Methods for preparing protein-based bio-feeds from maize by-products

Abstract

This invention discloses a method for preparing protein-based bio-feed from corn by-products, belonging to the field of bio-feed technology. The method uses corn germ meal and corn sugar residue as raw materials, which are dried, pulverized, and sieved, then mixed in a specific ratio as a solid-state fermentation substrate. Activated feed yeast is spray-inoculated, the moisture content is adjusted, and a composite carbon source is added. Aerobic solid-state fermentation is carried out under controlled temperature, humidity, stirring, and intermittent ventilation conditions. After fermentation, corn glycerin nano-dispersions are sprayed onto the surface of the material, followed by rapid drying, pulverization, and sieving to obtain the protein-based bio-feed. This invention can improve the protein conversion efficiency, protein digestibility, and product stability of corn by-products, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biofeed technology, specifically, it relates to a method for preparing protein biofeed based on corn by-products. Background Technology

[0002] The deep processing of corn generates various byproducts, such as corn germ meal, corn sugar residue, corn protein feed, and dry alcohol residue containing solubles. These byproducts are abundant and inexpensive, providing a basis for further development and utilization as feed ingredients. However, corn germ meal and other raw materials typically suffer from high fiber content, poor palatability, and limited protein utilization, thus restricting their application in high-value feeds.

[0003] In existing technologies, the main ways to utilize corn by-products include direct feeding, simple mixing, spray drying, and microbial fermentation modification. Among these, direct feeding or simple mixing, although simple in process, is difficult to significantly improve the digestibility and absorption efficiency of protein; while traditional products are usually attached to the alcohol production process, and the product quality is greatly affected by the state of raw materials, fermentation path, and heat drying process, resulting in defects such as fluctuations in protein quality, high fiber content, and high risk of mycotoxins.

[0004] Furthermore, existing fermentation modification technologies for corn germ meal mostly employ yeast, lactic acid bacteria, Bacillus, white-rot fungi, or complex enzymes for treatment, primarily focusing on increasing crude protein, improving palatability, or general detoxification. However, these technologies typically fail to establish specific carbon-nitrogen matching relationships, moisture content windows, aerobic turning regimes, and termination drying regimes around the specific substrate system of corn germ meal and corn sugar residue. They also do not prioritize protein digestibility as a core objective for process optimization, resulting in deficiencies in the product's nutritional value and quality stability.

[0005] On the other hand, zein, a corn gliadin, has been widely studied for its application in nanocarrier construction in recent years due to its amphiphilicity, self-assembly ability, biodegradability, and good interfacial interaction capabilities. Publicly available information indicates that zein can form nanoparticles in ethanol / water antisolvent systems or under specific pH conditions, providing encapsulation, protection, and sustained release of active substances. However, current zein nanotechnology mainly focuses on food, pharmaceutical, and agricultural delivery, and there is a lack of mature solutions that effectively integrate with solid-state fermentation feed processes using corn by-products, particularly lacking an integrated process design that balances the stability of the primary fermentation live bacteria system with the need for interfacial protection in post-processing.

[0006] Therefore, it is necessary to provide a new method for preparing protein bio-feed based on corn by-products. This method involves constructing a solid-state fermentation substrate using corn germ meal and corn sugar residue, improving protein digestibility through directional aerobic solid-state fermentation with feed yeast, and preferably combining it with corn homologous zein nano-interface composite technology to protect and stabilize the functional components in the fermentation products. This results in a bio-feed product with stable quality, high digestibility, and suitability for industrial implementation. Summary of the Invention

[0007] To address the problems of low protein utilization, poor palatability, and low added value when corn by-products are directly used as feed or simply mixed in existing technologies, and the lack of specific carbon-nitrogen matching, moisture content control, aerobic turning and ventilation regimes, and endpoint control in existing fermentation modification technologies for the specific substrate system of corn germ meal and corn sugar residue, resulting in insufficient protein conversion efficiency, digestibility, and product quality stability, this invention provides a method for preparing protein bio-feed based on corn by-products. Furthermore, to address the problems of heat damage, protein aggregation, accelerated browning, and poor stability of functional components in the subsequent drying process of fermentation products in existing technologies, this invention also introduces corn gliadin nano-dispersions for interfacial complexation after the primary fermentation, thereby improving the product's thermal stability, redispersibility, and protein digestibility.

