Method for preparing ovotransferrin hydrolysate with high immunocompetence based on adult gastrointestinal simulated digestion system

Abstract

The invention belongs to the field of food nutrition and functional factors, and particularly relates to a method for preparing ovotransferrin hydrolysate with high immunocompetence based on an adult gastrointestinal simulated digestion system. According to the method, ovotransferrin is treated through the stomach and intestine of a simulated adult, OVT hydrolysate is prepared, and the OVT hydrolysate contains HTEGSTT, TTSY and / or RTAGWVIPMG. The invention finds that the immunomodulatory activity of OVT is enhanced after gastrointestinal digestion and hydrolysis, the OVT is more easily hydrolyzed in simulated adult digestive juice than in infant digestive juice, and a contrast experiment shows that the nutrition intervention of OVT adult and infant simulated digestion products can promote the generation of short-chain fatty acids such as acetic acid, propionic acid and butyric acid; the swelling injury caused by inflammation to small intestine mucous membrane tissues is effectively relieved, so that the intestinal barrier immune function is obviously improved, and the OIA shows higher intestinal immune activity than the OIB.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food nutrition and functional factors, and particularly relates to a method for preparing high-immunity-activity ovotransferrin hydrolysate based on adult gastrointestinal simulated digestion system. BACKGROUND

[0002] Ovotransferrin (OVT), an iron-binding glycoprotein derived from chicken eggs, belongs to the transferrin family together with lactoferrin. Studies have shown that OVT has antibacterial, antiviral, antitumor, immunomodulatory, and iron absorption promoting activities, and has great potential in the fields of nutrition and health. However, the activity of ovotransferrin in the natural state is relatively low, and moderate hydrolysis of ovotransferrin can significantly improve its biological activity. Moderate hydrolysis can break its rigid structure and expose potential active sites, thereby improving its antibacterial and immunomodulatory activities and fully exerting its value in the fields of nutrition and health. Different populations have different digestion abilities, and hydrolysis can make OVT adapt to the characteristics of the digestive systems of different populations, generate small peptide segments that are easy to absorb, improve bioavailability, and enhance the effect of nutritional supplementation.

[0003] However, existing in vitro simulated digestion research uses uniform digestion conditions and does not fully consider the physiological differences in the digestive systems of different age groups. For example, the digestion abilities of newborns and adults are quite different, which can lead to significant differences in the digestion patterns and biological effects of OVT in different populations, thereby affecting its actual application effect.

[0004] For example, the prior art CN111072770A discloses an ovotransferrin antibacterial peptide and a preparation method thereof. The method involves heat treating ovotransferrin, hydrolyzing it with pepsin, separating and purifying it, and identifying three highly active antibacterial peptides.

[0005] For example, the prior art (In Vitro Simulated Digestion Characteristics of Egg Ovotransferrin Allergen, Jing-Shu Wang et al., Chinese Journal of Food Science, Vol. 21, No. 1) discloses using an in vitro static digestion mode to simulate the digestion of chicken ovotransferrin by gastric juice, small intestinal juice, and small intestinal brush border membrane enzyme of infants and adults, respectively, analyzing the digestion products using Tricine-SDS-PAGE and MALDI-TOF-MS, and studying the digestion characteristics of egg white allergen ovotransferrin.

[0006] Given the shortcomings of existing technologies and the necessity of hydrolysis, this invention innovatively uses a reaction system that simulates adult human gastrointestinal digestive fluid to prepare ovotransferrin hydrolyzate. This approach aims to produce an ovotransferrin hydrolyzate with high immunogenicity through moderate hydrolysis. Specific beneficial effects include: The bioactive peptides produced by hydrolysis of OVT can disrupt bacterial cell membranes, inhibit bacterial growth and reproduction, reduce the risk of harmful bacterial infection, and maintain human health. It also enhances immunomodulatory effects, stimulates immune cell proliferation and differentiation, enhances immune cell activity, regulates immune system function, improves immunity, and helps resist disease. It also promotes iron absorption. The hydrolysis process may release iron-binding peptides, which facilitate iron absorption and utilization, prevent iron-deficiency anemia, and improve human iron status.

