A method for preparing highly immunologically active ovotransferrin hydrolysate based on an adult gastrointestinal simulated digestive system

Abstract

This invention belongs to the field of food nutrition and functional factors, specifically relating to a method for preparing highly immunomodulatory ovotransferrin (OVT) hydrolysates based on an adult gastrointestinal simulated digestive system. The method involves processing OVT using a simulated adult gastrointestinal tract to prepare OVT hydrolysates containing HTEGSTT, TTSY, and / or RTAGWVIPMG. This invention reveals that OVT exhibits enhanced immunomodulatory activity after gastrointestinal digestion and hydrolysis, and is more readily hydrolyzed in simulated adult digestive fluids than in infant digestive fluids. Comparative experiments show that nutritional intervention with OVT simulated digestive products from adults and infants can promote the production of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, effectively alleviate swelling and damage to the small intestinal mucosa caused by inflammation, significantly improve intestinal barrier immune function, and OVT IA exhibits higher intestinal immune activity than OVT IB.

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] In view of the deficiencies of the prior art and the necessity of hydrolysis, the present application innovatively uses simulated adult gastrointestinal digestive juice as the reaction system to prepare egg transferrin hydrolysate. The purpose is to obtain egg transferrin hydrolysate with high immunological activity through moderate hydrolysis. The specific advantages are as follows: the bioactive peptides produced by the hydrolyzed OVT can destroy the bacterial cell membrane structure, inhibit bacterial growth and reproduction, reduce the risk of harmful bacterial infection, and maintain human health. Improve the immune regulation effect, stimulate immune cell proliferation and differentiation, enhance immune cell activity, regulate immune system function, improve human immunity, and help resist diseases. Promote iron absorption, release of iron-binding peptide fragments during the hydrolysis process, which is beneficial to the absorption and utilization of iron elements, prevent iron deficiency anemia, and improve the iron nutritional status of the human body.

[0007] The present application is expected to provide a scientific basis for the precise application of ovotransferrin in the field of nutrition and health, fill the gap in current research and application, and promote its effective use in different population nutrition supplementation. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a method for preparing high-immunological-activity ovotransferrin hydrolysate based on adult gastrointestinal simulation 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 application is a method for preparing high-immunological-activity ovotransferrin hydrolysate, which uses simulated adult gastrointestinal juice or simulated infant gastrointestinal juice to treat ovotransferrin.

[0010] In some embodiments, the method comprises the following steps:

[0011] (1) Raw material pretreatment: Fresh eggs are washed, and egg white and yolk are separated. The egg white is homogenized at 4°C for 20 min. The pH value of the egg white solution is adjusted to 6.0 using a pH 6.0, 50 mM sodium acetate buffer. The supernatant is saved for later use after 8000 rpm refrigerated centrifugation for 15 min and the precipitate is discarded. The pH of the supernatant is adjusted to 6.0 using 1 M NaOH. Weakly acidic cation exchange resin is added according to the ratio of egg white solution to resin of 4:1. After static adsorption for 2 h, filtration is performed to obtain a lysozyme-removed egg white solution. The pH of the lysozyme-removed egg white solution is adjusted to 6.0, and SP-Sepharose chromatography column is used for elution. The OVT-containing protein solution is collected, concentrated by ultrafiltration, and freeze-dried to obtain OVT.

[0012] (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);

[0013] (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);

[0014] (4) Simulated adult gastrointestinal digestion: 40 mL SGF and 50 mL, 10 mg / mL OVT solution are mixed, 5 mL pepsin (2000 U / mL) is added, HCl (6 mol / L) is used to adjust the pH value to 2.0, ultrapure water is added to make the volume reach 100 mL, and then incubation is carried out for 2 h (37°C, 150 rpm); the pH value is adjusted to 7.0 to terminate the gastric digestion; then 80 mL SIF solution, 25 mL bile salt (10 mM) and 5 mL trypsin (100 U / mL) are added, HCl (6 mol / L) is used to adjust the pH value to 7.0, and then ultrapure water is added to make the volume reach 200 mL, and incubation is carried out for 2 h (37°C, 150 rpm); after each step of digestion, boiling is carried out for 10 min to inactivate the enzyme, then centrifugation is carried out, dialysis (molecular weight cut-off of 300 Da) is carried out, and the liquid in the dialysis bag is collected as OVT adult hydrolysate (OIA) for digestion property and intestinal immune activity determination;

[0015] The second technical solution provided by the application is a high immune activity OVT hydrolysate prepared by the method of the first technical solution, wherein the hydrolysate contains HTEGSTT, TTSY and / or RTAGWVIPMG. Compared with the current research, the adult simulated digestion product is prepared, which is more comprehensive.

