A method for preparing protein hydrolyzed corn syrup feedstock by desulfurization homogenization viscosity reduction section oxygen control composite fermentation based on bacillus subtilis corn syrup homologous matrix adaptive activation and application

Abstract

The application relates to a method for preparing a hydrolyzed corn syrup feedstock based on Bacillus subtilis corn syrup homologous matrix adaptive activation desulfurization homogenization viscosity reduction section oxygen control composite fermentation and application, and relates to a method for preparing a protein hydrolyzed corn syrup feedstock and application. In order to solve the problems that nitrogen component accumulation is easy to be insufficient when the existing corn syrup is directly used as a feedstock and a fermentation end point is not easy to be controlled, the present application carries out buffer desulfurization treatment and homogenization viscosity reduction treatment on liquid corn syrup, then carries out resuspension, vortex dispersion and low-speed oscillation activation on sterilized corn syrup supernatant, centrifugation is carried out to discard the supernatant, pre-treatment sterilized corn syrup is connected, aerobic enzyme production hydrolysis fermentation is carried out, then Candida utilis bacteria liquid is connected to carry out micro-aerobic low-speed nutrition conversion fermentation, post-ripening enzymolysis and enzyme activity termination treatment are carried out, so that protein release and controlled hydrolysis in the corn syrup are promoted. The present application belongs to the technical field of microbial fermentation feed.

Description

Technical Field

[0001] This invention relates to a method for preparing protein hydrolyzed corn steep liquor feed raw materials and its application, belonging to the field of microbial fermentation feed technology. Background Technology

[0002] Corn steep liquor is an important byproduct of wet corn processing and starch production. It contains a variety of nutrients, including protein, soluble sugars, amino acids, organic acids, minerals, and vitamins, and has the potential to be developed and utilized as a feed ingredient. Compared with general plant protein raw materials, corn steep liquor has a wider range of sources, lower cost, and a richer nutritional composition, making it valuable for applications in fermented and liquid feeds.

[0003] Untreated corn steep liquor still contains a certain proportion of large-molecule proteins and nitrogenous components that are not easily absorbed and utilized by animals, thus limiting its protein utilization efficiency. Furthermore, the wet processing of corn may introduce or leave behind a certain amount of sulfites, which, if used directly as feed ingredients, may affect the microbial fermentation process and animal safety. In addition, corn steep liquor contains both solids and soluble components, resulting in a high viscosity system that is prone to sedimentation, stratification, and localized aggregation. When used directly as feed ingredients, it may lead to insufficient protein hydrolysis, inadequate nutrient release, and issues with palatability and stability that require improvement.

[0004] Microbial fermentation can improve the nutritional characteristics of feed ingredients through cell growth, extracellular enzyme secretion, and metabolic transformation. Bacillus subtilis has a strong protease production capacity; during fermentation, it secretes proteases to degrade large protein molecules in the feed ingredients into smaller peptides and amino acids, thereby increasing the degree of protein hydrolysis. Candida utilis can utilize sugars and other nutrients in the fermentation substrate for growth and reproduction, forming cell proteins and participating in the transformation of nitrogenous components in the fermentation system, thus playing a role in improving the nutritional level of the fermentation products.

[0005] Some existing technologies use solid byproducts such as soybean meal, soybean residue, soybean hulls, corn hulls, corn germ meal, or corn starch residue as the main fermentation substrate to prepare peptide-rich feed or microecological fermented feed through solid-state fermentation with compound microorganisms. The fermentation targets of this type of technology are usually solid or semi-solid materials with adjusted moisture content. Corn steep liquor is mostly used as a nutrient supplement or auxiliary component for adjusting moisture content, rather than systematically addressing the problems inherent in liquid corn steep liquor, such as sulfite residue, high viscosity, uneven solid dispersion, and insufficient protein hydrolysis.

[0006] Liquid corn steep liquor contains soluble proteins, amino acids, organic acids, sugars, minerals, and vitamins, but its high viscosity and solid content make it prone to clumping, floating, settling, or uneven distribution when powdered inoculants are added directly. While simple resuspending of the inoculum powder with sterile water or saline can improve dispersion, the bacteria still need to readjust to the substrate environment after entering the high-viscosity corn steep liquor system containing organic acids and sulfite residues, leading to uneven fermentation start-up and reduced batch repeatability. Furthermore, simultaneous co-culture fermentation of *Candida utilis* and *Bacillus subtilis* in corn steep liquor may result in insufficient enzyme production or low small peptide accumulation efficiency in the early stages due to differences in aerobic characteristics, substrate utilization, and functional stages between the two microorganisms.

[0007] Therefore, the key technical issues to be addressed are how to reduce the impact of sulfites in corn steep liquor on microbial growth and feed safety without relying on solid-state fermentation carriers, improve the dispersion uniformity of solids and microorganisms in high-viscosity corn steep liquor, avoid the secondary adaptation lag caused by direct addition or simple resuspension of Bacillus subtilis powder into the corn steep liquor system, shorten the adaptation period of Bacillus subtilis after entering the corn steep liquor system, and achieve the controlled conversion of corn steep liquor protein into soluble protein, small peptides and amino acid nitrogen through segmented oxygen-controlled fermentation and post-ripening enzymatic hydrolysis, in order to improve the feed utilization value of corn steep liquor. Summary of the Invention

[0008] This invention addresses the problems of insufficient accumulation of easily absorbed nitrogenous components and difficulty in controlling the fermentation endpoint when corn steep liquor is used directly as a feed ingredient. It proposes a method and application for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homogenous matrix, desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation.