[0007] The present invention is expected to provide a scientific basis for the precise application of ovotransferrin in the field of nutrition and health, fill the gaps in current research and application, and promote its effective use in nutritional supplementation for different populations. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the present invention provides a method for preparing a highly immunologically active ovotransferrin hydrolyzate based on simulated adult gastrointestinal digestion, which aims to solve the technical problem of how to improve the immunological activity of ovotransferrin.

[0009] The first technical solution provided by the present invention is a method for preparing a highly immunologically active ovotransferrin hydrolyzate, wherein the method comprises treating ovotransferrin with simulated adult gastrointestinal fluid or simulated infant gastrointestinal fluid.

[0010] In certain embodiments, the method comprises the following steps: (1) Raw material pretreatment: Wash fresh eggs, separate the egg white and yolk, and homogenize the egg white at 4°C for 20 minutes. Use pH 6.0, 50 mM sodium acetate buffer to adjust the pH of the egg white solution to 6.0, centrifuge at 8000 rpm for 15 minutes, discard the precipitate, and save the supernatant for later use. Use 1 M NaOH to adjust the pH of the supernatant to 6.0, add weak acidic cation exchange resin at a ratio of 4:1 between egg white solution and resin, and statically adsorb for 2 hours before filtering to obtain the egg white solution with lysozyme removed. Continue to adjust the pH of the egg white solution without lysozyme to 6.0, use SP-Sepharose chromatography column for elution, collect the protein solution containing OVT, concentrate by ultrafiltration, and freeze-dry to obtain OVT.

[0011] (2) Simulated gastric juice preparation: according to the proportion of 13.8 mL KCl (concentration of 0.5 mol / L), 1.8 mL KH2PO4 (concentration of 0.5 mol / L), 25 mL NaHCO3 (concentration of 1 mol / L), 23.6 mL NaCl (concentration of 2 mol / L), 0.8 mL MgCl2·6H2O (concentration of 0.15 mol / L), 1.0 mL (NH4)2CO3 (concentration of 0.5 mol / L), 2.6 mL HCl (concentration of 6 mol / L), 0.01 mL CaCl2·2H2O (concentration of 0.3 mol / L) to prepare simulated gastric juice (SGF); (3) Simulated intestinal juice preparation: according to the proportion of 13.6 mL KCl (concentration of 0.5 mol / L), 1.6 mL KH2PO4 (concentration of 0.5 mol / L), 85 mL NaHCO3 (concentration of 1 mol / L), 19.2 mL NaCl (concentration of 2 mol / L), 2.2 mL MgCl2·6H2O (concentration of 0.15 mol / L), 1.4 mL HCl (concentration of 6 mol / L), 0.08 mL CaCl2·2H2O (concentration of 0.3 mol / L) to prepare simulated intestinal juice (SIF); (4) Simulated adult gastrointestinal digestion: 40 mL SGF and 50 mL, 10 mg / mL OVT solution were mixed, 5 mL pepsin (2000 U / mL) was added, HCl (6 mol / L) was used to adjust the pH value to 2.0, ultrapure water was added to make the volume reach 100 mL, and then incubation was carried out for 2 h (37°C, 150 rpm), the pH value was adjusted to 7.0 to terminate the gastric digestion; then the above mixed solution was taken, 80 mL SIF solution, 25 mL bile salt (10 mM) and 5 mL trypsin (100 U / mL) were added, HCl (6 mol / L) was used to adjust the pH value to 7.0, and then ultrapure water was added to make the volume reach 200 mL, and then incubation was carried out for 2 h (37°C, 150 rpm); after each step of digestion, boiling was carried out for 10 min to inactivate the enzyme, then centrifugation was carried out, dialysis (molecular weight cut-off of 300 Da) was carried out, and the liquid in the dialysis bag was collected as OVT adult hydrolysate (OIA) for digestion property and intestinal immune activity determination; The second technical scheme provided by the application is a high immune activity OVT hydrolysate prepared by the method of the first technical scheme, and the hydrolysate contains HTEGSTT, TTSY and / or RTAGWVIPMG.

