Hydrolyzed protein for improving intestinal barrier and preventing diarrhea and method for preparing the same

Abstract

The application discloses a hydrolyzed protein for improving intestinal barrier and preventing diarrhea and a preparation method thereof, and belongs to the technical field of microorganisms and enzymes. The whey protein solution is first subjected to trypsin enzymolysis, then subjected to TG enzyme crosslinking, and finally subjected to secondary hydrolysis by using Tenyo protease M, so that the prepared hydrolyzed protein has the effect of preventing diarrhea; further, the peptide screened from the hydrolyzed protein also has the function of improving intestinal barrier. The protein peptide prepared by using the method can improve the intestinal barrier function of the organism, reduce the change of osmotic pressure, and achieve the effect of preventing diarrhea.

Description

Technical Field

[0001] This invention relates to a hydrolyzed protein that improves the intestinal barrier and prevents diarrhea, and its preparation method, belonging to the field of microbial and enzyme technology. Background Technology

[0002] Diarrhea is a common digestive symptom, mainly caused by a variety of factors such as intestinal barrier damage, osmotic imbalance, pathogen infection, or malabsorption. Finding safe and effective nutritional interventions is crucial for infants, the elderly with impaired digestive function, postoperative patients, and individuals with intestinal dysfunction.

[0003] In existing technologies, hydrolysis using a single protein source such as whey protein is a common method for preparing hypoallergenic and easily absorbed formula foods, such as those for special medical purposes. For example, hydrolysis of large milk protein molecules can significantly reduce their allergenicity and help lower the incidence of diarrhea in infants and young children. Whey protein, due to its high digestibility and rich bioactive components such as glycomacropeptides, is considered to have the potential to regulate the gut microbiota.

[0004] Currently, there are products on the market that reduce allergenicity by hydrolyzing single milk protein sources. However, these technologies mostly focus on reducing allergenicity, and research on how to reduce intestinal osmotic pressure and actively repair intestinal barrier function while supplementing nutrients to prevent and assist in the treatment of diarrhea is insufficient. Some products, after supplementing protein source nutrients, can easily lead to an increase in intestinal osmotic pressure, causing diarrhea in people with vulnerable intestines.

[0005] Patent CN109619358A discloses a solid functional beverage based on soybean peptide protein powder and its preparation method. However, this patent mainly achieves the effect of intestinal health by adding prebiotics such as polysaccharides and cellulose to promote the growth and reproduction of beneficial bacteria in the human body. The prebiotics used, such as lactitol, are not easily digested and absorbed in the gastrointestinal tract, and will still increase the osmotic pressure of intestinal contents, leading to diarrhea.

[0006] Therefore, supplementing nutrients while reducing the osmotic pressure of intestinal contents and actively repairing the intestinal barrier function to prevent and assist in the treatment of diarrhea is of great research significance. Summary of the Invention

[0007] To address the aforementioned problems, this application provides a hydrolyzed protein that improves the intestinal barrier and prevents diarrhea, and a method for preparing the same. In this invention, a whey protein solution is first hydrolyzed with trypsin, then cross-linked with TG enzyme, and finally hydrolyzed a second time using Amano proteinase M. The resulting hydrolyzed protein has an anti-diarrheal effect. Furthermore, peptides screened from this hydrolyzed protein also improve intestinal barrier function. The protein peptides prepared using this method can improve the body's intestinal barrier function and reduce osmotic pressure changes, thereby achieving an anti-diarrheal effect.

[0008] The first objective of this invention is to provide an engineered Escherichia coli strain that expresses an amino acid sequence as shown in SEQ ID NO: 1 (IVQKPTPEGDLEILLQK), an amino acid sequence as shown in SEQ ID NO: 2 (LGEYGFQKFKDLGEEHFK), or an amino acid sequence as shown in SEQ ID NO: 3 (HLVDEPQKHLVDEPQNLIK).

