Protein hydrolysate with gastric mucosa protection effect, small molecule protein hydrolysate and application

Abstract

The invention relates to a proteolysis product with a gastric mucosa protection effect, a micromolecular proteolysis product and application. The present invention relates to a proteolysis product having a gastric mucosa protective effect, which is obtained by performing enzymolysis on a mycoprotein dispersion liquid using papain to obtain a solution in which the proteolysis product having the gastric mucosa protective effect is dispersed. The proteolysis product provided by the invention has a gastric mucosa protection effect, and especially has obvious protection and repair effects on ethanol-induced gastric mucosa injury; in addition, the raw materials are easy to obtain, safe and reliable, the preparation method is simple, no harmful substance is introduced, and batch production is easy. The proteolysis product provided by the invention has homology of medicine and food, also has a gastric mucosa protection effect, and can be used as a health-care product with an auxiliary protection function on gastric mucosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteolysis, and particularly relates to a proteolytic product with gastric mucosa protection effect, a small molecule proteolytic product with gastric mucosa protection effect and their uses. Background Art

[0002] Gastric mucosal injury refers to pathological changes such as inflammation, erosion, ulcer, etc. caused by the invasion of various pathogenic factors on the gastric mucosa. Helicobacter pylori infection, long-term use of non-steroidal anti-inflammatory drugs (such as aspirin, ibuprofen, etc.), bad eating habits (such as overeating, long-term eating of spicy and stimulating foods, etc.), alcohol stimulation, stress states (such as severe trauma, major surgery, etc.) may all lead to damage to the gastric mucosa. After gastric mucosal injury, patients may experience symptoms such as upper abdominal pain, abdominal distension, nausea, vomiting, loss of appetite, acid reflux, belching, etc. If the injury is relatively severe, it can lead to complications such as gastric bleeding and gastric perforation, and even endanger life.

[0003] Gastric mucosal injury, as the core pathological manifestation of digestive tract diseases such as gastritis and gastric ulcer, its repair and protection mechanisms are the research focuses in the medical field. Commonly used drugs for treating gastrointestinal diseases, such as omeprazole, which is a proton pump inhibitor that can effectively inhibit gastric acid secretion, and is used for gastric and duodenal ulcers, reflux or erosive esophagitis, Zollinger-Ellison syndrome, etc. However, although similar gastric mucosal protectants can relieve symptoms, they have problems such as low bioavailability and doubts about long-term use safety.

[0004] There is a need in this field to develop a health product or food of food origin for preventing and / or repairing gastric mucosal injury and preventing gastrointestinal diseases. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, one of the purposes of the present invention is to provide a proteolytic product with gastric mucosa protection effect. By using papain to enzymatically hydrolyze the bacterial cell protein dispersion, a solution in which the proteolytic product with gastric mucosa protection effect is dispersed is obtained.

[0006] The proteolytic product provided by the present invention is derived from bacterial cell protein (such as the bacterial cell protein of yeast), and has the effect of repairing the damaged gastric mucosa of ethanol-induced rats, and can repair the damaged gastric mucosa, especially the gastric mucosa damaged by alcohol. It is speculated that papain may produce substances beneficial to gastric mucosa repair after enzymatically hydrolyzing bacterial cell protein, achieving the effect of repairing the damaged gastric mucosa.

[0007] Preferably, the bacterial cell protein includes yeast protein, preferably including Saccharomyces cerevisiae protein.

[0008] The microbial protein described in the present invention can be understood as microbial cells (or microorganisms), for example, yeast (exemplarily including Saccharomyces cerevisiae, etc.). After culturing, fermenting, and centrifuging, the microbial cell raw materials are collected, the yeast cells are broken, the cell wall fragments are removed, the protein is precipitated, and then dried to obtain the microbial protein.

[0009] Preferably, the concentration of the microbial protein dispersion is 5.8 - 6.7%, such as 5.9%, 6.1%, 6.2%, 6.3%, 6.4%, 6.6%, etc.