[0012] The third technical solution provided by the present application is a product containing the OVT hydrolysate of the second technical solution.

[0013] In some embodiments, the product comprises food, medicine.

[0014] The fourth technical solution provided by the present application is the use of the OVT hydrolysate of the second technical solution in the preparation of a product for improving intestinal health.

[0015] In some embodiments, the product comprises food, medicine or health care product.

[0016] In some embodiments, the medicine at least has one of the following effects: (1) promoting the generation of short-chain fatty acids in the individual; (2) relieving the swelling damage of the small intestinal mucosa tissue of the individual.

[0017] In some embodiments, the short-chain fatty acids comprise acetic acid, propionic acid and butyric acid.

[0018] Compared with the prior art, the technical effects of the present application are as follows: The present application constructs a simulated environment suitable for the human gastrointestinal tract, explores the differences in simulated digestion behavior of OVT at different ages, finds that the immune regulation activity of OVT is enhanced after gastric and intestinal digestion and hydrolysis, and that OVT is more easily hydrolyzed in simulated adult digestive juice than in simulated infant digestive juice. In OIA, two O-glycosylation sites T108 modified glycopeptides HTEGSTT and TTSY are found, and in OIB, one O-glycosylation site T480 modified decapeptide RTAGWVIPMG is found. By inducing immunosuppressed mice with cyclophosphamide, the intestinal immune activity of different simulated digestion products of OVT is evaluated. In order to verify the effectiveness of the present application, infant simulated digestion products (OIB) are also prepared as a comparison. Comparative experiments show that the nutritional intervention of OVT adult and infant simulated digestion products can promote the generation of short-chain fatty acids such as acetic acid, propionic acid and butyric acid, effectively relieve the swelling damage of the small intestinal mucosa tissue caused by inflammation, and significantly improve the intestinal barrier immune function, and OIA shows higher intestinal immune activity than OIB.

[0019] The preparation method of the infant simulated digestion product (OIB) is as follows: the simulated digestion gastric juice is mixed with the ovotransferrin solution, 250 U / mL pepsin is added, the pH value is adjusted to 2.0 by using HCl, and the incubation is performed for 2 h; the pH value is adjusted to 7.0 to terminate the gastric digestion, and the enzyme is inactivated by boiling; then the above-mentioned mixed solution is taken, the simulated digestion intestinal juice, 1.25 mM bile salt and 12.5 U / mL trypsin are added, the pH value is adjusted to 7.0 by using HCl, and the incubation is performed for 2 h; the enzyme activity is inactivated by boiling, and then the centrifugation, dialysis and collection of the liquid in the dialysis bag are performed, so as to obtain the OVT infant hydrolysate. The function of the OVT infant hydrolysate is compared with that of the OVT adult simulated digestion product protected in the application, so as to demonstrate the technical effectiveness. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is the hydrolysis site and polypeptide sequence diagram of ovotransferrin. In the figure, OVT is ovotransferrin.

[0021] Figure 2 The figure is the pathological change diagram of mouse small intestinal tissue sections.

[0022] Figure 3 The figure is the content change diagram of short-chain fatty acids in mouse feces. In the figure, OVT is ovotransferrin, OIA is the ovotransferrin in vitro simulated adult digestion product, and OIB is the ovotransferrin in vitro simulated infant digestion product.

[0023] Figure 4 The figure is the content change diagram of various acids in mouse feces. In the figure, OVT is ovotransferrin, OIA is the ovotransferrin in vitro simulated adult digestion product, and OIB is the ovotransferrin in vitro simulated infant digestion product. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application, and are not used to limit the present application.

