Complex preparation based on casein and rice protein enzymatic hydrolysate and its preparation and application

Abstract

The application particularly relates to a compound preparation based on casein and rice protein enzymolysis products and preparation and application thereof. The application provides a compound preparation based on casein and rice protein enzymolysis products, which is prepared by compounding casein enzymolysis products (C-T) and rice protein enzymolysis products (R-AT) at a ratio of 1:3-3:1. Both of them have a characteristic C-terminal nA-P-A' structure sequence (hydrophobic amino acids are enriched at the C-terminal of casein peptides, and positively charged amino acids are enriched at the C-terminal of rice protein peptides), and can significantly play intestinal cell protection activity and in-vivo intestinal barrier protection function by synergistically regulating Nrf2 antioxidant and NF-kappa B anti-inflammatory pathways.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biotechnology and food nutrition, specifically relating to compound preparations based on casein and rice protein hydrolysates, and their preparation and application. Background Technology

[0002] Impaired intestinal barrier function is a core pathological element in digestive system diseases such as inflammatory bowel disease (IBD), short bowel syndrome, and infectious enteritis. It manifests as multiple barrier dysfunctions, including mucosal structure destruction, loss of tight junction proteins, mucus layer degradation, and immune cell infiltration. The integrity of the intestinal barrier is fundamental to maintaining nutrient absorption, immune homeostasis, and microbial balance. Its damage can lead to decreased digestive and absorptive efficiency, loss of endogenous proteins, and systemic inflammatory responses, significantly impacting patients' nutritional status, quality of life, and clinical prognosis. The etiology of this damage is complex, involving multiple factors such as infection, drugs, immune abnormalities, and nutritional deficiencies. Its core pathological features include downregulation of tight junction protein expression (such as ZO-1, occludin, and claudin family), reduced mucus layer thickness, and impaired goblet cell function. These changes lead to increased intestinal epithelial permeability, macromolecular antigen translocation, and immune activation, further exacerbating intestinal inflammation and protein loss [Food Res Int. 2021 Sep;147:110485.]. Therefore, repairing the intestinal barrier structure and restoring its function is a key intervention strategy for these diseases.

[0003] Currently, nutritional interventions for intestinal barrier damage mainly include enteral nutrition support, specific nutrient supplementation, and the application of medical foods. Among them, enteral nutrition is a commonly used clinical method. Whole protein formulas (such as Ensure) are highly antigenic and require complete digestive function, which can easily increase the burden on the intestines [World J Gastrointest Surg. 2016 Oct 27;8(10):700-705.]; Amino acid formulas have high absorption efficiency, but their osmotic pressure is too high and they lack peptide-mediated mucosal repair stimulation, making it difficult to promote the recovery of barrier function [Expert Consensus on Nutritional Therapy for Inflammatory Bowel Disease (Third Edition) [J]. Gastroenterology, 2025,30(3):152-172]. Short peptide formulas (such as Peptone) overcome the above shortcomings to some extent. They can be directly absorbed through the peptide transporter PepT1, reducing the digestive burden. However, their protein source is singular (mainly based on whey protein), ignoring the physiological characteristics of East Asian populations with strong adaptability to plant proteins, and existing products lack precise design for active barrier repair function. Peptides have become a research hotspot in the field of intestinal barrier repair due to their small molecular weight, high bioavailability, low antigenicity, and various biological activities (such as anti-inflammatory, antioxidant, tight junction regulation, and mucus secretion promotion) [Food Res Int. 2021 Sep;147:110485.]. Currently, some studies have reported food-derived peptides with intestinal barrier protection functions, such as NPWDQ derived from casein (which can upregulate occludin expression) [Br J Nutr. 2010 Oct;104(7):951-6.], the tripeptide VPY from soybean protein hydrolysate (which can inhibit the TNF-α-mediated inflammatory pathway) [Biochim Biophys Acta. 2012 Nov;1820(11):1753-63.], and sulfur-containing oligopeptides derived from milk protein (which can enhance the expression of mucin MUC2). However, research on intestinal barrier protective peptides still faces challenges such as limited sources, limited activity, and poor species adaptability. In addition, existing peptide research is mostly focused on single protein sources and lacks synergistic design of animal and plant dual protein peptides, which cannot simultaneously meet the multiple barrier maintenance needs such as rapid absorption, mucosal repair, immune regulation, and microbial homeostasis.

[0004] Therefore, developing dual-source peptide combinations that are based on the nutritional and metabolic characteristics of East Asian populations and have both high absorption and barrier repair functions has significant clinical value and industrialization prospects. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a compound preparation based on casein and rice protein hydrolysate, and its preparation and application, wherein the above-mentioned hydrolysate has an intestinal barrier protection effect when used alone or in combination.

[0006] This invention provides a casein hydrolysate in which the short peptide has an nA-P-A' characteristic sequence at its C-terminus, wherein n ranges from 1 to 6, the A position is any amino acid, and the A' position is enriched with hydrophobic amino acids, including Pro, Leu, or Val.

