Production method for improving digestibility of feed soybean meal through synergism of bacteria and enzymes and application of production method

Abstract

The invention relates to a production method for improving the digestibility of feeding soybean meal through synergism of bacteria and enzymes and application. The production method comprises the following steps: a lignin embedding layer, a cross-linked polysaccharide-protein network and a phytic acid binding region in soybean meal jointly form a digestibility bottleneck; an infrared spectrum is combined with an enzyme activity site scanning technology, an area sensitive to a bacterium-enzyme synergistic reaction in soybean meal is identified, and a micro-treatment pre-reaction condition is designed to activate a structure; by means of metabolic flow analysis, a multi-strain cross-path metabolic map is established, one strain secretes a small molecule activator, and the other matched strain releases response type enzyme; the method comprises the following steps: constructing a synergistic metabolism system which takes lactic acid bacteria to reduce the pH value of the system in an anaerobic state and synchronously generate trace hydrogen peroxide as a signal, synergistically activates acid protease, acts on a soybean meal matrix together with yeast, and decomposes protein and non-starch polysaccharide in soybean meal through bacterium and enzyme complementation so as to improve the digestibility of the soybean meal; technical breakthrough and remarkable gain are realized from multiple dimensions of structure regulation and function release to animal utilization.

Description

Technical Field

[0001] This invention relates to a production method for improving the digestibility of soybean meal for feed, specifically a production method and application for improving the digestibility of soybean meal for feed through synergistic effects of microorganisms and enzymes. Background Technology

[0002] Current production methods for synergistically improving the digestibility of soybean meal for feed have a certain research foundation in theoretical construction and preliminary application. However, compared with published literature such as CN105510273A, "A Method for Authenticating and Identifying Soybean Meal Based on Micro-area Spectroscopic Characteristics," it can be seen that the current technical approach has significant shortcomings and systemic drawbacks in terms of precise structural identification, stratified reaction control, and actual nutrient release mechanisms, making it difficult to meet the actual needs of modern efficient animal nutrition processing. The technology described in the published literature mainly focuses on using micro-area near-infrared spectroscopy to authenticate and identify the origin of soybean meal samples. Its essence is a non-target screening method, focusing on macroscopic quality monitoring and processing consistency judgment, that is, determining whether the soybean meal to be tested belongs to a type already existing in the standard database. Its advantages are fast detection speed, high sensitivity, and non-destructive nature, making it suitable for use in grain, oil, or feed quality monitoring scenarios. However, the functionality of this technology is completely limited to the characterization level, lacking the ability to analyze the microscopic nutritional barrier structure of soybean meal, and unable to assess its conversion efficiency or nutrient release pathway in the actual digestive system. Therefore, when it is introduced into the complex biological reaction system of synergistically improving digestibility through bacteria and enzymes, it will be severely inadequate.

[0003] First, the spectral characterization used in the published literature completely fails to consider the spatial distribution differences of antinutritional structures in soybean meal. For example, phytic acid-polysaccharide complexes and lignin-protein cross-linking regions constitute key barriers that animals cannot digest, and these are the fundamental problems limiting soybean meal utilization. This technology only establishes the statistical distance (GH value) between the sample and the test sample based on the near-infrared spectral curve, without establishing a logical relationship between spectral characteristics and structural activity, enzyme response sites, and degradation pathways. It lacks a structure-function-biological response closed loop, therefore it can provide almost no effective data guidance or response control model for improving digestibility. Secondly, most common microbial-enzyme co-fermentation systems currently available are still based on homogeneous soybean meal raw materials for fermentation. Their core process lacks a stratified control design for the structure of soybean meal. Enzyme preparations or strains exhibit non-selective diffusion during fermentation, which can easily lead to the following drawbacks: First, the enzyme utilization efficiency is low, with some enzymes becoming inactive due to spatial barriers before entering the particles. Second, the structural reactions lack a gradient sequence, resulting in excessively strong early reactions and a lack of response areas in the later stages, leading to a sudden release of nutrients rather than a slow release. Third, the colonization of strains is chaotic, failing to achieve preferential occupation and tiered metabolism, ultimately resulting in low fermentation efficiency, a low proportion of small peptides in the finished product, and incomplete degradation of antigenic proteins.

[0004] Furthermore, in traditional processes, the particle size of soybean meal is poorly controlled and the porosity of the structure is uncontrollable. It is impossible to set up structural channels according to the diffusion characteristics of enzymes and the metabolic rhythm of microorganisms. Structural activation is often achieved through high temperature, high humidity or strong acid and alkali treatment. Although this method can destroy the structure, it often leads to thermal denaturation of soybean meal protein, allosteric inactivation of small peptides or release of minerals, which increases nutrient loss and goes against the original intention of green and efficient utilization.

[0005] Furthermore, while some literature has attempted to link infrared spectroscopy, enzyme response characteristics, and microbial metabolic features in analysis, most lack standardized response functions for modeling. For example, they lack the ability to couple and model variables such as enzyme dosage, structural resistance, time window, and porosity effects, making enzyme-microbial interactions reliant on empirical operations without theoretical support, and unable to precisely control the fermentation window and the timing of structural release. The Mahalanobis distance (GH) index provided in published literature can only be used for sample clustering and lacks time-domain response capabilities. Therefore, it cannot monitor changes in the enzymatic reaction state of structures at different time points, nor can it predict at which hour of fermentation a particular structure will open or which components will be released. Summary of the Invention

[0006] The purpose of this invention is to provide a production method and application for synergistic improvement of the digestibility of soybean meal for feed by bacteria and enzymes, thereby solving some of the drawbacks and shortcomings pointed out in the background art.

[0007] The first technical solution of the present invention: a production method for synergistically improving the digestibility of soybean meal for feed by microorganisms and enzymes, comprising,

[0008] (1) The lignin-encapsulated layer, cross-linked polysaccharide-protein network and phytic acid binding region in soybean meal together constitute the digestibility bottleneck; infrared spectroscopy combined with enzyme activation site scanning technology is used to identify the regions in soybean meal that are sensitive to the synergistic reaction of bacteria and enzymes, and microprocessing pre-reaction conditions are designed to activate the structure.

[0009] (2) Using metabolic flux analysis, a multi-species cross-pathway metabolic map was established, in which one species secretes a small molecule activator and another supporting species releases a responsive enzyme; a synergistic metabolic system was constructed in which lactic acid bacteria lower the pH of the system under anaerobic conditions and simultaneously produce trace amounts of hydrogen peroxide as a signal to synergistically activate acidic proteases, which work together with yeast on the soybean meal matrix. Through the complementary decomposition of proteins and non-starch polysaccharides in soybean meal by bacteria and enzymes, the digestibility of soybean meal was improved.

[0010] (3) Optimize the particle size and pore structure of soybean meal to gradually expose different functional layers during fermentation; through responsive microencapsulation technology, first release phytase to break the phytic acid structure, then release acidic protease to decompose the antigen protein, forming different enzyme release rhythms; use organic acids and activated polypeptide microbial metabolites to reverse regulate the enzyme reaction rate and achieve multi-level control.

[0011] Furthermore, the method for identifying regions in soybean meal sensitive to synergistic reactions with microorganisms and enzymes includes,

[0012] Infrared spectroscopy was used to scan the structural domains of raw soybean meal to identify high-density cross-linked regions, anti-nutritional factor encapsulation layers, and complex polysaccharide-protein structures. The obtained infrared characteristic spectra were matched with a pre-set enzyme activity site database to determine the regions in soybean meal that are sensitive to co-degradation by bacteria and enzymes.

[0013] Based on the spatial distribution and structural characteristics of the identified sensitive areas, targeted pretreatment conditions were formulated, including micro-acid treatment, mild humid heat treatment, and intermittent low-energy shear stirring.

[0014] Optionally, pre-enzyme introduction treatment can be performed by adding a low dose of structural pre-lysin to activate the structure of soybean meal, causing partial deconstruction while retaining nutritional activity; subsequently, the treated soybean meal can be used in a microbial-enzyme co-fermentation system to improve digestibility.

[0015] Furthermore, the infrared spectroscopy analysis employs Fourier transform infrared spectroscopy to identify amide bonds, glycosidic bonds, phosphate ester bonds, and structural groups that bind to anti-nutritional factors; the enzyme activity site database contains characteristic spectral information on the corresponding binding sites of phytase, β-glucanase, xylanase, and acidic protease with the anti-nutritional structure of soybean meal.

[0016] Furthermore, the microacid treatment is carried out in the pH range of 4.5 to 5.5 to loosen phosphate bonds and protein complexes;

[0017] Mild humid heat treatment is carried out at 45–55°C for a time of no more than 30 minutes.

[0018] Intermittent low-energy shear mixing treatment uses a low-speed intermittent mixing method to locally disrupt the dense structure of soybean meal particles and promote the exposure of surface pores.

[0019] Furthermore, the pretreatment involves structural analysis of the soybean meal raw material to identify non-starch polysaccharide structural regions that encapsulate nutrient factors, including xylan, glucan, and pectin cross-linked complex layers; and the addition of a low dose of structural pre-lysin for targeted activation; the structural pre-lysin is xylanase, glucanase, pectinase, or arabinosidase, and the amount added is limited to a subfunctional level that does not cause complete degradation of the overall structure, with the action time controlled between 10 and 30 minutes.

[0020] To express the dynamic activation efficiency, the following structured response function is adopted.