[0009] The second objective of this invention is to provide a protein peptide that has the function of preventing and assisting in the treatment of diarrhea, wherein the protein peptide has an amino acid sequence as shown in SEQ ID NO: 1 (IVQKPTPEGDLEILLQK), an amino acid sequence as shown in SEQ ID NO: 2 (LGEYGFQKFKDLGEEHFK), or an amino acid sequence as shown in SEQ ID NO: 3 (HLVDEPQKHLVDEPQNLIK).

[0010] A third objective of this invention is to provide a method for preparing hydrolyzed protein, wherein the hydrolyzed protein contains the protein peptides described above, comprising the steps of:

[0011] (1) Dissolve whey protein in deionized water to obtain a protein solution. Adjust the pH to 7-8 and add 4000-5000 U / g whey protein of trypsin. After enzymatic hydrolysis at 35-40℃ for 3-4 h, inactivate the enzyme to obtain trypsin hydrolyzed protein intermediate.

[0012] (2) Adjust the pH of the intermediate product of trypsin hydrolysis to 7-8, add 5-10 U / g whey protein TG enzyme for cross-linking for 3-4 h; adjust the pH to 8-9 again, add 500-1000 U / g whey protein Amano protease M for enzymatic hydrolysis and inactivation to obtain hydrolyzed protein.

[0013] In one embodiment, the trypsin in step (1) is purchased from Sigma-Aldrich Trading Ltd.

[0014] The TG enzyme mentioned in step (2) was purchased from MedChemExpress; the Amano protease M enzyme was purchased from Amano Enzyme, Japan.

[0015] In one embodiment, the concentration of the protein solution in step (1) is 2-8% (w / v).

[0016] A fourth object of the present invention is to provide hydrolyzed proteins prepared by any of the methods described above.

[0017] The fifth objective of this invention is to provide a method for improving the antidiarrheal properties of hydrolyzed protein, wherein the improvement of the antidiarrheal properties of hydrolyzed protein is achieved by increasing the abundance of the aforementioned protein peptides, comprising the steps of:

[0018] (1) Dissolve whey protein in deionized water to obtain a protein solution. Adjust the pH to 7-8 and add 4000-5000 U / g whey protein of trypsin. After enzymatic hydrolysis at 35-40℃ for 3-4 h, inactivate the enzyme to obtain trypsin hydrolyzed protein intermediate.

[0019] (2) Adjust the pH of the intermediate product of trypsin hydrolysis to 7-8, add 5-10 U / g whey protein TG enzyme for cross-linking for 3-4 h; adjust the pH to 8-9 again, add 500-1000 U / g whey protein Amano protease M for enzymatic hydrolysis and then inactivate the enzyme to obtain hydrolyzed protein.

[0020] The trypsin in step (1) was purchased from Sigma-Aldrich Trading Co., Ltd.; the TG enzyme in step (2) was purchased from MedChemExpress; and the Amano protease M enzyme was purchased from Amano Enzyme, Japan.

[0021] In one embodiment, the concentration of the protein solution in step (1) is 2-8% (w / v).

[0022] A sixth object of the present invention is to provide a medicine containing the protein peptides or hydrolyzed proteins described above.

[0023] In one embodiment, the drug may further contain a derivative of a protein peptide, which is a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation of the amino acid side chain group, amino terminus, or carbonyl terminus of the protein peptide.

[0024] In one embodiment, the drug further contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0025] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0026] In one embodiment, the dosage form of the drug includes oral dosage form, injectable dosage form, and inhaled dosage form.

[0027] In one embodiment, the oral dosage form includes tablets, capsules, granules, oral liquids, and oral suspensions;

[0028] The injectable dosage forms include injection solutions and injection powders for injection;

[0029] The inhalation formulations include aerosols and powder inhalers;

[0030] A seventh object of the present invention is to provide the use of the protein peptides described above, or the hydrolyzed proteins described above, or any of the methods described above, in the preparation of products for the prevention or adjunctive treatment of diarrhea, said products including pharmaceuticals.

