A method for producing threonine protein feed by using corn sugar residue

Abstract

The application discloses a kind of production of threonine protein feed using corn sugar residue, and belongs to the technical field of biological feed processing and agricultural byproduct resource utilization technology.Corn sugar residue, corn germ meal and loose structure auxiliary materials are mixed, and after pretreatment by composite enzyme, nano-composite regulating agent containing acidified nano-montmorillonite, chitosan and metal phenolic network nano-coating modified yeast cell wall polysaccharide is added, and Candida utilis and Bacillus subtilis coated with the coating are inoculated, and after shallow forced aeration solid state fermentation, Lactobacillus plantarum ripening, low-temperature drying and crushing screening, threonine protein feed is prepared.The application promotes protein conversion and threonine enrichment through oxygen supply and heat dissipation regulation during fermentation process and synergistic effect of metal phenolic network nano-coating, improves DON adsorption, stress resistance and antioxidant capacity of functional bacteria, and improves product protein digestibility, viable bacteria preservation rate and storage stability.

Description

Technical Field

[0001] This invention belongs to the technical field of biological feed processing and agricultural by-product resource utilization. Specifically, it relates to a method for preparing threonine protein feed using corn residue. Background Technology

[0002] The corn deep-processing industry generates byproducts such as corn residue, corn germ meal, and sprayed corn husks during the production of starch sugars, amino acids, and organic acids. These byproducts are stable in source and low in cost, and have the potential to be developed into protein feed ingredients. Corn residue contains residual sugars, fermentable carbon sources, fiber, and a small amount of protein, making it suitable as a substrate for microbial fermentation. Corn germ meal has a high protein content and can serve as a primary nitrogen source for solid-state fermentation systems. However, corn residue suffers from high moisture content, easy spoilage, poor flowability, low protein content, and an unbalanced amino acid composition. Corn germ meal, on the other hand, has a high fiber content, and its protein digestibility and small peptide content need improvement. Simple mixing, drying, or grinding is insufficient to obtain protein feed with high nutritional value and quality stability. Threonine is one of the important limiting amino acids in livestock and poultry diets and is closely related to protein utilization and animal growth. Existing corn byproduct protein feeds typically have insufficient threonine levels. Threonine fermentation broth contains free L-threonine, soluble nitrogen sources, and other fermentation metabolites. Introducing it into solid-state fermentation systems of corn sugar residue and corn germ meal can improve the carbon-nitrogen balance of the substrate and increase the threonine level in the product. However, current technologies for utilizing threonine fermentation broth mainly focus on recovery, concentration, or use as a general nitrogen source, and targeted conversion processes for solid-state fermentation of corn sugar residue still need to be improved.

[0003] Microbial fermentation is an important means of improving the feed quality of agricultural by-products. *Candida utilis* can utilize sugar substrates to form cell proteins, while *Bacillus subtilis* can produce proteases and polysaccharide degradation enzymes. Combined with pretreatment with complex enzymes, this can promote the degradation of macromolecular proteins and non-starch polysaccharides, increasing the content of readily available nutrients such as acid-soluble proteins and small peptides. However, corn residue and corn germ meal differ significantly in physical structure, moisture content, and sugar-nitrogen composition. During solid-state fermentation, improper material structure, moisture content, bed thickness, ventilation, and temperature control can easily lead to problems such as localized hypoxia, central heat accumulation, uneven fermentation, sugar residue, and viable cell attenuation. Oxygen transfer and metabolic heat removal in aerobic solid-state fermentation mainly depend on the material's pore structure, bed thickness, and ventilation conditions. Excessively thick bed or insufficient ventilation can easily cause hypoxia and heat accumulation in the central area, inhibiting microbial metabolism and potentially leading to protein thermal damage and decreased digestibility. Therefore, relying solely on empirical turning or intermittent ventilation is insufficient to guarantee the stability and repeatability of industrial fermentation. It is necessary to establish a shallow fermentation, forced ventilation, and monitoring system for temperature, exhaust oxygen content, and fermentation endpoints adapted to the material structure. Furthermore, corn byproducts pose a risk of mycotoxin contamination during storage, transportation, and processing, with deoxynivalenol (DON) being a significant factor affecting feed safety. Relying solely on microbial fermentation for detoxification is susceptible to influences from the microbial strain, substrate, and fermentation conditions. Conventional montmorillonite adsorbents suffer from problems such as large particle size, limited specific surface area, insufficient dispersibility, and inadequate adsorption stability. Acidification, nano-sizing, and composite modification with chitosan oligosaccharides and yeast cell wall polysaccharides hold promise for improving the adsorption performance and interfacial compatibility of montmorillonite, providing a new technical approach for the simultaneous control of DON during fermentation.

[0004] The viable cell count and stress resistance of functional bacteria also affect the quality of fermented feed. The metabolic heat generated during solid-state fermentation and the subsequent drying process can easily damage the microbial cells, and oxidative stress during storage further reduces the viable cell retention rate. Existing microencapsulation or coating technologies are mainly used for liquid-cultured bacterial powders, and their application in corn residue solid-state fermentation systems remains limited. Metal-Phenolic Networks (MPNs), formed by the coordination and self-assembly of polyphenolic compounds and metal ions, possess characteristics such as mild film-forming conditions, good biocompatibility, and antioxidant and interface modification properties. They can form nano-coatings on the surface of microorganisms or carriers, providing a technological basis for improving the heat resistance and stress resistance of functional bacteria and the stability of composite adsorption materials.

[0005] In summary, existing technologies for preparing corn by-product protein feed still suffer from problems such as insufficient substrate utilization, low threonine levels, unstable control of oxygen supply and heat dissipation during solid-state fermentation, limited DON control effects, insufficient stress resistance and preservation capabilities of functional bacteria, and loss of nutrients and active ingredients due to high-temperature drying. Therefore, it is necessary to establish a method for preparing threonine protein feed suitable for corn sugar residue. This method should utilize a combination of technologies including substrate conditioning, enzymatic pretreatment, solid-state fermentation with compound bacteria, toxin adsorption and regulation, stress resistance protection of functional bacteria, fermentation process control, and low-temperature post-treatment to improve the resource utilization level of corn sugar residue and the nutritional value, safety, and storage stability of the resulting protein feed. Summary of the Invention

[0006] To address the problems of high moisture content, easy spoilage, low protein and threonine content, insufficient digestibility, unstable mycotoxin control, low viable bacteria retention rate, and easy clumping in existing technologies for the direct utilization of corn residue, this invention provides a method for producing threonine protein feed from corn residue. This method constructs a solid fermentation substrate composed of corn residue, corn germ meal, and loosely structured auxiliary materials. It combines this with pretreatment with compound enzymes, fermentation with compound bacteria containing threonine fermentation broth, simultaneous toxin control and stress protection of functional bacteria using a metallophenolic network nanocoating modified carrier, shallow forced aerobic fermentation, post-ripening and stabilization with *Lactobacillus plantarum*, and low-temperature drying. This achieves high-value utilization of corn residue and improves the protein nutritional value, safety, viable bacteria retention rate, and storage stability of the resulting feed.