[0009] The technical solution adopted by the present invention to solve the above problems is as follows: The steps of the method of the present invention include: Step 1: Take liquid corn steep liquor, stir and mix it, add buffer desulfite treatment agent, and aerate it. Then, homogenize and reduce viscosity, and then sterilize it. After sterilization, cool it to the inoculation temperature to obtain pretreated sterilized corn steep liquor. Step 2: Take Bacillus subtilis powder and use sterilized corn steep liquor supernatant as the homologous matrix activation solution. Resuspend the powder at a ratio of 1g Bacillus subtilis powder to 10mL sterilized corn steep liquor supernatant. After initial dispersion by vortexing for 30s, activate the powder by low-speed shaking at 37℃ and 120rpm for 1h, so that the Bacillus subtilis powder changes from a dry powder aggregated state to a uniform suspension. Then, centrifuge the suspension at 6000r / min for 10min, discard the supernatant, and collect the wet Bacillus subtilis cells that have been adaptively activated by the corn steep liquor homologous matrix. Step 3: Inoculate the wet Bacillus subtilis cells obtained in Step 2 into the pretreated sterilized corn steep liquor obtained in Step 1. After thorough mixing, carry out the first stage of enzyme-producing hydrolysis fermentation under aerobic shaking conditions to obtain Bacillus subtilis pre-hydrolyzed corn steep liquor. Step 4: Inoculate the Bacillus subtilis pre-hydrolyzed corn steep liquor obtained in Step 3 with Candida utilis culture, and carry out the second stage of nutrient conversion fermentation under micro-aerobic low-speed shaking conditions to obtain segmented compound fermented corn steep liquor. Step 5: The segmented compound fermented corn liquor obtained in Step 4 is subjected to post-ripening enzymatic hydrolysis, followed by enzyme activation termination treatment to obtain protein hydrolyzed fermented corn liquor feed raw material.

[0010] Furthermore, in step 1, the buffer desulfite treatment agent is a compound of calcium carbonate and magnesium carbonate, and the amount of buffer desulfite treatment agent added is 0.2% to 1.5% of the mass of liquid corn steep liquor; The aeration time is 20-60 min, and the pH of the corn steep liquor system after treatment is 5.5-7.2; The homogenization and viscosity reduction treatment is a high-pressure homogenization treatment with a treatment pressure of 20~60Mpa and a treatment number of 1~3 times; The sterilization conditions are 100~121℃ for 10~30 minutes.

[0011] Furthermore, in step 2, the sterilized corn steep liquor supernatant is obtained by collecting the supernatant after centrifuging the liquid corn steep liquor at 6000 r / min for 10 min, sterilizing it at 121℃ for 15 min, and then cooling it. The conditions for discarding the supernatant after centrifugation are: centrifugation at 6000 r / min for 10 min; The ratio of Bacillus subtilis powder to sterilized corn steep liquor supernatant is 1g:10mL.

[0012] Furthermore, in step 3, the initial addition amount of Bacillus subtilis powder is 5% of the mass or volume of the liquid corn steep liquor; The first stage of enzyme production and hydrolysis fermentation is carried out at a temperature of 34~39℃ for 8~18 hours. The first stage of enzyme production and hydrolysis fermentation is carried out under the following conditions: 37°C and 150 rpm shaking fermentation.

[0013] Furthermore, in step 4, the amount of Candida utilis culture added is 5% to 15% of the volume of the liquid corn steep liquor; The second stage of compound fermentation is carried out at a temperature of 30~37℃ for 18~36 hours.

[0014] Furthermore, in step 5, the post-ripening enzymatic hydrolysis treatment involves heating the segmented compound fermented corn liquor to 45-55℃ and holding it at that temperature for 2-6 hours. The enzyme activity termination treatment involves treating the fermented corn steep liquor after post-ripening enzymatic hydrolysis at 75-85℃ for 10-20 minutes.

[0015] Furthermore, the specific steps also include: The protein hydrolyzed fermented corn syrup feed raw materials after enzyme activity termination treatment are vacuum packaged, low-temperature dried, spray dried, or drum dried.

[0016] Furthermore, the moisture content of the dried protein hydrolyzed fermented corn syrup feed raw material should not exceed 13%.

[0017] The protein hydrolyzed fermented corn steep liquor feed raw material prepared by the method of the present invention contains cell protein derived from Candida utilis, cell components derived from Bacillus subtilis, soluble protein, small peptides and amino acid nitrogen.

[0018] The protein hydrolyzed fermented corn steep liquor feed raw material described in this invention is used to prepare compound feed, fermented feed, liquid feed, concentrated feed or feed additive.

[0019] The beneficial effects of this invention are: 1. The method of the present invention involves buffering and desulfite treatment and homogenization to reduce viscosity of liquid corn steep liquor. Then, sterilized corn steep liquor supernatant is resuspended at a ratio of 1g of Bacillus subtilis powder to 10mL of sterilized corn steep liquor supernatant. After initial dispersion by vortexing for 30s, it is activated by low-speed shaking at 37℃ and 120rpm for 1h. After centrifugation at 6000r / min for 10min, the supernatant is discarded to obtain wet Bacillus subtilis cells. These cells are then inoculated into pretreated sterilized corn steep liquor for aerobic enzymatic hydrolysis fermentation. Subsequently, Candida utilis culture is inoculated for microaerobic low-speed nutrient conversion fermentation. After short-term after-ripening enzymatic hydrolysis and enzyme activity termination treatment, the release and controlled hydrolysis of proteins in the corn steep liquor are promoted, thereby increasing the content of soluble protein, small peptides and amino acid nitrogen in the fermentation products.

[0020] 2. The fermented corn syrup feed raw material of the present invention contains cell protein from Candida utilis, cell components from Bacillus subtilis, as well as soluble protein, small peptides and amino acid nitrogen produced by fermentation, and has a high degree of protein hydrolysis and good feed nutrient utilization potential.