[0021] ;

[0022] in,

[0023] : Indicates the structural activation response rate per unit time, reflecting the structure's ability to release enzyme responses; Enzyme reaction time; The sensitivity factor for enzyme dosing is determined by the strength of structural recognition matching; : Raw material porosity factor, reflecting the degree to which particle size and pore combination support the enzyme diffusion pathway; Enzyme stability activity coefficient represents the proportion of enzymes that retain effective activity under set temperature and humidity conditions; The structural resistance coefficient represents the cross-linking strength of the structure itself; the higher the value, the more difficult it is to break down. It acts as an inhibitor, controlling the degradation trend of enzymes over long periods of time, including enzyme inactivation or side reactions.

[0024] The first half of the function To activate the growth term, describe the rapid increase in the enzyme's response to structure in the early stages of its action; in the denominator... This introduces an inhibitory mechanism against excessively long-term action of structural resistance and enzyme activity to prevent structural disintegration; this function controls the optimal action time window, maintaining the opening of structural channels while avoiding protein denaturation or loss of target nutrients; when Reaching the threshold This indicates that the structure has been fully activated and can enter the synergistic stage between bacteria and enzymes.

[0025] Furthermore, the method for optimizing soybean meal particle size and pore structure includes,

[0026] (S1) Wet grinding of soybean meal to control the particle size to 300-800 micrometers, and hot pressing and instantaneous explosion treatment of the pulverized material to form porous microcracks on the surface, maintain a partially dense cross-linked structure in the middle layer, and retain micropores in the inner layer.

[0027] (S2) Regulate the drying rate and temperature to form a layered pore gradient from the outside to the inside: the surface layer has medium pore size, which is conducive to the initial contact of enzymes; the middle layer is a cross-linked structure enrichment area, which provides for the synergistic effect of bacterial enzymes; the inner layer is a microporous structure, which is used to retain small peptides and embed anti-nutritional factors.

[0028] (S3) Obtain soybean meal with a layered response structure for stepwise response and timed release in subsequent microbial-enzyme co-fermentation process.

[0029] Furthermore, the wet milling is carried out under conditions where the soybean meal moisture content is 18% to 25% to retain fiber flexibility and enhance micropore formation ability; the hot pressing and instantaneous explosion treatment is carried out under conditions of ≤0.3MPa and ≤15s to form a non-fully expanded structure.

[0030] Furthermore, the drying process employs a low-temperature slow drying method at 50–70°C. The resulting layered pore gradient structure can sequentially guide different enzymes and microorganisms to gradually invade from the surface to the inner layer during the synergistic fermentation of bacteria and enzymes, thereby increasing the release rate of proteins, small peptides, and phosphorus components.

[0031] To express the guiding effect of this layered structure on the permeation and action rate of bacterial enzymes, a synergistic osmotic response function was used.

[0032] ;

[0033] in:

[0034] : Structure in radius Placement, fermentation time The intensity of the synergistic response to bacterial enzyme penetration; : The depth within the particle radius; Time variable during fermentation; The initial porosity coefficient depends on the surface porosity density formed during the drying process. : Structure in The local porosity activity coefficient at a location reflects whether a layer at a certain depth has responsiveness; : The sensitivity index for synergistic growth of bacterial enzymes; the higher the value, the more the bacterial enzyme activity depends on the structure for release. : Structural compaction inhibition coefficient, reflecting the resistance of dense regions to enzyme diffusion; Permeability decay index: This represents the degree of nonlinear decay of enzyme permeability with structural depth.

[0035] denominator This controls the physical reality that the deeper the structure, the more difficult it is for the enzyme to penetrate; within the molecule This indicates a synergistic increase in enzyme activity during fermentation, but this increase must be supported by the pore structure to translate into effective permeability; when At a certain time Reaching the critical response value This indicates that the structural layer is open to the synergistic effect of bacterial enzymes and can be further degraded.

[0036] The second technical solution of the present invention is the application of a production method for improving the digestibility of soybean meal for feed through synergistic enhancement of microbial enzymes. The layered structure exposes different functional areas over a delayed period, enabling the microbial enzymes to form a synergistic gradient reaction from the outer layer to the inner layer during fermentation, thereby improving the digestibility of soybean meal in the animal intestine.

[0037] Furthermore, the outer layer response includes primary degrading enzymes such as phytase and β-glucanase, the middle layer response is synergistic with acidic proteases and polysaccharides, and the inner layer response is a late-stage degrading enzyme produced by specific bacterial species.

[0038] The production method for synergistic enhancement of feed soybean meal digestibility proposed in this invention has the following beneficial effects, achieving technological breakthroughs and significant gains from multiple dimensions, including structural regulation, functional release, and animal utilization:

[0039] By using infrared spectroscopy and enzyme activity site matching technology, high-density regions of anti-nutritional factors such as lignin-polysaccharide-protein cross-linking structures and phytate complex layers in soybean meal can be identified, thereby enabling targeted addition of enzyme preparations during the pretreatment stage, avoiding ineffective effects and excessive structural degradation, preserving nutritional activity, and creating a favorable channel for subsequent synergistic bacterial-enzyme reactions.

[0040] By utilizing wet pulverization, low-pressure instantaneous explosion, and low-temperature slow drying technologies, the three-layer structure of soybean meal (porous surface layer, cross-linked middle layer, and microporous inner layer) is reconstructed, and a gradient pore pathway is constructed. This allows different types of enzymes and microorganisms to be exposed and respond sequentially according to the structural layers during fermentation, effectively improving the selectivity and stability of enzyme diffusion pathways and enhancing overall degradation efficiency.

[0041] A time-sequential enzyme release strategy and a metabolic regulation mechanism of functional strains were established. The surface rapid-release phytase and glucanase, the middle layer acidic protease and polysaccharide enzyme synergistic degradation, and the late-stage enzymes induced by specific strains in the inner layer were matched to effectively promote the phased release and efficient transformation of key nutrients such as antigen proteins, small peptides, and phosphorus, and optimize the synergistic pathway between bacteria and enzymes.

[0042] By controlling the amount of pretreatment enzymes, treatment time, and drying temperature, the structure is partially loosened but not completely disintegrated. This maintains the overall particle stability of soybean meal while forming open channels, preventing protein thermal denaturation and inactivation of functional factors, and ensuring the sustainable progress of subsequent synergistic reactions. Attached Figure Description

[0043] Figure 1 This is a diagram showing the entire process and structural relationship of the synergistic effect of bacteria and enzymes in improving the digestibility of soybean meal in this invention.

[0044] Figure 2 This is a diagram showing the relationship between the multi-strain and signal-regulated stratified degradation function of soybean meal in this invention.

[0045] Figure 3 This is a schematic diagram of the three-in-one progressive regulation fermentation system for soybean meal of the present invention.

[0046] Figure 4 This is a flowchart comparing the soybean meal structure identification and targeted preprocessing in Example 1.

[0047] Figure 5 This is a flowchart of the activation of soybean meal microstructure and optimal pretreatment time in Example 1.

[0048] Figure 6This is a mind map of the preparation and co-fermentation of layered soybean meal in Example 2. Detailed Implementation

[0049] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0050] Production methods for synergistically improving the digestibility of soybean meal for animal feed using microorganisms and enzymes include:

[0051] (1) The lignin-encapsulated layer, cross-linked polysaccharide-protein network and phytic acid binding region in soybean meal together constitute the digestibility bottleneck; infrared spectroscopy combined with enzyme activation site scanning technology is used to identify the regions in soybean meal that are sensitive to the synergistic reaction of bacteria and enzymes, and microprocessing pre-reaction conditions are designed to activate the structure.

[0052] The method for identifying regions in soybean meal sensitive to synergistic reactions of microorganisms and enzymes includes...

[0053] Infrared spectroscopy was used to scan the structural domains of raw soybean meal to identify high-density cross-linked regions, anti-nutritional factor encapsulation layers, and complex polysaccharide-protein structures. The obtained infrared characteristic spectra were matched with a pre-set enzyme activity site database to determine the regions in soybean meal that are sensitive to co-degradation by bacteria and enzymes.

[0054] The infrared spectroscopy analysis employs Fourier transform infrared spectroscopy to identify amide bonds, glycosidic bonds, phosphate ester bonds, and structural groups that bind to anti-nutritional factors; the enzyme activity site database contains characteristic spectral information on the binding sites of phytase, β-glucanase, xylanase, and acidic protease to the corresponding anti-nutritional structures of soybean meal.

[0055] Based on the spatial distribution and structural characteristics of the identified sensitive areas, targeted pretreatment conditions were formulated, including micro-acid treatment, mild moist heat treatment, and intermittent low-energy shear stirring. The micro-acid treatment was carried out in the pH range of 4.5 to 5.5 to loosen phosphate bonds and protein complexes. The mild moist heat treatment was carried out at 45 to 55°C for a time controlled within 30 minutes. The intermittent low-energy shear stirring treatment used a low-speed intermittent stirring method to locally disrupt the dense structure of soybean meal particles and promote the exposure of surface pores.

[0056] The pretreatment involves structural analysis of soybean meal raw materials to identify non-starch polysaccharide structural regions that encapsulate nutrient factors, including xylan, glucan, and pectin cross-linked complex layers; and targeted activation by adding a low dose of structural pre-lysin; the structural pre-lysin is xylanase, glucanase, pectinase, or arabinosidase, and the amount added is limited to a subfunctional level that does not cause complete degradation of the overall structure, and the action time is controlled within 10–30 min;

[0057] Optionally, pre-enzyme introduction treatment can be performed by adding a low dose of structure pre-lysin to activate the structure of soybean meal, causing partial deconstruction while retaining nutritional activity; the treated soybean meal can then be used in a microbial-enzyme co-fermentation system to improve digestibility.