[0007] The present invention adopts the following technical solution: a method for preparing threonine protein feed using corn sugar residue, comprising the following steps: (1) Raw material conditioning: corn sugar residue, corn germ meal and granular pore-conditioning additives are mixed at a dry basis mass ratio of 1:(3.0-8.0):(0.2-1.5) to obtain mixed solid material; wherein, the total sugar content in the dry matter of the corn sugar residue is 18%-65%, and the granular pore-conditioning additives are selected from one or more of wheat bran, corn husk, sprayed corn husk, soybean meal, rice bran meal and corn. Multiple types, with a particle size of 0.3-4.0 mm; (2) Enzymatic pretreatment: Add a complex enzyme preparation including cellulase, xylanase and neutral protease to the mixed solid material, and treat it at 45-58℃ for 1-5 h to obtain pretreated material; (3) Preparation of nanocomposite regulator: After sodium montmorillonite is activated by organic acid and wet nano-sized, it is compounded with chitosan oligosaccharide and yeast cell wall polysaccharide to obtain nanocomposite regulator; The yeast cell wall polysaccharide is modified by metal phenolic network nano-coating, and the metal phenolic network is composed of tannic acid and Fe 3+ Zn 2+ or Ca 2+One or more metal ions in the mixture coordinate and self-assemble in a weakly alkaline aqueous phase to form the metal-phenolic network nanocoating; the thickness of the coating is 5-20 nm; the D50 particle size of the nanocomposite regulator is 0.08-0.80 μm, and the BET specific surface area is 60-260 m². 2 / g; (4) Preparation of fermentation broth: Candida utilis and Bacillus subtilis were activated and cultured in a culture medium containing threonine fermentation broth, and cultured with shaking or ventilation at 28-40℃ and pH 5.5-7.0 for 2-8 hours to obtain fermentation broth; the Candida utilis and Bacillus subtilis were coated with a metal phenolic network nanocoating; the viable count of Candida utilis in the fermentation broth was 1.0×10 7 -1.0×10 9 CFU / mL, the viable count of Bacillus subtilis was 1.0 × 10⁻⁶. 6 -1.0×10 8 CFU / mL; (5) Inoculation and mixing: The pretreated material, the nanocomposite regulator and the fermentation liquid are mixed to adjust the initial water content of the material to be fermented to 32%-44%, the initial pH to 5.0-6.2 and the free L-threonine content to 0.08%-0.60%; wherein, the amount of nanocomposite regulator added is 0.10%-1.50% of the dry basis mass of the material to be fermented; (6) Aerobic solid-state fermentation: The material to be fermented is laid in a shallow fermentation bed or a multi-layer shallow tray fermenter with bottom ventilation holes, so that the effective material layer thickness is 10-35cm, and fermented at 34-42℃ for 18-36h; during fermentation, air is continuously introduced from the bottom at a ventilation intensity of 0.02-0.30L / (kg·min), and the center temperature of the material layer, the surface temperature of the material layer and the volume fraction of exhaust oxygen are detected online; when the center temperature of the material layer exceeds 42℃ and the difference between the center temperature of the material layer and the surface temperature of the material layer exceeds 6℃. When any of the following conditions are met, the ventilation intensity is increased to 1.5-3.0 times the original ventilation intensity until the temperature at the center of the material layer is below 40℃ and the oxygen volume fraction in the exhaust is above 18%; (7) Post-fermentation stabilization: After the aerobic solid-state fermentation is completed, plant lactobacillus coated with metal phenolic network nano-coating is introduced and post-fermented for 4-12 hours under the conditions of 30%-40% moisture content and 30-37℃ temperature; (8) Low-temperature drying and crushing: the post-fermented material is dried at low temperature so that the moisture content is not higher than 10%, and then crushed and screened to obtain threonine protein feed; Among them, the aerobic solid-state fermentation endpoint meets the following conditions: the reducing sugar content is not higher than 3.0%; the temperature at the center of the material layer is at least 2℃ lower than the highest temperature at the center of the material layer during fermentation; the two consecutive carbon dioxide release rate detection values ​​obtained at 4-hour intervals are lower than their respective previous detection values; and the number of viable Candida utilis is not lower than 1.0×10 7CFU / g. The carbon dioxide release rate was measured at 4-hour intervals. The CO2 release rate showed a decreasing trend in two consecutive measurements (i.e., the later measurement value was lower than the previous measurement value).

[0008] The principle behind the activation of sodium montmorillonite by citric acid lies in the fact that citric acid provides H+. + Replacement of metal cations (such as Na) between montmorillonite layers + Ca 2+ (etc.), reducing interlayer van der Waals forces and increasing interlayer spacing; simultaneously, citrate ions can act as intercalating agents to enter the interlayer space of montmorillonite, further expanding the layered structure; in addition, acid treatment can also partially dissolve the Al in the montmorillonite framework. 3+ Mg 2+ Octahedral cations are added to increase the number of silanols and the specific surface area of ​​the montmorillonite sheets. After acidification pretreatment, combined with the mechanical action of wet nano-sizing (sand milling or ultrasonic exfoliation), the montmorillonite sheets are further exfoliated into nanoscale, forming nano-montmorillonite with a large specific surface area and abundant surface active sites. When subsequently compounded with chitosan oligosaccharides and yeast cell wall polysaccharides, the organic polysaccharides can be loaded onto the surface and interlayer of the nano-montmorillonite sheets through hydrogen bonding, electrostatic interactions, etc., forming an inorganic-organic hybrid adsorption interface. This interface synergistically exerts the dual effects of interlayer adsorption of montmorillonite and coordination binding of polysaccharides, which is beneficial to improving the adsorption capacity and adsorption stability of deoxynivalenol, while reducing the risk of desorption of toxins in the digestive tract environment.

[0009] The principle behind the modification of yeast cell wall polysaccharides and functional bacteria by a metal phenolic network nanocoating lies in the fact that tannic acid molecules contain abundant pyrogallol and catechol structural units, which can interact with Fe... 3+ Zn 2+ Ca 2+When metal ions coordinate and complex, a continuous nano-network coating is formed in situ on the surface of yeast cell wall polysaccharides or bacterial cell walls under weakly alkaline conditions (pH 7.0-9.0). This coating provides antioxidant activity through phenolic hydroxyl groups, scavenging free radicals during fermentation and storage, and inhibiting lipid oxidation and cell damage. Furthermore, in the weakly acidic environment of the digestive tract, partial dissociation of coordination bonds slowly releases polyphenols and metal ions, thereby exerting antibacterial and immunomodulatory effects. Coating the surfaces of Candida utilis, Bacillus subtilis, and Lactobacillus plantarum with a metal-phenolic network nanocoating can improve the cell's tolerance to aerobic solid-state fermentation metabolic heat, two-stage low-temperature drying thermal shock, and long-term storage oxidative environment, reducing the loss of viable cells. Simultaneously, when the yeast cell wall polysaccharides modified with the metal-phenolic network are combined with nano-montmorillonite and chitosan oligosaccharides, the polyphenolic groups can act as bridging groups, interacting with the silanol groups on the montmorillonite sheet surface and the amino groups of the chitosan oligosaccharides through hydrogen bonding and electrostatic interactions. This further stabilizes the inorganic-organic hybrid interface of the nanocomposite regulator, improves the adsorption stability of deoxynivalenol, and reduces the risk of desorption.

[0010] Preferably, the corn sugar residue is dehydrated by pressure filtration or centrifugation before conditioning to achieve a moisture content of 35%-65%; when the pH of the corn sugar residue is lower than 4.5, calcium carbonate is used to adjust its pH to 4.8-5.8.

[0011] Preferably, in the compound enzyme preparation, the amount of cellulase added is 50-600 U / g dry basis material, the amount of xylanase added is 30-500 U / g dry basis material, and the amount of neutral protease added is 100-1200 U / g dry basis material.

[0012] Preferably, step (3) includes: dispersing sodium montmorillonite in a citric acid solution with a mass fraction of 0.2%-2.0%, activating it at 50-80℃ for 0.5-3h; washing it with water until the pH is 5.5-7.0, and then obtaining nano-montmorillonite slurry by wet grinding or ultrasonic exfoliation; adding chitosan oligosaccharide and yeast cell wall polysaccharide to the nano-montmorillonite slurry, compounding it at 40-70℃ for 0.5-4h, and then spray drying or low-temperature drying at a material temperature not higher than 55℃ to obtain the nano-composite regulator.

[0013] Preferably, the dry basis mass ratio of the sodium-based montmorillonite, chitosan oligosaccharide, and yeast cell wall polysaccharide is 100:(1-15):(2-25); the degree of deacetylation of the chitosan oligosaccharide is not less than 80%, and the number average molecular weight is 0.5-10 kDa; the total content of β-glucan and mannan oligosaccharide in the yeast cell wall polysaccharide is not less than 40%; and the interlayer spacing of the nanocomposite regulator is 1.35-2.20 nm.

[0014] Preferably, the metal phenolic network is formed by the coordination self-assembly of tannic acid and metal ions, wherein the molar ratio of tannic acid to metal ions is (2:1) to (1:3), and the metal ions are Fe. 3+ Zn 2+ and Ca 2+ One or more of the following; preferably, the metal ion is Fe. 3+ Furthermore, tannic acid and Fe 3+ The molar ratio is 1.5:1-2.0:1; the coating rate of the yeast cell wall polysaccharide after modification by the metal phenolic network nanocoating is 60%-95%, and the coating thickness is 5-20nm; the coating rate of Candida utilis, Bacillus subtilis and Lactobacillus plantarum after coating by the metal phenolic network nanocoating is not less than 70%.

