplex stainless steel twin-screw extruder, and the corresponding process materials of Comparative Examples 2 and 3 within the corresponding retention time.

[0117] See appendix Figure 2 , Figure 2 The data reflects the distribution of ferulic acid chromatographic peaks in the extruded material of Example 1 via a duplex stainless steel twin-screw extruder, as well as the materials of Comparative Examples 2 and 3 at the corresponding retention times.

[0118] in conclusion:

[0119] According to Table 1, Figure 1 and Figure 2 According to the data, the release concentrations of acetic acid and ferulic acid in the material of Example 1 were relatively high.

[0120] Comparative Example 2 used deionized water instead of acidic conditioning solution during the conditioning stage. Its acetic acid release was 2.65 mg / g dry matter, and the ferulic acid release was also at a low level, indicating that the exogenous weak acid conditions affected the degree of hydrolysis of the acetyl groups of the hemicellulose side chain and the degree of lignin depolymerization.

[0121] Comparative Example 3, treated under normal pressure cooking and ordinary mechanical pulverization conditions, showed a ferulic acid release of 35.2 μg / g dry matter, which was lower than the data in Example 1. This indicates that the physical work and flash evaporation process provided by the duplex stainless steel twin-screw extruder help promote changes in plant cell wall structure and the release of internal substances.

[0122] A comprehensive comparison shows that Example 1, which combines acidic conditioning liquid with twin-screw extrusion, can promote the release of endogenous mediators in the material. The generated weak acidic substances can provide the corresponding pH environment for the system, and the released ferulic acid and other phenolic substances can provide the corresponding physicochemical conditions for the degradation of the depolymerase bacterial solution in the subsequent stages.

[0123] Test Example 2: Monitoring of Dynamic Phase Transition Characteristics of Fermentation Systems

[0124] (1) Sensor deployment. After the material in Example 1 was homogenized and discharged and before it entered the high-density silage hydraulic baler, a wireless in-situ pH sensor and a miniature carbon dioxide concentration detection probe were embedded inside the material. The carbon dioxide release rate was calculated based on the change in carbon dioxide concentration per unit time and the calibrated volume of the probe. For Comparative Example 5, after the material was homogenized and discharged and before conventional aerated composting and fermentation, a pH sensor and a miniature carbon dioxide concentration detection probe of the same specification were embedded at the same depth inside the compost pile.

[0125] (2) Dynamic data acquisition. The timing was taken as the moment when hydraulic mechanical force was applied for bundling and compaction in Example 1 and when conventional aerated pile fermentation began in Comparative Example 5. The system parameters were recorded from 0h to 72h during the fermentation cycle. The 60h time was the fermentation endpoint corresponding to Example 1, and the 72h data was used as extended monitoring data to observe the trend of system changes. The data acquisition terminal automatically recorded the pH value and carbon dioxide release rate in the system every 15 minutes.

[0126] (3) Data processing. The characteristic time point data of the fermentation process were extracted and averaged with the effective data collected before and after each characteristic time point to obtain the pH value and carbon dioxide release rate of Example 1 and Comparative Example 5 at each fermentation time point.

[0127] Table 2. Dynamic monitoring data of fermentation process of each group of materials

[0128] 0 5.82 14.23 5.86 13.95 2 5.71 6.12 5.79 15.11 4 5.48 1.84 5.68 17.02 8 4.95 0.43 5.43 19.34 12 4.62 0.21 5.21 18.76 24 4.18 0.15 4.95 16.42 36 3.96 0.11 4.78 12.55 48 3.81 0.12 4.61 9.32 60 4.00 0.08 4.54 7.18 72 3.94 0.06 4.45 5.61

[0129] See appendix Figure 3 , Figure 3 The data includes the monitoring data distribution under the conditions of homogenized discharge and hydraulic mechanical force applied for bundling and compaction in Example 1, and under the conditions of conventional permeable stacking in Comparative Example 5.

[0130] in conclusion:

[0131] According to Table 2 and Figure 3 According to the data, after being baled and compacted by a high-density silage hydraulic baler, the carbon dioxide release rate of the fermentation system in Example 1 decreased from 14.23 mmol / (L·h) to 1.84 mmol / (L·h) within 0h to 4h, and remained at a relatively low level in the subsequent period; the pH value of the system decreased from 5.82 to 4.18 within 0h to 36h, and decreased to 4.00 at 60h, which corresponds to the pH value at the 60h fermentation endpoint in Example 1.