[0007] The present invention also provides a method for preparing the above-mentioned casein hydrolysate, wherein the casein hydrolysate is prepared by a single-step enzymatic hydrolysis, and the single-step enzymatic hydrolysis is trypsin hydrolysis.

[0008] The present invention also provides a method for preparing the above-mentioned casein hydrolysate, wherein the casein hydrolysate is prepared by two-step enzymatic hydrolysis. The first step of the two-step enzymatic hydrolysis includes one of flavor protease, neutral protease, alkaline protease and papain hydrolysis, and the second step is trypsin hydrolysis.

[0009] The present invention also provides a rice protein hydrolysate, wherein the C-terminus of the short peptide in the rice protein hydrolysate has an nB-P-B' characteristic sequence, wherein n ranges from 1 to 6, the B position is any amino acid, and the B' position is enriched with positively charged amino acids, including Arg or His.

[0010] The present invention also provides a method for preparing the above-mentioned rice protein hydrolysate, wherein the rice protein hydrolysate is prepared by a single-step enzymatic hydrolysis, and the single-step enzymatic hydrolysis is alkaline protease hydrolysis.

[0011] The present invention also provides a method for preparing the above-mentioned rice protein hydrolysate. The rice protein hydrolysate is prepared by two-step enzymatic hydrolysis. The first step of the two-step enzymatic hydrolysis includes one of alkaline protease, trypsin, and neutral protease hydrolysis, and the second step includes one of trypsin, flavor protease, alkaline protease, and trypsin hydrolysis.

[0012] The present invention also provides a compound preparation based on casein and rice protein hydrolysate, the compound preparation comprising the above-mentioned casein hydrolysate and rice protein hydrolysate.

[0013] Furthermore, casein and rice protein hydrolysate showed a synergistic effect in evaluating intestinal cell protective activity, with the protein ratio of the mixture being 1:3 to 3:1.

[0014] Furthermore, the theoretical shelf life of the compound preparation stored at 25°C under vacuum packaging is not less than 170 days.

[0015] The present invention also provides the application of the above-mentioned casein hydrolysate, rice hydrolysate and compound preparation in the preparation of medicines for preventing or repairing damage to intestinal barrier function.

[0016] Furthermore, a medicine for preventing or repairing damage to the intestinal barrier function comprises a combination of casein hydrolysate, rice hydrolysate, or a compound preparation with at least one active ingredient that improves intestinal barrier function, and pharmacologically acceptable excipients, wherein pharmacologically acceptable excipients include at least one of fillers, stabilizers, and flavoring agents.

[0017] Furthermore, casein hydrolysate, rice hydrolysate, and compound preparations can restore body weight, maintain intestinal villi length, and improve nutritional health.

[0018] Furthermore, casein hydrolysate, rice hydrolysate, or compound preparations can exert their effects by inhibiting oxidative stress and inflammatory responses, specifically by binding to Keap1 and IKK-β proteins and regulating the Nrf2 and NF-κB signaling pathways to enhance the therapeutic effect on intestinal barrier damage.

[0019] The present invention also provides the application of the above-mentioned casein hydrolysate, rice hydrolysate and compound preparation in the preparation of health foods that aid digestion.

[0020] The technical solution of this invention has the following advantages: 1. Intestinal cell protection function: Both casein hydrolysate and rice hydrolysate have excellent intestinal cell protection effects; 2. High efficiency and synergistic effect: The dual-protein compound formulation showed superior protective effects compared to single enzyme hydrolysates in cell and animal models. For example, cell survival rate was increased by 13.67% (vs. casein hydrolysate) and 6.54% (vs. rice hydrolysate) compared to single enzyme hydrolysates, and it was able to effectively maintain body weight and intestinal integrity in in vivo models.

[0021] 3. Well-defined structure and strong targeting: The C-terminal nA-P-A' sequence characteristics of the enzymatic hydrolysate peptide ensure high affinity binding of the peptide to Keap1 and IKK-β, with a clear mechanism and well-defined structure-activity relationship.

[0022] 4. High industrial feasibility: The preparation process is simple and the conditions are controllable, making it easy to scale up production; the spray-dried powder has good stability under vacuum packaging, and the theoretical shelf life at 25°C is no less than 170 days, making it suitable for large-scale applications.

[0023] 4. Wide safety and applicability: The raw materials are widely available (both casein and rice protein are food grade), with high biosafety, making it suitable for different populations and product forms. Attached Figure Description

[0024] Figure 1 The effect of different concentrations of casein trypsin hydrolysate CT intervention on RKO cell survival rate in Example 1.

[0025] Figure 2The sequence characteristics of the short peptides in the casein trypsin hydrolysate CT from Example 1 are shown.

[0026] Figure 3 This study analyzes the binding strength of short peptides in the casein trypsin hydrolysate CT from Example 1 with Keap1 and IKK-β.