[0058] (2) Using metabolic flux analysis, a multi-species cross-pathway metabolic map was established, in which one species secretes a small molecule activator and another supporting species releases a responsive enzyme; a synergistic metabolic system was constructed in which lactic acid bacteria lower the pH of the system under anaerobic conditions and simultaneously produce trace amounts of hydrogen peroxide as a signal to synergistically activate acidic proteases, which work together with yeast on the soybean meal matrix. Through the complementary decomposition of proteins and non-starch polysaccharides in soybean meal by bacteria and enzymes, the digestibility of soybean meal was improved.

[0059] (3) Optimize the particle size and pore structure of soybean meal to gradually expose different functional layers during fermentation; through responsive microencapsulation technology, first release phytase to break the phytic acid structure, then release acidic protease to decompose the antigen protein, forming different enzyme release rhythms; use organic acids and activated polypeptide microbial metabolites to reverse regulate the enzyme reaction rate and achieve multi-level control.

[0060] The method for optimizing soybean meal particle size and pore structure includes,

[0061] (S1) Wet grinding of soybean meal to control the particle size to 300-800 micrometers, and hot pressing and instantaneous explosion treatment to form porous microcracks on the surface, maintain a partially dense cross-linked structure in the middle layer, and retain micropores in the inner layer; the wet grinding is carried out under the condition of soybean meal moisture content of 18%-25% to retain fiber flexibility and enhance micropore formation ability; the hot pressing and instantaneous explosion treatment is carried out under the conditions of ≤0.3MPa and ≤15s to form a non-fully expanded structure;

[0062] (S2) By controlling the drying rate and temperature, a layered pore gradient is formed from the outside to the inside. The surface layer has medium pore size, which is conducive to the initial contact of enzymes; the middle layer is a cross-linked structure enrichment zone, which provides for the synergistic effect of bacteria and enzymes; the inner layer is a microporous structure, which is used to retain small peptides and embed anti-nutritional factors. The drying is carried out at a low temperature of 50-70℃. The resulting layered pore gradient structure can guide different enzymes and microorganisms to gradually invade from the surface to the inner layer during the synergistic fermentation of bacteria and enzymes, thereby improving the release rate of proteins, small peptides and phosphorus components.

[0063] (S3) Obtain soybean meal with a layered response structure for stepwise response and timed release in subsequent microbial-enzyme co-fermentation process;

[0064] The application of a production method that synergistically enhances the digestibility of soybean meal for animal feed involves a layered structure that exposes different functional zones over a delayed period, allowing the enzymes to form a synergistic gradient reaction from the outer to the inner layers during fermentation, thereby improving the digestibility of soybean meal in the animal's intestines. The outer layer response includes primary degrading enzymes such as phytase and β-glucanase; the middle layer response involves the synergistic action of acidic proteases and polysaccharides; and the inner layer response involves late-stage degrading enzymes produced by specific bacterial species.

[0065] Combined with appendix Figure 1 As shown, this invention provides a production method for synergistically improving the digestibility of soybean meal for feed by combining microorganisms and enzymes, aiming to solve the problems of low digestibility and poor bioavailability of soybean meal due to its complex structural barriers. Soybean meal, as a high-protein feed resource, contains various anti-nutritional factors. These factors do not exist in a free state but are embedded in highly complex structures, specifically including lignin-polysaccharide-protein complexes, a dense network structure composed of cross-linked non-starch polysaccharides and globular proteins, and chelation regions formed by the binding of phytic acid and protein. These structures significantly limit the absorption and utilization of nutrients such as protein, small peptides, and phytic acid phosphorus in soybean meal by the animal intestine, becoming a key obstacle to improving its feed value.

[0066] To this end, this invention first introduces Fourier transform infrared spectroscopy (FTIR) analysis to scan the microstructure of soybean meal raw materials. By identifying the vibrational peak position changes of different chemical bonds, such as characteristic bands like C–O–C, C=O, and P–O–C, the embedded regions and their chemical cross-linking forms in soybean meal are determined. These are then matched with a pre-established enzyme activity site database, which covers the preferred recognition fragments and structural fingerprints of various feed enzymes (such as phytase, xylanase, glucanase, and acidic protease) on specific substrates. After systematic analysis, the co-sensitive regions in soybean meal that have potential responses to specific enzymes can be located, namely key degradation entry sites where the microstructure is not yet fully open but has a certain affinity and spatial contact conditions. After identification, a microprocessor pre-reaction scheme is formulated based on the distribution and structural density of the sensitive areas. Specifically, mild acid treatment within the pH range of 4.5–5.5 induces the initial dissociation of ester bonds in the composite structure. Simultaneously, short-term moist heat treatment is performed at a controlled temperature of 45–55℃ to loosen the structural entanglement, allowing the proteoglycan network to partially unfold while maintaining its complete functional activity. This is supplemented by low-speed intermittent mechanical shearing to generate surface microcracks, selectively destroying the embedded areas rather than completely pulverizing the soybean meal. Furthermore, to further enhance the accessibility of the sensitive structures, extremely low doses of structural pre-lysing enzymes (such as xylanase, pectinase, and dextranase) can be added at this stage. The enzyme dosage is controlled to not exceed 0.05% of the dry weight of the soybean meal, and the action time is limited to 10–30 minutes. The aim is to open the initial channel for synergistic bacterial-enzyme interaction at the structural level, rather than achieving complete hydrolysis. The innovation of this method lies in realizing the spatial access and initiation pathway construction of the bacterial-enzyme synergistic mechanism at the structural level, avoiding the problems of ineffective enzyme addition and delayed structural reactions in traditional methods.

[0067] Combined with appendix Figure 2 As shown, to achieve efficient and orderly degradation of proteins, phytic acid, phosphorus, and antigenic factors in soybean meal, a multi-species synergistic mechanism was constructed through metabolic flux analysis, forming a synergistic metabolic domain dominated by microorganisms and responded to by enzymes. This implementation first sets up a microbial combination based on the target function, and then screens core microbial groups with complementary metabolic characteristics using metabolic pathway tracking technologies (such as metabolic flux labeling and transcriptome data association analysis). These include precursor bacteria that secrete metabolic regulatory factors and responsive enzyme-expressing bacteria. The first category of microorganisms are regulatory-driven microorganisms, such as lactic acid bacteria, which produce lactic acid during early fermentation, rapidly lowering the local pH to the range of 4.2–5.5. This acidic environment helps inhibit the growth of other microorganisms and activates the activity of acidic proteases embedded in the soybean meal structure, achieving preliminary cleavage of heat-stable antigenic proteins.

[0068] The second type of strain uses aerobic bacteria that have a natural oxygen requirement and have long been recognized as safe in the feed industry, such as thermotolerant yeasts. During their vigorous metabolic phase, these strains fully utilize the dissolved oxygen supplemented in the fermentation system to secrete oxygen-tolerant hydrolases such as lipases and xylanases, thereby further decomposing the cell walls, polysaccharides, and oils of soybean meal in the middle and later stages of fermentation. This, combined with the lactic acid bacteria-acidic protease system from the earlier stage, completes the total degradation. If it is necessary to adjust the osmotic pressure of the fermentation broth and the stability of the bacterial film, a small amount of food-grade glycerol can be added exogenously to the formula (instead of relying on bacterial synthesis) to improve the persistence and stability of the synergistic fermentation of the bacterial community.

[0069] Through the dynamic accumulation of the aforementioned metabolic factors, another type of responsive microbial strain is guided to express structurally responsive enzyme systems in a timely manner, such as specific xylanase, acidic phytase, and small peptide transporters, thereby achieving a triple linkage between bacteria, enzymes, and structures at the metabolic level. To precisely control the construction process of this synergistic metabolic domain, this invention sets metabolic signal trigger points in the fermentation system, using the concentration thresholds of metabolic regulatory factors (such as lactic acid > 5 mmol / L and hydrogen peroxide < 0.5 mmol / L) as the initiation signal. When the system environment meets the triggering conditions, the metabolic mutual induction pathway between microbial strains is initiated, entering a stage of clear functional division and synergistic structural decomposition. The overall design of this process not only achieves the directional initiation of different types of enzyme systems according to functional levels, but also significantly improves the efficiency of stratified degradation of the internal structure of soybean meal. At the same time, it effectively avoids problems such as enzyme inactivation, reaction mismatch, and substrate waste in traditional single-strain or single-enzyme treatments, enabling the anti-nutritional barrier in soybean meal to be broken down layer by layer, protein conversion to be more thorough, and the release of small peptides to be significantly increased.