[0008] This invention adopts the following technical solution: a method for preparing protein bio-feed based on corn by-products, comprising the following steps: drying, pulverizing, and sieving corn germ meal and corn sugar residue separately, then mixing them at a dry weight ratio of 6:4 to 7:3 to obtain a solid fermentation substrate; preparing an activation solution containing 0.5-1.0% glucose and 0.1-0.2% yeast extract with water; adding feed yeast at a ratio of yeast powder to activation solution mass of 1:10 to 1:20; and activating at 30-35℃ for 3-4 hours to obtain... The activated bacterial solution is sprayed onto the solid fermentation substrate, and water and a composite carbon source are added to make the initial moisture content of the material 35-50%. Then, aerobic solid fermentation is carried out at 30-35℃, relative humidity 70-85%, and material layer thickness 8-12cm. During the fermentation process, the mixture is turned over once every 8-12 hours and intermittent ventilation is implemented. After fermentation for 48-72 hours, the material is immediately dried at 80-100℃ until the product moisture content is no higher than 12%. The product is then crushed and sieved to obtain the protein biological feed.

[0009] In the aerobic solid-state fermentation process, the inoculated fermentation material is spread into a layer with a thickness of 8-12cm. During the fermentation process, the material is turned over once every 8-12 hours and ventilated for 15 minutes every 2 hours to maintain the fermentation material layer in an aerobic state.

[0010] Preferably, the moisture content of the corn germ meal is no higher than 12%, and more than 95% of it passes through a 40-mesh sieve after being crushed; the corn sugar residue is pre-dried to a moisture content of no higher than 10%, crushed, and passed through a 40-60 mesh sieve before being mixed with the corn germ meal.

[0011] Preferably, the composite carbon source includes glucose and molasses, and the viable count of the feed yeast powder is not less than 2.0 × 10⁻⁶. 10 The activated bacterial solution was inoculated evenly by spraying, ensuring that the bacteria were evenly distributed in the solid substrate without any obvious bacterial accumulation points.

[0012] Preferably, the feed yeast is Candida utilis and / or Saccharomyces cerevisiae; the inoculation amount is 5-10% of the dry weight of the solid-state fermentation substrate.

[0013] Preferably, during the aerobic solid-state fermentation process, the pH of the material is controlled at 5.0-6.0, and the fermentation endpoint is determined by the material temperature reaching its peak and then falling back, and the pH decreasing from its initial value and then starting to rise again.

[0014] Preferably, the rapid drying is carried out using fluidized bed drying or an equivalent rapid drying method, with a drying temperature of 80-100℃. After drying, the moisture content of the material is no higher than 12%, and then it is pulverized and passed through a 60-mesh sieve.

[0015] Preferably, after the aerobic solid-state fermentation is completed and before rapid drying, the process further includes a step of combining the zein nano-dispersion with the fermentation material; the zein has the CAS number 9010-66-6, and is formed into nanoparticles by self-assembly using an ethanol-water solution-water anti-solvent system before being sprayed onto the surface of the fermentation material.

[0016] Preferably, the method for preparing the zein nanodispersion is as follows: zein is dissolved in a 70-90% volume fraction ethanol aqueous solution, the zein concentration is controlled at 5-20 mg / mL, and the solution is added to a water-antisolvent under stirring conditions, the volume ratio of solvent to antisolvent is controlled at 1:3 to 1:8, so that the zein undergoes self-assembly due to the change in solvent polarity, forming nanoparticles with an average particle size of 80-400 nm.

[0017] Preferably, the aqueous antisolvent further contains one or more of pectin, sodium caseinate, and chitosan as a stabilizer to improve the salt resistance, thermal stability, or redispersibility of zein nanoparticles through electrostatic interaction and / or steric hindrance.

[0018] Preferably, the zein nano-dispersion is sprayed onto the surface of the material after the main fermentation is completed, with a solid content equivalent to 1-5 parts by weight of the dry basis of the fermented material. After spraying, it is mixed for 3-10 minutes and aged for 15-30 minutes, and then rapidly dried. The composite mechanism between the zein nanoparticles and the fermented material is at least one of hydrophobic association, hydrogen bonding and electrostatic adsorption.