[0027] Figure 4 The effect of different concentrations of rice protein alkaline + trypsin hydrolysate R-AT intervention on RKO cell survival rate in Example 2.

[0028] Figure 5 The sequence characteristics of the short peptide in the rice protein alkaline + trypsin hydrolysate R-AT in Example 2 are shown.

[0029] Figure 6 This study analyzed the binding strength of short peptides in the rice protein alkaline + trypsin hydrolysate R-AT with Keap1 and IKK-β in Example 2.

[0030] Figure 7 The protective activity of the mixture of casein trypsin hydrolysate CT and rice protein alkaline + trypsin hydrolysate R-AT on RKO cells in Example 3 was demonstrated.

[0031] Figure 8 The change in body weight of mice modeled with indomethacin under the intervention of the enzymatic hydrolysate in Example 4.

[0032] Figure 9 This demonstrates the protective effect of the enzymatic hydrolysate intervention in Example 4 on maintaining serum albumin levels in mice.

[0033] Figure 10 This refers to the effect of enzymatic hydrolysate intervention on the maintenance of mouse jejunal villus length in Example 4. Detailed Implementation

[0034] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0035] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0036] 1. Methods for evaluating intestinal cell protective activity (1) Cell Culture Method: The RKO cell line is a type of human colon adenocarcinoma cell with typical epithelial cell morphology. It expresses multiple nutrient transporters, which can simulate the nutrient absorption process in intestinal cells and respond to inflammatory stimuli, thus simulating the process of intestinal inflammation. RKO cells were revived in 25T cell culture flasks and cultured in a complete medium prepared with 89% DMEM high glucose basal medium, 10% (v / v) fetal bovine serum, and 1% (v / v) double antibiotics. After cell attachment, the medium was immediately replaced. The culture environment was 37°C and 5% (v / v) CO2. When the cells reached about 80% confluence with the bottom of the culture flask, they were passaged. When they reached about 80% confluence again, subsequent experiments were performed.

[0037] (2) Cellular experimental intervention method: RKO cells that had reached approximately 80% confluence with the bottom of the culture flask were used for screening protease hydrolysates with intestinal cell protection activity. Cells were digested with trypsin, centrifuged, and resuspended at 5 x 10⁻⁶ ppm. 3 Cells were seeded at a density of [number] cells / well in 96-well plates. After 12 h of static incubation, the single protein hydrolysate and its compound preparation were prepared in DMEM complete medium at a total protein concentration of 150 mg / mL and added to the wells. After 24 h of intervention with the hydrolysate, the supernatant was discarded, and DMEM basal medium containing 3‰ (v / v) H2O2 was added for 2 h of intervention. Subsequently, the supernatant was discarded, and cell viability was detected by MTT assay.

[0038] (3) MTT assay for cell viability: 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) was prepared into a 5 mg / L stock solution using sterile PBS. Before intervention, the stock solution was prepared to a final concentration of 0.5 mg / mL using DMEM basal medium. The cell culture supernatant was carefully discarded, and medium containing MTT was added. The cells were then incubated at 37°C for 4 h. After incubation, the medium in the wells was discarded, and 100 μL of DMSO was added. The cells were shaken at low speed for 5 min on a shaker to dissolve the crystals completely. The absorbance of the wells was then measured at 570 nm using a microplate reader. Cell viability was calculated using the following formula:

[0039] 2. Peptide profiling identification and peptide characteristic structure characterization methods (1) Pretreatment methods for peptide profiling identification: 1) Peptide extraction: Use a 10kD ultrafiltration tube to remove protein and extract peptides; 2) Reductive alkylation: Take 100 μg of sample, dilute to 100 μL with water, accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, and reduce in a 56℃ water bath for 1 h; then, accurately pipette 2 μL of 1M IAM solution into the sample to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min; finally, accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L to neutralize unreacted IAA.

[0040] 3) C18 desalting: Desalting with C18 stage-tip and vacuum drying at 45℃.

[0041] (2) Peptide profiling identification: Liquid chromatography conditions: 1) Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm; 2) Mobile phase A: 0.1% (v / v) FA, mobile phase B: 0.1% (v / v) FA, 80% (v / v) ACN; 3) Flow rate: 600 nL / min; 4) Analysis time for each component: 66 min; 5) Specific chromatographic elution procedure: 0–2 min: 4%, 2–45 min: 8%, 45–55 min: 28%, 55–56%: 40%, 56–66 min: 95%.

[0042] Mass spectrometry conditions: Level 1 mass spectrometry parameters: 1) Resolution: 70,000; 2) AGC target: 3e6; 3) Maximum IT: 100 ms; 4) Scan range: 100 to 1500 m / z; Secondary mass spectrometry parameters: 1) Resolution: 17,500; 2) AGCtarget: 1e5; 3) MaximumIT: 50ms; 4) TopN: 20; 5) NCE / steppedNCE: 28.