[0070] Combined with appendix Figure 3The flowchart utilizes microbial metabolites as feedback regulators to achieve multi-stage linkage reactions. In practice, soybean meal is first processed to a particle size range of 300–800 micrometers. Through wet granulation and low-temperature slow drying, a progressively decreasing pore structure is formed from the outside in. The outer layer is a medium-sized porous structure, facilitating rapid colonization of macromolecular enzymes and lead bacteria. The middle layer is a dense cross-linked region, primarily storing antigenic proteins and complex phytic acid complexes. The inner layer is a microporous encapsulation region, rich in small peptides and residual anti-nutritional factors. This structure provides the spatial basis for staged fermentation. Based on this, the present invention employs responsive microcapsules to encapsulate various feed enzymes. Phytase is encapsulated in acid-sensitive capsules that dissolve easily at low pH. In the early stages of fermentation, lactic acid produced by lactic acid bacteria rapidly lowers the environmental pH, preferentially releasing phytase to act on the phytate complex region in the middle layer structure, breaking the cross-linking between proteins and phosphate groups. Subsequently, in the later stages of fermentation, the gradual accumulation of activated peptides, amino acids, and organic acids released by the bacterial community's metabolism not only serves as a nutritional supplement for synergistic bacterial development but also has a regulatory effect. When their concentration reaches a set threshold, they can activate the outer layer of the enzyme capsule encapsulated with acidic protease (e.g., based on peptide bond cleavage responsive materials), causing the acidic protease to be released over time and concentrated on the antigen protein region, thus achieving the effect of first breaking cross-links and then breaking down the enzyme. The main chain undergoes a layer-by-layer degradation process. Furthermore, these metabolites, such as lactic acid, peptides, and short-chain fatty acids, can further regulate the rate of enzyme-catalyzed reactions by adjusting local pH, metal ion concentration, and enzyme structural stability. This enhances or inhibits the catalytic efficiency of enzymes at a certain stage, enabling the entire co-fermentation process to have adaptive regulatory capabilities and avoiding excessive early enzyme dissipation or delayed enzyme failure in later stages. This strategy achieves multi-dimensional linkage from physical structural configuration (particle size and pore size) to the rhythm of biological enzyme release and metabolic signal feedback control, thus constructing a three-in-one fermentation regulation system of structural response – enzyme rhythm – metabolic feedback. This effectively promotes the stepwise release and complete conversion of target nutrients such as protein, small peptides, and phytic acid phosphorus in soybean meal, significantly improving feed digestibility and the bioavailability of functional substances, demonstrating strong practicality and industrial-scale promotion value.

[0071] Example 1:

[0072] Combined with appendix Figure 4 As shown, a medium-sized feed mill conducted a trial production of fermented soybean meal for feed. The raw material used was non-GMO defatted soybean meal from Northeast China (batch number: DBP202501), with a sample size of 100 kg. Preliminary testing showed a protein content of 47.8%, crude fiber content of 5.2%, phytic acid content of 1.5%, and antigenic protein (β-conglycinin + glycoprotein) accounting for 18.7% of the crude protein. Preliminary animal simulation digestibility was 67.2%. To precisely improve its nutritional utilization rate, the following structural identification and pre-activation process of this invention was implemented:

[0073] The first step involved structural analysis of the soybean meal samples using Fourier transform infrared spectroscopy (FTIR). A Bruker Vertex 70 FTIR spectrometer was used, and the samples were pressed into KBr pellets. Wavenumber measurements were performed in the range of 4000–400 cm⁻¹. In the characteristic spectra, absorption peaks at 1742 cm⁻¹, 1243 cm⁻¹, and 1027 cm⁻¹ corresponded to the C=O and P=O ester bonds and the C–O–C glycosides, respectively. Combined with the sample density distribution and separation chromatography results, the soybean meal exhibited a strong phytate-protein complex and a dextran cross-linked layer. The spectral characteristics matched the pattern of initial cleavage by phytase followed by delayed degradation by acidic proteases in the enzyme site database of this project with a match rate of 87.6%. Further scanning electron microscopy analysis after sample sectioning confirmed that the embedded region was mainly concentrated in the middle layer of the soybean meal particles, with a thickness of approximately 20–40% of the particle radius, providing a basis for subsequent targeted pretreatment design.

[0074] The second step, based on the above identification results, is to formulate the following pretreatment plan: Soybean meal is added to the reaction vessel, the material-to-water ratio is controlled at 1:1.2, the pH is adjusted to 5.0, and 0.2 mol / L citric acid is added for a slightly acidic treatment for 20 minutes. Then, the temperature is raised to 50℃ for a constant-temperature moist heat treatment for 30 minutes, during which low-speed (45 rpm) intermittent stirring is implemented, stopping for 3 minutes every 5 minutes of operation to promote gradual softening of the encapsulated layer structure rather than overall disintegration. At the 20th minute, a pre-lysin complex solution (containing 50 U / g xylanase and 30 U / g pectinase) is added, with the enzyme amount controlled at 0.05% of the dry weight of the soybean meal. This step aims to slightly shear the cross-linked structure, exposing potential enzyme sites, but avoiding excessive protein degradation. After treatment, the soybean meal showed a 23.1% increase in surface porosity under a scanning electron microscope, and obvious stratification gaps appeared in the middle layer encapsulated structure. The protein SDS-PAGE spectrum showed little change in the intensity of the main band, indicating that the nutritional components were well preserved.

[0075] The third step involves adjusting the pretreated soybean meal to a moisture content of 65% and placing it in a 38℃ fermentation chamber. A bacterial-enzyme synergistic system is then added for verification: 3 × 10⁻⁶ Lactiplantibacillus plantarum bacteria are inoculated with this lactic acid-producing strain. 7 CFU⁻¹ with safe yeast Saccharomyces cerevisiae 5×10 6CFUg⁻¹ was used to rapidly generate lactic acid and maintain a slightly acidic environment. Phytase 300Ug⁻¹ and acidic protease 800Ug⁻¹ were added simultaneously (with xylanase 6000Ug⁻¹ added if necessary). The pH drop triggered the enzymes to gradually open the structural channels of the soybean meal. Fermentation lasted 48 hours, with samples taken and analyzed every 6 hours. The results showed that phytic acid content decreased by 42% after 12 hours, free small peptides increased significantly after 24 hours, and hydrolyzed small peptides accounted for 21.4% of total protein at 48 hours. Antigen protein decreased to 15.8% of the initial level, and the in vitro simulated digestibility increased to 82.6%. This verified the technical effect of the synergistic fermentation of lactic acid bacteria, yeast, and acidic protease / phytase in significantly improving the digestibility and utilization of soybean meal.

[0076] This experiment used a Bruker Vertex 70 Fourier transform infrared spectrometer to scan defatted soybean meal samples for structural chemical bond identification. The samples were vacuum dried (60℃, 6 hours) and then ground through a 100-mesh sieve. 2 mg of the sample was mixed with 200 mg of anhydrous KBr and compressed into tablets. The samples were then detected within the scanning range of 4000–400 cm⁻¹. Several key absorption peaks were observed in the infrared spectrum, including the amide I band (C=O stretching) at 1655 cm⁻¹ and the amide II band (N–H bending and C–N stretching) at 1540 cm⁻¹, indicating the presence of a high content of heat-stable antigenic protein structures in soybean meal. Obvious glycosidic bond peaks were detected in the 1050–1150 cm⁻¹ range, especially at 1078 cm⁻¹ and 1043 cm⁻¹, showing the presence of a large number of arabinoxylan and β-mannan fragments linked by glycosidic bonds. At the same time, P=O and P–O–C bond absorption peaks appeared near 1240 cm⁻¹ and 970 cm⁻¹, proving that phytic acid complexes are widely present in soybean meal and stably embedded in the protein-polysaccharide cross-linked structure.

[0077] The aforementioned infrared spectral characteristic peaks were imported into the soybean meal antinutritional enzyme active site database (version number: FDB-V3.1) established in this project. The database contains structural target information and characteristic spectral image segments of 28 commercial feed enzymes, including the frequency range of phytase's action on P–O–C cleavage bonds, the absorption peak response range of xylanase to 1,4-β-xylosidic bonds, the spectral segment of β-glucanase recognizing β-1,4 linkage sites, and the corresponding absorption peak positions of acidic proteases recognizing amide bonds (especially cysteine–glutamate binding sites). Through characteristic spectrum superposition analysis, the infrared spectrum matched 91.3% with phytase, 87.2% with xylanase, 83.9% with β-glucanase, and 89.6% with acidic proteases (represented by neutral acidic proteases). Based on the multiple matching intensity of the spectra, the system identified that the sensitive regions in soybean meal with the potential for synergistic degradation by bacteria and enzymes are mainly concentrated in the following three categories: first, the phytic acid-protein chelate region, whose P-O-C structure response enzyme recognition index R1 reaches 0.89; second, the xylan-protein complex region, whose glycosidic bond active region R2 is 0.86; and third, the heat-stable antigen protein embedding region, whose amide bond structure activation response R3 is 0.91. The spatial overlap of the three regions reaches 58.4%, providing a basis for structural activation.

[0078] Based on the above structural identification results, the following strategy was set for verification in conjunction with the fermentation path simulation model: 50 kg of soybean meal raw material was divided into three groups according to three pretreatment strategies: physical wet heat group (Group A), physical + enzyme identification guided group (Group B), and the spectral identification + targeted enzyme site matching synergistic pretreatment group described in this invention (Group C). Group C used phytase (300 U / g), xylanase (500 U / g), and neutral acidic protease (700 U / g) selected under infrared spectral guidance. After pretreatment with slightly acidic + wet heat + low dose of pre-cracking enzyme, it was inoculated with synergistic bacterial strains (lactic acid bacteria + yeast) for fermentation. Samples were taken and analyzed after 48 hours of fermentation in all three groups. Digestibility results showed that small peptides in group A increased to 16.4% of total protein, group B increased to 19.8%, and group C significantly increased to 23.5%. Meanwhile, the antigen protein degradation rate in group C reached 85.2%, higher than group A's 63.4% and group B's 72.9%. The residual phytic acid in fermented soybean meal decreased to 0.34%. Animal in vitro digestion simulation experiments showed that the protein digestibility of group C was 84.1%, which was 16.9 percentage points higher than that of untreated raw materials.