[0019] Compared with existing technologies, this invention has at least the following beneficial effects: First, by constructing a specific solid-state fermentation substrate system using corn germ meal and corn sugar residue, this invention can better match carbon and nitrogen sources and improve the looseness and aeration of the material pile, thereby facilitating the proliferation and metabolism of feed yeast and the accumulation of microbial protein, and improving the protein conversion efficiency and nutritional utilization value of corn by-products. Second, by activating yeast and combining it with spray inoculation, moisture content control, aerobic turning, intermittent ventilation, and fermentation endpoint control, this invention can effectively suppress problems such as local anaerobic digestion, overheating, and uneven fermentation, improving the stability of the fermentation process and the consistency of product quality. Third, by introducing corn gliadin nano-dispersions for interfacial composite after the main fermentation, this invention can protect the surface proteins and functional components of the fermentation products during subsequent drying, reducing the risk of heat damage, protein aggregation, and browning, and improving the thermal stability and redispersibility of the product. Fourth, the present invention uses a rapid drying method to terminate fermentation in time and reduce water activity, which helps to reduce the adverse effects of excessive heat exposure on protein quality, thereby obtaining protein biological feed with suitable moisture, good flowability, stable quality and high protein digestibility. Attached Figure Description

[0020] Figure 1 This is a photograph of the solid-state fermentation substrate prepared in Example 1; Figure 2 This is a photograph of the activated bacterial solution prepared in Example 1; Figure 3 This is a photograph of the solid-state fermentation product prepared in Example 1; Figure 4 These are the infrared spectrum and transmission electron microscope image of the zein nanodispersion prepared in Example 1; Figure 5 This is a photograph of the light yellowish-brown powdered protein biological feed prepared in Example 1. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. However, these embodiments are only for illustrating the present invention and do not constitute any limitation on the scope of protection of the present invention. For those skilled in the art, simple deductions or substitutions made without departing from the concept of the present invention should be considered as falling within the scope of protection defined by the claims of the present invention. In the present invention, unless otherwise specified, intermediate values ​​are used for undefined parameter ranges by default.

[0022] The corn germ meal and corn sugar residue used in this invention are both derived from corn deep-processing byproducts. The parameters (mass percentage) of the corn germ meal are as follows: crude protein ≥18%, crude fat ≤2%, crude ash ≤4%, crude fiber ≤8%, moisture ≤12%, and it is a yellowish-brown granular powder, for example, from COFCO Biochemical Technology Co., Ltd. (50kg / bag). The parameters (mass percentage) of the corn sugar residue are as follows: protein 20-25%, crude fat 18-22%, crude ash 7-10%, and sugar 30-35%, for example, from Shouguang Baijulong Biological Feed Co., Ltd. The corn gliadin is zein, CAS number 9010-66-6, purity >99%, nitrogen content 13.1-17%, loss on drying ≤8.0%, ether-soluble matter <2.0%, residue on ignition <0.3%, and heavy metals <20ppm; pectin, CAS number 9000-69-5, is derived from grapefruit peel, with a density of 1.5±0.1g / cm³. 3 Sodium caseinate CAS No. 9005-46-3; Chitosan CAS No. 9012-76-4, density 1.75 g / cm³ 3 Unless otherwise stated, glucose, molasses (CAS No. 8061-53-8), and yeast extract (CAS No. 8013-01-2) are all feed-grade or food-grade raw materials, and the water is purified water or process water that meets the requirements for feed processing water. The main equipment used in the embodiments is as follows: hammer mill, Jiangyin Hongda SF-20; twin-shaft paddle mixer, Changzhou Lima WZ-50; pressure spray inoculation device, Shanghai Yanzheng YZ-SW-2; constant temperature and humidity solid fermentation chamber, Shanghai Yiheng HWS-500; fluidized bed dryer, Changzhou Yibu FG-5; high-speed disperser, IKAT25digital; dynamic light scattering particle size analyzer, Malvern Zetasizer NanoZS; colorimeter, Konica Minolta CR-400.