[0132] Comparative Example 5 was fermented using conventional aerated stacking after homogenization. Its carbon dioxide release rate showed an upward trend in the early stage of fermentation, reaching 19.34 mmol / (L·h) at 8h and then tending to decline. The corresponding pH value of the system decreased relatively slowly. The pH value was measured at 4.54 at 60h and still at 4.45 at 72h, which were significantly higher than the pH values ​​at the corresponding time points of Example 1.

[0133] Analysis of the experimental process in Example 1 shows that applying hydraulic mechanical force to bundle and compact the materials reduced the porosity between them, limiting the entry of external oxygen. Combined with the early oxygen consumption by microorganisms, this promoted the transition of the fermentation system from an aerobic to an anaerobic state. This environmental change facilitated the metabolic acid production of the introduced *Lactobacillus plantarum* culture, leading to a decrease in carbon dioxide release rate and system pH value in the early monitoring stages.

[0134] Comparative Example 5 used conventional aerated stacking, which maintained a certain degree of oxygen exchange inside the material. The system maintained respiratory metabolism for a longer period of time, which made the overall acid production and pH reduction process relatively delayed.

[0135] The above test results reflect the actual impact of the hydraulic mechanical force applied for bundling and compaction on the changes in biochemical parameters within the fermentation system.

[0136] Test Example 3: Comparative Test of Lignocellulose Cascade Degradation Rate

[0137] (1) Sampling and pretreatment. The final fermentation products of Examples 1 to 4 and Comparative Examples 1 to 5 after the fermentation cycle were collected respectively, while the corresponding untreated mixed substrate was retained as a blank control sample. Each group of samples was placed in an electric thermostatic drying oven at 65°C and dried to constant weight. The dried samples were crushed by a plant micro pulverizer, passed through a 40-mesh sieve, and the sieve-passing material was collected, placed in a self-sealing bag, and stored in a desiccator for later use.

[0138] (2) Composition determination. The contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) in each group of samples were determined by Van der Waals detergent fiber analysis. 1.00 g of sample powder was weighed and placed in a filter bag. Neutral detergent, a small amount of sodium sulfite, and α-amylase were added. The mixture was boiled at 100 °C for 1 h, washed, and dried. The NDF content was calculated. Subsequently, the residue was boiled at 100 °C for 1 h with acid detergent (a mixture of 0.5 mol / L sulfuric acid and hexadecyltrimethylammonium bromide) and washed and dried. The ADF content was calculated. The residue was hydrolyzed with a 72% sulfuric acid solution at 20 °C for 3 h. After washing with water until neutral, the residue was dried and ashed. The ADL content was calculated.

[0139] (3) Data calculation. The absolute contents of cellulose and lignin in each group of samples were calculated by the difference method, where the cellulose content was the difference between the ADF content and the ADL content, and the lignin content was equal to the ADL content.

[0140] Based on the corresponding component content in the initial blank control sample, the lignin degradation rate and cellulose degradation rate of each group of materials were calculated after the fermentation cycle.

[0141] Table 3. Results of Lignocellulose Degradation Rate Measurement for Each Group of Materials

[0142] Example 1 45.12 52.34 Example 2 41.05 48.71 Example 3 46.88 54.62 Example 4 43.27 50.15 Comparative Example 1 8.45 18.53 Comparative Example 2 25.66 32.14 Comparative Example 3 19.34 28.92 Comparative Example 4 11.21 35.67 Comparative Example 5 36.54 41.23

[0143] in conclusion:

[0144] According to the data in Table 3, the lignin degradation rate in the final fermentation products after the fermentation cycle of Examples 1 to 4 ranged from 41.05% to 46.88%, and the cellulose degradation rate ranged from 48.71% to 54.62%.

[0145] Comparative Example 1, which was treated with conventional mechanical crushing without the use of acidic conditioning solution or extrusion puffing, showed that the degradation rates of lignin and cellulose were 8.45% and 18.53%, respectively. The data showed that the single treatment method had a relatively limited impact on the lignin-carbohydrate complex structure in the plant cell wall.

[0146] Comparative Example 2 did not use an acidic conditioning solution during the conditioning stage, and Comparative Example 3 did not use a duplex stainless steel twin-screw extruder for treatment. The degradation rate of these two groups was lower than that of Example 1. The test results show that the lack of weak acid-induced or thermo-mechanical physical phase change deconstruction process affected the increase of substrate specific surface area and the release of internal substances, which is not conducive to the entry of enzyme system and microorganisms into the substrate for reaction in subsequent stages.

[0147] Comparative Example 4 lacked the stages of inoculation with depolymerase bacteria and aerobic fermentation in a shallow trough turning fermentation bed, and its lignin degradation rate was 11.21%, while the corresponding cellulose degradation rate was 35.67%.