[0010] If the concentration of the microbial protein dispersion is too high, the contact between papain and the microbial protein is likely to be uneven, affecting the yield of the proteolysate; if the concentration of the microbial protein dispersion is too low, the contact between papain and the microbial protein is insufficient, also affecting the yield of the proteolysate.

[0011] Preferably, the molecular weight of the papain is 23 - 25 kDa, such as 23.3 kDa, 23.8 kDa, 24.2 kDa, 24.6 kDa, 24.9 kDa, etc.

[0012] Preferably, the activity of the papain is 15 - 250,000 u / g, such as 170,000 u / g, 190,000 u / g, 230,000 u / g, 250,000 u / g, 290,000 u / g, etc.

[0013] Preferably, the addition amount of the papain is 0.28% - 0.32% of the mass of the microbial protein, such as 0.29%, 0.30%, 0.31%, etc.

[0014] An appropriate addition amount of papain can improve the effect of the proteolysate on protecting the gastric mucosa, probably because an appropriate addition amount of papain can increase the content of the effective substances in the proteolysate that protect the gastric mucosa.

[0015] Preferably, the temperature of the enzymatic hydrolysis is 50 - 55 °C, such as 52 °C, 53 °C, 54 °C, 55 °C, etc., and the enzymatic hydrolysis time is 1.2 - 1.8 h, such as 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, etc.

[0016] Appropriate enzymatic hydrolysis temperature and time can also improve the effect of the proteolysate on protecting the gastric mucosa, probably because appropriate enzymatic hydrolysis conditions can increase the content of the effective substances in the proteolysate that protect the gastric mucosa.

[0017] Preferably, the proteolysate contains any one or at least two combinations of the following peptide chains: LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, or TGVIKPGMVVT.

[0018] Under normal circumstances, in the protease hydrolysate, the peptide chains LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, and TGVIKPGMVVT all exist.

[0019] Preferably, the solution dispersed with the protease hydrolysate having a gastric mucosa protection effect is dried to obtain a protease hydrolysate powder having a gastric mucosa protection effect.

[0020] Preferably, the drying temperature ≤ 90 °C, such as 85 °C, 80 °C, 76 °C, 72 °C, 68 °C, 60 °C, 57 °C, 53 °C, 47 °C, 40 °C, 35 °C, 32 °C, 28 °C, 23 °C, 18 °C, 12 °C, 8 °C, 3 °C, -4 °C, -10 °C, -14 °C, -18 °C, -25 °C, -28 °C, -35 °C, -38 °C, -45 °C, -50 °C, -60 °C, -80 °C, -90 °C, -100 °C, -120 °C, etc., preferably 10 to -80 °C.

[0021] Preferably, the drying includes any one or a combination of at least two of freeze-drying, drying, and air-drying.

[0022] As a specific embodiment, the protease hydrolysate having a gastric mucosa protection effect described in the present application is prepared by the following steps:

[0023] (1) Add the cell protein to water at a material-liquid mass ratio of 1:15, and heat it in a water bath to 50 - 55 °C to obtain a cell protein aqueous dispersion.

[0024] (2) Add papain to the cell protein aqueous dispersion, maintain the temperature at 50 - 55 °C for enzymatic hydrolysis. After enzymatic hydrolysis for 1.2 - 1.8 h, quickly raise the temperature to 85 - 90 °C and maintain it for 10 - 20 min for inactivation to obtain a cell protein protease hydrolysate solution, which is the solution dispersed with the protease hydrolysate having a gastric mucosa protection effect. The addition amount of papain is 0.28% - 0.32% of the mass of the cell protein.

[0025] Optionally, (3) freeze-dry the solution dispersed with the protease hydrolysate having a gastric mucosa protection effect to obtain a protease hydrolysate having a gastric mucosa protection effect.