[0023] Example 1 This embodiment is a preferred implementation, and fully includes the steps of substrate pretreatment, yeast activation, aerobic solid-state fermentation, preparation of zein nanodispersion, post-fermentation nanocomposite, rapid drying, and pulverization and sieving.

[0024] Preparation of solid-state fermentation substrate: 600.0g of corn germ meal was pulverized using a hammer mill and passed through a 40-mesh standard sieve. The sieving results showed that 95.6% of the material passed through the 40-mesh sieve. 800.0g of wet corn sugar residue (dry weight 346.8g) was pre-dried in a 100℃ hot air dryer to a moisture content of 9.2%, then pulverized and passed through a 50-mesh sieve. The above corn germ meal and corn sugar residue were added to a twin-shaft paddle mixer at a dry weight ratio of 6:4 and dry-mixed at 25℃ and 80rpm for 5 minutes to obtain the solid-state fermentation substrate (e.g., corn germ meal and corn sugar residue). Figure 1 (As shown).

[0025] Table 1. Solid substrate composition of Example 1

[0026] Activation of feed yeast: Weigh 46.8g of freeze-dried Candida utilis (commercially sourced, catalog number: CICC31494, China Industrial Microbial Culture Collection Center; other similar isolates can also be used) with a viable count of 3.1 × 10⁻⁶ cells. 10 CFU / g, the moisture content of the bacterial powder is 6.4%. Separately, 468.0g of purified water was added, along with 3.7g of glucose and 0.7g of yeast extract, to prepare an activation solution containing 0.8% glucose and 0.15% yeast extract. The activation solution was placed in a sterile mixing tank, and the temperature was adjusted to 32℃. Yeast powder was added at a ratio of 1:10 (batch weight of bacterial powder to activation solution weight), and stirred at 120 rpm for 5 minutes to ensure uniform dispersion. Then, it was allowed to stand at 32℃ for 3.5 hours to activate, yielding the activated bacterial solution (e.g., CFU / g). Figure 2 (As shown). The total mass of the activated bacterial solution was approximately 519.2g.

[0027] Table 2 Composition of bacterial suspension activation in Example 1

[0028] Inoculation, water conditioning, and aerobic solid-state fermentation: The obtained solid-state fermentation substrate was placed in a mixer, which was started and maintained at 80 rpm. The activated bacterial solution obtained in step 2 was evenly sprayed onto the solid-state substrate using a pressure spray device at a pressure of 0.25 MPa and a flow rate of 120 mL / min. The inoculation amount was 5.0% based on the dry weight of the solid-state fermentation substrate. Subsequently, a composite carbon source aqueous solution consisting of 10.0 g glucose, 25.0 g molasses, and 216.6 g purified water was added to adjust the initial moisture content of the material to 38.0%. Mixing was continued for 5 minutes to obtain a loose, uniform fermentation material without obvious bacterial accumulation points. The fermentation material was transferred to a constant temperature and humidity solid-state fermentation chamber with a material layer thickness of 10 cm. The fermentation temperature was controlled at 32℃, and the relative humidity at 80%. Intermittent ventilation was implemented during fermentation, with a ventilation frequency of 15 minutes every 2 hours and a ventilation volume of 0.20 m³ / min. 3 / (kg dry basis·h). In this embodiment, the inoculated fermentation material is laid in a 10cm thick layer for aerobic solid-state fermentation. During the fermentation process, the material is turned over once every 10 hours and ventilated for 15 minutes every 2 hours. Due to the small thickness of the material layer, combined with periodic turning and intermittent ventilation, air exchange can be maintained inside the material layer, avoiding the formation of a long-term anaerobic zone, thereby ensuring the normal metabolic activity of yeast during the fermentation process.

[0029] Stir the mixture every 10 hours for 5 minutes. During fermentation, maintain the pH between 5.0 and 6.0. If the pH is higher than 5.5, adjust it to 5.5 using feed-grade citric acid or lactic acid; if the pH is lower than 5.0, adjust it to 5.0 using calcium carbonate aqueous dispersion. At 60 hours of fermentation, the core temperature of the material drops from a peak of 34.8℃ to 32.5℃ (temperature probe located at the geometric center of the material layer, approximately 5cm from the bottom; recorded every 2 hours; maximum recorded value was 34.8℃), the pH decreases from the initial 5.9 to 5.1 and then rises back to 5.4, and the material exhibits a distinct sour and yeasty aroma. Fermentation is then considered complete, and the product is as follows: Figure 3 As shown.