[0031] The eighth object of the present invention is to provide a method for constructing the above-described engineered Escherichia coli, comprising the steps of: chemically synthesizing a DNA fragment encoding an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and introducing Nde I and Xho I restriction enzyme sites at its 5' and 3' ends, respectively; double-digesting the synthesized fragment and the pET22b(+) vector with the corresponding restriction endonuclease and then recovering the fragment; ligating the fragment with T4 DNA ligase to construct recombinant plasmids pET22b-SEQ1, pET22b-SEQ2 or pET22b-SEQ1;

[0032] The above recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, and positive transformants were selected to obtain engineered Escherichia coli strains expressing the sequences IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, or HLVDEPQKHLVDEPQNLIK.

[0033] Beneficial effects

[0034] (1) The hydrolyzed protein obtained by first hydrolyzing with trypsin, then cross-linking with TG enzyme, and then performing secondary enzymatic hydrolysis with Amano protease M can enhance the TEER value of intestinal epithelial cells, reduce the fluorescence transmittance of cells, and increase the expression of tight junction protein ZO-1, thereby improving intestinal barrier function, benefiting intestinal health, and thus achieving the purpose of adjuvant treatment and prevention of diarrhea.

[0035] (2) The present invention further analyzes the peptides in the hydrolyzed protein and screens out peptides IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, and HLVDEPQKHLVDEPQNLIK. These peptides have anti-digestive properties when they first enter the intestine. After effectively stimulating intestinal epithelial cells and improving intestinal function, they are recognized and hydrolyzed by intestinal peptidase. They have a high affinity for PepT1, which improves protein bioavailability and reduces the proportion of unused amino acid deamination, thereby avoiding a sharp increase in osmotic pressure and having excellent anti-diarrheal effect. Attached Figure Description

[0036] Figure 1 This refers to the ability of intestinal peptidases to recognize and degrade long peptides. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0038] Raw material source:

[0039] The whey protein was purchased from Fonterra, New Zealand, and the product name was WPC 80.

[0040] Trypsin was purchased from Sigma-Aldrich Trading Ltd.

[0041] Amano Protease M was purchased from Amano Enzyme, Japan, and the model name is Protease M "Amano" SD.

[0042] The TG enzyme was purchased from MedChemExpress.

[0043] The measurement methods involved in the examples are as follows:

[0044] 1. Osmotic pressure measurement

[0045] Hydrolyzed protein samples were digested using the INFOOGEST 2.0 in vitro digestion model. 50 μL samples were taken every 30 minutes. Using a freezing point osmometer, the sample was added to the sample tube, the instrument automatically froze and measured the freezing point, and the osmotic pressure was directly read. The formula for the colloid osmotic pressure reduction rate is as follows:

[0046]

[0047] Wherein, P0 is the colloidal osmotic pressure of the sample that is not cross-linked with PBA-CS during the same period, and P1 is the colloidal osmotic pressure of the sample that is cross-linked with PBA-CS.

[0048] 2. TEER value determination

[0049] Caco-2 cells were cultured and seeded into Transwell chambers. Using a Millicell ERS-2 epithelial physiometer, electrodes were inserted into the upper and lower chambers of the Transwell to measure the barrier tightness by reading the resistance values.

[0050] 3. Transmittance of fluorescent yellow in cells

[0051] Fluorescein was used as a marker. After incubation at the top of the Transwell, the basal side liquid was collected, and the absorbance was measured using a fluorescence spectrophotometer to detect the effect of the cross-linked proteolytic solution on the permeability of the Caco-2 monolayer epithelial barrier. The concentration of fluorescein was calculated based on the standard curve. The formula is as follows:

[0052]

[0053] 4. ZO-1 expression detection

[0054] Total protein was extracted from different groups of Caco-2 cells using a total protein extraction kit; the samples were quantified using a BCA protein detection kit; and the expression of tight junction protein ZO-1 was detected using Western blotting.

[0055] 5. Diarrhea performance test

[0056] Mice were divided into groups to establish the model, and different samples were administered by gavage. After 1 hour, the number of excluded particles and the time of glass bead excretion were counted.