[0021] 3. The residual sulfite content in the protein hydrolysis fermented corn steep liquor feed raw material of the present invention is lower than that of corn steep liquor without buffer desulfite treatment, the apparent viscosity is lower than that of corn steep liquor without homogenization and viscosity reduction treatment, and the soluble protein content, amino acid nitrogen content, protein hydrolysis degree, crude protein content and small peptide ratio are higher than those of the control group of uninoculated corn steep liquor or the control group of synchronous mixed fermented corn steep liquor; the protease activity in the fermentation supernatant before enzyme activity termination is higher than that of the control group of uninoculated corn steep liquor or the control group of synchronous mixed fermented corn steep liquor. Attached Figure Description

[0022] Figure 1 This is a process flow diagram of the present invention; Figure 2 This is a graph showing the changes in soluble protein content in fermented corn steep liquor under different treatment groups; Figure 3 This is a graph showing the changes in amino acid nitrogen content in fermented corn steep liquor under different treatment groups; Figure 4 This is a graph showing the changes in protease activity in fermented corn steep liquor under different treatment groups; Figure 5 This is a graph showing the changes in the degree of protein hydrolysis in fermented corn steep liquor under different treatment groups; Figure 6 This is a graph showing the crude protein content at the 48-hour endpoint of fermented corn steep liquor in different treatment groups. Figure 7 This is a graph showing the changes in the proportion of small peptides in fermented corn steep liquor under different treatment groups; Figure 8 This is a graph showing the effect of different pretreatment methods on the residual sulfite content and apparent viscosity of corn steep liquor.

[0023] Example The *Candida utilis* broth described in this invention can be a commercially available *Candida utilis* broth, and the strain is *Candida utilis*; the *Bacillus subtilis* powder can be a commercially available feed-grade *Bacillus subtilis* powder, and the strain is *Bacillus subtilis*. Both of the above strains are conventional fermentation strains that can be obtained commercially in this field.

[0024] Corn steep liquor (CSL) Candida utilis, CU; Bacillus subtilis, BS; Desulfitation (DS) Homogenization (H); Simultaneous fermentation of corn steep liquor by compound bacteria: CU-BS; Segmented compound microbial fermentation of corn steep liquor: BS→CU; Desulfite-homogenization and viscosity reduction-segmented compound bacteria fermentation of corn steep liquor: DS-H-BS→CU.

[0025] Example 1 The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homogenous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation, as described in this embodiment, includes the following specific steps: Step 1: Take 50 mL of corn steep liquor, stir and mix thoroughly, add calcium carbonate and magnesium carbonate compound, and perform micro-nano aeration for 40 min, followed by high-pressure homogenization at 40 MPa twice. Transfer the processed corn steep liquor to a sterile Erlenmeyer flask and sterilize it at 121°C for 15 min. After sterilization, the corn steep liquor that has been treated with desulfite and homogenization to reduce viscosity is cooled to approximately 37°C for later use. Step 2: Weigh 2.5g of Bacillus subtilis powder and place it in a sterile centrifuge tube; Take liquid corn steep liquor, centrifuge at 6000 r / min for 10 min, collect the supernatant, sterilize at 121℃ for 15 min, and cool it to use as sterilized corn steep liquor supernatant for later use; Add 25 mL of sterile corn steep liquor supernatant to 2.5 g of Bacillus subtilis powder, according to the ratio of 1 g of Bacillus subtilis powder to 10 mL of sterile corn steep liquor supernatant. First, vortex for 30 seconds to fully wet and initially disperse the bacterial powder; then place the bacterial suspension in a constant temperature shaker at 37°C and activate it by low-speed shaking at 120 rpm for 1 hour. After activation, the bacterial suspension was centrifuged at 6000 r / min for 10 min, the supernatant was discarded, and the wet Bacillus subtilis cells that had been adapted and activated by corn steep liquor homologous matrix were collected for later use. Step 3: Add the Bacillus subtilis wet cells obtained in Step 2 to 50 mL of pretreated, sterilized, and cooled corn steep liquor. The initial addition amount of Bacillus subtilis powder is 2.5 g, which is approximately 5% of the mass or volume of the corn steep liquor. After inoculation, mix thoroughly to ensure that the Bacillus subtilis wet cells are evenly distributed in the liquid corn steep liquor system. Then, carry out the first stage of enzyme-producing hydrolysis fermentation for 12 hours at 37℃ and 150 rpm shaking conditions to obtain Bacillus subtilis pre-hydrolyzed corn steep liquor. Step 4: Add 5.0 mL of Candida utilis culture to the Bacillus subtilis pre-hydrolyzed corn steep liquor obtained in Step 3. The amount added is 10% of the volume of corn steep liquor. After mixing thoroughly, place it in a constant temperature shaker at 37℃ and shake at a low speed of 80 rpm for the second stage of microaerobic nutrient conversion fermentation for 36 h. Step 5: After the second stage of fermentation is completed, the fermented corn syrup is heated to 45-55℃ and kept at this temperature for 2-6 hours for post-ripening enzymatic hydrolysis. Then, it is treated at 75-85℃ for 10-20 minutes to terminate enzyme activity, resulting in a segmented compound fermented corn syrup containing Candida utilis and Bacillus subtilis, which is a protein hydrolyzed fermented corn syrup feed ingredient.