[0030] Table 3 Process parameters for the fermentation stage in Example 1

[0031] Preparation and post-fermentation compounding of zein nanodispersions: 20.0 g of zein was weighed and added to 2000 mL of 80% (v / v) ethanol aqueous solution. The mixture was stirred at 600 rpm for 40 min at 25 °C to completely disperse the zein and form a 10.0 mg / mL zein ethanol aqueous solution. Separately, 8000 mL of purified water was used as the water-to-antisolvent mixture. 4.0 g of pectin and 6.0 g of sodium caseinate were added, and the pH was adjusted to 6.8. The mixture was stirred at 800 rpm for 30 min to fully hydrate the stabilizer. The zein ethanol aqueous solution was added to the water-to-antisolvent mixture at a rate of 20 mL / min, maintaining a stirring speed of 1000 rpm, and controlling the solvent-to-antisolvent volume ratio at 1:4. After the addition was complete, stirring was continued for 30 min to allow the zein to self-assemble due to the change in solvent polarity, resulting in zein nanodispersions. The infrared spectrum and transmission electron microscopy image are shown below. Figure 4As shown. The obtained dispersion showed no visible precipitation after standing at 25℃ for 24 hours. After the main fermentation and before rapid drying, the zein nano-dispersion was sprayed onto the surface of the fermentation material at a solids content equivalent to approximately 3.4 parts by weight of the dry basis of the fermentation material. Based on approximately 882.0 g of dry basis fermentation material, the solids content of the zein nano-dispersion was 28.1 g, including 20.0 g of zein, 4.0 g of pectin, and 6.0 g of sodium caseinate, which was converted to a solids content of 30.0 g. After spraying, it was mixed at 60 rpm for 6 min, and then aged at 25℃ for 20 min. During this stage, the zein nanoparticles mainly aggregated on the surface of the fermentation material through hydrophobic association, hydrogen bonding, and electrostatic adsorption.

[0032] Table 4. Composition and parameters of zein nanodispersion in Example 1

[0033] Rapid drying and pulverization / sieving: The compounded material is immediately fed into a fluidized bed dryer. The inlet air temperature is set to 90℃, the outlet air temperature is controlled at 58℃, the fluidizing velocity is 1.8 m / s, and the drying time is 38 min. Heating is stopped when the product moisture content reaches 9.6%, and the material is cooled to room temperature. The dried material is then pulverized by a hammer mill and passed through a 60-mesh sieve to obtain a light yellowish-brown powdered protein feed. Figure 5 As shown. The resulting product has no obvious burnt smell, but has a sour and yeasty aroma, good powder flowability, and a moisture content not exceeding 12%.

[0034] In this embodiment, pH detection was performed using a method of "pre-adjustment detection, post-adjustment verification, and endpoint criterion based solely on naturally monitored pH before adjustment." For each pH test, samples were taken from the upper, middle, and lower parts of the material layer; samples were taken at 0.75H, 0.50H, and 0.25H, based on the actual material layer thickness H. 20.0g of sample was taken from each height layer. After thoroughly mixing the three layers, 10.0g was taken as the pH test sample, added to 50.0mL of deionized water, shaken at 25°C for 10min, and allowed to stand for 2min before pH measurement. The pH meter was calibrated at three points using standard buffer solutions at pH 4.00, pH 6.86, and pH 9.18 before testing. In this embodiment, the naturally monitored pH of the material after inoculation, water adjustment, and thorough mixing was measured at 0h as 5.9. Since the pH was higher than 5.5, a 10% (w / w) feed-grade lactic acid aqueous solution was used for spray correction. The cumulative amount of lactic acid aqueous solution added was 6.0 g. After mixing for 5 minutes, the adjusted control pH was measured to be 5.5. This adjusted control pH was only used to confirm that the material had entered the suitable pH range for yeast fermentation and was not used as the endpoint criterion for "pH starting to rise after decreasing from the initial value". At 12h, 24h, 36h, 48h, and 60h of fermentation, pH was measured before the addition of any acid-base regulators and recorded as the naturally monitored pH. In this example, the naturally monitored pH was measured to be 5.4 at 12h, 5.1 at 24h, 5.2 at 36h, 5.3 at 48h, and 5.4 at 60h. During the aforementioned 12h to 60h period, the naturally monitored pH did not fall below 5.0, therefore no calcium carbonate aqueous dispersion was added; the naturally monitored pH also did not rise above 5.5, therefore no lactic acid or citric acid was added again. Therefore, in this embodiment, "pH decreased from the initial 5.9 to 5.1 and then rose back to 5.4" refers to the trend of the naturally monitored pH before adjustment, where 5.1 at 24h was the lowest point of the naturally monitored pH, and the pH rises at 36h, 48h, and 60h were not caused by the adjustment with calcium carbonate aqueous dispersion. In this embodiment, when the pH rise is used as an auxiliary criterion for the endpoint, only naturally monitored pH data that is not directly affected by recent acid-base regulators are used; the pH control after adjustment is only used as a process control record and not as a criterion for the fermentation endpoint.