[0057] 6. Peptide abundance detection

[0058] The relative abundance of specific peptides, i.e., the response value of liquid chromatography-tandem mass spectrometry, was identified using an ultra-high performance liquid chromatography-tandem mass spectrometer and PEAKS Studio.

[0059] 7. Protein digestion assay

[0060] The in vitro digestion model INFOOGEST 2.0 was used to digest the hydrolyzed protein samples, and the digestibility was determined by Kjeldahl nitrogen determination.

[0061] 8. Intestinal peptidase recognition ability

[0062] The brush border membrane sacs of the mouse small intestine were extracted and prepared into a 1.0 mg / mL suspension. 1 mg / mL of peptide was added and incubated at 37℃ for 15 min. The degradation rate of the peptide was detected by LC-MS / MS.

[0063] 9. Determination of PepT1 affinity

[0064] Caco-2 cells were cultured on Transwell plates to form a dense monolayer. The peptide solution was hydrolyzed with 0.2% (w / v) intestinal peptidase and then mixed with 3 mM glycyl-sarcosine (Gly-Sar) to inhibit the peptide concentration required for 50% Gly-Sar transport.

[0065] 10. Determination of antioxidant capacity

[0066] The antioxidant capacity of medium- and long peptides was determined using DPPH and ABTS kits.

[0067] Example 1: Preparation of hydrolyzed protein for adjuvant treatment and prevention of diarrhea

[0068] The preparation method of hydrolyzed protein for adjuvant treatment and prevention of diarrhea is as follows:

[0069] Whey protein was dissolved in deionized water to prepare a 5% (w / v) protein solution, and the pH was adjusted to 7.0. Trypsin with a concentration of 4500 U / g whey protein was added, and the mixture was enzymatically hydrolyzed at 37°C for 4 h and then treated at 100°C for 10 min to inactivate the enzyme, thus obtaining the trypsin-hydrolyzed protein intermediate.

[0070] Adjust the pH of the trypsin-hydrolyzed protein intermediate to 7.0, and add TG enzyme at 200 rpm for 3 h with 8 U / g whey protein.

[0071] The pH of the system was then adjusted to 8.5, and Amano protease M with a whey protein content of 800 U / g was added. After enzymatic hydrolysis at 40°C for 1 h, the enzyme was inactivated by treatment at 100°C for 10 min to obtain hydrolyzed protein.

[0072] Comparative Example 1: No TG enzyme treatment

[0073] The specific implementation method is the same as in Example 1, except that TG enzyme is not used in the enzymatic hydrolysis process, while the other steps remain the same to prepare hydrolyzed protein.

[0074] Comparative Example 2: Amano protease M was replaced with alkaline protease.

[0075] The specific implementation method is the same as in Example 1, except that the flavor protease is replaced with alkaline protease during the enzymatic hydrolysis process, while the other steps remain the same, to prepare hydrolyzed protein.

[0076] Comparative Example 3: Without Amano protease M

[0077] The specific implementation method is the same as in Example 1, except that flavor protease is not used in the enzymatic hydrolysis process, while the other steps remain the same to prepare hydrolyzed protein.

[0078] Example 2: Performance Measurement

[0079] The hydrolyzed proteins prepared in Example 1 and Comparative Examples 1-3 were used to determine their properties.

[0080] 1. Osmotic pressure measurement

[0081] The osmotic pressure measurement results of the hydrolyzed proteins prepared in Example 1 and Comparative Examples 1-3 are shown in Table 1.

[0082] Table 1

[0083]

[0084] Table 1 shows that: In Comparative Example 1, without cross-linking treatment, the hydrolyzed protein exposed more sites to Amano protease M, resulting in a higher osmotic pressure; In Comparative Example 2, after replacing Amano protease M with alkaline protease, the alkaline protease, with its wider hydrolysis sites, could still recognize many sites after cross-linking treatment, leading to an increase in osmotic pressure; In Comparative Example 3, omitting the secondary enzymatic hydrolysis step, the hydrolyzed protein contained fewer peptides and free amino acids, resulting in a lower osmotic pressure.