[0015] Preferably, the threonine fermentation broth is a yeast fermentation broth, filtrate, concentrate, or a combination thereof obtained after Corynebacterium glutamicum ferments to produce L-threonine. The L-threonine content of the threonine fermentation broth is 5-80 g / L, and the total nitrogen content is 0.5%-8.0%. The culture medium containing the threonine fermentation broth includes the threonine fermentation broth, glucose, molasses, yeast extract, and water, wherein the ratio of viable cells of Candida utilis to Bacillus subtilis is (5-50):1.

[0016] Preferably, in the multi-layer shallow tray fermenter, the effective material layer thickness of each shallow tray is 12-28 cm, the bottom opening rate of the shallow tray is 8%-35%, and the vertical spacing between adjacent shallow trays is 8-25 cm; the inoculum amount of *Lactobacillus plantarum* in step (7) is 1.0 × 10⁻⁶ cm. 6 -1.0×10 8 CFU / g wet material.

[0017] Preferably, the low-temperature drying includes: a first drying section with a hot air temperature of 65-90℃ and a material temperature not exceeding 55℃, drying to a moisture content of 14%-18%; a second drying section with a hot air temperature of 45-65℃ and a material temperature not exceeding 50℃, drying to a moisture content not exceeding 10% and a water activity not exceeding 0.65; after crushing and sieving, the mass percentage of particles with a diameter of 0.25-2.00mm is not less than 70%, and the mass percentage of particles with a diameter greater than 8mm is not more than 2%.

[0018] Compared to existing technologies, this invention has at least the following beneficial effects: This invention constructs a solid-state fermentation substrate using corn residue, corn germ meal, and loosely structured auxiliary materials. Pretreatment with cellulase, xylanase, and neutral protease improves the material's pore structure, enhances the availability of fiber and macromolecular proteins, and promotes the formation of easily digestible protein components such as acid-soluble proteins. A culture medium containing threonine is used to activate *Candida utilis* and *Bacillus subtilis*. Through the synergistic utilization of fermentable sugars and nitrogenous components by the two bacteria, the formation of cell proteins and acid-soluble proteins is promoted, increasing the L-threonine content and protein nutritional value of the product. This invention uses acidified nano-montmorillonite, chitosan oligosaccharides, and a metal-phenolic network nanocoating to modify yeast cell wall polysaccharides to construct a nanocomposite regulator. Through the formation of a composite adsorption interface between inorganic sheets and organic polysaccharides, the adsorption stability of deoxynivalenol is improved, and the desorption risk is reduced. Simultaneously, the formation of a metal-phenolic network nanocoating on the surface of functional bacteria enhances the bacteria's antioxidant, heat-resistant, and stress-resistant capabilities, reducing bacterial damage and viable cell loss during fermentation, drying, and storage, thereby improving product viable cell retention and storage stability. This invention employs shallow-layer forced-ventilation aerobic solid-state fermentation, adjusting ventilation based on the core temperature of the substrate layer and the volume fraction of oxygen in the exhaust gas. This reduces localized hypoxia and central heat accumulation, improving fermentation uniformity and repeatability. The fermentation endpoint is determined by a combination of reducing sugar content, core temperature drop, carbon dioxide release rate, and viable cell count, enhancing the objectivity of process control. After aerobic fermentation, the bacteria are stabilized through post-ripening with *Lactobacillus plantarum*, combined with low-temperature drying and pulverization / sieving. This reduces product moisture and water activity while maintaining protein digestibility, active microbial stability, flowability, and particle size uniformity. Attached Figure Description

[0019] Figure 1 This is a characterization diagram of the nanocomposite regulator in this invention; Figure 2 These are images of threonine protein feed products from the embodiments and comparative examples of this invention; Figure 3 This is a normalized, noise-reduced liquid chromatogram of the changes in DON content in the embodiments and comparative examples of this invention. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments. These embodiments are used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, all raw materials used are feed-grade or food-grade, and the water used is purified water that meets the requirements for feed processing; the amount of each component is expressed in grams (g). When involving water-containing raw materials, the table lists both the actual weighed mass and the dry basis converted mass. The dry basis mass ratio is calculated based on the mass of each material after deducting moisture. In this specification, CFU stands for Colony Forming Unit, used to indicate the number of viable bacteria; DON stands for Deoxynivalenol, also known as vomitoxin; BET stands for Brunauer-Emmett-Teller, a specific surface area measurement method; D50 is the particle size corresponding to a cumulative distribution of 50%; DLS stands for Dynamic Light Scattering; HPLC stands for High Performance Liquid Chromatography; XRD stands for X-ray Diffraction; ICP-MS stands for Inductively Coupled Plasma Mass Spectrometry; r / min stands for revolutions per minute; and CAS stands for Chemical Abstracts Service registration number. In this manual, MPN stands for Metal-Phenolic Network; TEM stands for Transmission Electron Microscope; and DPPH stands for 1,1-diphenyl-2-picrylhydrazine, a reagent used in the determination of free radical scavenging activity.

[0021] Supplementary notes on MPN coating operation: This application preferably uses tannic acid (CAS No. 1401-55-4) and ferric chloride hexahydrate (CAS No. 10025-77-1) to construct a metallophenolic network. Based on the dry weight of the object to be coated, the amount of tannic acid added is 0.5%-12.0%, and the amount of ferric chloride hexahydrate added is 0.3%-8.0%. The object to be coated is dispersed in purified water to form a 0.1%-5.0% solid dispersion. Tannic acid is added first, and after stirring for 5-30 min, the metal salt is added. The pH is adjusted to 7.5-8.5 with 0.1-1.0 mol / L sodium hydroxide solution, and the reaction is carried out at 20-35℃ and 100-400 r / min for 20-90 min. After the reaction is complete, the mixture is centrifuged at 3000-8000 r / min, washed 1-3 times with purified water, and then freeze-dried or vacuum-dried at a material temperature not exceeding 45℃. The coating thickness was determined by TEM, the Fe-O / polyphenol coordination characteristics were confirmed by XPS or FTIR, and the coating efficiency was calculated by thermogravimetric analysis and the characteristic peak area of ​​polyphenols.