[0079] The above examples illustrate that the present invention uses Fourier transform infrared spectroscopy to identify structural groups in soybean meal, such as amide bonds, glycosidic bonds, and phosphate ester bonds, that bind to anti-nutritional factors. By matching these groups with an enzyme activity site database, the invention can efficiently locate the regions in the soybean meal structure that are most sensitive to the synergistic degradation response of bacteria and enzymes, guide the selection of enzymes and the timing of their administration, and establish a scientific basis for structural-level degradation.

[0080] In this embodiment, 30 kg of soybean meal was divided into three groups: a conventional heat treatment control group (Group A), a slightly acidic + moist heat treatment group (Group B), and a slightly acidic + moist heat + shear triple treatment group proposed in this invention (Group C), to compare the effects of different treatment combinations on the degree of structural loosening, porosity improvement, and subsequent fermentation efficiency.

[0081] Group C, as the implementing entity, first added warm water at a mass ratio of 1:1.1 for mixing, and then slowly added citric acid solution to adjust the pH to 5.0. This acidic environment was maintained for 20 minutes. Monitoring data showed that this pH value induced a preliminary charge destabilization reaction in the phosphate ester bonds of the soybean meal, particularly significantly increasing the loosening of the binding sites between phytic acid and protein. Structural bond energy model calculations showed a decrease in the binding strength of the P–O–C binding region of approximately 17.2%. No enzyme preparations were added at this stage; the aim was to induce stress-induced loosening of the structure through a slightly acidic environment to activate enzyme accessibility in the areas to be cleaved. Subsequently, the system was heated to 50°C and maintained at a constant temperature for 25 minutes of moist heat treatment, controlling the risk of protein thermal denaturation within a limited range not exceeding 30 minutes. During heat treatment, the slurry was shear-stirred using a propeller-type agitator at a stirring intensity of 45 rpm, employing an intermittent strategy of 5 minutes of stirring followed by 3 minutes of settling. The aim was to break down locally dense areas of the surface particles, creating shallow cracks and structural channels, while avoiding overall pulverization and structural disintegration. Scanning electron microscopy data showed that the average pore size of the soybean meal particles after this treatment increased from the original 1.2 μm to 2.6 μm, the micropore density increased by 34.7%, the crack length distribution was concentrated between 5–20 μm, and the degree of structural rupture was controlled within the allowable range.

[0082] Subsequently, groups A, B, and C were inoculated into the same bacterial-enzyme co-fermentation system, containing L. plantarum (3 × 10⁻⁶). 7 CFU / g), B. subtilis (1×10) 8 The fermentation formula consisted of CFU / g, phytase (300 U / g), and acidic protease (800 U / g). The fermentation temperature was 37℃, humidity 65%, and fermentation time was 48 hours. Samples were collected every 12 hours during the fermentation process to measure parameters including phytic acid residue, the proportion of soluble small peptides in total protein, residual antigen protein, and in vitro simulated digestibility. Results showed that group C achieved a phytic acid degradation rate of 73.8% by 24 hours, compared to 59.3% for group B and only 46.5% for group A. The proportion of hydrolyzed small peptides in group C ultimately reached 22.6%, an increase of 4.2% compared to group B and 7.8% compared to group A. The residual antigen protein rate in group C was only 13.1%, significantly lower than that of groups B (19.7%) and A (26.5%). In the simulated digestion experiment, the digestibility of group C reached 84.9%, significantly higher than that of groups B (78.2%) and A (71.6%).

[0083] The above results clearly verify the feasibility and effectiveness of the micro-acid-moist heat-shear triple structure activation strategy proposed in this invention. The micro-acid treatment precisely controls the pH within the range of 4.5–5.5, selectively loosening the phosphate ester bond binding region and the protein cross-linking network. The moist heat treatment within the range of 45–55℃ promotes the softening of the structural chains without causing thermal inactivation of the protein. The shearing and stirring, in a low-speed intermittent manner, guides the formation of porous channels at the microscale. The combination of the three factors produces a good structural response.

[0084] Combined with appendix Figure 5 As shown, this embodiment will next conduct a comprehensive microstructural analysis of this batch of soybean meal. Infrared spectroscopy analysis confirmed that it has strong phytate phosphate bond peaks (1240 cm⁻¹, 970 cm⁻¹), amide bond peaks (1655 cm⁻¹), and glycoside bond peaks (1043 cm⁻¹), indicating the presence of a large number of anti-nutritional factor structural complex regions. Combined with image optical density scanning and BET surface area analysis results, the outer porosity of this soybean meal sample is 18.6%, the cross-linking density value of the middle layer (expressed as optical density difference ΔD) is 0.42, and the average particle size is 486 μm. Based on the database feature spectrum matching and enzyme target distribution prediction, it can be judged that its structural resistance to decomposition is moderate to strong, making it most suitable for entering the bacterial-enzyme co-process after activation. Based on the analysis results, the following enzyme activation strategy was formulated: a compound system of structure pre-cleavage enzymes xylanase (50 U / g), dextranase (40 U / g), and pectinase (30 U / g) was selected, with the total amount added controlled at 0.09% of the dry weight of soybean meal. The reaction times were set at 10 minutes, 20 minutes, and 30 minutes, respectively, to investigate the differences in structure activation efficiency corresponding to the three time durations. During the pretreatment stage, the temperature was set at 50℃, the pH was controlled at 5.0, and the humidity was approximately 65%. The stirring speed of the reaction system was set at 50 rpm, with intermittent operation (5 minutes of stirring, 3 minutes of stillness). To accurately express the degree of structure activation, the structure response function was substituted:

[0085] ;

[0086] The values ​​of each parameter in this experiment are as follows:

[0087] This indicates that under conditions of 50℃ and pH 5.0, the enzyme activity retention rate is 85%;

[0088] The score is derived from the matching score between the enzyme and the target site in the infrared characteristic spectrum (matching rate 83% × standardized coefficient).

[0089] The result was calculated by normalizing the particle size of 486 μm and the surface porosity of 18.6%.

[0090] The structural resistance coefficient is calculated based on the optical density difference and infrared intensity value.

[0091] , is the long-term inhibition coefficient, taking into account that the enzyme activity decreases by about 15% within 30 minutes at this temperature, and includes side reaction interference factors.

[0092] Substitute the above parameters into the response function to calculate the structural activation values ​​at different times:

[0093] when minute:

[0094] ;

[0095] when minute:

[0096] ;

[0097] when minute:

[0098] ;

[0099] The calculation results show that the structural activation response reached its peak (0.042) at 10 minutes. Subsequently, due to the increase of the enzyme inhibition term in the denominator with the square of time, the overall response value rapidly decreased, indicating that completing the initial initiation of the structural channel within 10 minutes is the most economical and effective processing window. Combined with electron microscopy image analysis, the 10-minute pretreated samples showed obvious structural gaps, with the outer pore size increasing to an average of 2.3 μm. Furthermore, no main band breakage was observed in protein electrophoresis, indicating that the trophic structure remained intact and the activation level was optimal.

[0100] The 10-minute treatment protocol was introduced into an actual co-fermentation system. A combination of lactic acid bacteria (L. plantarum) and yeast (B. subtilis), along with xylanase (300 U / g) and acidic protease (800 U / g), was used for co-fermentation for 48 hours. The proportion of small peptides reached 21.9%, the residual rate of antigen protein decreased to 13.8%, and the in vitro digestibility increased to 83.7%. The results were better than the fermentation control group without pre-cleavage enzyme activation (small peptide proportion 16.5%, digestibility 74.1%), which fully verified the guiding and forward-looking nature of the structure-response function in actual production.

[0101] In summary, this invention, through infrared spectroscopy and microstructural parameter extraction, combined with multi-factor structural response function modeling and time window control, not only achieves precise control of local structural activation, but also avoids structural collapse and nutrient loss caused by excessive enzymatic hydrolysis, ensuring the smooth implementation of subsequent synergistic effects of bacteria and enzymes. Ultimately, it significantly improves the protein digestibility and bioavailability of soybean meal for feed, demonstrating strong technological originality and industrial-scale promotion value.

[0102] Example 2:

[0103] Combined with appendix Figure 6 As shown in the flowchart, a pilot-scale fermentation workshop is used as the scenario to carry out structural control design and response performance verification experiments on defatted soybean meal raw material from Northeast China (batch number: DBP202501). The present invention first implements step (S1), which involves wet grinding of soybean meal using a high-shear rotor wet mill with a moisture content controlled at 20%, so that the particle size distribution is controlled within the range of 300 to 800 micrometers, with a D50 of 476 μm. The purpose is to balance permeability and structural stability through medium particle size. Subsequently, hot-pressing instantaneous explosion treatment is carried out within 3 hours after grinding, with a pressure set at 0.25 MPa and an action time of 10 seconds, rapidly releasing steam to cause instantaneous thermal expansion and micro-cracking of the soybean meal particle surface. Microscopic observation shows that irregular cracks in the range of 0.5–3 μm are formed on the surface, with an average pore size increase of 22%. The middle layer structure retains some polysaccharide-protein cross-linking bands without obvious breakage, while the inner layer maintains relatively intact microporosity.

[0104] In step (S2), the raw material is dried in a controlled manner using a low-temperature flatbed dryer with a humidity-controlled air duct. The temperature is set at 55℃, the air velocity at 0.3 m / s, and the total drying time is 5 hours. Throughout the process, a multi-stage control logic is implemented, prioritizing drying of the outer layer, drying of the middle layer, and slow drying of the inner layer. The final measured moisture content is 9.7%. BET surface area measurement data shows that the average pore size of the surface layer is 2.5 μm, and that of the middle layer is 1.4 μm. The inner layer is 0.6 μm in diameter, and the pore gradient shows a significant decreasing trend from the outside to the inside along the radial direction. Particle slice images show that the middle layer is a dense zone containing xylan and protein complexes, which, according to near-infrared imaging, account for 28.6% of the total particle mass and is the main battlefield for subsequent synergistic degradation. The inner layer has a stable microporous region, which is presumably containing residual anti-nutritional factors and potential small peptide components.