[0026] The second object of the present application is to provide a small molecule protease hydrolysate having a gastric mucosa protection effect, which is prepared by the following method:

[0027] Obtain a solution dispersed with the protease hydrolysate having a gastric mucosa protection effect described in the first object, and then intercept the component with a molecular weight ≤ 5 kDa to obtain a solution dispersed with a small molecule protease hydrolysate having a gastric mucosa protection effect;

[0028] The solution of the proteolysate with gastric mucosa protection effect described in one of the purposes includes the solution of the proteolysate with gastric mucosa protection effect dispersed as described in one of the purposes, or the reconstitution solution of the small molecule proteolysate with gastric mucosa protection effect described in one of the purposes.

[0029] Optionally, in order to smoothly intercept the molecular weight, it is preferred to centrifuge the solution of the proteolysate before intercepting the molecular weight, and then subject the supernatant to the treatment of intercepting the molecular weight ≤ 5 kDa.

[0030] Preferably, the solution of the small molecule proteolysate with gastric mucosa protection effect is dried to obtain a proteolysate powder with gastric mucosa protection effect.

[0031] Preferably, the drying temperature ≤ 90 °C, such as 85 °C, 80 °C, 76 °C, 72 °C, 68 °C, 60 °C, 57 °C, 53 °C, 47 °C, 40 °C, 35 °C, 32 °C, 28 °C, 23 °C, 18 °C, 12 °C, 8 °C, 3 °C, -4 °C, -10 °C, -14 °C, -18 °C, -25 °C, -28 °C, -35 °C, -38 °C, -45 °C, -50 °C, -60 °C, -80 °C, -90 °C, -100 °C, -120 °C, etc.

[0032] Preferably, the drying includes any one or a combination of at least two of freeze-drying, spray-drying, baking, and air-drying.

[0033] Preferably, the proteolysate contains any one or a combination of at least two of the following peptide chains: LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, or TGVIKPGMVVT;

[0034] Preferably, the sum of the relative mass contents of LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, and TGVIKPGMVVT in the proteolysate ≥ 6%, such as 6.23%, 6.58%, 6.83%, 7.05%, 7.36%, 7.55%, 7.72%, 7.86%, etc.

[0035] The third purpose of this application is to provide a use of a proteolysate with gastric mucosa protection effect as described in one of the purposes or a small molecule proteolysate with gastric mucosa protection effect as described in the second purpose, and the proteolysate or small molecule enzymolysate is used as any one or a combination of at least two of health product additives, healthy food additives, or drugs.

[0036] Preferably, the health products include health products with auxiliary protection function for gastric mucosa.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The proteolytic product provided by this application has a gastric mucosa protection effect, especially for ethanol-induced gastric mucosa injury, there is an obvious protection and repair effect; in addition, the raw materials of the present invention are easily available, safe and reliable, the preparation method is simple, no harmful substances are introduced, and it is easy to mass-produce.

[0039] (2) The proteolytic product provided by this application has the homology of medicine and food, and at the same time has a gastric mucosa protection effect, and can be used as a health product with an auxiliary protection function for the gastric mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Presents macroscopic observation photos of the gastric tissue damage of rats in the blank control group, model group, positive control group, experimental group A and experimental groups 1-6;

[0041] Figure 2 Presents macroscopic observation photos of the gastric tissue damage of rats in comparative experimental groups 1-2. DETAILED DESCRIPTION OF THE INVENTION

[0042] The following further explains the technical solution of the present invention in conjunction with the specific embodiments. It should be noted that the specific embodiments are only a specific implementation and interpretation of the essence of the technical solution of the present invention, and should not be understood as a limitation of the protection scope of the present invention.

[0043] The reagents and instruments used in the examples can all be purchased from commercially available products, and the detection methods are conventional methods well known in the art.