[0022] Corn sugar residue is a byproduct of corn sugar production. After pressure filtration, it is used. The dry matter contains 32.4% total sugar, 17.8% crude protein, 1.86 mg / kg initial DON content, and a pH of 4.9. Corn germ meal contains 19.6% crude protein and 12.0% moisture. Wheat bran (its commercially available moisture content is approximately 13%, and dry matter content is approximately 87%; on a virgin basis, crude protein is typically 15%, crude fat approximately 3%, crude fiber approximately 12%, crude ash approximately 6%, and starch generally not exceeding 15%), sprayed corn husks (crude protein not less than 11%, crude fiber not more than 10%, crude ash not more than 8%, and moisture not more than 12%), rice bran meal (its dry matter content is approximately 91.6%, average crude protein content is approximately 6.7%, crude fiber approximately 30.8%, NDF approximately 51.7%, ADF approximately 35.4%, lignin approximately 11.8%, crude ash approximately 19.1%, and crude fat approximately 4.8%), and corn DDGS (containing soluble... Corn distillers' dried grains with solubles (DDGS) are the main solid byproducts of ethanol production from corn via dry fermentation. They are obtained by mixing and drying solids from distilled grains with concentrated distilled solubles in a specific ratio. The dry matter content of conventional commercial corn DDGS is approximately 89.6%, with average contents of crude protein, crude fat, crude fiber, NDF, ADF, ash, residual starch, and total phosphorus (approximately 30.4%, 14.0%, 7.9%, 35.6%, 10.5%, 4.9%, 5.0%, and 0.85%, respectively) on a dry matter basis. These are all conventional feed ingredients. Sodium-based montmorillonite is feed grade, with a montmorillonite content of 92.5%. The degree of deacetylation of chitosan oligosaccharides is 91.2%, and the number-average molecular weight is 3.2 kDa. The total content of β-glucan and mannan oligosaccharides in yeast cell wall polysaccharides is 58.6%. The threonine fermentation broth was the membrane filtrate from the fermentation of L-threonine by Corynebacterium glutamicum, with an L-threonine content of 30.0 g / L and a total nitrogen content of 2.4%. The lyophilized Candida utilis ATCC22023 bacterial powder had a viable count of 5.0 × 10⁻⁶ cells / mL. 10 CFU / g; The viable count of Bacillus subtilis (ATCC23857) freeze-dried bacterial powder was 5.0 × 10⁻⁶. 10 CFU / g; The viable count of the freeze-dried Lactobacillus plantarum (ATCC8014*) bacterial powder is 1.0 × 10⁻⁶. 11CFU / g. Cellulase (CAS No.: 9012-54-8) activity was 100,000 U / g, xylanase (CAS No.: 9025-57-4) activity was 200,000 U / g, and neutral protease (CAS No.: 9068-59-1) activity was 100,000 U / g. Sodium selenite was a feed-grade sodium selenite premix with a selenium content of 0.020%. Tannic acid (CAS No.: 1401-55-4, food grade, purity not less than 98%) and ferric chloride hexahydrate (FeCl3·6H2O, CAS No.: 10025-77-1, analytical grade) were used to prepare the metal phenolic network nanocoating, and sodium hydroxide (CAS No.: 1310-73-2, analytical grade) was used to adjust the pH of the metal phenolic network self-assembly system. The main equipment includes: a hammer mill (Jiangyin Hongda SF-20 model); a twin-shaft paddle mixer (Changzhou Lima WZ-50 model); a constant temperature enzymatic hydrolysis chamber (Shanghai Yiheng HWS-500 model); a wet sand mill (Netzsch MiniCer model); a spray dryer (Shanghai Yacheng YC-015 model); a multi-layer shallow tray solid-state fermenter (self-made, effective size of each tray 400mm×300mm×300mm, bottom opening rate 18%); a fluidized bed dryer (Changzhou Yibu FG-5 model); and a dynamic light scattering particle size analyzer (MalvernZ). The following instruments were used: etasizer Nano ZS; Micromeritics ASAP2460 surface area analyzer; Shenzhen Cornuo GT-903 oxygen / carbon dioxide analyzer; Konica Minolta CR-400 colorimeter; AquaLab 4TE water activity analyzer; FOSS Kjeltec 8400 nitrogen analyzer; Agilent 1260 high-performance liquid chromatograph; and Shimadzu UV-2600 UV-Vis spectrophotometer. All yields are on a dry basis and include washing losses.

[0023] Example 1: This example is a preferred embodiment, including raw material conditioning, enzymatic pretreatment, preparation of nanocomposite regulators (modification with a metal phenolic network nanocoating containing yeast cell wall polysaccharides), preparation of fermentation broth (coated with a metal phenolic network nanocoating containing Candida utilis and Bacillus subtilis), inoculation and mixing, shallow forced ventilation aerobic solid-state fermentation, post-ripening and stabilization of Lactobacillus plantarum (coated with a metal phenolic network nanocoating containing Lactobacillus plantarum), two-stage low-temperature drying and pulverization and sieving steps, and preparation of selenium-containing threonine protein feed in the middle stage of fermentation.

[0024] Raw material conditioning and enzymatic pretreatment: Take 300.0g of pressed corn sugar residue with a moisture content of 50.0% and a dry weight of 150.0g; take 852.3g of corn germ meal with a moisture content of 12.0% and a dry weight of 750.0g; take 111.1g of wheat bran with a moisture content of 10.0% and a dry weight of 100.0g. The dry basis weight ratio of the three is 1:5.0:0.67. After adjusting the pH of corn sugar residue to 5.2 with calcium carbonate, it was mixed with corn germ meal and wheat bran to form a mixed solid material. Specifically, after thoroughly mixing the corn sugar residue, feed-grade calcium carbonate was added in batches with continuous stirring, using the pH of its aqueous extract (material-to-liquid ratio of 1:5, g / mL) as the detection index, to adjust the pH to 5.2±0.1. After standing for equilibration for 15 minutes, the pH was measured again. Subsequently, the adjusted corn sugar residue was mixed with corn germ meal and wheat bran at a predetermined dry basis mass ratio to obtain a mixed solid fermentation substrate. The wheat bran was pulverized to a particle size of 0.5-2.0 mm to improve the pore structure of the solid fermentation material. 3.0 g of cellulase, 1.3 g of xylanase, and 6.0 g of neutral protease were added to the above mixed solid material, followed by purified water to adjust the moisture content of the material to approximately 32.0%. The material was placed in a constant temperature enzymatic hydrolysis incubator and treated at 52℃ for 3 hours, with stirring at 60 r / min for 3 minutes every 45 minutes. After enzyme treatment, the temperature was reduced to 38°C at a cooling rate of 1.0°C / min to obtain the pretreated material.

[0025] Table 1. Raw material conditioning and enzymatic pretreatment formulation for Example 1

[0026] Preparation of nanocomposite regulators: 100.0 g of feed-grade sodium montmorillonite was weighed and added to 1500.0 g of a 1.0% (w / w) citric acid aqueous solution. The mixture was stirred and activated for 2 h at 65℃ and 500 r / min. After activation, the filter cake was washed with purified water until the pH of the filtrate reached 6.2. The resulting wet filter cake was then added to 600.0 g of purified water and ground for 90 min using a wet sand mill. The milling media consisted of zirconia beads with a diameter of 0.5 mm and a spindle speed of 2200 r / min, yielding a nano-montmorillonite slurry. Separately, 15.0 g of yeast cell wall polysaccharide was dispersed in 300.0 g of purified water, and 2.4 g of tannic acid and 1.4 g of ferric chloride hexahydrate were added (tannic acid and Fe...). 3+The molar ratio of the tannins was approximately 1.7:1 (based on the molar ratio of effective phenolic hydroxyl groups to metal ions in the tannic acid molecule). The pH was adjusted to 8.0 with sodium hydroxide solution, and the mixture was self-assembled for 1.0 h at 30℃ and 400 r / min. After centrifugation and washing three times at 4000 r / min, a wet material of yeast cell wall polysaccharide modified with a metal-phenolic network nanocoating was obtained (the coating thickness was approximately 12 nm as measured by transmission electron microscopy, with a coverage rate of 86.2%). 8.0 g of chitosan oligosaccharide and the aforementioned yeast cell wall polysaccharide modified with a metal-phenolic network nanocoating (based on a dry basis) were added to the above nano-montmorillonite slurry, and compounded for 2 h at 55℃ and 600 r / min. Subsequently, spray drying was performed at an inlet air temperature of 135℃, an outlet air temperature of 72℃, and a feed rate of 8.0 g / min to obtain 122.5 g of nanocomposite regulator. This was then pulverized and passed through a 100-mesh sieve for later use. Figure 1 As shown, the particle size of the nanocomposite regulator D50 is 0.30 μm, and its BET specific surface area is 176.8 m². 2 / g, the interlayer spacing was measured to be 1.76nm by XRD, the in vitro adsorption capacity of DON was 1.52mg / g, the desorption rate of DON after 2h shaking at pH 6.5 was 8.9%, and the DPPH free radical scavenging rate was 71.3%.

[0027] Table 2 Formulation for Preparation of Nanocomposite Regulator in Example 1

[0028] Preparation of fermentation broth: Weigh 80.0g of threonine fermentation broth, 3.0g of glucose, 5.0g of molasses, 1.2g of yeast extract, and 130.8g of purified water. Mix them and incubate at 50℃ for 10min. Cool to 37℃ to obtain the activation culture medium. Separately, take 2.0g of freeze-dried *Candida utilis* powder and 0.15g of freeze-dried *Bacillus subtilis* powder, and disperse them in 40.0g of purified water containing 0.10g of tannic acid and 0.06g of ferric chloride hexahydrate (tannic acid and Fe...). 3+ The molar ratio of the two components was approximately 1.6:1. The pH was adjusted to 8.0 with sodium hydroxide solution, and the cells self-assembled for 30 min at 30℃ and 150 r / min. After centrifugation and washing at 4000 r / min, cells coated with a metal-phenolic network nanocoating (coating rate 82.5%) were obtained. The coated cells were added to an activation medium and activated at 37℃ and 120 r / min for 4 h to obtain the fermentation broth. The viable count of *Candida utilis* in the fermentation broth was 4.0 × 10⁻⁶. 8 CFU / g, Bacillus subtilis viable count was 3.0 × 10⁻⁶. 7 The ratio of CFU / g to viable bacteria is approximately 13:1.