[0026] Example 2: Preparation of fermented corn steep liquor from different treatment groups Step 1: Thoroughly mix the corn steep liquor to ensure even dispersion of solids, soluble proteins, sugars, and other nutrients. Measure 50 mL of the corn steep liquor and transfer it to an autoclaved fermentation flask. To avoid contamination or changes in system volume caused by sampling at different time points, this embodiment preferably uses independent fermentation bottles for grouping, that is, a separate fermentation bottle is set up for each fermentation time point; Step 2: Set up a blank control group, a single strain of Candida utilis, a single strain of Bacillus subtilis, a synchronous compound strain, a segmented compound strain, and a desulfite-homogenization viscosity reduction-segmented compound strain for evaluating fermentation effect; and set up an untreated group, a desulfite-treated group, a homogenization viscosity reduction group, and a desulfite-homogenization viscosity reduction group for evaluating pretreatment effect. The blank control group was designated as the CK group, which contained only 50 mL of sterile corn steep liquor and was not inoculated with any bacterial strains. The single-strain group of Candida utilis was designated as group CU. 5.0 mL of Candida utilis culture was added to 50 mL of sterile corn steep liquor. The Bacillus subtilis single-strain group was designated as the BS group. 2.5g of Bacillus subtilis powder that had been resuspended was added to 50mL of sterilized corn steep liquor. The synchronous compound bacterial group was designated as the CU-BS group. 5.0 mL of Candida utilis culture and 2.5 g of Bacillus subtilis powder that had been resuspended were added simultaneously to 50 mL of sterilized corn steep liquor. The segmented compound bacterial group was designated as BS→CU group. First, Bacillus subtilis wet cells were added to 50 mL of sterilized corn steep liquor. After quantitative resuspension of the supernatant of sterilized corn steep liquor according to step 2, activation was carried out at 37℃ and 120 rpm for 1 h, and centrifugation was performed to discard the supernatant. After the first stage of enzyme-producing hydrolytic fermentation, 5.0 mL of Candida utilis culture was added to carry out the second stage of nutrient conversion fermentation. The desulfite-homogenization and viscosity reduction-segmented compound bacterial group is designated as DS-H-BS→CU group. First, the corn steep liquor is subjected to buffer desulfite treatment and homogenization and viscosity reduction treatment. Then, compound fermentation is carried out according to the Bacillus subtilis corn steep liquor homologous matrix adaptive activation method and BS→CU segmented fermentation method described in step 2. After the fermentation is completed, post-ripening enzymatic hydrolysis and enzyme activation termination treatment are performed. Step 3: After each group of samples is inoculated, mix them thoroughly and place them in a 37℃ constant temperature incubator or a constant temperature shaker for fermentation; Samples were taken from each treatment group at 0h, 12h, 24h, 36h and 48h. Before sampling, the fermented corn steep liquor was thoroughly mixed. The undiluted original sample was used to determine the residual sulfite content and apparent viscosity. The whole sample was dried or digested and used to determine the crude protein content. Another part of the sample was diluted, shaken and centrifuged, and the supernatant was used to determine the soluble protein content, amino acid nitrogen content, protease activity before enzyme activity termination, degree of protein hydrolysis and small peptide ratio.

[0027] In this embodiment, the CK group was used to evaluate the changes in corn steep liquor itself during the cultivation process; the CU group was used to evaluate the effect of Candida utilis fermentation alone on the nitrogenous components of corn steep liquor; the BS group was used to evaluate the effect of Bacillus subtilis fermentation alone on the hydrolysis of corn steep liquor proteins; the CU-BS group was used to evaluate the comprehensive effect of simultaneous co-fermentation of two strains; the BS→CU group was used to evaluate the effect of staged fermentation with Bacillus subtilis fermentation followed by Candida utilis inoculation; and the DS-H-BS→CU group was used to evaluate the synergistic effects of desulfite removal, homogenization and viscosity reduction, staged oxygen-controlled fermentation, and post-ripening enzymatic hydrolysis. By comparing the changes in various indicators of the above treatment groups under the same fermentation time, it can be determined whether the process of the present invention is more conducive to the conversion of corn steep liquor proteins into soluble proteins, small peptides, and amino acid nitrogen than single-strain fermentation or simultaneous mixed-strain fermentation.

[0028] Comparative Example 1: Uninoculated corn steep liquor control group Take 50 mL of corn steep liquor, mix thoroughly, sterilize at 121°C for 15-20 min, cool to 37°C, and treat without adding any bacterial strains for 0-48 h under the same conditions as in Example 1 to obtain an uninoculated corn steep liquor control sample. This comparative example is used to eliminate the influence of the corn steep liquor itself on protein hydrolysis indicators during heating, storage, or incubation.

[0029] Comparative Example 2: Fermentation of corn steep liquor by a single strain of Candida utilis Step 1: Take 50 mL of corn steep liquor, mix thoroughly, sterilize at 121℃ for 15-20 min, cool to 37℃, add 5.0 mL of Candida utilis culture, mix well, and ferment at 37℃ for 0-48 h to obtain a single-strain fermented corn steep liquor sample of Candida utilis.

[0030] This comparative study was used to evaluate the effects of Candida utilis fermentation alone on the content of soluble protein, small peptides, amino acid nitrogen, and crude protein in corn steep liquor.

[0031] Comparative Example 3: Bacillus subtilis single-strain fermentation of corn steep liquor Take 50 mL of corn steep liquor, mix thoroughly, and sterilize at 121℃ for 15-20 min. After sterilization, cool to approximately 37℃. Weigh 2.5 g of Bacillus subtilis powder and resuspend it in a small amount of sterile physiological saline to obtain a Bacillus subtilis suspension or slurry. Add the Bacillus subtilis suspension or slurry to the sterilized corn steep liquor, mix thoroughly, and ferment at 37℃ for 0-48 h to obtain a single-strain fermented corn steep liquor sample of Bacillus subtilis, referred to as the BS group sample. This comparative example is used to evaluate the effects of Bacillus subtilis fermentation alone on corn steep liquor protease activity, degree of protein hydrolysis, small peptide formation, and amino acid nitrogen accumulation.