[0044] Example 1

[0045] A proteolytic product with a gastric mucosa protection effect is prepared by the following steps:

[0046] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to the material-liquid mass ratio of 1:15, and stir evenly to obtain a cell protein aqueous dispersion;

[0047] (2) Add papain (enzyme activity 200,000 u / g, commercially purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.) to the cell protein aqueous dispersion, maintain the temperature at 55 °C for enzymatic hydrolysis, after enzymatic hydrolysis for 1.5 h, quickly raise the temperature to 90 °C, and maintain for 15 min for inactivation to obtain a cell protease hydrolysis solution, which is a solution in which a proteolytic product with a gastric mucosa protection effect is dispersed. The addition amount of papain is 0.30% of the cell protein mass;

[0048] (3) Centrifuge the solution in which the proteolysate with gastric mucosa protection effect is dispersed at 4 °C (10 min, 8000 r / min) to obtain the supernatant after centrifugation. Then filter the supernatant through a 5 kDa filter membrane (20 °C, 0.2 MPa), and intercept the fraction with a molecular weight ≤ 5 kDa to obtain the proteolysate solution with gastric mucosa protection effect. Freeze-dry this solution (drying temperature -80 °C) to obtain the small molecule proteolysate powder 1# with gastric mucosa protection effect (yield 15.0%, calculated based on yeast protein as 100%).

[0049] Example 2

[0050] A proteolysate with gastric mucosa protection effect is prepared by the following steps:

[0051] (1) Add Saccharomyces cerevisiae protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, and add potable water according to the solid-liquid mass ratio of 1:16, and stir evenly to obtain the cell protein aqueous dispersion.

[0052] (2) Add papain (enzyme activity 200,000 u / g) to the cell protein aqueous dispersion, maintain the temperature at 50 °C for enzymatic hydrolysis. After enzymatic hydrolysis for 1.8 h, quickly raise the temperature to 90 °C and maintain it for 15 min for inactivation to obtain the cell protease hydrolysis solution. The addition amount of papain is 0.32% of the mass of the cell protein.

[0053] (3) Centrifuge the solution in which the proteolysate with gastric mucosa protection effect is dispersed at 4 °C (10 min, 8000 r / min) to obtain the supernatant after centrifugation. Then filter the supernatant through a 5 kDa filter membrane (20 °C, 0.2 MPa), and intercept the fraction with a molecular weight ≤ 5 kDa to obtain the proteolysate solution with gastric mucosa protection effect. Freeze-dry this solution (drying temperature -80 °C) to obtain the small molecule proteolysate powder 2# with gastric mucosa protection effect (yield 15.5%, calculated based on yeast protein as 100%).

[0054] Example 3

[0055] A proteolysate with gastric mucosa protection effect is prepared by the following steps:

[0056] (1) Add Saccharomyces cerevisiae protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, and add potable water according to the solid-liquid mass ratio of 1:14, and stir evenly to obtain the cell protein aqueous dispersion.

[0057] (2) Add papain (enzyme activity: 200,000 u / g) to the mycoprotein aqueous dispersion, maintain the temperature at 55 °C for enzymatic hydrolysis. After 1.2 h of enzymatic hydrolysis, quickly raise the temperature to 90 °C and maintain for 15 min for inactivation to obtain the mycoprotein hydrolysate. The addition amount of papain is 0.32% of the mass of mycoprotein.

[0058] (3) Centrifuge (10 min, 8000 r / min) the solution in which the proteolytic product with gastric mucosa protection effect is dispersed at 4 °C to obtain the supernatant after centrifugation. Then filter the supernatant through a 5 kDa filter membrane (20 °C, 0.2 MPa), and intercept the components with a molecular weight ≤ 5 kDa to obtain the proteolytic product solution with gastric mucosa protection effect. Freeze-dry this solution (drying temperature: -80 °C) to obtain the small-molecule proteolytic product powder 3# with gastric mucosa protection effect (yield: 14.5%, calculated based on yeast protein as 100%).

[0059] Example 4

[0060] A proteolytic product with gastric mucosa protection effect is prepared by the following steps:

[0061] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to the material-liquid mass ratio of 1:15, and stir evenly to obtain the mycoprotein aqueous dispersion.

[0062] (2) Add papain (enzyme activity: 200,000 u / g, commercially purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.) to the mycoprotein aqueous dispersion, maintain the temperature at 55 °C for enzymatic hydrolysis. After 1.5 h of enzymatic hydrolysis, quickly raise the temperature to 90 °C and maintain for 15 min for inactivation to obtain the mycoprotein hydrolysate, which is the solution in which the proteolytic product with gastric mucosa protection effect is dispersed. The addition amount of papain is 0.30% of the mass of mycoprotein.