[0025] Test method: (1) Analysis of digestion characteristics: the digestion product is diluted to 5 mg / mL with deionized water, filtered through a 0.45 μm membrane, and analyzed by using ESI-MS / MS combined with online HPLC. The instrument parameter settings are shown in Table 1. The chromatogram of the ovotransferrin digestion product is analyzed by using MassLynx V4.1 software combined with proteomics difference analysis method, and the structural differences of the digestion products of the two are analyzed. Then the chromatogram is introduced into Progenesis QI combined with a shared database (ChemSpider Search and Share Chemistry) to analyze the difference peptide sequence, combined with a mass spectrum database (Swissprot-1.0), and the potential immunomodulatory peptide confidence sequence is analyzed and verified from the difference peptide sequence of the simulated infant and adult digestion products of OVT.

[0026] Table 1 HPLC-ESI-MS / MS working parameter settings

[0027] (2) Short-chain fatty acid determination: The frozen fecal sample was slowly thawed at 4°C, and 80 ± 2 mg of feces was accurately weighed in a 1.5 mL sterile EP tube, 0.5 mL of 4°C pre-cooled saturated NaCl solution was added, vortexed for 30 s, and then ice-bathed for 30 min. The sample was homogenized at 4°C using a tissue grinder, then 40 μL of 4°C pre-cooled 10% (v / v) sulfuric acid solution was added for acidification, vortexed for 30 s. After acidification, 1 mL of 4°C pre-cooled anhydrous ether (containing 1 mmol / L 2-ethylbutyric acid as an internal standard) was added, vortexed for 30 s, and centrifuged at 4°C, 14000 g for 15 min. The supernatant was transferred to a 1.5 mL sterile EP tube, and 0.25 g of anhydrous sulfuric acid was added to remove water. After ice-bath standing for 15 min, centrifugation was performed at 4°C, 14000 g for 15 min, and the supernatant was used for GC-MS analysis. The test parameter settings are shown in Table 2. The short-chain fatty acid content was statistically analyzed using GC-MS Post-run software.

[0028] Table 2 GC-MS analysis parameter settings

[0029] (3) Histopathological observation: The small intestine tissue fixed with 4% paraformaldehyde for more than 24 h was sequentially dehydrated with ethanol gradient, transparentized with xylene, and embedded with paraffin. Thin sections of 5 μm were cut using a tissue sectioning machine, and stained with hematoxylin-eosin. After dehydration, the sections were mounted, and the pathological changes of the immune tissue were observed under a microscope, and image acquisition and analysis were performed.

[0030] (4) Data statistics and analysis: All data were expressed as mean ± standard error (MEAN ± SEM), and the experiment was repeated at least 3 times (n ≥ 3). Single factor analysis of variance (ANOVA) was performed using SPSS 22.0 software, and Duncan's significant difference (HSD) post-test was used to evaluate the statistical significance between different groups (P < 0.05). Graphpad Prism 8.0 software and Origin Pro 2022 were used for plotting.

[0031] Raw materials used in the examples: 1. Pepsin (P7000) source: purchased from Sigma-Aldrich Company, USA.

[0032] 2. Pancreatin (P7545) was purchased from Sigma-Aldrich, USA.

[0033] 3. BALB / c mice (license number for the use of laboratory animals: SYXK(SU)2016-0045) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were bred in the specific pathogen-free (SPF) barrier environment of the animal experiment center of Jiangnan University.

[0034] Example 1. Preparation of OVT hydrolysate: The specific steps are as follows: (1) Raw material pretreatment: Fresh eggs were washed, and the egg white and yolk were separated. The egg white was homogenized at 4°C for 20 min. The pH value of the egg white solution was adjusted to 6.0 using a 50 mM sodium acetate buffer. The solution was centrifuged at 8000 rpm for 15 min, and the precipitate was discarded. The supernatant was stored for use. The pH value of the supernatant was adjusted to 6.0 using 1 M NaOH. Weak acid cation exchange resin was added at a ratio of 4:1 (egg white solution:resin), and static adsorption was performed for 2 h. The solution was filtered to obtain a lysozyme-free egg white solution. The pH value of the lysozyme-free egg white solution was adjusted to 6.0, and SP-Sepharose chromatography column was used for elution. The OVT-containing protein solution was collected, concentrated by ultrafiltration, and freeze-dried to obtain OVT.