[0029] Table 3. Fermentation broth formulation for Example 1

[0030] Inoculation mixing and shallow forced aeration aerobic solid-state fermentation: The pretreated material was placed in a biaxial paddle mixer and stirred at 80 r / min. 9.0 g of the obtained nanocomposite regulator, 3.0 g of chitosan oligosaccharide-coated calcium carbonate microparticles, and the obtained fermentation broth were all added to the material. The chitosan oligosaccharide-coated calcium carbonate microparticles, consisting of 2.7 g of calcium carbonate and 0.3 g of chitosan oligosaccharide, were used to maintain the pH within the range of 4.6-6.5 during fermentation and were not used as the fermentation endpoint criterion. After mixing for 8 min, the initial moisture content of the material was measured to be 39.5%, the initial pH was 5.6, and the free L-threonine content was 0.21%. The material to be fermented was evenly spread in a multi-layer shallow tray solid-state fermenter, with each layer being 22 cm thick, the bottom opening rate of the shallow trays being 18%, and the vertical spacing between adjacent shallow trays being 15 cm. The fermenter temperature was controlled at 38℃, the relative humidity at 82%, and the fermentation time at 26 h. Forced bottom ventilation was used during fermentation at a rate of 0.12 L / (kg·min). The material was turned over every 4 hours for 4 minutes each time. The center temperature, surface temperature, exhaust oxygen volume fraction, and carbon dioxide release rate of the material were monitored online. At the 12th hour of fermentation, 1.0 g of feed-grade sodium selenite premix was added, equivalent to 0.20 mg / kg dry basis selenium, to prepare selenium-containing threonine protein feed. During fermentation, if any of the following conditions occurred: center temperature exceeding 42°C, temperature difference between center and surface exceeding 6°C, or exhaust oxygen volume fraction falling below 16%, the material was immediately turned over, and the ventilation rate was increased to 2.0 times the original rate until the center temperature dropped below 40°C and the exhaust oxygen volume fraction recovered to above 18%. In this embodiment, the highest center temperature was 40.6°C and the lowest exhaust oxygen volume fraction was 18.7%, with no abnormal anaerobic conditions or sustained heat accumulation observed. The fermentation endpoint was not determined by pH recovery, but by the following indicators: reducing sugar content was 2.1%, below 3.0%; the core temperature of the substrate decreased from a peak of 40.6℃ to 38.0℃, a decrease of 2.6℃; the carbon dioxide release rate decreased from 0.72 g / (kg·h) to 0.51 g / (kg·h) and 0.38 g / (kg·h) in two consecutive testing periods; and the viable count of *Candida utilis* was 3.6 × 10⁻⁶. 7 CFU / g. Aerobic solid-state fermentation is terminated after the above conditions are met.

[0031] Post-ripening and stabilization of *Lactobacillus plantarum*: After aerobic solid-state fermentation, 0.20g of freeze-dried *Lactobacillus plantarum* powder was dispersed in 20.0g of purified water containing 0.02g of tannic acid and 0.012g of ferric chloride hexahydrate (tannic acid and Fe...). 3+The molar ratio of the two components was approximately 1.6:1. The pH was adjusted to 8.0 with sodium hydroxide solution, and the mixture was self-assembled for 30 min at 30℃ and 150 r / min to obtain *Lactobacillus plantarum* coated with a metal-phenolic network nanocoating (coating rate 79.8%). The coated *Lactobacillus plantarum* was then uniformly sprayed into the fermentation material, with an initial inoculum size of 1.2 × 10⁻⁶. 7 CFU / g wet material. The moisture content of the material was controlled at 36.0%, the temperature at 33℃, and the post-ripening time was 8 hours. At the end of the post-ripening, the pH of the material was 4.8, with a yeast aroma and a slightly sour aroma, and no putrid odor.

[0032] Two-stage low-temperature drying and pulverization / screening: The post-ripened material is fed into a fluidized bed dryer. In the first stage, the hot air temperature is set at 78℃, and the material temperature is controlled to not exceed 52℃, drying to a moisture content of 16.2%. In the second stage, the hot air temperature is set at 58℃, and the material temperature is controlled to not exceed 48℃, continuing drying to a moisture content of 8.3% and a water activity of 0.55. After drying, the material is pulverized by a hammer mill and screened by a vibrating screen, ensuring that particles with a diameter of 0.25-2.00mm account for 78.4% of the total mass, and particles larger than 8mm account for 0.0%, yielding threonine protein feed.

[0033] Table 4. Fermentation, ripening, and drying process parameters for Example 1

[0034] Examples 2-4 Examples 2-4 follow the same basic steps as Example 1, including raw material conditioning, enzymatic pretreatment, preparation of nanocomposite regulators (modification with a metal-phenolic network nanocoating containing yeast cell wall polysaccharides), preparation of fermentation broth (coated with a metal-phenolic network nanocoating), inoculation and mixing, aerobic solid-state fermentation, post-ripening and stabilization of *Lactobacillus plantarum* (coated with a metal-phenolic network nanocoating), low-temperature drying, and pulverization and sieving. Differences are shown in Table 5; equipment, detection methods, and operating procedures not listed are the same as in Example 1.

[0035] Table 5. Main differences between Examples 2-4 and Example 1

[0036] In Example 2, the proportion of corn sugar residue was increased, the amount of nanocomposite regulator added was reduced, and the fermentation time was extended to 36 hours, illustrating that qualified products could still be obtained under conditions of low nanocomposite regulator dosage and low ventilation intensity. In Example 3, the proportion of corn germ meal, the amount of compound enzyme, the amount of threonine fermentation broth added, and the fermentation temperature and ventilation intensity were increased, illustrating the applicability under conditions of higher protein substrate and higher ventilation. Example 4 used corn DDGS as a loosely structured auxiliary material, and added 1.5g of sodium selenite premix during the middle of fermentation, equivalent to adding 0.30mg / kg dry basis material based on selenium, to prepare selenium-containing threonine protein feed. In addition, the temperature at the center of the material layer briefly exceeded 42℃ during fermentation (reaching a maximum of 42.7℃), which triggered ventilation enhancement measures (increasing to 2.0 times the original ventilation intensity), and the temperature dropped back below 40℃. The process parameters for the modification and coating of the metal phenolic network nanocoating and the dosage of tannic acid / metal salt in Examples 2-4 are shown in Table 5.

[0037] Comparative Examples 1-8 All comparative examples were based on Example 1 with single-factor or few-factor modifications. Unlisted raw material sources, equipment, testing methods, and operating procedures were the same as in Example 1. The purpose of these comparative examples was to investigate the effects of composite enzymes, nanocomposite regulators, metal-phenolic network nanocoatings, threonine fermentation broth, aerated substrate, post-ripening stabilization, and two-stage low-temperature drying on product quality.

[0038] Table 6 shows the main differences between the comparative examples and Example 1.

[0039] Comparative Example 1 was used to verify the effects of the complex enzyme on fiber degradation, protein peptide release, and fermentation substrate availability. Comparative Examples 2 and 3 were used to verify the contribution of nano-montmorillonite modified with chitosan oligosaccharides and yeast cell wall polysaccharides to DON adsorption stability and desorption control. Comparative Example 4 was used to verify the necessity of threonine fermentation broth as an organic nitrogen source and L-threonine source. Comparative Example 5 was used to verify the necessity of shallow substrate layer and forced aeration for oxygen supply and metabolic heat removal in aerobic solid-state fermentation. Comparative Example 6 was used to verify the effects of *Lactobacillus plantarum* post-ripening and stabilization on product pH stability, viable cell structure, and storage stability. Comparative Example 7 was used to verify the effect of two-stage low-temperature drying on protein thermal damage, browning, and digestibility retention. Comparative Example 8 was used to verify the contribution of the metal-phenolic network nanocoating to DON adsorption stability, cell heat resistance, and product storage stability.