[0063] (3) Freeze-dry the solution in which the proteolytic product with gastric mucosa protection effect is dispersed (drying temperature: -80 °C) to obtain the proteolytic product powder 4# with gastric mucosa protection effect (yield: 96.2%, calculated based on yeast protein as 100%).

[0064] Example 5

[0065] A proteolytic product with gastric mucosa protection effect is prepared by the following steps:

[0066] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to the material-liquid mass ratio of 1:10, and stir evenly to obtain the mycoprotein aqueous dispersion.

[0067] (2) Add papain (enzyme activity: 200,000 u / g, commercially purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.) to the mycelial protein aqueous dispersion, maintain the temperature at 50 °C for enzymatic hydrolysis. After 2.5 h of enzymatic hydrolysis, quickly raise the temperature to 90 °C and maintain it for 15 min for inactivation to obtain the mycelial protein hydrolysate. The addition amount of papain is 0.35% of the mass of the mycelial protein;

[0068] (3) Freeze-dry the dispersed mycelial protein hydrolysate (drying temperature: -80 °C) to obtain the protease hydrolysate powder 5# with gastric mucosa protection effect (yield: 96.8%, calculated based on yeast protein as 100%);

[0069] Example 6

[0070] A protease hydrolysate with gastric mucosa protection effect is prepared by the following steps:

[0071] (1) Add Saccharomyces cerevisiae protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to the material-liquid mass ratio of 1:25, and stir evenly to obtain the mycelial protein aqueous dispersion;

[0072] (2) Add papain (enzyme activity: 200,000 u / g, commercially purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.) to the mycelial protein aqueous dispersion, maintain the temperature at 55 °C for enzymatic hydrolysis. After 1 h of enzymatic hydrolysis, quickly raise the temperature to 90 °C and maintain it for 15 min for inactivation to obtain the mycelial protein hydrolysate. The addition amount of papain is 0.25% of the mass of the mycelial protein;

[0073] (3) Freeze-dry the dispersed mycelial protein hydrolysate (drying temperature: -180 °C) to obtain the protease hydrolysate powder 6# with gastric mucosa protection effect (yield: 95.2%, calculated based on yeast protein as 100%);

[0074] Comparative Example 1

[0075] A protease hydrolysate with gastric mucosa protection effect is prepared by the following steps:

[0076] (1) Add Saccharomyces cerevisiae protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to the material-liquid mass ratio of 1:15, and stir evenly to obtain the mycelial protein aqueous dispersion;

[0077] (2) Add bromelain (enzyme activity: 500,000 u / g, commercially purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.) to the mycelial protein aqueous dispersion, maintain the temperature at 55 °C for enzymatic hydrolysis. After 1.5 h of enzymatic hydrolysis, quickly raise the temperature to 90 °C and maintain it for 15 min for inactivation to obtain the mycelial protein hydrolysate, which is the solution of the protease hydrolysate with gastric mucosa protection effect after dispersion. The addition amount of papain is 0.30% of the mass of the mycelial protein;

[0078] (3) Freeze-dry the solution in which the proteolysate with gastric mucosa protection effect is dispersed (drying temperature: -180°C) to obtain proteolysate powder X1 (yield: 96.2%, calculated based on yeast protein as 100%).

[0079] Comparative Example 2

[0080] A proteolysate with gastric mucosa protection effect is prepared by the following steps:

[0081] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to the mass ratio of material to liquid of 1:15, and stir evenly to obtain a dispersed liquid of cell protein in water.

[0082] (2) Add flavorzyme and lipase to the dispersed liquid of cell protein in water (the dosage ratio of enzymes is 2:3, commercially purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.), maintain the temperature at 55°C for enzymatic hydrolysis. After 1.5 h of enzymatic hydrolysis, quickly raise the temperature to 90°C and maintain it for 15 min for inactivation to obtain cell protease hydrolyzate solution, which is the solution in which the proteolysate with gastric mucosa protection effect is dispersed. The addition amount of papain is 0.30% of the mass of cell protein.