[0035] (2) Preparation of simulated gastric juice: According to the ratio of 13.8 mL KCl (concentration of 0.5 mol / L), 1.8 mL KH2PO4 (concentration of 0.5 mol / L), 25 mL NaHCO3 (concentration of 1 mol / L), 23.6 mL NaCl (concentration of 2 mol / L), 0.8 mL MgCl2·6H2O (concentration of 0.15 mol / L), 1.0 mL (NH4)2CO3 (concentration of 0.5 mol / L), 2.6 mL HCl (concentration of 6 mol / L), and 0.01 mL CaCl2·2H2O (concentration of 0.3 mol / L), simulated gastric juice (SGF) was prepared; (3) Preparation of simulated intestinal juice: According to the ratio of 13.6 mL KCl (concentration of 0.5 mol / L), 1.6 mL KH2PO4 (concentration of 0.5 mol / L), 85 mL NaHCO3 (concentration of 1 mol / L), 19.2 mL NaCl (concentration of 2 mol / L), 2.2 mL MgCl2·6H2O (concentration of 0.15 mol / L), 1.4 mL HCl (concentration of 6 mol / L), and 0.08 mL CaCl2·2H2O (concentration of 0.3 mol / L), simulated intestinal juice (SIF) was prepared; (4) Simulating adult gastrointestinal digestion: 40 mL of SGF was mixed with 50 mL of 10 mg / mL OVT solution, 5 mL of pepsin (2000 U / mL) was added, and HCl (6 mol / L) was used to adjust the pH value to 2.0. After the volume was made up to 100 mL with ultrapure water, the mixture was incubated for 2 h (37°C, 150 rpm). The pH value was adjusted to 7.0 to terminate the gastric digestion. Then, 80 mL of SIF solution, 25 mL of bile salt (10 mM), and 5 mL of trypsin (100 U / mL) were added to the mixture, and HCl (6 mol / L) was used to adjust the pH value to 7.0. The volume was made up to 200 mL with ultrapure water, and the mixture was incubated for 2 h (37°C, 150 rpm). After each step of digestion, the enzymes were inactivated by boiling for 10 min. After centrifugation, the mixture was dialyzed (molecular weight cut-off: 300 Da), and the liquid in the dialysis bag was collected as the OVT adult hydrolysate (OIA);

[0036] Example 2: Digestion property analysis: To explore the structure-activity relationship between the OVT digestion product structure and immune activity, potential immunomodulatory peptide sequences were analyzed and verified based on the characteristics of immunomodulatory peptides, i.e., rich in hydrophobic and branched-chain amino acids, in combination with the Swissprot-1.0 mass spectrometry database. Considering that polypeptides with a molecular weight of 500-1000 Da have high antioxidant and immune activity, and the number of characteristic peptides in the digestion product is large, tetrapeptides and decapeptides with a confidence score of more than 85 were selected for in-depth analysis. Figure 1 Figure 2 shows the hydrolysis sites and polypeptide sequences of OVT. Further analysis showed that OIA (OVT in vitro simulated adult digestion product) had a total of 15 highly predicted active polypeptides, with an integral ratio of 1184.42. Table 4 shows that OIB (OVT in vitro simulated infant digestion product) not only has a lower degree of hydrolysis than OIA, but also has a lower number of predicted immune active peptides and peak area than OIA. There are 11 polypeptides in OIB that meet the conditions, accounting for 945.05, and there are 12 polypeptides in LIB (lactoferrin hydrolysate after simulated infant gastrointestinal digestion), with a total peak area of 1157.25. The content of decapeptides in OIB increased significantly, and the number was much higher than that in OIA. These results confirmed that OVT has a higher degree of hydrolysis in the adult digestion environment, and also revealed the influence of different digestion environments on the length distribution of polypeptides.