[0148] Comparative Example 5 was fermented using conventional aerated stacking after homogenization. Its lignin degradation rate was 45.68%, which was basically the same as that of Example 1, but its cellulose degradation rate dropped to 32.85%.

[0149] Data shows that although a continuous aerobic environment ensures the degradation of lignin, the lack of mechanical compaction by high-density hydraulic baling limits the close contact between cellulase and the internal structure of the material, and fails to quickly establish a closed acidic microenvironment suitable for the synergistic effect of cellulase and lactic acid bacteria, thus affecting the degradation efficiency of cellulose.

[0150] The data comparison above reflects that the pre-treatment step, combined with the aerobic fermentation stage, has a synergistic effect on improving the steric hindrance of biomass and promoting the degradation of cellulose substances.

[0151] Test Example 4: Comparative Test of Enrichment and Nutrient Conversion Efficiency of Acid-Soluble Small Peptides

[0152] (1) Sampling and pretreatment. The final fermentation products after the fermentation cycle of each example and comparative example were collected as experimental samples, and the corresponding untreated mixed substrate was taken as blank control samples. Each group of samples was placed in a 60℃ constant temperature drying oven and baked to constant weight. The samples were then crushed by a pulverizer and passed through a 60-mesh sieve to obtain dry basis powder samples of each group of materials for later use.

[0153] (2) Determination of crude protein content. The crude protein content of the sample was determined by the Kjeldahl method. 0.50 g of dry powder sample was accurately weighed and placed in a digestion tube. A mixed catalyst of potassium sulfate and copper sulfate was added, followed by the injection of concentrated sulfuric acid.

[0154] The digestion tubes were placed on a digestion furnace and digested at 420°C until the liquid turned a transparent blue-green color. After cooling, the digestate was distilled and titrated using an automated Kjeldahl nitrogen analyzer. The volume of standard acid consumed was recorded, the total nitrogen content of the sample was calculated, and multiplied by the protein conversion factor of 6.25 to calculate the crude protein content of the final fermentation product for each group.

[0155] (3) Determination of small peptide proportion. The proportion of small peptides was determined by a combination of trichloroacetic acid precipitation and biuret colorimetric method. 2.00 g of dry powder sample was weighed and deionized water was added at a ratio of 1:10 (g / mL). After extraction for 2 h, the supernatant was collected by centrifugation. An appropriate amount of the supernatant was added to an equal volume of 15% trichloroacetic acid solution and allowed to stand to precipitate large molecular weight proteins. After centrifugation at 10000 r / min for 10 min, the supernatant containing acid-soluble small peptides and a small amount of free amino acids was collected. The supernatant was reacted with biuret reagent and the absorbance was measured at a wavelength of 540 nm. The content of acid-soluble peptide components was calculated by combining the standard curve, and finally, its mass percentage of total crude protein was calculated.

[0156] Table 4. Results of Enrichment and Nutritional Conversion Efficiency of Acid-Soluble Small Peptides in Each Group

[0157] Example 1 21.42 32.76 Example 2 19.85 28.53 Example 3 22.18 35.21 Example 4 20.57 30.14 Comparative Example 1 11.24 4.82 Comparative Example 2 16.53 15.34 Comparative Example 3 15.81 12.77 Comparative Example 4 14.16 9.45 Comparative Example 5 13.52 8.21

[0158] in conclusion:

[0159] According to the data in Table 4, the crude protein content in the final fermentation products of Examples 1 to 4 ranged from 19.85% to 22.18%, and the proportion of small peptides in the crude protein ranged from 28.53% to 35.21%.

[0160] Comparative Example 1 used a single closed anaerobic fermentation method, with a crude protein content of 11.24% and a small peptide ratio of 4.82%. The data comparison showed that, without the pre-treatment step of biophysical coupling, the fermentation system had relatively limited conversion of nitrogen sources in high-fiber raw materials and degradation efficiency of macromolecular proteins.

[0161] Comparative Example 5 was fermented using conventional aerated stacking after homogenization, without the use of a high-density silage hydraulic baler to apply hydraulic mechanical force for compaction. Its crude protein content was 13.52%, and the proportion of small peptides was 8.21%. Because Comparative Example 5 did not undergo a physical compaction process, the oxygen mass transfer of the system was not interrupted, and the microenvironment required for acid production was not quickly established.