[0083] (3) Freeze-dry the solution in which the proteolysate with gastric mucosa protection effect is dispersed (drying temperature: -80°C) to obtain proteolysate powder X2 (yield: 94.7%, calculated based on yeast protein as 100%).

[0084] Performance test:

[0085] (1) Polypeptide identification:

[0086] Use a nano liquid chromatography-mass spectrometry instrument (liquid chromatography model: Vanquish Horizon UHPLC, mass spectrometer model: Orbitrap Exploris 480) to identify the polypeptide components of the proteolysate solutions in Examples 1-4. The test conditions are as follows:

[0087] (a1) Extraction of sample protein: Weigh 200 mg of proteolysate powder, add 800 μL of 0.1 M HCl solution, grind and homogenize the sample, centrifuge the ground sample for 20 min (12000 rpm, 4°C), aspirate the supernatant, add 3 times the volume of ethanol, shake well, and precipitate the protein overnight at 4°C. Then centrifuge the sample again for 20 min (12000 rpm, 4°C), accurately aspirate the supernatant and freeze-dry it. Dissolve the freeze-dried sample with an appropriate volume of ultrapure water (desalting volume) to fully dissolve the freeze-dried peptide, and filter it through an ultrafiltration tube (centrifuge at 14000 rpm, 4°C for 10 min) to remove macromolecular proteins in the sample.

[0088] (a2) Desalting of the polypeptide component: Activate and equilibrate the C18 solid-phase extraction column, enrich the polypeptide solution obtained in the above step into the extraction column, aspirate 100 μL of 2% acetonitrile and 0.1% formic acid to wash the salt in the sample; then aspirate 50 μL of 50% acetonitrile and 0.1% formic acid, repeat 2 times, and collect the eluate with an EP tube. Place the collected eluate in a rotary vacuum dryer, under the condition of not heating, vacuum dry it, and store it at -80 °C for standby. Repeat 3 times.

[0089] (a3) UHPLC-MS / MS analysis: Dissolve the polypeptide component in 10 μL of 0.1% FA solution. The U3000 reversed-phase liquid phase conditions are as follows: nanoViper C18 chromatographic column (75 μm × 250 mm, 2 μm), mobile phase A is 99.9% H2O and 0.1% formic acid, mobile phase B is 80% acetonitrile, 19.9% H2O, and 0.1% formic acid. The elution gradient is 0 - 5 min, 0 - 1% B; 5 - 6 min, 1 - 5% B; 6 - 36 min, 5 - 10% B; 36 - 52 min, 10 - 25% B; 52 - 56 min, 25 - 35% B; 56 - 58 min, 35 - 90% B; 58 - 60 min, 90% B; the elution time is 60 min, and the flow rate is 1.2 μL / min. Use an Orbitrap Exploris 480 mass spectrometer to analyze and identify the polypeptide mixture, in high-sensitivity mode, and set the instrument parameters: each full scan is a high-speed signal-dependent scan, and the scan time is 60 min. The resolution of the first-level full scan is 120,000, the scan range is 350 - 1500 m / z, AGC is 3e6, and the maximum injection time is 25 ms; the resolution of the second-level scan is 15,000, AGC is 5e4, the collision energy is 30%, charge state screening (including precursors with +2 to +6 charges), and dynamic exclusion is 30 s.

[0090] To ensure the stability of the instrument and reduce systematic errors (such as temperature, humidity, cleanliness of the instrument, etc.), it is necessary to do a good job in quality control guarantee, including the total ion current of the mass spectrum Figure 1 Consistency assessment and parallel quality control of experimental processing.