[0016] The third technical solution provided by the present application is a product containing the OVT hydrolysate described in the second technical solution.

[0017] In some embodiments, the product comprises a pharmaceutical product.

[0018] The fourth technical solution provided by the present application is the use of the OVT hydrolysate described in the second technical solution in the preparation of a product for improving intestinal health.

[0019] In some embodiments, the pharmaceutical product at least has one of the following effects:

[0020] (1) promoting the generation of short-chain fatty acids in the individual;

[0021] (2) relieving the swelling damage of the small intestinal mucosa tissue of the individual.

[0022] In some embodiments, the short-chain fatty acids include acetic acid, propionic acid and butyric acid.

[0023] Compared with the prior art, the technical effects of the present application are as follows:

[0024] The present application constructs a simulated in-vitro 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 in-vitro 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.

[0025] Preparation method of infant simulated digestion product (OIB): mixed simulated gastric juice and ovotransferrin solution, added 250 U / mL pepsin, adjusted pH value to 2.0 with HCl, incubated for 2 h, adjusted pH value to 7.0 to terminate gastric digestion, and boiled to inactivate the enzyme; then took the above mixture, added simulated intestinal juice, 1.25 mM bile salt and 12.5 U / mL trypsin, adjusted pH value to 7.0 with HCl, incubated for 2 h, boiled to inactivate the enzyme, and then centrifuged, dialyzed, and collected the liquid in the dialysis bag as OVT infant hydrolysate. It is used for comparison with the function of the OVT adult simulated digestion product protected by the application to demonstrate the technical effectiveness. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure is a hydrolysis site and polypeptide sequence diagram of ovotransferrin. In the figure, OVT is ovotransferrin.

[0027] Figure 2 Figure is a pathological change diagram of mouse small intestine tissue section.

[0028] Figure 3 Figure is a diagram of the content change of short-chain fatty acids in mouse feces. In the figure, OVT is ovotransferrin, OIA is ovotransferrin in vitro simulated adult digestion product, and OIB is ovotransferrin in vitro simulated infant digestion product.

[0029] Figure 4 Figure is a diagram of the content change of various acids in mouse feces. In the figure, OVT is ovotransferrin, OIA is ovotransferrin in vitro simulated adult digestion product, and OIB is ovotransferrin in vitro simulated infant digestion product. DETAILED DESCRIPTION

[0030] 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.

[0031] Test method: (1) analysis of digestion characteristics: the digestion product was diluted to 5 mg / mL with deionized water, filtered through a 0.45 μm membrane, and analyzed by ESI-MS / MS combined with online HPLC. The instrument parameter settings are shown in Table 1. The chromatogram of ovotransferrin digestion product was analyzed using MassLynx V4.1 software combined with proteomics difference analysis method, and the structural differences of the digestion products of the two were analyzed. Then the chromatogram was imported into Progenesis QI combined with shared database (ChemSpider Search and Share Chemistry) to analyze the difference peptide sequence, combined with mass spectrometry database (Swissprot-1.0), and the potential immunomodulatory peptide confidence sequence was analyzed and verified from the difference peptide sequence of the simulated infant and adult digestion products of OVT.

[0032] Table 1 HPLC-ESI-MS / MS working parameter settings

[0033]

[0034] (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, and 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.

[0035] Table 2 GC-MS analysis parameter settings

[0036]

[0037] (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.

[0038] (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.

[0039] Raw materials used in the examples: 1, pepsin (P7000) from Sigma-Aldrich Company, USA.

[0040] 2. Pancreatin (P7545) was purchased from Sigma-Aldrich, USA.

[0041] 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.