[0035] Example 2 This embodiment is basically the same as Embodiment 1, except for the dry weight ratio of corn germ meal to corn sugar residue, the inoculation amount, and the fermentation time. Except for the parameters listed in Table 5, the remaining steps, equipment, and testing methods are the same as in Embodiment 1.

[0036] Table 5. Parameters that differ between Example 2 and Example 1

[0037] In Example 2, the core temperature of the fermented material decreased from 34.5℃ to 33.1℃ after 48 hours, the pH decreased from the initial 5.8 to 5.2 and then rose back to 5.3, and the final odor was normal.

[0038] Example 3 This embodiment is basically the same as Example 1, except for the yeast inoculation amount, substrate thickness, fermentation time, zein concentration, and stabilizer composition. Except for the parameters listed in Table 6, the remaining steps are the same as in Example 1.

[0039] Table 6. Parameters that differ between Example 3 and Example 1

[0040] In Example 3, chitosan was first dissolved in a 0.5% acetic acid aqueous solution, and then the pH was adjusted to 5.8 with a 1 mol / L sodium hydroxide solution before being used as a water-solvent stabilizer. The resulting zein nanodispersion had a zeta potential of +28.6 mV, and the coating on the material surface was good after spraying. The temperature probe was located at the geometric center of the material layer, approximately 6 cm from the bottom.

[0041] Example 4 This embodiment is basically the same as Example 1, except that the drying temperature of the zein nanodispersion is different. Except for the parameters listed in Table 7, the other steps are the same as in Example 1.

[0042] Table 7. Parameters that differ between Example 4 and Example 1

[0043] The product obtained in Example 4 has a lighter color, but due to the lower amount of zein solids, the protective effect of the nano-interface layer on the surface proteins of the fermented material is weaker than that in Example 1.

[0044] Comparative Example 1 This comparative example is essentially the same as Example 1, except that corn residue is not added; only corn germ meal is used as the solid-state fermentation substrate. The remaining inoculation, fermentation, zein compounding, and drying steps are the same as in Example 1. This comparative example is used to investigate the effect of the missing specific corn by-product compounding ratio on fermentation efficiency and protein digestibility.

[0045] Comparative Example 2 This comparative example is essentially the same as Example 1, except that the dry weight ratio of corn germ meal to corn sugar residue is adjusted to 8:2, exceeding the range of 6:4 to 7:3 defined in the claims. The remaining steps are the same as in Example 1. This comparative example is used to investigate the effects of deviations in the substrate carbon-nitrogen ratio and physical aeration structure on the fermentation effect.

[0046] Comparative Example 3 This comparative example is essentially the same as Example 1, except that after fermentation, the zein nanodispersion spraying and compounding process is not performed; instead, fluidized bed drying is carried out directly. The remaining steps are the same as in Example 1. This comparative example is used to investigate the effect of the nano-interface compounding step after primary fermentation on the product's thermal stability, redispersibility, and protein digestibility.