[0043] (3) Library search peptide comparison: The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: 1) Database: Uniprot_Bostaurus; 2) Peptide Mass Tolerance: 20 ppm; 3) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da.

[0044] 3. Molecular docking Peptide molecules obtained from peptide mapping were molecularly docked with proteins related to oxidative stress (Keap1) and the inflammation regulatory pathway (IKK-β). Keap1 protein is a core pivot molecule in the regulation of oxidative stress; peptides can exert antioxidant functions by binding to it, blocking the Keap1-Nrf2 interaction, promoting Nrf2 nuclear translocation, and enhancing superoxide dismutase (SOD) expression. The NF-κB pathway is an important mechanism for regulating inflammation. I-κB kinase (IKK) promotes IκB phosphorylation, leading to the dissociation of IκB from NF-κB protein, which then translocates to the nucleus, enhancing the expression of pro-inflammatory factors. IKK-β is the main catalytic unit in the IKK complex; therefore, IKK-β can serve as an anti-inflammatory target in the NF-κB signaling pathway to regulate the inflammatory response. The crystal structures of Keap1 (PDB: 2FLU) and IKK-β (PDB: 3RZF) were obtained from the PDB database. After removing water molecules and ligand structures, hydrogen atoms were added and charges were assigned. The positive ligand for Keap1 is TX6 (PubChem CID: 121488089), and the positive ligand for IKK-β is XNM(4-((4-(4-(chlorophenyl) pyrimidin-2-yl) amino) phenyl) (4-(2-hydroxyethyl) piperazin-1-yl) methanone). The peptides were used to construct three-dimensional structures using ChemDraw, and energy minimization was performed using the MMFF94 force field algorithm. Finally, the output was in pdbqt format using AutoDock Tools 1.5.7. The coordinates of the Keap1 protein docking active site are X: 89, Y: -26 and Z: 57, and the docking box size is 100 Å × 100 Å × 100 Å; the coordinates of the IKK-β protein docking active site are X: 5, Y: 9, Z: 2, and the docking box size is 30 Å × 30 Å × 30 Å.

[0045] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0046] Example 1: Preparation process, activity evaluation, and structural characterization of casein hydrolysate from a dual-protease hydrolysate formulation with intestinal barrier protection function. Casein hydrolysis method: Weigh casein powder and add it to distilled water at a material-to-liquid ratio of 1:20 (i.e., 0.05 g / mL). Slowly adjust the pH to 6.8 while in suspension. Prepare an enzyme stock solution by taking trypsin, flavor protease, neutral protease, alkaline protease, and papain, and activate it at 37°C before use.

[0047] Two methods were used for enzymatic hydrolysis: 1) Add trypsin to the preheated casein solution, with a final enzyme activity concentration of 5000 (U / g prot), and hydrolyze for 4 hours at a temperature of 55℃. 2) In the first step of enzymatic hydrolysis, flavor protease, neutral protease, alkaline protease or papain are added to achieve a final enzyme activity concentration of 5000 (U / g prot). After 2 hours, trypsin is added to continue enzymatic hydrolysis for another 2 hours, with a final trypsin activity concentration of 5000 (U / g prot). The temperature throughout the process is 55℃.

[0048] After inactivation, the enzyme was inactivated at 100℃ for 15 min, cooled, and centrifuged at 20℃, 4000 rpm for 15 min. The casein hydrolysate obtained after centrifugation was stored at -80℃.

[0049] In this embodiment, the casein hydrolysate was evaluated using the method described above for evaluating intestinal cell protective activity, with glutathione and casein as controls. The five hydrolysates obtained above showed improved intestinal cell survival rates, as shown in Table 1. Trypsin hydrolysate (CT) exhibited the best effect in improving survival rates.

[0050] The effect of different concentrations of casein trypsin hydrolysate CT intervention on RKO cell survival rate, such as Figure 1 As shown. By Figure 1 It can be seen that when the concentration of trypsin hydrolysate (CT) intervention is 25-500 mg / mL, the cell survival rate of the intervention group is increased by 17.90%-30.31% compared with the model group.

[0051] Table 1. Intestinal cell protective activity of casein hydrolysates

[0052] Note: The presence of the same letter between two groups indicates that the difference is not significant. p >0.05), with no overlapping letters between the two groups indicating a significant difference ( p <0.05).

[0053] Peptide profiling and molecular docking function prediction were performed on casein trypsin hydrolysate (CT), which has the strongest cell protective activity.

[0054] Figure 2The sequence characteristics of short peptides in casein trypsin hydrolysate (CT) are shown. The six images represent the peptide characteristics of peptides 3-8 in casein trypsin hydrolysate (CT). In each image, the left side of the X-axis represents the N-terminus and the right side represents the C-terminus. The stacked letter symbols on the Y-axis and their relative sizes reflect the amino acid composition dominance of that site.