[0037] Table 3: Polypeptide identification results of OVT simulated adult gastrointestinal digestion product (OIA)

[0038] Note: * indicates glycopeptide Table 4: Polypeptide identification results of OVT simulated infant gastrointestinal digestion product (OIB)

[0039] Note: *marked as glycopeptides Example 3 OVT simulated infant and adult digestion products on the pathological histomorphology of the mouse intestine: The immunosuppressed mouse model was constructed after being reviewed and approved by the Experimental Animal Management and Animal Welfare Ethics Committee of Jiangnan University, with the ethical approval number JN.No20230415b1500616, and strictly in accordance with the International Guide for Experimental Animals. The experimental animals were 6-8-week-old male BALB / c mice (experimental animal use license number: SYXK (Su) 2016-0045) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised in the specific pathogen-free (SPF) barrier environment of the Animal Experiment Center of Jiangnan University. The barrier environment meets the Chinese experimental animal environment facility detection standard, and the specific environmental conditions are: temperature 25°C ± 2°C, relative humidity 50% ± 5%, 12 h light / dark cycle, air cleanliness 100,000 levels, static pressure 20-50 Pa. BALB / c mice were used as objects, and cyclophosphamide was injected intraperitoneally to establish an immunosuppressed mouse model.

[0040] Animal grouping: 40 mice were randomly divided into 5 groups (8 mice per group): normal group, cyclophosphamide model group, OVT group, OIA group and OIB group. The dosages of OVT, OIA and OIB were 200 mg / kg / d. The normal group and the model group were given the same volume of normal saline as a control. To ensure the reliability of the experimental results, all mice were adaptively fed for 7 days before experimental operation. During the experiment, all mice freely ingested standard animal feed and drank water. Dietary intervention was achieved by gavage, lasting for 25 days. To simulate the influence of external stress on the immune system, at the end of the 25-day intervention period, all mice except the normal control group received intraperitoneal injection of cyclophosphamide at a dose of 80 mg / kg / d for 3 consecutive days. The normal control group was given intraperitoneal injection of the same volume of normal saline. 12 hours after the last cyclophosphamide injection, the mice were fasted for 12 hours and euthanized using carbon dioxide. The small intestine tissue (about 1 cm long) was immersed in 4% paraformaldehyde solution for fixation for subsequent histopathological examination.

[0041] The intestinal mucosal immune system is the largest immune organ of the body and plays a key role in maintaining overall immune balance. The intestinal epithelium is not only a physical barrier but also a site for the interaction of immune cells with foreign antigens. Therefore, maintaining the structural integrity of the intestine is crucial for normal immune function. Figure 2The effects of OVT and its digestion products on the intestinal histomorphology of immunosuppressed mice were demonstrated. The model group mice showed significant intestinal damage, including atrophy of villi, disordered arrangement, necrosis of epithelial cells, vacuolar deformation of villi, and shortening of crypt length, which reflected the cytotoxic effects of cyclophosphamide. Compared with the model group, the intestinal histomorphology of the mice was improved after the nutritional intervention of OVT, OIA, and OIB, and the arrangement of villi gradually became orderly, but a certain degree of vacuolar deformation was still observed. Compared with the OVT group, the intestinal histomorphology of the mice in the OIB group was slightly improved, while the OIA group showed a significant improvement. This result indicates that OVT, after digestion, may enhance the overall immune status by improving the mucosal immune function, and OIA shows a more effective immune-enhancing effect than OIB.

[0042] Example 4: Effects of OVT simulated infant and adult digestion products on short-chain fatty acids in mouse feces: Animal model construction and grouping: Refer to Example 3, after the last intraperitoneal injection of cyclophosphamide, the fresh feces of the mice were collected before fasting. The fecal samples were quickly transferred to pre-cooled 1.5 mL nuclease-free EP tubes and then stored in a -80°C refrigerator.