[0085] 2. Determination of intestinal permeability marker content

[0086] After Caco-2 cells formed a monolayer epithelial cell barrier, the cells were randomly divided into 6 groups:

[0087] The normal group consisted of 200 μL of blank serum.

[0088] The diarrhea group was given 100 μL of blank serum and 100 μL of 1 μmol / L trypsin.

[0089] Implementation 1 involved adding 100 μL of serum containing 0.015 g / mL of hydrolyzed protein from Example 1 and 100 μL of 1 μmol / L trypsin.

[0090] Comparison groups 1-3 were prepared by adding 100 μL of serum containing 0.015 g / mL of hydrolyzed protein from comparison groups 1, 2, and 3, and 100 μL of trypsin at 1 μmol / L, respectively.

[0091] After 24 h of cell treatment, the TEER value, fluorescence transmittance, and expression level of the surface tight junction protein ZO-1 of the Caco-2 cell monolayer model were measured.

[0092] The TEER values ​​of the Caco-2 cell monolayer model are shown in Table 2. The results show that no cross-linking treatment was performed in Comparative Example 1, and no secondary enzymatic digestion was performed in Comparative Example 3. The hydrolyzed protein obtained had little effect on the TEER value of the cells. In Comparative Example 2, alkaline protease was used instead of Amano protease M, and the hydrolyzed protein obtained had a limited increase in the TEER value.

[0093] Table 2 TEER value determination

[0094]

[0095] Note: P < 0.01 compared with the normal control group; ## P < 0.01, compared with the diarrhea group; x ± s, n = 3

[0096] The results of measuring the transmittance of fluorescent yellow and the expression of the surface tight junction protein ZO-1 in the Caco-2 cell monolayer model are shown in Table 3. The results show that the hydrolyzed protein obtained by non-crosslinking treatment in Comparative Example 1 could not stimulate ZO-1 expression and achieve the function of repairing intestinal barrier function. The hydrolyzed proteins obtained by Comparative Examples 2 and 3 had limited stimulation of ZO-1 expression and the effect was not as significant as that of Example 1.

[0097] Table 3

[0098]

[0099] Note: P < 0.01 compared with the normal control group; ## P < 0.01, compared with the diarrhea group; x ± s, n = 3

[0100] 3. Detection of a rat IBS diarrhea model

[0101] The rat IBS diarrhea model was constructed by referring to the literature "The effect of paeoniflorin on irritable bowel syndrome and the influence of intestinal epithelial cell barrier function". Abnormal defecation in rats was induced by restraint stress stimulation method.

[0102] Specifically, healthy male SD rats were housed separately in cages for one week in a room with varying day and night light conditions and a room temperature of 22°C, with free access to food and water. The rats were fasted for 24 hours prior to the experiment. After being anesthetized with ether, their anterior shoulders, forearms, and chests were restrained with paper tape. A 3 mm diameter glass bulb was placed in the rat's rectum, 3 cm from the anus. The rat was then quickly moved to its cage, which was lined with clean filter paper. Timing began after the rat awoke and spontaneously rolled into a supine position; the restraint time was 1 hour. The number of fecal pellets and the time it took for the glass bulb to be expelled were recorded within 1 hour. An increase in the frequency of defecation or a decrease in the defecation time indicated successful model establishment.

[0103] The rats were divided into 7 groups:

[0104] The normal group consisted of healthy rats, who were administered 10 mL / kg of physiological saline by gavage each time.

[0105] The diarrhea group consisted of rats with diarrhea, which were administered 10 mL / kg of physiological saline by gavage each time.

[0106] The treatment group consisted of rats with diarrhea, who were administered loperamide hydrochloride at a concentration of 0.17 mg / mL via gavage each time.

[0107] Group 1 consisted of rats with diarrhea, who were administered 10 mL / kg of the hydrolyzed protein from Example 1 via gavage each time.

[0108] Group 1 consisted of rats with diarrhea, who were given 10 mL / kg of hydrolyzed protein from Comparative Example 1 by gavage each time.