[0040] Test methods: Determination of particle size, specific surface area, interlayer spacing, and viscosity of the nanocomposite regulator: The nanocomposite regulator was prepared into a 0.10% (w / w) dispersion with purified water. After sonication at 25℃ for 5 min, the D50 particle size was determined using a dynamic light scattering particle size analyzer. Each sample was measured in triplicate. The BET specific surface area was determined using a specific surface area analyzer. After vacuum degassing at 80℃ for 12 h, the specific surface area was calculated using the nitrogen adsorption method. The interlayer spacing was determined using X-ray diffraction, with a scanning angle 2θ of 3°-30° and a scanning speed of 2° / min. The apparent viscosity of the nanocomposite regulator slurry was determined using a Brookfield DV2T rotational viscometer, rotor No. 2, 60 r / min, 25℃. Determination of DON in vitro adsorption and desorption rates: Simulated gastrointestinal fluid with a DON concentration of 2.0 mg / L was prepared, and the pH was adjusted to 2.5, 5.0, and 7.0, respectively. Weigh 0.1000 g of the nanocomposite regulator and add 50.0 g of the simulation solution. Shake at 37℃ and 180 r / min for 2 h. After centrifugation, collect the supernatant and determine the residual DON content using high-performance liquid chromatography (HPLC). Calculate the DON adsorption capacity per unit mass of adsorbent. Then, transfer the adsorbed precipitate to pH 7.0 phosphate buffer and shake at 37℃ for another 2 h. Measure the amount of DON released into the supernatant and calculate the desorption rate. Fermentation process monitoring: Record the center temperature of the substrate, surface temperature, exhaust oxygen volume fraction, and carbon dioxide release rate every 4 h during fermentation. Temperature was measured using a Pt100 platinum resistance probe inserted at the geometric center of the substrate. Oxygen and carbon dioxide were measured using an online gas analyzer. Reducing sugars were determined using the 3,5-dinitrosalicylic acid colorimetric method, expressed as glucose. pH was measured after mixing the sample and purified water at a mass ratio of 1:5 and shaking at 25℃ for 10 min. pH was used only as a process control indicator and not as a criterion for the fermentation endpoint. Determination of routine nutritional and safety indicators of the finished product: Moisture content was determined according to the feed moisture determination method, and dried to constant weight at 105℃. Crude protein was determined using the Kjeldahl method, with a nitrogen conversion factor of 6.25. Acid-soluble protein was determined using the trichloroacetic acid precipitation method, and the results were expressed as a percentage of the finished product's dry weight. L-Threonine was determined using high-performance liquid chromatography after acid hydrolysis. DON was determined using immunoaffinity column purification-high-performance liquid chromatography. Selenium content was determined using microwave digestion-inductively coupled plasma mass spectrometry. Viable cell count determination: Candida utilis was counted using yeast extract peptone glucose agar plates and incubated at 28℃ for 48 h. Before counting Bacillus subtilis, the sample suspension was incubated at 80℃ for 10 min to inactivate non-spore-forming bacteria, and then inoculated onto nutrient agar plates and incubated at 37℃ for 24 h. Lactobacillus plantarum was cultured on MRS agar medium and anaerobically at 37℃ for 48 h. Results are expressed as CFU / g. In vitro crude protein digestibility, particle size, color, flowability and stability were determined: In vitro crude protein digestibility was determined using a two-step digestion method involving pepsin and trypsin.In the first stage, pepsin was added and digested for 4 hours at pH 2.0 and 39℃. In the second stage, the pH was adjusted to 6.8, trypsin was added, and digestion continued at 39℃ for 16 hours. The digestion residue was washed, dried, and the residual crude protein was measured to calculate the in vitro crude protein digestibility. Particle size was determined using the standard sieve method, recording the mass percentage of particles between 0.25-2.00 mm and the mass percentage of particles larger than 8 mm. Color was determined using a colorimeter, measuring L, a, and b* values; five measurements were taken for each sample, and the average value was recorded. Flowability was characterized using the angle of repose method. The dry basis yield was calculated as the percentage of the final dried product's dry basis mass to the total dry basis mass of the substrate and added solids before fermentation. Storage stability was tested by sealing the samples and placing them at 37℃ and 75% relative humidity for 30 days, measuring the clumping rate, changes in DON content, and the retention rate of *Candida utilis* viable cells. Determination of the thickness and coverage of the metallophenolic network coating: Yeast cell wall polysaccharides modified with the metallophenolic network or coated cells were fixed with glutaraldehyde and dehydrated with a gradient of ethanol. The morphology of the coating was observed and the coating thickness was measured using transmission electron microscopy. The coverage was determined by UV-Vis spectrophotometry, using the characteristic absorption peak of tannic acid (280 nm) as a basis, and the ratio of the difference in polyphenol content in the supernatant before and after self-assembly to the initial amount added was calculated. Determination of DPPH free radical scavenging rate: A 0.1 mmol / L DPPH-ethanol solution was prepared. 2.0 mL of the sample dispersion was mixed with 2.0 mL of the DPPH solution, and the mixture was reacted in the dark for 30 min. The absorbance was measured at 517 nm, and the free radical scavenging rate was calculated. Determination of cell heat resistance survival rate: Uncoated and metallophenolic network-coated cells were treated in an 80℃ water bath for 30 min, and plate counting was performed to calculate the relative survival rate. The MPN coating thickness was determined by TEM observation of at least 50 particle or bacterial cell cross sections and the average value was taken; tannic acid-metal coordination was confirmed by the shift of the phenolic hydroxyl and carbonyl related peaks in FTIR and the Fe2p peak in XPS; the bacterial cell coating rate was determined by flow cytometry after fluorescent labeling of tannic acid, and the coating rate = number of fluorescent positive bacteria / total number of bacteria × 100%; the heat resistance survival rate = number of viable bacteria after heat treatment / number of viable bacteria before heat treatment × 100%.

[0041] Test Results Table 7. Test results of the performance of nanocomposite modifiers and metal phenolic network coatings.

[0042] Table 8 Key Indicators of Fermentation Process

[0043] Table 9 Results of tests on the nutritional, safety, and processing performance of the finished product

[0044] Table 10 Results of tests on viable bacteria, particle size, and storage stability of the finished product.