[0055] Depend on Figure 2 It can be seen that the peptide identification results show that the short peptides in the trypsin hydrolysate CT have common characteristics. The C-terminus of the short peptides in the casein trypsin hydrolysate CT is characterized by nA-P-A' (that is, the abundance of Pro in the second to last position of the C-terminus is significantly dominant), and the last amino acid (A' position) is enriched with hydrophobic amino acids (Pro, Leu and Val).

[0056] Figure 3 The binding strength analysis of short peptides to Keap1 and IKK-β in casein trypsin hydrolysate CT is shown.

[0057] Depend on Figure 3 It was found that in the molecular docking results, the top 40 abundant peptides (accounting for 87.44% of the total response value) exhibited good Keap1 and IKK-β binding activity. Among them, peptides with high Keap1 affinity (binding affinity < -7.0 kcal / mol) accounted for 35.00% (total mean -6.10 kcal / mol), and peptides with high IKK-β affinity accounted for 45.00% (total mean -6.22 kcal / mol). In addition, the C-terminus hydrophobic amino acids (Ile, Val, and Leu) in the casein trypsin hydrolysate CT showed significantly high Keap1 binding activity, such as the peptide shown in SEQ ID NO.1 (-10.62 kcal / mol, binding strength greater than the positive molecule TX6) and the peptide shown in SEQ ID NO.2 (-9.83 kcal / mol). Moreover, the "FP" sequence often appeared in the peptides, and these peptides mainly bind to the protein through hydrophobic interactions. The C-segment of the peptide with high IKK-β binding activity in CT also strongly favors hydrophobic amino acids (Pro and Phe), such as the peptide shown in SEQ ID NO.3 (-9.75 kcal / mol) and the peptide shown in SEQ ID NO.4 (-9.86 kcal / mol). These two peptides can bind to IKK-β through π-π stacking.

[0058] Example 2: Preparation process and activity evaluation of rice protein hydrolysate in a dual-protease hydrolysate formulation with intestinal barrier protection function. Rice protein hydrolysis method: Weigh rice protein powder and add it to distilled water at a material-to-liquid ratio of 1:10 (0.1 g / mL). Prepare a 200 mg / mL enzyme stock solution using the required amount of protease, and activate it at 37°C before use.

[0059] Four methods were used for enzymatic hydrolysis: 1) Add alkaline protease to the preheated casein solution, with a final enzyme activity concentration of 5000 (U / g prot), and hydrolyze for 4 hours at a temperature of 55℃. 2) In the first step of enzymatic hydrolysis, alkaline protease is added to a final enzyme activity concentration of 5000 (U / g prot). After 2 hours, trypsin or flavor protease is added to continue enzymatic hydrolysis for another 2 hours. In the second step, the final enzyme activity concentration of the protease is 5000 (U / g prot), and the temperature throughout the process is 55℃. 3) In the first step of enzymatic hydrolysis, trypsin or neutral protease is added to achieve a final enzyme activity concentration of 5000 (U / g prot). After 2 hours, alkaline protease is added to continue enzymatic hydrolysis for another 2 hours, with the final enzyme activity concentration also reaching 5000 (U / g prot). The temperature throughout the process is 55℃. 4) In the first step of enzymatic hydrolysis, neutral protease is added to a final enzyme activity concentration of 5000 (U / g prot). After 2 hours, trypsin is added to continue enzymatic hydrolysis for another 2 hours, with a final enzyme activity concentration of 5000 (U / g prot). The temperature throughout the process is 55℃.

[0060] After enzymatic hydrolysis, the enzyme was inactivated at 100℃ for 15 min, cooled, and centrifuged at 20℃, 4000 rpm for 15 min. The hydrolysate obtained after centrifugation was stored at -80℃.

[0061] In this embodiment, the rice protein hydrolysates were evaluated using the aforementioned method for assessing intestinal cell protective activity, with glutathione and rice protein used as controls. The five protein hydrolysates obtained above showed improved intestinal cell survival rates, as shown in Table 2. The alkaline + trypsin hydrolysate (R-AT) exhibited the best improvement in survival rate.

[0062] Figure 4 The effect of different concentrations of rice protein alkaline + trypsin hydrolysate R-AT intervention on RKO cell survival was shown. Figure 4 It was found that when the concentration of alkaline + trypsin hydrolysate (R-AT) intervention was 25-500 mg / mL, the cell survival rate of the intervention group was increased by 27.76%-37.58% compared with the model group.

[0063] Table 2. Intestinal cell protective activity of rice protein hydrolysates

[0064] Note: The presence of the same letter between two groups indicates that the difference is not significant. p>0.05), with no overlapping letters between the two groups indicating a significant difference ( p <0.05).

[0065] Peptide protease + trypsin hydrolysate (R-AT) of rice protein, which has the strongest cell-protective activity, was identified and its molecular docking function was predicted.