[0109] The two groups of rats were treated with diarrhea, and each time they were administered 10 mL / kg of hydrolyzed protein from the control group 2 via gavage.

[0110] The three groups of rats were diarrheal rats, and each time they were given 10 mL / kg of hydrolyzed protein from the control group 3 by gavage.

[0111] Mice in each group were administered the medication twice daily by gavage, with an 8-hour interval between doses, for 7 consecutive days. The number of fecal spots and the time of glass bulb expulsion were recorded for each group. The results for each group are shown in Table 4.

[0112] Table 4

[0113]

[0114] Note: P < 0.01, compared with the normal group; # P < 0.05 ## P < 0.01, compared with the diarrhea group; x ± s, n = 7

[0115] As shown in Table 4, compared with the normal group, the number of fecal particles in the diarrhea group was significantly increased (P<0.01), the excretion time was significantly reduced (P<0.01), and the time to excrete glass beads was shortened from (34.55±9.32) min to (14±3.68) min. Compared with the diarrhea group, the number of fecal particles excreted in 1 hour in group 1 was significantly reduced, and the time to excrete glass beads was significantly prolonged. The results of comparison group 1 were similar to those of the diarrhea group. The treatment effects of comparison groups 2 and 3 were not significant.

[0116] 4. Protein digestibility

[0117] The results are shown in Table 5. The results show that Example 1, which underwent secondary enzymatic hydrolysis after cross-linking, was easily digested by the intestines; Comparative Example 1, which did not undergo cross-linking treatment, did not expose more enzymatic hydrolysis sites and had a lower digestibility; Comparative Example 3, which lacked a secondary enzymatic hydrolysis step, was even less easily digested.

[0118] Table 5 Protein digestibility

[0119]

[0120] Example 3: Identification and Performance Determination of Peptides in Antidiarrheal Hydrolyzed Protein

[0121] 1. Identification and synthesis of polypeptide sequences

[0122] The hydrolyzed protein prepared in Example 1 was subjected to peptidomics analysis.

[0123] The hydrolyzed protein obtained in Example 1 was centrifuged at 10000×g for 20 min at 4°C, and the supernatant was obtained by filtration. The supernatant was then centrifuged at 5000×g for 15 min using an ultrafiltration tube, and the molecular weight was cut off to 5000 Da. The filtrate was then dialyzed in 100 mL of ultrapure water for 24 h using a dialysis bag.

[0124] The hydrolyzed protein obtained by dialysis was chromatographically analyzed using a HiLoad 16 / 600 Superdex 30 pg column with 50 mM phosphate buffer (pH 7.0–7.5) as the mobile phase at a flow rate of 1 mL / min and an elution time of 90 min. The UV absorption peak was monitored at 220 nm. The fraction containing the target molecular weight range (1500–2500 Da) was collected and lyophilized to obtain a powder.

[0125] Fine purification was performed using a C18 column (250 mm × 4.6 mm, 5 μm particle size). The aqueous phase consisted of ultrapure water + 0.1% TFA; the organic phase consisted of acetonitrile + 0.1% TFA. The gradient elution program was as follows: initially, 5% B phase was maintained for 5 minutes to equilibrate; within 30-40 minutes, the B phase was linearly increased to 50%; during washing, the B phase was increased to 90% within 2 minutes and maintained for 5 minutes; finally, the B phase was rapidly reduced to 5% and reequilibrated for 10-15 minutes.

[0126] The lyophilized sample obtained from gel filtration was dissolved and loaded with a small amount of aqueous phase, and the above gradient was run. Fractions were collected manually or automatically based on the UV absorption peak (220 nm). Only the central portion of one peak was collected each time to ensure purity. The collected fractions were lyophilized to obtain white powdered peptides. Each peptide was validated using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0127] The UniProtr whey protein database was used for comparison to screen for components with high peptide abundance. Target components were usually eluted in organic phases of 20% to 40%, and their amino acid sequences were confirmed by tandem mass spectrometry (MS / MS).