[0047] Comparative Example 4 This comparative example is essentially the same as Example 1, except that the drying method was changed from rapid fluidized bed drying at 90°C to static drying in a hot air box at 120°C for 120 minutes, until the product moisture content dropped to 9.8%. The remaining steps are the same as in Example 1. This comparative example is used to investigate the effects of excessively high drying temperature and prolonged heat exposure time on protein browning, available lysine, and digestibility.

[0048] Table 8. Main differences between the comparative examples and Example 1

[0049] Test methods: Moisture content determination: Following GB / T6435-2014, a Shanghai Precision Scientific DHG-9070A electric thermostatic drying oven was used to dry to constant weight at 105℃. Crude protein content determination: Following GB / T6432-2018, the Kjeltec nitrogen determination method was used, employing a FOSSKjeltec 8400 fully automatic Kjeltec nitrogen analyzer to determine total nitrogen, with a conversion factor of 6.25. Acid-soluble protein content determination: Following the acid-soluble protein determination method in GB / T22492-2008, 2.000 g of sample was weighed, 50 mL of 15% trichloroacetic acid solution was added, and the mixture was shaken at 25℃ for 30 min, then filtered. The acid-soluble nitrogen in the filtrate was determined using the Kjeltec nitrogen determination method and converted to acid-soluble protein. The result is expressed as a percentage of acid-soluble protein in the crude protein mass. In vitro crude protein digestibility determination: A two-stage enzymatic hydrolysis method simulating the pig gastrointestinal tract was used. Weigh 1.000 g of sample, add 50 mL of pH 2.0 hydrochloric acid-potassium chloride buffer and 3000 U of pepsin, and digest at 39 °C and 120 rpm for 2 h with shaking. Then adjust the pH to 6.8 with 1 mol / L sodium hydroxide, add 2000 U of trypsin and 0.5 g of bile salts, and continue digestion at 39 °C for 4 h. After digestion, centrifuge, wash, dry the residue, and determine the residual nitrogen to calculate the in vitro crude protein digestibility. Each group was measured in triplicate. Live yeast count: Following GB / T22547-2008, the YPD agar plate counting method was used. Yeasts were incubated at 30 °C for 48 h and the counts were expressed as CFU / g. Vomitoxin content determination: High performance liquid chromatography (HPLC) was used. The instrument was an Agilent 1260 Infinity II with a C18 column. The mobile phase was water-acetonitrile (90:10, v / v), the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. Samples were purified using an immunoaffinity column before injection. The detoxification rate was calculated as follows: Reduction rate (%) = (Toxin content in substrate before fermentation - Toxin content in product) / Toxin content in substrate before fermentation × 100%. Zein nanoparticle Zeta potential determination: The nanodispersion was diluted 10-fold and measured using a Malvern Zetasizer NanoZS at 25℃, with three parallel measurements recorded, and the average PDI or Zeta potential was recorded. Viscosity determination: The apparent viscosity of the zein nanodispersion was measured using a Brookfield DV2T rotational viscometer, rotor #2, 60 rpm, 25℃. Color determination: L*, a*, and b* values ​​were measured using a Konica Minolta CR-400 colorimeter, with five measurements taken for each sample and the average value recorded. Product yield determination: Calculated as the ratio of the final dried product dry weight to the total dry weight of the substrate and added solids before fermentation. Thermal stability evaluation: The product was placed in a 90℃ oven for 30 min, and the retention rate of acid-soluble protein and color change ΔE before and after treatment were measured to characterize the effects of rapid drying and zein compounding on protein thermal damage.

[0050] Table 9. Test Results of Examples and Comparative Examples

[0051] Results analysis: Deviations in substrate ratio, lack of zein complex, or deviations in drying conditions can lead to insufficient crude protein increase, decreased acid-soluble protein ratio, reduced live yeast count, increased browning, or decreased digestibility, demonstrating that the technical solution of this invention has clear process necessity and stable technical effects.