[0105] In step (S3), the prepared stratified responsive soybean meal raw material is used in combination with the microbial-enzyme synergistic system, and the synergistic strain L. plantarum (3×10⁻⁶) is prepared. 7 CFU / g) and B. subtilis (1×10) 8The fermentation process involved adding phytase (initial release, 300 U / g) and acidic protease (released 6 hours later, 800 U / g) at different time intervals to respond to the functional zones of the middle and inner layers, respectively. The fermentation temperature was set at 38℃, humidity at 65%, and the total fermentation time was 48 hours, with samples taken every 12 hours. Experimental observations showed that at 12 hours, the surface pores were fully utilized, leading to rapid colonization of the microbial community, and a 54.1% reduction in phytate under the action of phytase. At 24 hours, enzymatic activity in the dense middle layer peaked, with a significant increase in small peptide content, reaching 17.3% of the total protein. At 36 hours, the acidic protease response was activated, penetrating deeper into the inner layer, increasing the proportion of small peptides to 21.9%, reducing the residual antigen protein rate to 12.7%, and achieving a final digestibility of 83.8%, an improvement of 11.2% compared to the un-layered ground soybean meal (control group digestibility 72.6%).

[0106] To further quantify the degree to which particle size and structure support the reaction release pathway, a structural penetration coefficient is introduced. As an evaluation indicator, it is defined as the active penetration capacity of bacterial enzymes into structural layers of different depths per unit time, and is calculated using the following expression:

[0107] ;

[0108] in: For the first Reaction release rate of structural layer (% / h) This represents the effective working area (cm²) of the layer. The average structural layer thickness is (mm). The fermentation time is given in hours (h). In the stratified response structure system, the measured λ value is 0.076 (strong response), while the λ value of traditional whole-crushed soybean meal is 0.045 (weak response), further demonstrating that the structure designed in this invention has a better spatial response path and time release gradient.

[0109] This embodiment uses controlled moisture content and burst pressure-time parameters to form a functional particle structure that is not fully expanded but has active pores. The initial moisture content of the experimental raw material, soybean meal, was 9.8%, the protein content was 47.5%, and the antigen protein content was approximately 18.4%. According to the design requirements, the soybean meal was first humidified to the target moisture range and divided into three groups for comparative testing: Group A had 18% moisture, Group B had 22%, and Group C had 25%. During humidification, an atomizing spray system was used, and the mixture was stirred in stages and allowed to stand for 3 hours to ensure that moisture evenly penetrated the soybean meal particles rather than just adsorbing onto the surface. A high-shear wet milling equipment (FZ-600 type) was used to wet-mill the samples in each group, screening for a target particle size range of 300–800 micrometers, with a D50 controlled at 480–500. Within a certain range, after wet grinding, the process immediately proceeds to the next step to prevent structural collapse caused by moisture fluctuations.

[0110] The hot-pressing instantaneous explosion treatment uses a modified puffing silo system, with the steam pressure controlled between 0.25 and 0.3 MPa. After continuous pressurization and heating, the pressure is rapidly released, with the actual steam pressure maintained at 0.28 MPa. The treatment time is set to 13 seconds to ensure that the pressure can trigger micro-fractures in the surface cell walls to form cracks, without causing protein thermal denaturation or starch gelatinization that leads to structural gelatinization and pore sealing. After the instantaneous explosion treatment, the three groups of raw materials are pre-dehydrated with 30°C hot air to about 14% moisture content, and then slowly dried in a 55°C flat plate dryer until the final moisture content is controlled below 10%, completing the basic raw material treatment.

[0111] Structural analysis showed that the optimal micropore formation effect was achieved when the moisture content was controlled at 22% (Group B). BET surface area analysis revealed a total porosity of 23.1%, approximately 18% higher than the sample treated with 18% moisture content, with an average pore size of 2.1 μm. Density imaging also showed obvious shallow cracks on the surface, a dense cross-linked structure in the middle layer, and no collapse in the inner layer, maintaining the desired micropore layout. Group A, with its low moisture content, experienced brittle cracks during crushing, resulting in suboptimal micropore formation and a total porosity of only 16.7%. Group C, due to its high moisture content, showed a tendency for some particles to semi-gelatinize after the instantaneous explosion, leading to pore structure collapse and localized adhesion, resulting in a decrease in porosity to 21.3%. The 13-second instantaneous explosion time for heat treatment was also confirmed as the optimal window through multiple batches of experiments. Extending the time to 15 seconds resulted in excessive surface swelling, causing disordered enzyme penetration and a decrease in subsequent synergistic degradation efficiency.

[0112] The three groups of treated soybean meal raw materials were inoculated into the same microbial-enzyme co-fermentation system, using Lactobacillus plantarum (3×10⁻⁶). 7 CFU / g) and Bacillus subtilis (1×10 8 A dual-strain system (CFU / g) supplemented with xylanase (300 U / g) and acidic protease (800 U / g) was used under standard solid-state fermentation conditions for 48 hours at 37°C and 65% humidity, with sampling every 12 hours. Results showed that group B (22% moisture + 13 seconds of 0.28 MPa instantaneous burst) achieved a small peptide content of 18.2% at 24 hours, a phytic acid degradation rate of 69.4%, a final small peptide percentage of 22.8%, a decrease in antigen protein residue to 13.1%, and an overall digestibility of 84.2%. Control groups A and C achieved 77.4% and 79.3%, respectively, further demonstrating that optimal moisture control and the non-expansion instantaneous burst time-pressure ratio are crucial for pore formation and subsequent enzyme penetration and bacterial colonization pathways.

[0113] To further quantify the impact of the instantaneous detonation treatment parameters on the structural reactivity, the structure formation activity coefficient λ' is defined as follows:

[0114] ;

[0115] in:

[0116] The effective crack density (cracks / mm²) on the structural surface was measured to be 28.4 for group B, 19.7 for group A, and 21.1 for group C. The average pore size (μm) is 2.1 for group B; Moisture correction factor (normalized based on the ratio of actual moisture to the optimal 22%). Let λ be the heat treatment time (s). Substituting the data, we calculated λ'=2.85 for group B, 1.68 for group A, and 2.12 for group C, clearly verifying that the instantaneous explosion response of group B is closest to the ideal structure that is not fully expanded but fully activated.

[0117] Using defatted soybean meal sample DBP202501 as the model material, a low-temperature drying condition control experiment and structure-response coupling modeling analysis were designed and carried out. First, after standardized operations including wet grinding and instantaneous explosion pretreatment, soybean meal with an initial moisture content of 22.6% was subjected to slow drying treatment. Drying temperatures were set at 50℃, 60℃, and 70℃, and the differences in surface pores and internal structure differentiation formed at different temperatures were compared. All samples were dried using a hot air plate dryer (wind speed 0.3 m / s), with a thickness of 1.5 cm, for drying times of 6 h (50℃), 4.5 h (60℃), and 3.5 h (70℃), with the final moisture content controlled below 10%. BET surface area and pore size analysis results showed that the porosity of the sample dried at 50℃ was 24.5% on the surface, 15.8% in the middle layer, and 11.2% in the inner layer. Although the sample dried at 70℃ had a high porosity of 29.3% on the surface, the middle and inner layers showed structural collapse and the average pore size was only 7.4 nm, which did not meet the characteristics of layered response. Finally, the sample dried slowly at 60℃ was selected as the optimal structural representative because it has a significant gradual transition characteristic from surface porosity to middle cross-linking to inner micropores, and has the best structural stability and layered uniformity.

[0118] Subsequently, this three-layered soybean meal raw material is used in a microbial-enzyme co-fermentation process, with the following microbial strain combination:

[0119] Lactobacillus plantarum (3×10 7 CFU / g) and Bacillus subtilis (1×10 8 The fermentation conditions were controlled at 37°C, 65% humidity, and 48 hours, with pore structure differentiation extraction and response rate sampling analysis performed every 6 hours during the fermentation. To quantify the layer-by-layer delay in enzyme permeation due to the layered structure, a synergistic permeation response function was proposed.

[0120] ;

[0121] The specific values ​​of each coefficient in this experiment are as follows:

[0122] Initial surface pore induction coefficient, normalized based on an average surface pore density of 26.8 pores / mm²;

[0123] Local structural activity coefficients: 1.0 for the surface layer, 0.68 for the middle layer, and 0.35 for the inner layer;

[0124] : A sensitivity index for synergistic growth of bacteria and enzymes, derived from previous kinetic modeling estimation;

[0125] : The structural compaction inhibition coefficient is derived from the measured middle layer density and inner layer molecular flow rate;

[0126] The permeation attenuation index is derived from the fitting results of the particle size progressive sample group and is controlled within the range of 1.3–1.8.