[0042] Example 1 Preparation of OVT hydrolysate: The specific steps are as follows:

[0043] (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. Weakly acidic cation exchange resin was added according to the ratio of egg white solution to resin of 4:1. After static adsorption for 2 h, the solution was filtered to obtain egg white solution without lysozyme. The pH value of the egg white solution without lysozyme was adjusted to 6.0, and the solution was eluted using an SP-Sepharose chromatographic column. The protein solution containing OVT was collected, concentrated by ultrafiltration, and freeze-dried to obtain OVT.

[0044] (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.

[0045] (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.

[0046] (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 the pH value was adjusted to 2.0 with HCl (6 mol / L). 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, the pH value was adjusted to 7.0 with HCl (6 mol / L), and the volume was made up to 200 mL with ultrapure water. 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 liquid in the dialysis bag was collected as the OVT adult hydrolysate (OIA) after dialysis (molecular weight cut-off: 300 Da);

[0047] 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 15 high-predictive-activity 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.

[0048] Table 3: Polypeptide identification results of OVT simulated adult gastrointestinal digestion product (OIA)

[0049]

[0050] Note: * indicates glycopeptide

[0051] Table 4. Polypeptide identification results of OVT simulated infant gastrointestinal digestion products (OIB)

[0052]

[0053] Note: *marked as glycopeptides

[0054] Example 3. Effects of OVT simulated infant and adult digestion products on intestinal pathological histomorphology in mice: 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 strict compliance with the international guidelines for laboratory 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 indicators of the barrier environment meet 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 the object, and an immunosuppressed mouse model was established by intraperitoneal injection of cyclophosphamide.

[0055] 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 were free to ingest standard animal feed and drinking 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 were given 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. Twelve 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.

[0056] 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 neat, but a certain degree of vacuolar deformation was still observed. Compared with the OVT group, the mice in the OIB group showed slight improvement in intestinal histomorphology, while the OIA group showed significant improvement. This result indicates that OVT, after digestion, may enhance the overall immune status by improving mucosal immune function, and OIA shows a more effective immune-enhancing effect than OIB.

[0057] Example 4 Effects of OVT simulated infant and adult digestion products on short-chain fatty acids in mouse feces: animal model construction and grouping

[0058] Short-chain fatty acids, as the main metabolites of intestinal microorganisms, 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 Figure 4 Figure 4 ​​As 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.

[0059] 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 adult gastrointestinal simulated digestion system, characterized by, Ovotransferrin is processed by the following steps: a) mixing simulated gastric juice with ovotransferrin solution, adding pepsin, adjusting pH to 2.0-3.5, and incubating at 37°C for 2-4 hours; b) adjusting pH to 7.0 to terminate gastric digestion, and boiling to inactivate the enzyme; c) adding simulated intestinal juice, bile salts and pancreatin, adjusting pH to 7.0-8.0, and incubating at 37°C for 2-4 hours, and then boiling to inactivate the enzyme; d) centrifuging, dialyzing with a molecular weight cut-off of 300 Da, and collecting the liquid in the dialysis bag to obtain OVT adult hydrolysate, and the polypeptide identification results of the hydrolysate are as follows: KTSCHTGLG, QAFEAGL, HTG, GST*, KDEKS, DGKGDV, LDGGQAF, DLTTQOE, SMRKD, TTSY*, RIS LTKCLFKVR VPSL, DLF, LDKGKGDV, CDRWSV, wherein the * marks a glycopeptide; the simulated gastric juice 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, CaCl2·2H2O 0.01-0.02 mL; the simulated intestinal juice 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, CaCl2·2H2O 0.08-0.12 mL; the ovotransferrin is a purified protein isolated from fresh chicken eggs, and is obtained by elution from an SP-Sepharose column after removal of lysozyme by a weakly acidic cation exchange resin.

2. A highly immunologically active ovotransferrin hydrolysate prepared based on an adult gastrointestinal model digestion system prepared by the method of claim 1, characterized in that, The hydrolysate has the functions of promoting the generation of short-chain fatty acids and relieving the swelling and damage of small intestinal mucosal tissue.

3. Use of a high immunologically active ovotransferrin hydrolysate prepared on the basis of an adult gastrointestinal simulated digestion system according to claim 2 for the preparation of a medicinal product for improving intestinal health, characterized in that, The drug product at least has one of the following effects: promoting the generation of short-chain fatty acids in an individual, and the short-chain fatty acids include acetic acid, propionic acid and butyric acid.

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