[0091] After identification, Examples 1 - 6 all contain the peptide chains LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, and TGVIKPGMVVT, and the contents of the peptide chains LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, and TGVIKPGMVVT in Examples 1 - 3 are relatively high. Table 1 shows the relative contents obtained by normalized calculation:

[0092] Table 1

[0093] peptide chain Example 1 Example 2 Example 3 LPLQDVYKIG 2.56 2.93 2.10 LPTFGAKDYETHRVA 2.06 2.25 1.97 RVETGVIKPGMVVT 2.03 1.98 1.96 TGVIKPGMVVT 1.59 2.03 1.78

[0094] As can be seen from Table 1, the sum of the relative mass contents of LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, and TGVIKPGMVVT in Examples 1 to 3 is ≥ 6%.

[0095] (2) Gastric mucosa repair activity test

[0096] With reference to the "Notice No. 107

[2012] of the State Food and Drug Administration on the Evaluation Method for the Auxiliary Protection Function against Gastric Mucosal Injury", the gastric mucosa repair activity test was carried out, and the test is as follows:

[0097] Step (1) Animal experiment on gastric mucosal injury:

[0098] (1a) Animal feeding conditions and ethics

[0099] The experiment complied with the "Regulations on the Use of Laboratory Animals of the Beijing Laboratory Animal Management Committee" and was approved by the Animal Welfare and Ethics Committee of Beijing Technology and Business University (approval number: BTBU2024208). Rats (SD, male, 6 weeks old) were housed in IVC (independent ventilation system) cages in an SPF-class animal room, with a feeding temperature of 20 - 26°C, a humidity of 40 - 70%, and a light cycle of 12 hours of light and 12 hours of darkness alternating. After adaptive feeding, the rats started the formal gavage experiment. During the experiment, the rats had free access to food and water. After the feeding ended, they were randomly grouped according to body weight, including a blank control group (fed with rat breeding feed), a model group (induced acute gastric mucosal injury in rats with ethanol), a positive control group (gavaged with omeprazole), experimental group A (gavaged with the aqueous reconstituted solution F80 of yeast protein), experimental group 1 (gavaged with the aqueous reconstituted solution of the small molecule proteolysate powder 1# of Example 1), experimental group 2 (gavaged with the aqueous reconstituted solution of the small molecule proteolysate powder 2# of Example 2), experimental group 3 (gavaged with the aqueous reconstituted solution of the small molecule proteolysate powder 3# of Example 3), experimental group 4 (gavaged with the aqueous reconstituted solution of the proteolysate powder 4# of Example 4), experimental group 5 (gavaged with the aqueous reconstituted solution of the proteolysate powder 5# of Example 5), experimental group 6 (gavaged with the aqueous reconstituted solution of the proteolysate powder 6# of Example 6), comparative experimental group 1 (gavaged with the aqueous reconstituted solution of the proteolysate powder X1 of Comparative Example 1), and comparative experimental group 2 (gavaged with the aqueous reconstituted solution of the proteolysate powder X2 of Comparative Example 2); each group had 8 rats, and the results in Table 2 later were the numerical averages of 8 rats.

[0100] (1b) Rat gavage experiment

[0101] Research method: Based on the evaluation method for the auxiliary protection function against gastric mucosal injury in the document No. 107

[2012] of the State Food and Drug Administration of China for Health Food and Cosmetics, and further modified on this basis, the beneficial effects of yeast protein hydrolysate in gastric mucosal protection were demonstrated.

[0102] After 7 days of adaptive feeding, the rats started drug intervention and were continuously gavaged for 14 days. Specifically: The rats in the blank control group and the model group were gavaged with normal saline once a day; the rats in the positive control group were gavaged with normal saline once a day for the first 12 days, and gavaged with omeprazole drug once a day (140 mg / kg w) on the 13th and 14th days; the rats in experimental group A were gavaged with an aqueous reconstituted solution of yeast protein F80 (1500 mg / kg); the rats in experimental groups 1 - 3 were gavaged with an aqueous reconstituted solution of the small molecule protein hydrolysate powder obtained from the corresponding examples (300 mg / kg calculated as protein hydrolysate); the rats in experimental groups 4 - 6 were gavaged with an aqueous reconstituted solution of the protein hydrolysate powder obtained from the corresponding examples (1500 mg / kg calculated as protein hydrolysate); the rats in the comparative experimental groups were gavaged with an aqueous reconstituted solution of the protein hydrolysate powder obtained from the corresponding comparative examples (1500 mg / kg calculated as protein hydrolysate). After fasting for 12 h at night on the 14th day (without water restriction), on the 15th day, except for the blank control group, the rats in other groups (model group, positive control group, experimental groups 1 - 6, comparative experimental groups 1 - 2) were gavaged with 1 mL of absolute ethanol to induce acute gastric mucosal injury. After gavaging with absolute ethanol, meloxicam, an analgesic, was subcutaneously injected (dose 1 - 2 mg / kg). 1 h later, the rats were anesthetized with isoflurane by respiration.