[0045] From Table 7, Figure 2 and Figure 3 It can be seen that the nanocomposite regulators obtained in Examples 1, 3, and 4 all have smaller D50 particle size, higher BET specific surface area, and increased interlayer spacing. This indicates that after sodium-based montmorillonite is activated by citric acid, wet-processed nano-sizing, and combined with chitosan oligosaccharide and yeast cell wall polysaccharide, the lamellar structure is partially exfoliated and surface modified, forming a porous interface conducive to DON adsorption. Comparative Example 2 uses only unmodified sodium-based montmorillonite, with a larger D50 particle size and lower BET specific surface area, resulting in a DON adsorption capacity of only 0.52 mg / g and a desorption rate of 38.7%. Comparative Example 3 does not contain montmorillonite nanocarriers and relies solely on chitosan oligosaccharide and yeast cell wall polysaccharide to provide organic adsorption sites, further reducing the DON adsorption capacity. This indicates a synergistic effect between the lamellar structure of the nano-montmorillonite and the organic polysaccharide modified layer. Table 8 shows that in Examples 1-4, under the conditions of a shallow material layer of 10-35 cm and forced ventilation, the highest temperature at the center of the material layer did not exceed 43°C, and the lowest exhaust oxygen volume fraction was higher than 16%, indicating that the process can avoid the central heat accumulation and local anaerobic problems commonly found in thick-layer solid-state fermentation. In Comparative Example 5, the material layer thickness was increased to 60 cm, while the ventilation intensity was reduced and the abnormal triggering measures were removed. The highest temperature at the center of the material layer rose to 47.8°C, the exhaust oxygen volume fraction decreased to 11.6%, the endpoint reducing sugar remained at 5.2%, and the viable count of *Candida utilis* decreased to 4.5 × 10⁻⁶. 5CFU / g. This result indicates that bed thickness, forced ventilation, and temperature rise / low oxygen triggering control are key conditions for maintaining the reproducibility of aerobic solid-state fermentation. Table 9 shows that the crude protein content of the product from Example 1 was 30.8%, the acid-soluble protein content was 9.6%, the L-threonine content was 1.56%, and the DON content decreased to 0.46 mg / kg, with an in vitro crude protein digestibility of 89.2%. This indicates that corn residue, corn germ meal, and loosely structured auxiliary materials formed a suitable carbon-nitrogen ratio and pore structure. Pretreatment with the composite enzyme released available sugars and small-molecule nitrogen sources, and *Candida utilis* and *Bacillus subtilis* further converted the substrate into cell protein, small peptides, and digestible protein components. In Example 2, under conditions of lower nanocomposite regulator addition and lower fermentation temperature, the DON control and acid-soluble protein content were slightly lower, but a qualified product was still obtained. Example 3 increased the dosage of compound enzyme, threonine fermentation broth, and nanocomposite regulator, further increasing the content of crude protein, acid-soluble protein, and L-threonine. However, due to the higher fermentation temperature, the color L* value was slightly lower. Example 4 used corn DDGS as a loosely structured admixture and added sodium selenite premix, achieving a selenium content of 0.27 mg / kg in the product, which can be used as an implementation method for selenium-containing threonine protein feed. Comparative Example 1, omitting the compound enzyme pretreatment, showed a higher endpoint reducing sugar content and a significant decrease in acid-soluble protein content and in vitro crude protein digestibility, indicating that cellulase, xylanase, and neutral protease can improve substrate fermentability and promote the conversion of macromolecular proteins into acid-soluble proteins. Comparative Example 4 replaced the threonine fermentation broth with ammonium sulfate and water. Although it still provided an inorganic nitrogen source, the L-threonine content in the finished product decreased to 0.74%, and the in vitro crude protein digestibility was also lower than in Example 1. This indicates that the threonine fermentation broth not only provides a nitrogen source but also directly provides L-threonine and fermentation metabolism-related nutrients, which is beneficial for forming high-threonine protein feed. In Comparative Example 6, after omitting the *Lactobacillus plantarum* post-ripening and stabilization process, the initial nutritional indicators of the finished product did not change significantly, but the clumping rate increased after 30 days, and the viable *Lactobacillus plantarum* count was below 1.0 × 10⁻⁶. 4 The CFU / g of yeast viability decreased. This result indicates that the post-ripening and stabilization step can improve the storage stability of the product through moderate acidification and microbial community stabilization. Comparative Example 7, subjected to long-term static hot air drying at 110℃, showed a product L* value decreasing to 52.0, a significant decrease in acid-soluble protein content and in vitro crude protein digestibility, and a reduction in the viable count of *Candida utilis* to 3.6 × 10⁻⁶. 5 CFU / g indicates that excessively high temperatures and prolonged heat exposure can cause excessive protein denaturation, deepen browning, and loss of viable bacteria. Therefore, two-stage low-temperature drying can better maintain protein digestibility and product color while reducing moisture and water activity. Furthermore, as... Figure 2As shown, the threonine protein feeds obtained in Examples 1 to 4 were all in relatively uniform granular form, with an overall color ranging from yellowish-brown to slightly light brown, and no obvious clumping, mold spots, or abnormal discoloration were observed. Among them, Examples 1 and 4 had a more obvious yeast aroma and a slightly sour aroma, Example 2 had a yeast aroma, and Example 3 had a strong yeast aroma, indicating that under the conditions of compound enzyme pretreatment, compound bacterial aerobic solid-state fermentation, post-ripening with Lactobacillus plantarum, and two-stage low-temperature drying, the particle shape, color, and sensory properties of the obtained materials were relatively stable. Comparative Example 1, omitting enzymatic pretreatment, resulted in uneven particle size, darker color, and a slight under-fermentation odor. Comparative Example 2, using unmodified sodium montmorillonite instead of the nanocomposite regulator, produced finer particles with a grayish color. Comparative Example 3, omitting the montmorillonite nanocarrier, resulted in looser particles and a lighter color. Comparative Example 4, after removing the threonine fermentation broth, produced more uniform particles but a lighter color and a weaker yeast aroma. Comparative Example 5, using an excessively thick material layer and low ventilation intensity, resulted in severe clumping, inconsistent particle size, and a noticeable off-odor. Comparative Example 6, omitting Lactobacillus plantarum post-ripening stabilization, resulted in uneven particle size, darker color, and a more pronounced sour taste. Comparative Example 7, using high-temperature static hot air drying, resulted in harder particles, a darker color, and a burnt smell. The above appearance results corroborate the results in Tables 9 and 10 regarding color L* values, angle of repose, particle size distribution, agglomeration rate, and viable cell retention rate, indicating that the combined effects of compound enzyme pretreatment, nanocomposite regulators, shallow forced ventilation, *Lactobacillus plantarum* post-ripening, and two-stage low-temperature drying collectively improve product morphology, color, and storage stability. Meanwhile, as... Figure 3As shown, each sample exhibited a characteristic DON response peak at a retention time of approximately 7.2 min; this characteristic peak corresponds to the chromatographic response of DON. The DON content in Examples 1 to 4 was no higher than 1.0 mg / kg. Among them, Example 3 had the lowest DON content, followed by Example 1. As shown in Table 7, the nano-montmorillonite modified with a combination of chitosan oligosaccharide and yeast cell wall polysaccharide used in the examples had a high DON adsorption capacity and a low desorption rate, which helped reduce the DON content in the finished product. The DON content in Comparative Examples 1 to 7 was relatively high. Among them, the DON content in Comparative Examples 3 and 5 was higher than 1.0 mg / kg; this indicates that omitting the montmorillonite nanocarrier or using a thick layer, low ventilation, and without temperature rise and low oxygen control significantly reduced the DON control effect. As shown in Table 7, the coating thickness, coverage rate, and DPPH free radical scavenging rate of the metal phenolic network nanocoatings are high. The yeast cell wall polysaccharides modified by the metal phenolic network nanocoatings in Examples 1-4 all have high coverage rates (72.5%-91.8%) and strong antioxidant activity (DPPH free radical scavenging rate of 58.6%-79.2%). In contrast, the DPPH free radical scavenging rates of Comparative Examples 2 and 3, which were not modified by the metal phenolic network, were only 25.7% and 19.3%, respectively. This indicates that the metal phenolic network nanocoating imparts significant antioxidant function to the carrier. As shown in Tables 9 and 10, in Comparative Example 8, after omitting the metal phenolic network nanocoating, the nano-montmorillonite, chitosan oligosaccharide, and yeast cell wall polysaccharide were still compounded in the original proportions. However, the particle size of its nanocomposite regulator D50 increased, the DON adsorption amount decreased to 1.21 mg / g, and the desorption rate increased to 15.6%. In Comparative Example 8, the DON content of the finished product increased to 0.63 mg / kg, the 30-day agglomeration rate increased to 3.4%, and the 30-day yeast viability retention rate decreased to 64.8%, all of which were worse than in Example 1 (DON content 0.46 mg / kg, agglomeration rate 0.8%, viability retention rate 78.6%). The results indicate that the metal phenolic network nanocoating enhances the adsorption stability of deoxynivalenol and reduces the risk of desorption by bridging the interface between the montmorillonite sheets and the chitosan oligosaccharide / yeast cell wall polysaccharide through polyphenol groups. At the same time, the coating endows the cells with heat resistance, stress resistance and antioxidant protection, reducing the loss of viable cells during fermentation, drying and storage. This is a key feature for achieving the integration of adsorption enhancement and cell stress resistance protection.

[0046] In summary, this invention achieves simultaneous improvements in threonine content, small peptide content, in vitro protein digestibility, DON control, viable cell retention, and product processing stability through a combination of substrate construction (corn sugar residue-corn germ meal-loose auxiliary material), pretreatment with a compound enzyme, fermentation with a compound bacteria containing threonine-containing fermentation broth, simultaneous control of toxins and stress protection by a nanocomposite regulator modified with a metallophenolic network nanocoating, shallow forced aeration aerobic solid-state fermentation, post-ripening and stabilization with *Lactobacillus plantarum*, and two-stage low-temperature drying. The comparative examples show that the absence of key components or exceeding process parameters (including the omission of the metallophenolic network nanocoating) leads to incomplete fermentation, insufficient DON control, decreased protein digestibility, increased viable cell loss, aggravated browning, or reduced storage stability, demonstrating a clear synergistic relationship and process necessity among the technical features of this invention.