[0032] Comparative Example 4: Simultaneous Fermentation of Corn Stem Cells with Compound Microorganisms Take 50 mL of corn steep liquor, mix thoroughly, and sterilize at 121℃ for 15-20 min. After cooling to 37℃, simultaneously add 5.0 mL of Candida utilis culture and 2.5 g of Bacillus subtilis powder that has been resuspended. Mix thoroughly and ferment at 37℃ and 150 rpm for 0-48 h to obtain a corn steep liquor sample fermented by a concurrent compound culture, referred to as the CU-BS group sample. This comparative example is used to evaluate the technical effect of the segmented inoculation method compared to the concurrent inoculation method.

[0033] Comparative Example 5: Fermented corn steep liquor without desulfite treatment and homogenization for viscosity reduction Take 50 mL of corn steep liquor, without buffering desulfite treatment or homogenization and viscosity reduction treatment, and sterilize only. Then, perform segmented co-fermentation using Bacillus subtilis first, followed by Candida utilis inoculation, to obtain an untreated segmented co-fermented corn steep liquor sample. This comparative example is used to evaluate the effects of desulfite treatment and homogenization and viscosity reduction pretreatment on cell dispersion, apparent viscosity, degree of protein hydrolysis, and small peptide formation.

[0034] Effect Experiment: 1. Pretreatment of fermentation samples Take the fermented corn steep liquor samples obtained in the examples and comparative examples, mix them thoroughly, and then take the samples. Since the corn steep liquor system has a certain viscosity and contains solids, it is necessary to shake or stir thoroughly before sampling to ensure the system is homogeneous. Dilute the samples with distilled water or phosphate buffer at a certain ratio, and shake thoroughly for 5-10 minutes to fully release the soluble components in the fermentation broth.

[0035] The diluted sample was then centrifuged at 8000 rpm for 10-15 minutes. The supernatant was collected after centrifugation to determine soluble protein content, amino acid nitrogen content, protease activity, small peptide ratio, and degree of protein hydrolysis. A separate, uncentrifuged fermentation sample was dried or directly digested to determine total nitrogen and crude protein content. For evaluation of the pretreatment effect, samples before and after fermentation were used to determine residual sulfite content and apparent viscosity.

[0036] 2. Determination of residual sulfite content and apparent viscosity The residual sulfite content can be determined by iodometric titration, distillation titration, or a commercial sulfite detection kit, and the result is expressed as mgSO2 / L. Apparent viscosity can be determined using a rotational rheometer or rotational viscometer at a temperature of 25℃ and a shear rate of 50 s⁻¹. - ¹, the results are expressed in mPa·s.

[0037] like Figure 8As shown, the residual sulfite content in untreated corn steep liquor was high, approximately 819.33 mg SO2 / L; after desulfite treatment alone, the residual sulfite content decreased to approximately 269.67 mg SO2 / L; homogenization treatment alone had little effect on the residual sulfite content; the residual sulfite content was further reduced after desulfite treatment combined with homogenization; the residual sulfite content in the DS-H-BS→CU group decreased to approximately 104.33 mg SO2 / L. These results indicate that buffered desulfite treatment can significantly reduce the residual sulfite content in corn steep liquor, and the combined fermentation process can further promote the decrease in sulfite levels in the system.

[0038] like Figure 8 As shown, the apparent viscosity of untreated corn steep liquor was approximately 994.67 mPa·s. The apparent viscosity did not change significantly after desulfite treatment alone, but it decreased significantly to approximately 454.00 mPa·s after homogenization alone. The apparent viscosity decreased to approximately 405.67 mPa·s after desulfite treatment combined with homogenization, and further decreased to approximately 291.33 mPa·s in the DS-H-BS→CU group. These results indicate that homogenization and viscosity reduction treatment can effectively improve the flowability and dispersion of liquid corn steep liquor, providing more favorable system conditions for uniform cell distribution and subsequent protein hydrolysis.

[0039] 3. Determination of soluble protein content The Bradford method was used to determine the soluble protein content in the supernatant of each group of fermentation samples. A standard curve was plotted using bovine serum albumin as a standard. An appropriate amount of fermentation supernatant was mixed with Bradford's colorimetric reagent, reacted at room temperature, and the absorbance was measured at 595 nm. The soluble protein content in the samples was calculated based on the standard curve, and the results are expressed in mg / mL.

[0040] like Figure 2 As shown, the soluble protein content in the CK group at 0h, 12h, 24h, 36h, and 48h was approximately 8.15 mg / mL, 7.85 mg / mL, 7.69 mg / mL, 7.38 mg / mL, and 7.05 mg / mL, respectively, showing a slight decreasing trend overall. This indicates that uninoculated corn steep liquor does not produce significant protein release or hydrolysis during cultivation. The soluble protein content in the CU group at the same time points was approximately 8.24 mg / mL, 8.76 mg / mL, 9.41 mg / mL, 9.71 mg / mL, and 9.57 mg / mL, respectively, indicating that Candida utilis fermentation can slightly increase the soluble nitrogenous components in the system, but the increase is limited.