[0128] The results are shown in Table 6. The protein hydrolysate includes peptides IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, and HLVDEPQKHLVDEPQNLIK.

[0129] Table 6

[0130]

[0131] 2. Detection of peptide properties

[0132] The peptides with the highest abundance, IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, and HLVDEPQKHLVDEPQNLIK, were synthesized and their performance was tested.

[0133] (1) Intestinal peptidase recognition ability

[0134] The results are as follows Figure 1 As shown, the results indicate that most of the target peptide was converted into dipeptides and tripeptides within 120 min, suggesting that the aforementioned long peptides can exist in the intestine for a period of time and can eventually be recognized and digested by intestinal peptidase.

[0135] (2) PepT1 affinity assay

[0136] The results are shown in Table 7. The results indicate that the IC values ​​of IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, HLVDEPQKHLVDEPQNLIK are...50 The concentrations were 0.30, 0.52, and 0.68 mM, respectively, indicating that low concentrations can occupy the Gly-Sar transport channel, suggesting that the above-mentioned long peptides have strong affinity after hydrolysis and can be rapidly absorbed.

[0137] Table 7

[0138]

[0139] (3) Determination of antioxidant capacity

[0140] The results are shown in Table 8. The results indicate that IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, HLVDEPQKHLVDEPQNLIK all exhibit good antioxidant capacity, with IVQKPTPEGDLEILLQK showing the best antioxidant capacity and exhibiting the best resistance to DPPH and ABTS. 50 The concentrations were 1.2 mg / mL and 0.9 mg / mL, respectively.

[0141] Table 8

[0142]

[0143] (4) Determination of intestinal permeability markers

[0144] After Caco-2 cells formed a monolayer epithelial cell barrier, the cells were randomly divided into 5 groups:

[0145] The normal group consisted of 200 μL of blank serum.

[0146] The diarrhea group was given 100 μL of blank serum and 100 μL of 1 μmol / L trypsin.

[0147] The IVQKPTPEGDLEILLQK group consisted of 100 μL of serum containing 0.05 g / mL IVQKPTPEGDLEILLQK and 100 μL of 1 μmol / L trypsin.

[0148] The LGEYGFQKFKDLGEEHFK group consisted of 100 μL of serum containing 0.05 g / mL LGEYGFQKFKDLGEEHFK and 100 μL of 1 μmol / L trypsin.

[0149] The HLVDEPQKHLVDEPQNLIK group consisted of 100 μL of serum containing 0.05 g / mL HLVDEPQKHLVDEPQNLIK and 100 μL of 1 μmol / L trypsin.

[0150] After 24 h of cell treatment, the TEER value, fluorescence transmittance, and expression level of the surface tight junction protein ZO-1 of the Caco-2 cell monolayer model were measured.

[0151] The results of the cell TEER value measurement are shown in Table 9, and the results of the fluorescent yellow transmittance and the relative expression level of ZO-1 are shown in Table 10. The cell experiment results show that the target peptide can effectively stimulate the expression of tight junction protein ZO-1, increase the cell TEER value, and reduce the fluorescent yellow transmittance.

[0152] Table 9

[0153]

[0154] Note: P < 0.01 compared with the normal control group; ## P < 0.01, compared with the diarrhea group; x ± s, n = 3

[0155] Table 10

[0156]

[0157] Note: P < 0.01 compared with the normal control group; ## P < 0.01, compared with the diarrhea group; x ± s, n = 3

[0158] (5) Detection of rat IBS diarrhea model

[0159] The model establishment method was the same as the rat IBS diarrhea model in Example 2. Rats were divided into 6 groups:

[0160] The normal group consisted of healthy rats, who were administered 10 mL / kg of physiological saline by gavage each time.

[0161] The diarrhea group consisted of rats with diarrhea, which were administered 10 mL / kg of physiological saline by gavage each time.

[0162] The treatment group consisted of rats with diarrhea, who were administered loperamide hydrochloride at a concentration of 0.17 mg / mL via gavage each time, at a dose of 10 mL / kg.