[0066] Figure 5 The sequence characteristics of short peptides in rice protein alkaline + trypsin hydrolysate R-AT are shown. The six images are peptide characteristics of peptides 4-9 in rice protein alkaline + trypsin hydrolysate R-AT. In each image, the left side of the X-axis is the N-terminus and the right side is the C-terminus. The stacked letter symbols on the Y-axis and their relative sizes reflect the amino acid composition dominance of that site.

[0067] Depend on Figure 5 It can be seen that the peptide profile identification results show that the short peptides in the R-AT enzymatic hydrolysate have common characteristics. Their C-terminus is characterized by nB-P-B' (that is, the second to last position of the C-terminus is Pro), and the last amino acid (B' position) is enriched with positively charged amino acids (Arg and His).

[0068] Figure 6 The binding strength analysis of short peptides in rice protein alkaline + trypsin hydrolysate R-AT with Keap1 and IKK-β is shown.

[0069] Depend on Figure 6 It was found that in the molecular docking results, the top 40 abundant peptides accounted for 60.55% of the total response value, and tended to have high Keap1 affinity. Among them, peptides with high Keap1 affinity accounted for 57.50% (total mean -7.42 kcal / mol), and peptides with high IKK-β affinity accounted for 37.50% (total mean -6.06 kcal / mol). Their C-terminal characteristic amino acids are basic amino acids (Arg, His), which can promote the formation of strong electrostatic forces between the peptides and the Keap1 binding pocket, thus tending to have higher binding strength.

[0070] Example 3: Evaluation of the intestinal cell protective activity of the dual-protein combination of casein hydrolysate and rice hydrolysate. CT and R-AT were prepared as interventions with CT:R-AT mass ratios of 1:3, 2:2, and 3:1, and finally, a total protein concentration of 150 mg / mL was prepared in DMEM complete medium. Using CT and R-AT enzymatic hydrolysates as controls, after 24 h of intervention, the supernatant was discarded, and DMEM basal medium containing 3‰ (v / v) H2O2 was added to the cells for another 2 h. Subsequently, the supernatant was discarded, and cell viability was detected by the MTT assay.

[0071] Figure 7The protective activity of a mixture of casein trypsin hydrolysate CT and rice protein alkaline trypsin hydrolysate R-AT on RKO cells was demonstrated.

[0072] Depend on Figure 7 It can be seen that in the enzymatic hydrolysates of CT and R-AT, the mixed samples with a CT:R-AT mass ratio of 3:1, 1:1, and 1:3 all showed a trend of higher activity than CT and R-AT alone, and at a 1:1 ratio, the activity was increased by 13.67% and 6.54% respectively compared to the CT group and the R-AT group. p <0.05).

[0073] Example 4: Effects of dual protease hydrolysates on the prevention of intestinal barrier damage and nutritional function in mice. Six-week-old male C57BL / 6J mice were used in the experiment and housed in an SPF-barrier environment with 25 ± 1°C, 50% ± 5% humidity, and a 12-hour light-dark cycle daily. During the rearing period, mice had free access to food and water and were fed a standard maintenance diet. After being quarantined in an isolation area for one week, the mice were transferred to the experimental room for acclimatization. Once the mice reached eight weeks of age, they were randomly assigned to groups for the experiment. Mice were administered the intervention via gavage and were divided into a control group, a model group, a casein hydrolysate CT group (CT group), a rice hydrolysate R-AT group (R-AT group), and a casein hydrolysate + rice hydrolysate group (CT + R-AT group, 1:1 mass ratio), with 6-7 mice in each group. The control group received 0.1 mL / 10 g body weight saline via gavage, while the intervention group received 400 mg / kg body weight via gavage. Mice were administered enzymatic hydrolysate via gavage for 1-50 days after the start of the experiment. From day 51 to day 55, the enzymatic hydrolysate intervention was stopped, and indomethacin (4 mg / kg bw) was administered via gavage for 5 consecutive days to induce intestinal injury. Body weight was recorded during the experiment. On the evening of day 55, mice were fasted but given free access to water. Mice were sacrificed on day 56, and blood was collected for serum albumin analysis. Jejunal tissue was dissected, prepared into paraffin sections, and stained with hematoxylin and eosin (HE). Subsequently, the length of jejunal villi was measured using ImageJ software. Figure 8 The changes in body weight of mice modeled with indomethacin under the intervention of enzymatic hydrolysate are shown. Figure 9 This study demonstrated the protective effect of enzymatic hydrolysate intervention on maintaining serum albumin levels in mice. Figure 10 The study demonstrated the effect of enzymatic hydrolysate intervention on the maintenance of jejunal villus length in mice.

[0074] Depend on Figure 8 It can be seen that the C-T+R-AT group of mice experienced the smallest decrease in body weight; from Figure 9 It can be seen that the decrease in serum albumin concentration was effectively prevented; by Figure 10It can be seen that the C-T+R-AT group had the most intact intestinal mucosal structure and a significant increase in villus length. All of these indicators were superior in the dual-enzyme hydrolysate intervention group to those in the single-enzyme hydrolysate intervention group.