[0041] The soluble protein content in the BS group at 0h, 12h, 24h, 36h, and 48h was approximately 8.67 mg / mL, 10.11 mg / mL, 12.37 mg / mL, 13.89 mg / mL, and 14.04 mg / mL, respectively, significantly higher than that in the CK and CU groups. This indicates that Bacillus subtilis can produce proteases during fermentation, promoting the release and initial hydrolysis of proteins in corn steep liquor. The soluble protein content in the CU-BS group at the same time points was approximately 8.89 mg / mL, 11.23 mg / mL, 14.46 mg / mL, 16.31 mg / mL, and 17.20 mg / mL, respectively, higher than that of the single-strain fermentation group. The soluble protein content in the BS→CU group at 48h was approximately 19.87 mg / mL, higher than that of the simultaneous co-fermentation group. The soluble protein content in the DS-H-BS→CU group at 48h was approximately 23.24 mg / mL, the highest among all groups. Therefore, it can be seen that the combination of desulfite treatment, homogenization and viscosity reduction, pre-enzyme production by Bacillus subtilis and subsequent transformation by Candida utilis can further increase the soluble protein content in the corn steep liquor system, which is beneficial to improving the utilization of corn steep liquor protein.

[0042] 4. Determination of amino acid nitrogen content The amino acid nitrogen content in fermentation samples was determined using the ninhydrin colorimetric method or an amino acid nitrogen kit. An appropriate amount of fermentation supernatant was reacted with the colorimetric reagent, and the absorbance was measured at the corresponding wavelength. The amino acid nitrogen content was calculated based on the standard curve, and the results were expressed as mg / 100 mL.

[0043] like Figure 3 As shown, the amino acid nitrogen content in the CK group remained relatively stable and decreased slightly during fermentation, reaching approximately 107.80 mg / 100 mL at 48 h. The amino acid nitrogen content in the CU group was approximately 168.03 mg / 100 mL at 48 h, indicating that *Candida utilis* fermentation can promote the accumulation of soluble nitrogenous substances to a certain extent, but its direct proteolytic ability is limited. The amino acid nitrogen content in the BS group was approximately 265.00 mg / 100 mL at 48 h, showing a significant upward trend, indicating that *Bacillus subtilis* can promote further decomposition of corn steep liquor protein through protease secretion, generating more free amino acids and small-molecule nitrogenous components. The amino acid nitrogen content in the CU-BS group, BS→CU group, and DS-H-BS→CU group increased sequentially at 48 h, reaching approximately 331.83 mg / 100 mL, 391.90 mg / 100 mL, and 455.77 mg / 100 mL, respectively. Therefore, the synergistic process of segmented compound fermentation and pretreatment can more effectively promote the conversion of corn steep liquor protein into amino acid nitrogen and increase the content of easily absorbed nitrogen-containing components in the fermentation products.

[0044] 5. Determination of protease activity The protease activity in fermented samples was determined using the casein-Folin method. The fermentation supernatant was used as the crude enzyme solution, and casein was used as the substrate. The reaction was carried out at 37°C for a certain time, followed by termination of the reaction with trichloroacetic acid. After centrifugation, the supernatant was collected and reacted with sodium carbonate solution and Folin phenol reagent for color development. The absorbance was measured near 680 nm. A standard curve was plotted using tyrosine as a standard, and the protease activity was calculated. One enzyme activity unit (U) was defined as the amount of enzyme required to release 1 μg of tyrosine per minute under specific reaction conditions. Results were expressed as U / mL.

[0045] like Figure 4 As shown, the protease activity in the CK group remained at a low level throughout, indicating that the uninoculated corn steep liquor itself did not possess significant protease activity. The protease activity in the CU group increased slightly with fermentation, but remained low overall. The protease activity in the BS group increased significantly with fermentation time, indicating that *Bacillus subtilis* was the main contributor to the increased protease activity in the fermentation system. The protease activity in the CU-BS group was higher than that in the single-strain fermentation group, indicating that the combined fermentation of the two strains could further improve the protease activity of the system. The BS→CU group preferentially inoculated with *Bacillus subtilis* in the first stage, which was beneficial for its full enzyme production under aerobic or shaking conditions; therefore, its protease activity was higher than that of the simultaneous combined fermentation group. The protease activity in the DS-H-BS→CU group was approximately 301.70 U / mL at 48 h, the highest among all treatment groups. Therefore, desulfite treatment and homogenization to reduce viscosity improved the fermentation substrate environment, and segmented aerobic fermentation enhanced the enzyme production of *Bacillus subtilis*, providing a more sufficient enzymatic basis for the hydrolysis of corn steep liquor proteins.

[0046] 6. Determination of the degree of protein hydrolysis The degree of protein hydrolysis in fermented corn steep liquor was evaluated using the TCA soluble nitrogen method. The specific steps are as follows: First, corn steep liquor samples from different fermentation time points were taken, thoroughly mixed, and accurately pipetted 10 mL. 40 mL of distilled water was added for dilution, and the mixture was shaken and mixed at room temperature for 10 min to fully release soluble proteins, small peptides, and free amino acids. The samples were then centrifuged at 9000 rpm for 10 min, and the supernatant was collected for later use.

[0047] Take 5 mL of the supernatant and add an equal volume of 10% trichloroacetic acid (TCA) solution. Mix thoroughly and let stand at room temperature for 30 min. Trichloroacetic acid can precipitate large molecular weight proteins and some high molecular weight peptides that have not been fully hydrolyzed, while small molecular weight peptides, free amino acids, and other soluble nitrogenous substances remain in the supernatant. After standing, centrifuge the sample again at 10000 r / min for 10 min, and use the supernatant to determine the soluble nitrogen content of TCA. At the same time, the total nitrogen content of the fermentation sample is determined using the Kjeldahl method.

[0048] The degree of protein hydrolysis (DH) is calculated using the following formula:

[0049] TCA soluble nitrogen refers to the soluble nitrogenous substances contained in the supernatant after trichloroacetic acid treatment, while total nitrogen refers to the total nitrogen content in the fermentation sample.