[0163] The IVQKPTPEGDLEILLQK group consisted of rats with diarrhea, who were administered 10 mL / kg of 50 mg / mL IVQKPTPEGDLEILLQK peptide solution by gavage each time.

[0164] The LGEYGFQKFKDLGEEHFK group consisted of rats with diarrhea, who were administered 10 mL / kg of 50 mg / mL LGEYGFQKFKDLGEEHFK peptide solution via gavage each time.

[0165] The HLVDEPQKHLVDEPQNLIK group consisted of rats with diarrhea, who were administered 10 mL / kg of 50 mg / mL HLVDEPQKHLVDEPQNLIK peptide solution by gavage each time.

[0166] Mice in each group were administered the medication twice daily by gavage, with an 8-hour interval between each administration, for 7 consecutive days. The number of fecal spots and the time of glass bulb expulsion were recorded for each group. The results for each group are shown in Table 11.

[0167] Table 11

[0168]

[0169] Note: P < 0.01, compared with the normal group; # P < 0.05 ## P < 0.01, compared with the diarrhea group; x ± s, n = 7

[0170] The results showed that peptides IVQKPTPEGDLEILLQK, LGEYGFQKFKDLGEEHFK, and HLVDEPQKHLVDEPQNLIK could effectively alleviate diarrhea symptoms in rats and had a certain anti-diarrheal effect.

[0171] (6) Relative abundance of peptides

[0172] The abundance of the target peptide in the hydrolyzed proteins of Example 1 and Comparative Examples 1-3 was further determined, and the results are shown in Table 12.

[0173] Table 12

[0174]

[0175] The results showed that in Comparative Example 1, the content of the target peptide in the secondary enzymatic hydrolysis product obtained without cross-linking of the hydrolyzed protein was extremely low, and therefore it could hardly improve the intestinal barrier function; in Comparative Example 2, the secondary enzymatic hydrolysis using alkaline protease resulted in a wider range of cleavage sites, leading to a low abundance of the target peptide in the product; in Comparative Example 3, the abundance of the target peptide obtained by omitting the secondary enzymatic hydrolysis was low.

[0176] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A protein peptide with preventive and adjunctive therapeutic effects on diarrhea, characterized in that, The protein peptide is an amino acid sequence such as IVQKPTPEGDLEILLQK as shown in SEQ ID NO: 1, LGEYGFQKFKDLGEEHFK as shown in SEQ ID NO: 2, or HLVDEPQKHLVDEPQNLIK as shown in SEQ ID NO:

3.

2. A method for preparing hydrolyzed protein, characterized in that, The hydrolyzed protein contains the protein peptide of claim 1, and includes the following steps: (1) Dissolve whey protein in deionized water to obtain a protein solution. Adjust the pH to 7-8 and add 4000-5000 U / g whey protein of trypsin. After enzymatic hydrolysis at 35-40℃ for 3-4 h, inactivate the enzyme to obtain trypsin hydrolyzed protein intermediate. (2) Adjust the pH of the intermediate product of trypsin hydrolysis to 7-8, add 5-10 U / g whey protein TG enzyme for cross-linking for 3-4 h; adjust the pH to 8-9 again, add 500-1000 U / g whey protein Amano protease M for enzymatic hydrolysis and then inactivate the enzyme to obtain hydrolyzed protein. The whey protein in question was purchased from Fonterra, New Zealand, and is designated as WPC 80.

3. The hydrolyzed protein prepared by the method of claim 2.

4. A medicine, characterized in that, The drug contains the protein peptide of claim 1 or the hydrolyzed protein of claim 3.

5. The medicine according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

6. The pharmaceutical product according to claim 4, characterized in that, The dosage forms of the medicine include oral dosage forms, injectable dosage forms, and inhaled dosage forms.

7. The use of the protein peptide of claim 1 or the hydrolyzed protein of claim 3 in the preparation of products for the prevention or adjunctive treatment of diarrhea, characterized in that, The products include pharmaceuticals.

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