[0052] 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 protein-rich bio-feed based on corn by-products, characterized in that, The process includes the following steps: Corn germ meal and corn sugar residue are dried, crushed, and sieved separately, then mixed at a dry weight ratio of 6:4 to 7:3 to obtain a solid-state fermentation substrate; an activation solution containing 0.5-1.0% glucose and 0.1-0.2% yeast extract is prepared with water; feed yeast is added at a ratio of 1:10 to 1:20 (mass of yeast powder to activation solution), and activated at 30-35℃ for 3-4 hours to obtain an activated bacterial solution; the activated bacterial solution is sprayed onto the solid-state fermentation substrate, and water and a composite carbon source are added to bring the initial moisture content of the material to 35-50%. Aerobic solid-state fermentation is then carried out at 30-35℃, relative humidity 70-85%, and a substrate layer thickness of 8-12cm. During fermentation, the substrate is turned over every 8-12 hours with intermittent ventilation. After 48-72 hours of fermentation, the substrate is immediately rapidly dried at 80-100℃ until the product moisture content is no higher than 12%, then crushed and sieved to obtain the protein-based biological feed.

2. The method for preparing protein bio-feed based on corn by-products according to claim 1, characterized in that, The moisture content of the corn germ meal is no higher than 12%, and more than 95% of it passes through a 40-mesh sieve after being crushed. The corn sugar residue is pre-dried to a moisture content of no higher than 10%, crushed, and passed through a 40-60 mesh sieve before being mixed with the corn germ meal.

3. The method for preparing protein bio-feed based on corn by-products according to claim 1, characterized in that, The composite carbon source includes glucose and molasses, and the viable count of the feed yeast powder is not less than 2.0 × 10⁻⁶. 10 The activated bacterial solution was inoculated evenly by spraying, ensuring that the bacteria were evenly distributed in the solid substrate without any obvious bacterial accumulation points.

4. The method for preparing protein bio-feed based on corn by-products according to claim 1, characterized in that, The feed yeast is Candida utilis and / or Saccharomyces cerevisiae; the inoculation amount is 5-10% of the dry weight of the solid fermentation substrate.

5. The method for preparing protein bio-feed based on corn by-products according to claim 1, characterized in that, During the aerobic solid-state fermentation process, the pH of the material is controlled between 5.0 and 6.0, and the fermentation endpoint is determined by the material temperature reaching its peak and then falling back, and the pH decreasing from its initial value and then starting to rise again.

6. The method for preparing protein bio-feed based on corn by-products according to claim 1, characterized in that, The rapid drying is carried out using fluidized bed drying or equivalent rapid drying methods, with a drying temperature of 80-100℃. After drying, the moisture content of the material is no higher than 12%, and then it is crushed and passed through a 60-mesh sieve.

7. The method for preparing protein bio-feed based on corn by-products according to claim 1, characterized in that, After the aerobic solid-state fermentation is completed and before rapid drying, the process includes a step of combining the zein nano-dispersion with the fermentation material; the zein has the CAS number 9010-66-6, and it is formed into nanoparticles by self-assembly in an ethanol-water solution-water anti-solvent system, and then sprayed onto the surface of the fermentation material.

8. The method for preparing protein bio-feed based on corn by-products according to claim 7, characterized in that, The method for preparing the zein nanodispersion is as follows: zein is dissolved in a 70-90% volume fraction ethanol aqueous solution, and the concentration of zein is controlled at 5-20 mg / mL. Under stirring conditions, the solution is added to a water-antisolvent, and the volume ratio of solvent to antisolvent is controlled at 1:3 to 1:8, so that the zein undergoes self-assembly due to the change in solvent polarity, forming nanoparticles with an average particle size of 80-400 nm.

9. The method for preparing protein bio-feed based on corn by-products according to claim 8, characterized in that, The aqueous antisolvent also contains one or more of pectin, sodium caseinate, and chitosan as stabilizers to improve the salt resistance, thermal stability, or redispersibility of zein nanoparticles through electrostatic interaction and / or steric hindrance.

10. The method for preparing protein bio-feed based on corn by-products according to claim 9, characterized in that, The zein nano-dispersion is sprayed onto the surface of the material after the main fermentation at a solid content equivalent to 1-5 parts by weight of the dry basis of the fermented material. After spraying, it is mixed for 3-10 minutes and aged for 15-30 minutes, and then rapidly dried. The composite mechanism between the zein nanoparticles and the fermentation material is at least one of hydrophobic association, hydrogen bonding, and electrostatic adsorption.

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