[0050] like Figure 5 As shown, the degree of protein hydrolysis in the CK group remained at a low level, approximately 8.98% at 48 h. The degree of protein hydrolysis in the CU group increased slightly, reaching approximately 16.20% at 48 h, indicating that *Candida utilis* had a certain promoting effect on the hydrolysis of corn steep liquor protein, but the degree of hydrolysis was limited. The degree of protein hydrolysis in the BS group increased significantly, reaching approximately 29.32% at 48 h, indicating that *Bacillus subtilis* could effectively promote the conversion of corn steep liquor protein into TCA-soluble nitrogenous components. The degrees of protein hydrolysis at 48 h were approximately 35.63%, 39.91%, and 44.48% for the CU-BS, BS→CU, and DS-H-BS→CU groups, respectively. Therefore, segmented oxygen-controlled fermentation and post-ripening enzymatic hydrolysis can further enhance the degradation effect of corn steep liquor protein, converting more large protein molecules into small peptides and free amino acids, and other soluble nitrogenous substances.

[0051] 7. Determination of crude protein content The total nitrogen content of fermented corn steep liquor samples was determined using the Kjeldahl method, and the crude protein content was calculated based on the total nitrogen content. The crude protein content was calculated using the following formula:

[0052] Results are expressed as a percentage on a dry basis.

[0053] like Figure 6 As shown, after 48 hours of fermentation, there were certain differences in the crude protein content among the different treatment groups. The crude protein content of the CK group was approximately 41.68%, indicating that the uninoculated treatment could not improve the total nitrogen content of the corn steep liquor. The crude protein contents of the CU and BS groups were approximately 46.46% and 46.38%, respectively. This increase may be related to the accumulation of microbial protein, nitrogenous substances introduced by the microbial powder itself, and the transformation of protein components during fermentation. The crude protein content of the CU-BS group was approximately 48.67%, higher than that of the single-strain fermentation group. The crude protein contents of the BS→CU group and the DS-H-BS→CU group were approximately 49.62% and 50.99%, respectively, indicating that the staged compound fermentation and its combination with the pretreatment process can further improve the total nitrogen content of the fermented corn steep liquor.

[0054] It should be noted that the crude protein content is calculated from total nitrogen, and this indicator cannot represent the degree of protein hydrolysis alone. Therefore, when evaluating the fermentation products of this invention, the improvement effect of the compound microbial fermentation on the utilization of corn steep liquor protein should be judged by combining soluble protein, amino acid nitrogen, degree of protein hydrolysis, and small peptide ratio. Furthermore, the improvement effect of the pretreatment and segmented oxygen control process on the stability of industrial applications should be judged by combining residual sulfite content, apparent viscosity, and batch stability.

[0055] 8. Determination of the proportion of small peptides The proportion of small peptides in fermented samples was evaluated using a trichloroacetic acid (TCA) precipitation method combined with a soluble peptide content determination method. The specific steps are as follows: First, corn steep liquor samples treated at different fermentation time points or endpoints were taken, thoroughly shaken and mixed, and then centrifuged at 8000-10000 rpm for 10-15 min. The supernatant was collected as the sample to be tested. A certain volume of the fermentation supernatant was taken, and 10% trichloroacetic acid solution was added at a 1:1 volume ratio. After thorough mixing, the mixture was allowed to stand at room temperature for 30 min to allow large molecular weight proteins and unhydrolyzed high molecular weight peptides in the system to precipitate. Subsequently, the mixture was centrifuged again at 8000-10000 rpm for 10 min. The supernatant after centrifugation was used to determine the TCA-soluble peptide content.

[0056] The components retained in the supernatant after TCA treatment are mainly small peptides with low molecular weight and some free amino acids. Therefore, it can be used to reflect the degree of protein conversion into small peptides during fermentation. The soluble peptide content in the supernatant can be determined using the biuret method, OPA method, or commercial small peptide detection kits, and the peptide content data can be obtained by converting the data using a standard curve. The total amount of soluble protein and peptides in the fermentation sample is also determined. The proportion of small peptides is calculated using the following formula:

[0057] like Figure 7 As shown, the proportion of small peptides in different treatment groups exhibited different patterns of change with fermentation time. The proportion of small peptides in the CK group remained at a low level overall and decreased slightly, indicating that uninoculated corn steep liquor did not significantly accumulate small peptides. The proportion of small peptides in the CU group gradually increased from 0 to 36 hours, reaching approximately 18.73% at 36 hours, and decreased slightly at 48 hours, indicating that *Candida utilis* could promote a certain degree of small peptide production, but some small peptides may have been further utilized by microorganisms in the later stages.

[0058] The proportion of small peptides in the BS group was significantly higher than that in the CU group, reaching approximately 36.56% at 36 hours, indicating that the protease produced by Bacillus subtilis effectively promoted the conversion of corn steep liquor protein into small peptides. The proportion of small peptides in the CU-BS group reached approximately 48.67% at 36 hours, higher than the single-strain fermentation group. The proportion of small peptides in the BS→CU group was approximately 54.58% at 36 hours, higher than the simultaneous co-fermentation group, indicating that the pre-fermentation hydrolysis by Bacillus subtilis improved the efficiency of small peptide accumulation. The DS-H-BS→CU group had the highest proportion of small peptides, reaching approximately 61.82% at 36 hours, slightly decreasing to approximately 56.71% at 48 hours. This trend aligns with the phased changes in protein hydrolysis during actual fermentation, namely, rapid hydrolysis of macromolecular proteins to form small peptides in the early and middle stages of fermentation, and further decomposition of some small peptides into free amino acids or utilization by microorganisms in the later stages of fermentation. Therefore, the proportion of small peptides peaked at 36h, while the content of amino acid nitrogen continued to increase at 48h. Both of these factors indicate that the process of this invention can promote the gradual conversion of corn steep liquor protein from macromolecular protein to small peptides and amino acid nitrogen.