[0075] Example 5 Preparation and stability of dual protease hydrolysates The CT and R-AT enzymatic hydrolysates prepared in Examples 1 and 2 were spray-dried (inlet temperature 180℃, outlet temperature 80℃, feed rate 30mL / min). The resulting powders were individually vacuum-packed and then mixed in vacuum-packed for accelerated storage stability testing: the powders were placed in constant temperature and humidity chambers at 40℃, 50℃, and 60℃ with 75% relative humidity. Samples were taken at the end of 0, 3, 7, and 14 days to evaluate changes in protective activity using a cell model, and the theoretical shelf life was calculated using the Arrhenius model. The activity retention rate was calculated based on cell viability and the results of the unpreserved samples. Then, the rate constant (k) of degradation kinetics at different temperatures and packaging groups was calculated using the first-order reaction model of Equation 1. Finally, the activation energy E was obtained by linear regression of ln k against 1 / T (T being the Kelvin temperature) using Equation 2. a Then, the k value for storage at 25°C is calculated backwards, and finally, the theoretical shelf life is calculated based on Formula 3, which tells us the time during which more than 90% of the activity is retained at 25°C.

[0076] Formula 1:

[0077] Formula 2:

[0078] Formula 3:

[0079] The powder obtained in this embodiment has a good shape and no unpleasant odor. The accelerated preservation results are shown in Table 3. The theoretical shelf life of vacuum-packed CT powder, R-AT powder, and mixed-package powder are approximately 172 days, 75 days, and 170 days, respectively, which meets the requirements of industrial application of the product and provides evidence for the selection of industrial packaging materials and packaging methods.

[0080] Table 3 Theoretical shelf life of enzymatic hydrolysate dry powder

[0081] Comparative Example 1: Comparison of intestinal cell protective activities of different casein protease hydrolysates Casein hydrolysis method: Weigh casein powder and add it to distilled water at a material-to-liquid ratio of 1:20 (i.e., 0.05 g / mL). Slowly adjust the pH to 6.8 while in suspension. Prepare an enzyme stock solution using trypsin, flavor protease, neutral protease, alkaline protease, or papain, and activate it at 37°C before use. Add flavor protease, neutral protease, alkaline protease, or papain to the preheated casein solution to a final enzyme activity concentration of 5000 U / g prot. Hydrolyze for 4 hours at 55°C. After hydrolysis, inactivate the enzyme at 100°C for 15 minutes, cool, and centrifuge at 20°C, 4000 rpm for 15 minutes. Store the casein hydrolysate obtained after centrifugation at -80°C.

[0082] In this comparative example, the casein hydrolysates were evaluated using the methods described above for assessing intestinal cell protective activity, with glutathione as a positive control. The effects of the four obtained protein hydrolysates on intestinal cell survival are shown in Table 4; none of them showed an improvement compared to the model group.

[0083] Table 4 Comparison of cell-protective activities of other casein hydrolysates

[0084] Note: The presence of the same letter between two groups indicates that the difference is not significant. p >0.05), with no overlapping letters between the two groups indicating a significant difference ( p <0.05).

[0085] Comparative Example 2: Comparison of intestinal cell protective activities of different protease hydrolysates of rice protein Rice protein hydrolysis method: Weigh rice protein powder and add it to distilled water at a material-to-liquid ratio of 1:10 (i.e., 0.1 g / mL). Prepare a 200 mg / mL enzyme stock solution using the required amount of protease, and activate it at 37°C before use.

[0086] Two methods were used for enzymatic hydrolysis: 1) In the first step of enzymatic hydrolysis, alkaline protease is added to a final enzyme activity concentration of 5000 (U / g prot). After 2 hours, neutral protease or papain is added to continue enzymatic hydrolysis for another 2 hours. In the second step, the final enzyme activity concentration of the protease is 5000 (U / g prot), and the temperature throughout the process is 55℃. 2) In the first step of enzymatic hydrolysis, flavor protease was added to a final enzyme activity concentration of 5000 U / g prot. After 2 hours, alkaline protease was added and hydrolysis continued for another 2 hours, with the final enzyme activity concentration also at 5000 U / g prot. The temperature throughout the process was 55℃. After hydrolysis, the enzyme was inactivated at 100℃ for 15 minutes, cooled, and centrifuged at 20℃, 4000 rpm, for 15 minutes. The hydrolysate obtained after centrifugation was stored at -80℃.

[0087] In this comparative example, the rice protein hydrolysates were evaluated using the methods described above for assessing intestinal cell protective activity, with glutathione and rice protein used as controls. The effects of the three protein hydrolysates obtained above on intestinal cell survival are shown in Table 5; none of them showed an improvement compared to the model group.