[0127] Substituting the above parameters into the function, we simulate the coordinated response values ​​Φ of the surface layer (r=0.2mm), middle layer (r=0.6mm), and inner layer (r=1.0mm) at different time points τ:

[0128] surface layer, ;

[0129] Middle layer, ;

[0130] Inner layer, ;

[0131] A critical response value Φn = 5.0 was set, representing that the structural layer possesses sufficient conditions for synergistic action between bacteria and enzymes. The above calculations show that the surface layer opened at 12 hours of fermentation, the middle layer reached the response threshold at 24 hours, while the inner layer gradually opened only after 36 hours, which fully conforms to the enzyme penetration and delayed function initiation pathway under structural control proposed in this invention. Further measurements of phytic acid content, small peptide release rate, and antigen protein degradation at each stage showed that the phytic acid degradation rate reached 61.3% at 12 hours, small peptides accounted for 16.4% of the total protein at 24 hours, and the small peptide content reached 22.3% and the digestibility increased to 84.7% after 48 hours of fermentation, significantly better than the traditional rapid drying treatment group (control digestibility was 74.1%).

[0132] This experiment, based on the aforementioned optimized batch of soybean meal raw material DBP202501, conducted batch validation on a continuous pilot-scale fermentation line. Previously, three-layered soybean meal particles with an average particle size of 480 μm, a surface pore size of approximately 2.4 μm, a dense middle layer, and an inner layer retaining micropores (<1 μm) were obtained through wet milling, instantaneous explosion pressure control treatment, and low-temperature slow drying at 60℃. The pore layer thickness ratio was approximately 3:4:3, and BET analysis confirmed its significant structural stratification characteristics. To evaluate the promoting effect of the delayed exposure mechanism on bacterial-enzyme synergy, key sampling time points were designed during the fermentation process to dynamically track the nutrient degradation pathway, structural response progress, and enzyme activity distribution area.

[0133] The fermentation system consisted of Lactobacillus plantarum (3 × 10⁻⁶) 7 CFU / g) and Bacillus subtilis (1×10 8 CFU / g), exogenous xylanase (300 U / g) was added at hour 0 in the form of encapsulated microcapsules, and acidic protease (800 U / g) was coated with a double-layer polysaccharide membrane for delayed release, starting to release from hour 18 after fermentation. The temperature was controlled at 37℃, humidity at 65%, and the fermentation period was 48 hours. The surface response threshold (CFU / g) was calculated based on the co-osmotic function model. =5.0) was achieved in 12 hours, the middle layer completed the response in 24 hours, and the inner layer gradually opened in 36 hours. In 12 hours, sampling analysis showed that the phytic acid degradation rate in the surface layer of soybean meal reached 61.7%, while the enzyme activity in the middle layer region had not yet penetrated significantly, indicating that the structural delay barrier played a role in inhibiting premature hydrolysis. In 24 hours, the antigen protein content in the middle layer region decreased by 42.5% compared with the initial value, corresponding to the release time of acidic protease, verifying that the middle layer is the main reaction layer. In the inner layer release stage from 36 to 48 hours, the accumulation of small peptides increased significantly, accounting for 22.9% of the total protein, while the residual phytic acid decreased to 0.36%, and the final residual antigen protein rate was 11.8%, which is consistent with the sequential response of the structural layers.

[0134] To further verify the digestive effect of this stratified structure in animals, 30 growing-finishing pigs with an average weight of 18.6 kg were selected and divided into an experimental group (using fermented structured soybean meal) and a control group (using untreated soybean meal). The experiment lasted for 21 days. Fecal sample analysis showed that the daily average nitrogen excretion in the experimental group decreased by 18.4%, the true protein digestibility increased to 82.1% (compared to 69.7% in the control group), and the serum total small peptide level was 26.2% higher, demonstrating that the structure-guided synergistic degradation products have better absorption efficiency in the intestine. To quantify the contribution of the stratified structure to the synergistic response efficiency, a stratified response index was established. , and defined as:

[0135] ;

[0136] in These are the response function values ​​for each layer of the structure. To determine the effective functional weights for the corresponding structural layers, the surface layer W1 = 0.3, the middle layer W2 = 0.4, and the inner layer W3 = 0.3. Substituting the actual data, the calculated... The value was 15.26, higher than the 8.42 measured in the unstructured treatment group (homogenized soybean meal treatment), indicating that the structure-delayed response mechanism improved the synergistic reaction efficiency by nearly 81%, demonstrating a significant optimization effect on the degradation pathway.

[0137] This embodiment of the experiment uses a defatted soybean meal sample (batch number: DBP202501) that has undergone prior structural optimization as the basis for research. A combined process of wet milling (22% water content), low-pressure instantaneous explosion (0.28 MPa, 13 seconds), and low-temperature slow drying (60℃, 4.5 h) was employed to prepare a layered responsive soybean meal with a distinct surface porous structure, a middle cross-linked structure, and an inner microporous structure. The structural diagram shows that the average pore size of the surface layer is 2.4 mm. The middle layer density is enhanced but retains 1.1. The average channel diameter shrinks to 0.7 mm in the inner layer. The three layers are clearly defined. Based on the requirements of the synergistic fermentation function, different enzyme systems are matched to the three layers: the surface layer is mainly treated with phytase (300 U / g) and β-glucanase (250 U / g), aiming to degrade the phytate complex salts and glucan fragments enriched in the outer layer of soybean meal; the middle layer is treated with acidic protease (800 U / g) and neutral xylanase (150 U / g), aiming to break down the dense cross-linked regions and release proteins; the inner layer relies on yeast (1 × 10⁻⁶). 8 CFU / g) and Bacteroides enzyme-producing strains naturally release delayed-activation late-stage enzymes, including acidic protease and pectinase, which target deep antigen proteins and residual polysaccharide structures.

[0138] To enhance the differentiation of interlayer interaction rhythms, the enzyme preparations were microencapsulated to control their release time windows. Phytase and β-glucanase were designed for immediate release, while acidic protease was released 6–8 hours into fermentation via pH-sensitive coating. Bacteroides-specific enzymes were expressed after a 16-hour delay following the natural growth cycle of the strain, thus forming a multi-rhythmic response mechanism. The total fermentation time was 48 hours at 37°C and 65% humidity. Samples were taken every 8 hours, and sections were prepared to obtain local samples of the three-layer structure for enzyme activity detection and degradation assessment.

[0139] The results showed that at 8 hours, the phytic acid degradation rate in the surface region reached 62.3%, and the glucan content decreased by 35.1%, with the β-mannan backbone length decreasing by 1.6 kDa, indicating that the surface enzymes had entered a highly active state; while the reactions in the middle and inner layers were not yet significant. At 24 hours, the reaction in the middle layer significantly increased, with a small peptide release rate of 18.5%, a protein residue rate decreasing by 42.7%, and phytic acid residue further decreasing to 0.38%, and β-xylan cleavage products beginning to penetrate into the inner layer; at 36 hours, pectin lyase and endopeptidase activity were detected in the inner layer, and the corresponding residual antigen protein content decreased to 14.2%, indicating that the specific bacterial strain had completed colonization and initiated the deep reaction. Finally, at 48 hours, small peptides accounted for 23.4% of the total protein, the total residual antigen protein rate decreased to 11.6%, and the in vitro digestibility of soybean meal reached 85.1%, significantly better than the control group (72.8%) without stratification.

[0140] To quantify the contribution ratio of each layer of response, the Cooperative Stratified Degradation Index (CI) is defined as follows:

[0141] ;

[0142] in:

[0143] The initial enrichment degree of the target structure for each layer (expressed as dry basis anti-nutritional factor content) was set as follows: surface layer 0.36 g / g, middle layer 0.41 g / g, inner layer 0.31 g / g; The cumulative enzyme activity units (U / g) of this layer were measured to be 320, 810, and 195, respectively. The completion rates of the reactions in this layer were 92.7%, 81.2%, and 68.4%, respectively. The total fermentation time was 48 hours.

[0144] Substitute into the calculation:

[0145] ;

[0146] The CI value of the unstratified control group was 5.96, indicating that the stratified structure improved the efficiency of the synergistic bacterial-enzyme reaction by approximately 46.6% per unit time. Furthermore, animal feeding experiments further corroborated this: feeding fattening pigs with this fermented soybean meal resulted in an average daily weight gain increase of 11.3% after 21 days, a nitrogen conversion rate increase of 14.8%, plasma small peptide levels that were 26.1% higher than the control group, and a 35.5% reduction in fecal antigen protein content.

[0147] In summary, this invention, by clearly constructing a three-stage gradient reaction pathway—outer layer phytic acid and dextran response → middle layer protein cross-linking region degradation → inner layer deep-embedded residual structure activation—not only strengthens the synergistic rhythm of the bacterial-enzyme combination but also effectively controls the spatial order and time window of nutrient release, transforming soybean meal from a structural blockade into a nutrient delivery platform, ultimately achieving high bioavailability. This constitutes the core essence and unique innovation of the technical solution of this invention.

[0148] Example 3:

[0149] This paper discloses a soybean meal treatment method centered on "structural pre-activation + microecological acidification + time-sequential enzyme synergy," aiming to significantly improve the protein digestibility and bioavailability of soybean meal. The technical means employed include an integrated process encompassing domain recognition, targeted enzyme release, synergistic regulation of metabolic signals, and intelligent setting of the solid-state fermentation environment.

[0150] Step 1: Raw material structure identification and targeted pretreatment

[0151] 1.1 Fourier transform infrared spectroscopy (FTIR) was used to scan the raw defatted soybean meal samples (particle size <1 mm), and characteristic absorption peaks in the 800–1800 cm⁻¹ region were collected. By comparing with an enzyme activity spectrum library, the lignin-pectin-protein complex region, the phosphate ester (1160 cm⁻¹) and amide bond (1650 cm⁻¹) dense region in the soybean meal were identified as enzyme-sensitive sites.