[0059] Working principle The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing hydrolyzed corn steep liquor feed ingredients based on Bacillus subtilis-based corn steep liquor homologous matrix adaptive activation, desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation, characterized in that... The specific steps include: Step 1: Take liquid corn steep liquor, stir and mix it, add buffer desulfite treatment agent, and aerate it. Then, homogenize and reduce viscosity, and then sterilize it. After sterilization, cool it to the inoculation temperature to obtain pretreated sterilized corn steep liquor. Step 2: Take Bacillus subtilis powder and use sterilized corn steep liquor supernatant as the homologous matrix activation solution. Resuspend the powder at a ratio of 1g Bacillus subtilis powder to 10mL sterilized corn steep liquor supernatant. After initial dispersion by vortexing for 30s, activate the powder by low-speed shaking at 37℃ and 120rpm for 1h, so that the Bacillus subtilis powder changes from a dry powder aggregated state to a uniform suspension. Then, centrifuge the suspension at 6000r / min for 10min, discard the supernatant, and collect the wet Bacillus subtilis cells that have been adaptively activated by the corn steep liquor homologous matrix. Step 3: Inoculate the wet Bacillus subtilis cells obtained in Step 2 into the pretreated sterilized corn steep liquor obtained in Step 1. After thorough mixing, carry out the first stage of enzyme-producing hydrolysis fermentation under aerobic shaking conditions to obtain Bacillus subtilis pre-hydrolyzed corn steep liquor. Step 4: Inoculate the Bacillus subtilis pre-hydrolyzed corn steep liquor obtained in Step 3 with Candida utilis culture, and carry out the second stage of nutrient conversion fermentation under micro-aerobic low-speed shaking conditions to obtain segmented compound fermented corn steep liquor. Step 5: The segmented compound fermented corn liquor obtained in Step 4 is subjected to post-ripening enzymatic hydrolysis, followed by enzyme activation termination treatment to obtain protein hydrolyzed fermented corn liquor feed raw material.

2. The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homologous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation according to claim 1, characterized in that, In step 1, the buffer desulfite treatment agent is a compound of calcium carbonate and magnesium carbonate, and the amount of buffer desulfite treatment agent added is 0.2% to 1.5% of the mass of liquid corn steep liquor. The aeration time is 20-60 min, and the pH of the corn steep liquor system after treatment is 5.5-7.

2. The homogenization and viscosity reduction treatment is a high-pressure homogenization treatment with a treatment pressure of 20~60Mpa and a treatment number of 1~3 times; The sterilization conditions are 100~121℃ for 10~30 minutes.

3. The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homologous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation according to claim 1, characterized in that, In step 2, the sterilized corn steep liquor supernatant is obtained by centrifuging liquid corn steep liquor at 6000 r / min for 10 min, collecting the supernatant, sterilizing it at 121 ℃ for 15 min, and then cooling it. The conditions for discarding the supernatant after centrifugation are: centrifugation at 6000 r / min for 10 min; The ratio of Bacillus subtilis powder to sterilized corn steep liquor supernatant is 1g:10mL.

4. The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homologous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation according to claim 1, characterized in that, In step 3, the initial addition amount of Bacillus subtilis powder is 5% of the mass or volume of the liquid corn steep liquor. The first stage of enzyme production and hydrolysis fermentation is carried out at a temperature of 34~39 ℃ for 8~18 h. The first stage of enzyme production and hydrolysis fermentation was carried out under the following conditions: 37 ℃ and 150 rpm shaking fermentation.

5. The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homologous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation, as described in claim 1, is characterized in that... In step 4, the amount of Candida utilis culture added is 5% to 15% of the volume of liquid corn steep liquor; The second stage of compound fermentation is carried out at a temperature of 30~37 ℃ for 18~36 h.

6. The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homologous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation, as described in claim 1, is characterized in that... In step 5, the post-ripening enzymatic hydrolysis treatment involves heating the segmented compound fermented corn liquor to 45-55 ℃ and holding it at that temperature for 2-6 hours. The enzyme activity termination treatment involves treating the fermented corn steep liquor after post-ripening enzymatic hydrolysis at 75-85℃ for 10-20 minutes.

7. The method for preparing hydrolyzed corn steep liquor feed ingredients based on the adaptive activation of Bacillus subtilis corn steep liquor homologous matrix through desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation, as described in claim 1, is characterized in that... The specific steps also include: The protein hydrolyzed fermented corn syrup feed raw materials after enzyme activity termination treatment are vacuum packaged, low-temperature dried, spray dried, or drum dried.

8. The method for preparing hydrolyzed corn steep liquor feed ingredients based on Bacillus subtilis-based corn steep liquor homologous matrix adaptive activation, desulfite homogenization, viscosity reduction, segmented oxygen-controlled compound fermentation, as described in claim 7, is characterized in that... The moisture content of the dried protein hydrolyzed fermented corn steep liquor feed raw material should not exceed 13%.

9. A protein hydrolyzed fermented corn steep liquor feed ingredient prepared by the method of any one of claims 1 to 8, characterized in that, The protein hydrolyzed fermented corn syrup feed ingredient contains cell protein derived from Candida utilis, cell components derived from Bacillus subtilis, soluble protein, small peptides, and amino acid nitrogen.

10. The application of the protein hydrolyzed fermented corn steep liquor feed ingredient according to claim 9, characterized in that, The protein hydrolyzed fermented corn syrup feed raw material is used to prepare compound feed, fermented feed, liquid feed, concentrated feed or feed additive.

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