[0088] Table 5 Comparison of cell-protective activities of other rice protein hydrolysates

[0089] Note: The presence of the same letter between two groups indicates that the difference is not significant. p >0.05), with no overlapping letters between the two groups indicating a significant difference ( p <0.05).

[0090] Comparative Example 3: Comparison of intestinal cell protective activities of oat protein hydrolysates produced by the same enzymatic hydrolysis method Weigh oat protein powder and add it to distilled water at a material-to-liquid ratio of 1:20 (i.e., 0.05 g / mL). Prepare a 200 mg / mL enzyme stock solution using the desired protease, and activate it at 37°C before use. The enzymatic hydrolysis method is the same as that used for rice protein hydrolysates with optimal activity. In the first step, add alkaline protease to a final enzyme activity concentration of 5000 U / g prot. After 2 hours, add trypsin and continue hydrolysis for another 2 hours, with a final trypsin activity concentration of 5000 U / g prot. The temperature throughout the process is 55°C. After hydrolysis, inactivate the protein at 100°C for 15 minutes, then cool and centrifuge at 20°C, 4000 rpm for 15 minutes. Store the hydrolysate obtained after centrifugation at -80°C.

[0091] In this comparative example, the oat protein hydrolysate was evaluated using the aforementioned method for assessing intestinal cell protective activity. The experimental results showed that the cell survival rate in the model group was 71.44 ± 0.54%, while the cell survival rate after intervention with the hydrolysate was 70.71 ± 3.11%, which was not significantly improved compared to the model group.

[0092] This invention provides a dual-protein formulation composed of casein hydrolysate (CT) and rice hydrolysate (R-AT) in an optimal ratio (e.g., 1:1). The formulation demonstrated significant intestinal protective activity in both in vitro cell models (RKO intestinal epithelial cells) and in vivo mouse models (indomethacin-induced intestinal injury), specifically manifested in improved cell viability, maintenance of serum albumin levels, and protection of jejunal villus structure. Its mechanism of action is related to the regulation of the peptides through the Keap1-Nrf2 antioxidant pathway and the IKK-β-NF-κB anti-inflammatory pathway. Furthermore, both the casein hydrolysate and rice hydrolysate peptides exhibit distinct C-terminal structural features (e.g., nA-P-A', with hydrophobic amino acids enriched at the A' position of the casein hydrolysate peptide and positively charged amino acids enriched at the B' position of the rice hydrolysate peptide), confirming that these features can effectively bind to Keap1 and IKK-β proteins, thereby promoting intestinal barrier repair. In summary, this invention is expected to provide a novel nutritional support solution for improving intestinal barrier function, laying the foundation for its large-scale production and commercial application.

[0093] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A casein hydrolysate, characterized in that, The short peptides in the casein hydrolysate have an nA-P-A' characteristic sequence at their C-terminus, where n ranges from 1 to 6, A is any amino acid, and A' is enriched with hydrophobic amino acids, including Pro, Leu, or Val.

2. A method for preparing the casein hydrolysate as described in claim 1, characterized in that, The casein hydrolysate was prepared by a single-step enzymatic hydrolysis, which was a trypsin hydrolysis.

3. A method for preparing the casein hydrolysate as described in claim 1, characterized in that, The casein hydrolysate is prepared by a two-step enzymatic hydrolysis. The first step of the enzymatic hydrolysis includes one of the following: flavor protease, neutral protease, alkaline protease, and papain hydrolysis. The second step is trypsin hydrolysis.

4. A rice protein hydrolysate, characterized in that, The short peptides in the rice protein hydrolysate have an nB-P-B' characteristic sequence at their C-terminus, where n ranges from 1 to 6, B is any amino acid, and B' is enriched with positively charged amino acids, including Arg or His.

5. A method for preparing soybean protein hydrolysate as described in claim 4, characterized in that, The soybean protein hydrolysate was prepared by a single-step enzymatic hydrolysis, which was an alkaline protease hydrolysis.

6. A method for preparing soybean protein hydrolysate as described in claim 4, characterized in that, The soybean protein hydrolysate is prepared by a two-step enzymatic hydrolysis. The first step of the enzymatic hydrolysis includes one of alkaline protease, trypsin, and neutral protease, while the second step includes one of trypsin, flavor protease, alkaline protease, and trypsin hydrolysis.

7. A compound preparation based on casein and rice protein hydrolysate, characterized in that, The compound preparation includes the casein hydrolysate of claim 1 and the rice hydrolysate of claim 4.

8. The compound preparation according to claim 7, characterized in that, The protein ratio of the casein hydrolysate to the rice hydrolysate is 1:3 to 3:

1.

9. The use of the casein hydrolysate of claim 1, the rice hydrolysate of claim 4, or the compound preparation of claim 7 in the preparation of a medicine for preventing or repairing damage to the intestinal barrier function.

10. The use of the casein hydrolysate of claim 1, the rice hydrolysate of claim 4, or the compound preparation of claim 7 in the preparation of health foods that aid digestion.

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