[0047] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection defined by the claims. Furthermore, for any issues that may arise with the claims, the embodiments described in the specification shall prevail.

Claims

1. A method for preparing threonine protein feed using corn residue, characterized in that, The process includes the following steps: (1) Raw material conditioning: Corn sugar residue, corn germ meal and granular pore conditioning additives are mixed at a dry basis mass ratio of 1:(3.0-8.0):(0.2-1.5) to obtain mixed solid material; (2) Enzymatic pretreatment: A complex enzyme preparation including cellulase, xylanase and neutral protease is added to the mixed solid material and treated at 45-58℃ for 1-5 hours to obtain pretreated material; (3) Preparation of nanocomposite regulators: Sodium-based montmorillonite was activated with organic acids and nano-sized by wet process, and then combined with chitosan oligosaccharide and yeast cell wall polysaccharide to obtain nanocomposite regulators; yeast cell wall polysaccharide was modified with a metal phenolic network nanocoating, the metal phenolic network being composed of tannic acid and Fe 3+ Zn 2+ or Ca 2+ One or more metal ions in the mixture are coordinated and self-assembled in a weakly alkaline aqueous phase to form a metal phenolic network nanocoating with a thickness of 5-20 nm; (4) Preparation of fermentation broth: Candida utilis and Bacillus subtilis are activated and cultured in a culture medium containing threonine fermentation broth to obtain fermentation broth; Candida utilis and Bacillus subtilis are coated with a metal phenolic network nanocoating; (5) Inoculation and mixing: The pretreated material, nanocomposite regulator and fermentation broth are mixed; (6) Aerobic solid-state fermentation: The material to be fermented is laid on a substrate with bottom ventilation holes. In a shallow fermentation bed or multi-layer shallow tray fermenter, the effective material layer thickness is 10-35cm, and fermentation is carried out at 34-42℃ for 18-36h; (7) Post-maturation and stabilization: After the aerobic solid fermentation is completed, plant lactobacillus coated with metal phenolic network nano-coating is introduced, and post-maturation is carried out for 4-12h under the conditions of 30%-40% moisture content and 30-37℃; (8) Low temperature drying and crushing: the post-maturated material is dried at low temperature so that the moisture content is not higher than 10%, and then crushed and screened to obtain threonine protein feed.

2. The method for preparing threonine protein feed using corn residue according to claim 1, characterized in that, In step (1), the total sugar content in the dry matter of the corn sugar residue is 18%-65%. The granular pore-conditioning auxiliary material is selected from one or more of wheat bran, corn husk, sprayed corn husk, soybean meal, rice bran meal and corn, with a particle size of 0.3-4.0 mm. Before conditioning the raw materials, the corn sugar residue is dehydrated by pressure filtration or centrifugation to make its moisture content 35%-65%. When the pH of the corn sugar residue is lower than 4.5, calcium carbonate is used to adjust its pH to 4.8-5.

8.

3. The method for preparing threonine protein feed using corn residue according to claim 1, characterized in that, In step (2), the amount of cellulase added to the compound enzyme preparation is 50-600 U / g dry basis material, the amount of xylanase added is 30-500 U / g dry basis material, and the amount of neutral protease added is 100-1200 U / g dry basis material; in step (3), the D50 particle size of the nanocomposite regulator is 0.08-0.80 μm, and the BET specific surface area is 60-260 m². 2 / g.

4. The method for preparing threonine protein feed using corn residue according to claim 1, characterized in that, Step (3) includes: dispersing sodium montmorillonite in a citric acid solution with a mass fraction of 0.2%-2.0% and activating it at 50-80℃ for 0.5-3h; washing with water until the pH is 5.5-7.0, and then obtaining nano-montmorillonite slurry by wet grinding or ultrasonic exfoliation; dispersing yeast cell wall polysaccharide in an aqueous solution containing tannic acid and metal ions, and self-assembling it at pH 7.0-9.0 and temperature 20-40℃ for 0.5-2h, and obtaining yeast cell wall polysaccharide modified with metal phenolic network nanocoating after centrifugation and washing; adding chitosan oligosaccharide and yeast cell wall polysaccharide modified with metal phenolic network nanocoating to the nano-montmorillonite slurry, compounding it at 40-70℃ for 0.5-4h, and obtaining nano-composite regulator by spray drying or low-temperature drying at a material temperature not higher than 55℃.

5. The method for preparing threonine protein feed using corn residue according to claim 4, characterized in that, In step (3), the dry basis mass ratio of sodium montmorillonite, chitosan oligosaccharide, and yeast cell wall polysaccharide is 100:(1-15):(2-25); the degree of deacetylation of chitosan oligosaccharide is not less than 80%, and the number average molecular weight is 0.5-10 kDa; the total content of β-glucan and mannan oligosaccharide in yeast cell wall polysaccharide is not less than 40%; the metal phenolic network is formed by the coordination self-assembly of tannic acid and metal ions, the molar ratio of tannic acid to metal ions is (2:1)-(1:3), and the metal ion is Fe. 3+ Zn 2+ and Ca 2+ One or more of the following; the thickness of the metal phenolic network nanocoating is 5-20 nm, and the coverage rate is 60%-95%; the interlayer spacing of the nanocomposite regulator is 1.35-2.20 nm.

6. The method for preparing threonine protein feed using corn residue according to claim 1, characterized in that, In step (4), the threonine fermentation broth is the yeast broth, filtrate, concentrate, or a combination thereof obtained after *Corynebacterium glutamicum* ferments to produce L-threonine. The L-threonine content of the threonine fermentation broth is 5-80 g / L, and the total nitrogen content is 0.5%-8.0%. The culture medium containing the threonine fermentation broth includes threonine fermentation broth, glucose, molasses, yeast extract, and water. The ratio of viable counts of *Candida utilis* to *Bacillus subtilis* is (5-50):

1. Before activation culture, *Candida utilis* and *Bacillus subtilis* are coated with a metal-phenolic network nanocoating. The mass fraction of polyphenolic compounds in the coating solution is 0.02%-0.5%, the mass fraction of metal ions is 0.005%-0.2%, the coating temperature is 20-40℃, and the coating time is 0.5-2 h. The viable count of *Candida utilis* in the fermentation broth is 1.0 × 10⁻⁶. 7 -1.0×10 9 CFU / mL, the viable count of Bacillus subtilis was 1.0 × 10⁻⁶. 6 -1.0×10 8 CFU / mL; the amount of nanocomposite regulator added in step (5) is 0.10%-1.50% of the dry basis mass of the material to be fermented; step (5) also includes adding chitosan oligosaccharide to coat calcium carbonate microparticles to maintain the pH in the range of 4.6-6.5 during fermentation.

7. The method for preparing threonine protein feed using corn residue according to claim 1, characterized in that, In step (6), the effective material layer thickness of each shallow tray in the multi-layer shallow tray fermenter is 12-28 cm, the bottom opening rate of the shallow tray is 8%-35%, and the vertical spacing between adjacent shallow trays is 8-25 cm. Before inoculation in step (7), *Lactobacillus plantarum* is coated with a metal-phenolic network nano-coating at a temperature of 20-40℃ for 0.5-2 hours, with an inoculation amount of 1.0 × 10⁻⁶. 6 -1.0×10 8 CFU / g wet material; During fermentation, air is continuously introduced from the bottom at a ventilation intensity of 0.02-0.30 L / (kg·min), and the center temperature of the material layer, the surface temperature of the material layer, and the volume fraction of exhaust oxygen are monitored online; When any of the following conditions are met, the ventilation intensity is increased to 1.5-3.0 times the original ventilation intensity, until the center temperature of the material layer is below 40℃ and the volume fraction of exhaust oxygen is above 18%.

8. The method for preparing threonine protein feed using corn residue according to claim 1, characterized in that, The low-temperature drying in step (8) includes: the hot air temperature of the first drying section is 65-90℃, the material temperature is not higher than 55℃, and the moisture content is dried to 14%-18%; the hot air temperature of the second drying section is 45-65℃, the material temperature is not higher than 50℃, and the moisture content is dried to 10% and the water activity is not higher than 0.65; after crushing and sieving, the mass proportion of particles with a particle size of 0.25-2.00mm is not less than 70%, and the mass proportion of particles with a particle size greater than 8mm is not higher than 2%.

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