[0152] 1.2 Based on the above structural characteristics, the following structural activation preprocessing conditions are set:

[0153] Slight acid treatment: Soak soybean meal in a citrate buffer solution with pH=5.2 for 30 min.

[0154] Moist heat treatment: Hold at 45℃ for 20 minutes to loosen the cross-linked structure.

[0155] Intermittent shearing: Use a paddle mixer to gently stir for 30 minutes at a frequency of 30 seconds every 5 minutes.

[0156] 1.3 Introduction of pre-lysin for structural cleavage: Add [the following ingredient] per 100 kg of soybean meal.

[0157] Xylanase (1000U / g) 20g,

[0158] Pectinase (800U / g) 10g,

[0159] The reaction temperature was 50℃, the reaction time was 20 min, and no enzyme inactivation treatment was performed afterwards.

[0160] Step 2: Particle size control and hierarchical structure construction

[0161] 2.1 The raw materials are conditioned to a moisture content of 22% and then processed to a particle size of 500–700 μm using a wet grinding equipment.

[0162] 2.2 Hot-press instantaneous explosion treatment: The material is fed into a steam explosion chamber at 0.25MPa and held for 15s. After exiting the chamber, it expands and releases instantly, forming a porous microcrack on the surface, a cross-linked composite zone in the middle, and a microporous state in the inner layer.

[0163] 2.3 Drying settings: Slowly dry at 60℃ hot air until the moisture content is ≤10%, retaining the "inner-outer" gradient structure of soybean meal particles, which is beneficial for subsequent bacterial and enzyme gradient invasion.

[0164] Step 3: Construction of the microbial-enzyme co-fermentation system

[0165] 3.1 Strain Combination and Metabolic Regulation Design:

[0166] Lactobacillus plantarum (10¹¹ CFU / g) 0.08%, function: secrete lactic acid for acid regulation and release glycerol signaling molecules;

[0167] Yeast 0.05%, function: secretes trace amounts of hydrogen peroxide, guiding the expression of aerobic enzyme systems.

[0168] 3.2 Enzyme Combination Design and Microcapsule Release Control: Three enzyme preparations were encapsulated using multi-segment release microcapsules:

[0169] Phase 1 (first 4 hours): Phytase is released and acts on the surface layer.

[0170] Phase 2 (8–24h): Xylanase + β-glucanase release, targeting the middle cell wall structure.

[0171] Phase 3 (24–48h): Acidic protease release, targeted degradation of deep macromolecular antigen proteins.

[0172] 3.3 Fermentation process setup:

[0173] First stage (microbial fermentation): After mixing the treated soybean meal with water at a mass ratio of 1:0.9, inoculate with microbial inoculum and ferment in a sealed container at 35℃ for 36 hours to lower the pH to 4.2±0.2, forming a lactic acid environment and killing and inhibiting other bacteria.

[0174] Second stage (enzyme synergy): Start the heating program to raise the temperature to 52°C, add hot water to bring the total water content to 95%, and perform enzymatic hydrolysis for 20 hours.

[0175] Step 4: Evaluation of Fermentation Products and Verification of Effects

[0176] After the above treatment, the soybean meal contains:

[0177] The free amino acid content reaches 2.9%, which is 9 times higher than that of the raw material;

[0178] Oligosaccharide residue <0.1%, essentially eliminated;

[0179] The total dissolved nitrogen ratio was increased to 2.7 times the original, and the protein molecular weight profile showed that most of the protein was distributed in the 2–6 kDa range, making it suitable for animal absorption; the viable count of lactic acid bacteria reached 10. 7 CFU / g, with probiotic potential.

[0180] Animal diet experiments have shown that replacing 8% of ordinary soybean meal with soybean meal increased the daily weight gain of pigs by 6.3% and decreased the feed conversion ratio by 4.8%, demonstrating significant value for widespread application.

Claims

1. A production method for synergistically improving the digestibility of soybean meal for feed using microorganisms and enzymes, characterized in that: include, (1) The lignin-encapsulated layer, cross-linked polysaccharide-protein network and phytic acid binding region in soybean meal together constitute the digestibility bottleneck; infrared spectroscopy combined with enzyme activation site scanning technology is used to identify the regions in soybean meal that are sensitive to the synergistic reaction of bacteria and enzymes, and microprocessing pre-reaction conditions are designed to activate the structure. (2) Using metabolic flux analysis, a multi-species cross-pathway metabolic map was established, in which one species secretes a small molecule activator and another supporting species releases a responsive enzyme; a synergistic metabolic system was constructed in which lactic acid bacteria lower the pH of the system under anaerobic conditions and simultaneously produce trace amounts of hydrogen peroxide as a signal to synergistically activate acidic proteases, which work together with yeast on the soybean meal matrix. Through the complementary decomposition of proteins and non-starch polysaccharides in soybean meal by bacteria and enzymes, the digestibility of soybean meal was improved. (3) Optimize the particle size and pore structure of soybean meal to gradually expose different functional layers during fermentation; through responsive microencapsulation technology, first release phytase to break the phytic acid structure, then release acidic protease to decompose the antigen protein, forming different enzyme release rhythms; use organic acids and activated polypeptide microbial metabolites to reverse regulate the enzyme reaction rate and achieve multi-level control.

2. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 1, characterized in that: The method for identifying regions in soybean meal sensitive to synergistic reactions of microorganisms and enzymes includes... Infrared spectroscopy was used to scan the structural domains of raw soybean meal to identify high-density cross-linked regions, anti-nutritional factor encapsulation layers, and complex polysaccharide-protein structures. The obtained infrared characteristic spectra were matched with a pre-set enzyme activity site database to determine the regions in soybean meal that are sensitive to co-degradation by bacteria and enzymes. Based on the spatial distribution and structural characteristics of the identified sensitive areas, targeted pretreatment conditions were formulated, including micro-acid treatment, mild humid heat treatment, and intermittent low-energy shear stirring. Optionally, pre-enzyme introduction treatment can be performed by adding a low dose of structural pre-lysin to activate the structure of soybean meal, causing partial deconstruction while retaining nutritional activity; subsequently, the treated soybean meal can be used in a microbial-enzyme co-fermentation system to improve digestibility.

3. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 2, characterized in that: The infrared spectroscopy analysis employs Fourier transform infrared spectroscopy to identify amide bonds, glycosidic bonds, phosphate ester bonds, and structural groups that bind to anti-nutritional factors. The enzyme activity site database contains characteristic spectral information on the binding sites of phytase, β-glucanase, xylanase, and acidic protease with the corresponding anti-nutritional structures of soybean meal.

4. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 3, characterized in that: The microacid treatment is carried out in the pH range of 4.5 to 5.5 to loosen phosphate bonds and protein complexes; Mild humid heat treatment is carried out at 45–55°C for a time of no more than 30 minutes. Intermittent low-energy shear mixing treatment uses a low-speed intermittent mixing method to locally disrupt the dense structure of soybean meal particles and promote the exposure of surface pores.

5. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 2, characterized in that: The pretreatment involves structural analysis of soybean meal raw materials to identify non-starch polysaccharide structural regions that encapsulate nutrient factors, including xylan, glucan, and pectin cross-linked complex layers; and the addition of a low dose of structural pre-lysin for targeted activation; the structural pre-lysin is xylanase, glucanase, pectinase, or arabinosidase, and the amount added is limited to a subfunctional level that does not cause complete degradation of the overall structure, with the action time controlled between 10 and 30 minutes.

6. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 1, characterized in that: The method for optimizing soybean meal particle size and pore structure includes, (S1) Wet grinding of soybean meal to control the particle size to 300-800 micrometers, and hot pressing and instantaneous explosion treatment of the pulverized material to form porous microcracks on the surface, maintain a partially dense cross-linked structure in the middle layer, and retain micropores in the inner layer. (S2) Regulate the drying rate and temperature to form a layered pore gradient from the outside to the inside. The surface layer has medium pore size, which is conducive to the initial contact of enzymes; the middle layer is a cross-linked structure enrichment area, which provides for the synergistic effect of bacterial enzymes; the inner layer is a microporous structure, which is used to retain small peptides and encapsulate anti-nutritional factors. (S3) Obtain soybean meal with a layered response structure for stepwise response and timed release in subsequent microbial-enzyme co-fermentation process.

7. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 6, characterized in that: The wet milling is carried out under conditions where the soybean meal moisture content is 18% to 25% to retain fiber flexibility and enhance micropore formation ability; the hot pressing and instantaneous explosion treatment is carried out under conditions of ≤0.3MPa and ≤15s to form a non-fully expanded structure.

8. The production method for synergistically improving the digestibility of soybean meal for feed according to claim 6, characterized in that: The drying process employs a low-temperature slow drying method at 50–70°C. The resulting layered pore gradient structure can sequentially guide different enzymes and microorganisms to gradually invade from the surface to the inner layer during the synergistic fermentation of bacteria and enzymes, thereby increasing the release rate of proteins, small peptides, and phosphorus components.

9. The application of the production method for synergistic enhancement of feed soybean meal digestibility by microbial enzymes as described in any one of claims 1 to 8, characterized in that: The layered structure, by delaying the exposure of different functional zones, enables the enzymes to form a synergistic gradient reaction from the outer layer to the inner layer during fermentation, thereby improving the digestibility of soybean meal in the animal intestine.

10. The application of the production method for synergistic improvement of feed soybean meal digestibility according to claim 9, characterized in that: The outer layer response includes primary degrading enzymes such as phytase and β-glucanase, the middle layer response is synergistic with acidic proteases and polysaccharides, and the inner layer response is a late-stage degrading enzyme produced by specific bacterial species.

Images