[0043] Short-chain fatty acids, as the main products of intestinal microbial metabolism, play a key role in maintaining intestinal health and regulating the host immune system. To further investigate the effects of OVT and its simulated digestion products on the intestinal microecology of CP-induced immunosuppressed mice, the levels (results shown in Figure 3 ) and distribution (results shown in Figure 4 ) of short-chain fatty acids in the feces of mice were comprehensively analyzed. The results are shown in Figure 4As shown, compared with the normal group, cyclophosphamide treatment significantly reduced the content of acetic acid, propionic acid, butyric acid, valeric acid and isovaleric acid in mice (P < 0.05). This phenomenon reflects the broad inhibition of cyclophosphamide on intestinal microbial community and its metabolic function, which may be one of the important factors leading to the decline of intestinal immune function. After nutritional intervention, all treatment groups were significantly higher than the model group, indicating that these digestion products can improve the production of short-chain fatty acids to varying degrees. The total acid content of the OIA group was 72.34 mmol / L, much higher than that of the OIB group (68.02 mmol / L). At the same time, the acetic acid content in the OIA group was 53.31 mmol / L, significantly higher than the acetic acid content of 43.34 mmol / L in the OIB group (P < 0.05). Overall, OIA and OIB can improve the SCFAs level of immunosuppressed mice to varying degrees, which indicates that OVT digestion products can play a role by regulating the intestinal microbiome. OIA is better at improving the total acid level, especially acetic acid, which may have a positive impact on intestinal health and immune function. This result is consistent with the effect of OIA and OIB nutritional intervention on intestinal tissue repair.

[0044] Although the present application has been disclosed in preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing a highly immunologically active ovotransferrin hydrolysate based on an adult gastrointestinal simulated digestive system, characterized in that: Ovotransferrin was treated by simulating the gastrointestinal environment of adults to obtain OVT hydrolysate containing HTEGSTT, TTSY and / or RTAGWVIPMG.

2. The method according to claim 1, characterized in that Simulated digestive gastric juice is mixed with ovotransferrin solution, and after adding pepsin, the pH value is adjusted to 2.0-3.5 with HCl and incubated for 2-4 hours; then the pH value is adjusted to 7.0 to terminate gastric digestion, and the enzyme is boiled to inactivate. Then, simulated digestive intestinal juice, bile salts and pancreatic enzymes are added, the pH value is adjusted to 7.0-8.0 with HCl, and the enzyme is boiled to inactivate after incubation for 2-4 hours; then, after centrifugation and dialysis, the liquid in the dialysis bag is collected as the OVT adult hydrolysate.

3. The method according to claim 2, wherein The simulated digestive gastric fluid contains the following components: KCl 13.8-14.2 mL, KH2PO4 1.8-2.3 mL, NaHCO3 25-28 mL, NaCl 23.6-24.8 mL, MgCl2·6H2O 0.8-1.2 mL, (NH4)2CO3 1.0-1.5 mL, HCl 2.6-3.2 mL, and CaCl2·2H2O 0.01-0.02 mL.

4. The method according to claim 2, wherein The simulated digestive intestinal fluid contains the following components: KCl 13.6-14.8 mL, KH2PO4 1.6-2.2 mL, NaHCO3 85-89 mL, NaCl 19.2-20.6 mL, MgCl2·6H2O 2.12-2.34 mL, HCl 1.4-1.8 mL, and CaCl2·2H2O 0.08-0.12 mL.

5. A highly immunologically active OVT hydrolyzate prepared by the method of claims 1 to 4, characterized in that: The hydrolysate contains HTEGSTT, TTSY and / or RTAGWVIPMG fragments.

6. A food containing the OVT hydrolyzate according to claim 5, which comprises infant formula or special medical food.

7. Use of the OVT hydrolyzate according to claim 5 in the preparation of a medicine for improving intestinal health.

8. The use according to claim 7, wherein the drug has at least one of the following effects: (1) Promote the production of individual short-chain fatty acids, including acetate, propionate, and butyrate; (2) Relieve swelling and damage to individual small intestinal mucosal tissue.

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