[0162] The process in this embodiment utilizes mechanical forced compaction to restrict oxygen, causing a decrease in the system's pH. The neutral protease added with the phase-change accelerator primarily exerts its hydrolytic effect during the near-neutral or weakly acidic stage from homogenization to the early stages of fermentation. As the system pH further decreases, the acidic environment helps inhibit contaminating microorganisms and stabilize acid-soluble small peptides. Simultaneously, endogenous acidic proteases and microbial metabolism-related proteases continue to participate in the protein degradation process. The synergistic effect of these proteases promotes the cleavage of large protein molecules and the enrichment of small peptide substrates.

[0163] The test results show that there is a direct correlation between the mechanical forced compaction process and the peptide conversion process in the fermentation system.

Claims

1. A biophysical coupled pretreatment method for high-fiber raw materials, characterized in that, Includes the following steps: The raw materials, including high-fiber raw materials, corn flour, soybean meal and molasses, are mixed to form a mixed base material, which is fed into a continuous twin-shaft paddle conditioner and introduced with acidic conditioning liquid for conditioning treatment. The mixture is then transported to a buffer silo for heaping treatment. The acidic conditioning liquid is made of citric acid or lactic acid aqueous solution. The bottom material after the slumping treatment is continuously fed into a duplex stainless steel twin-screw extruder for extrusion. The bottom material is extruded through the die hole to a normal pressure environment to achieve vaporization and flash evaporation, and is collected to form a porous sponge-like extruded bottom material. After being cooled, the puffed base material is introduced into the depolymerization enzyme liquid through an atomizing spraying device. The depolymerization enzyme liquid is made by mixing liquid mycelial fermentation liquid of *Amanita muscaria*, industrial laccase and xylanase. It is spread on a shallow trough turning fermentation bed with bottom ventilation for aerobic fermentation to obtain aerobic fermented material. The aerobic fermentation material is transferred to a high-shear biaxial paddle mixer for homogenization. During homogenization, a phase change accelerator is continuously sprayed via atomization, and the overall moisture content is adjusted. The phase change accelerator contains industrial cellulase, neutral protease, Bacillus subtilis culture, and Saccharomyces cerevisiae culture. After homogenization, the material is baleed and compacted using a high-density silage hydraulic baler. The compaction density of the bale is controlled at 600–1000 kg / m³. 3 ; During the sealing process, Lactobacillus plantarum culture is introduced through a synchronous inoculation device, and the mixture is sealed with a stretch film and allowed to ferment in a temperature-controlled chamber to obtain functional fermented feed, thus completing the pretreatment.

2. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The raw materials, by weight percentage, include: The ingredients consist of 70%–85% high-fiber raw materials, 7%–15% corn flour, 7%–12% soybean meal, and 1%–3% molasses.

3. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The specific parameters for the conditioning process are as follows: The amount of acidic conditioning solution sprayed is controlled to ensure that the overall moisture content of the mixed base material system is 25% to 35%, and the time for the heaping treatment is 15 to 20 minutes.

4. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The specific parameters of the duplex stainless steel twin-screw extruder are as follows: The temperature of the heating zone inside the extrusion chamber is set to 105-130℃, and the residence time of the material in the chamber is maintained at 20-45s by adjusting the screw speed.

5. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The aerobic fermentation steps specifically include: The puffed base material is cooled to 35-37°C by cold air convection before being inoculated with the depolymerization enzyme solution. The bottom ventilation volume of the shallow trench turning fermentation bed is 0.5 to 1.0 vvm, the material thickness is controlled at 30 to 40 cm, and the duration of the aerobic fermentation stage is 24 to 36 hours.

6. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The specific parameters for the homogenization process are as follows: After high-shear homogenization in the high-shear biaxial impeller mixer for 1-2 minutes, the overall moisture content of the aerobic fermentation material system after replenishment is stable at 50%-55%.

7. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The static fermentation step specifically includes: The inoculum amount of *Lactobacillus plantarum* culture is 0.5% to 1.5% based on the wet weight of the material. The sealed package is placed in a temperature-controlled chamber at 30-32°C and allowed to ferment for 48-72 hours.

8. The biophysical coupling pretreatment method for high-fiber raw materials according to claim 1, characterized in that, The preparation steps of the phase change enhancing liquid include: Dissolve 1.0%–2.0% by mass of soluble starch and 0.5%–1.0% by mass of yeast extract in deionized water, sterilize, and cool. Add industrial cellulase and neutral protease at a concentration of 1000-2000 U / g based on the dry weight of the aerobic fermentation material. The phase change activating solution was prepared by inoculating Bacillus subtilis culture and Saccharomyces cerevisiae culture at a volume ratio of 1.5:1 to 2:1 and mixing them evenly.

9. The application of a biophysical coupling pretreatment method for high-fiber raw materials as described in any one of claims 1-8 in the preparation of functional fermented feeds made from high-fiber raw materials.

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