[0103] (1c) Collection of rat gastric tissue

[0104] After the rats were euthanized, the abdominal cavity of the rats was opened, the intact stomach was exposed, the pylorus was ligated, the esophagus and duodenum were cut at both ends of the ligature, and the whole stomach was removed. After 10 min, it was cut along the greater curvature of the stomach. The gastric mucosal contents were washed clean with pre-cooled normal saline and then blotted dry with filter paper. The inner wall of the stomach was turned out to observe the damage of the gastric mucosa. At the same time, the bleeding points and bleeding bands, the length and width of the damage were measured, and pictures of the gastric mucosal damage were taken under a stereomicroscope to observe the gastric mucosal damage.

[0105] (1d) Determination of rat gastric mucosal injury

[0106] Under a stereomicroscope, the bleeding points and the lengths and widths of the bleeding bands in the gastric tissues were measured with a vernier caliper. The judgment methods for bleeding points and bleeding bands were as follows: ① Punctate bleeding: The hemorrhagic erosion points were less than 1 mm, and the number was calculated; ② Linear bleeding: When the length and width of the bleeding band were greater than 1 mm, measurement was required. Since the width represents the severity of the injury much more than the length, double scoring was used, and the scoring criteria are shown in Table 1. The degrees of gastric mucosal injury in each experimental group of rats were expressed by the injury incidence rate (%), the injury integral index, and the injury inhibition rate (the results are shown in Table 3), and the corresponding calculation formulas are shown as (1), (2), and (3):

[0107] Injury incidence rate (%) = the number of rats with bleeding or ulcers in a certain group / the number of rats in that group × 100% (1)

[0108] Injury integral index = the total injury score of the group / the number of animals in the group (2)

[0109] Injury inhibition rate (%) = (a - b) / a × 100% (3)

[0110] In formula (3), a and b are the injury integrals of the model group and the experimental group, respectively.

[0111] According to the above formulas, with Table 2 as the scoring criteria, through the measured gastric mucosal bleeding results of each group of rats, the injury data in Table 3 were calculated.

[0112] Table 2

[0113]

[0114] Results of the gastric mucosal injury and recovery experiment of rats gavaged with yeast protease hydrolysate in step (2)

[0115] (2a) Macroscopic observation results of gastric mucosal injury

[0116] The macroscopic observation results of gastric tissue injury in rats gavaged with different samples under a stereomicroscope were as Figure 1 ( Figure 1 Macroscopic observation photos of gastric tissue injury in rats of the blank control group, the model group, the positive control group, experimental group A, and experimental groups 1 - 6 were given, Figure 2 (macroscopic observation photos of gastric tissue injury in rats of comparative experimental groups 1 - 2 were given). The surface of the gastric mucosal tissue of rats in the blank control group was smooth, the folds were complete and the orientation was regular, and the color was light red. After ethanol induction, the gastric mucosa of rats in the model group showed severe bleeding, erosion, and the color turned red. Compared with the model group and comparative experimental groups 1 - 2, although the rats in the positive control group and experimental groups 1 - 6 were induced by ethanol, the hemorrhagic injury was significantly reduced, which played a protective role in gastric mucosal injury. However, the gastric mucosal injury of rats gavaged with the protease hydrolysate complex solution of comparative ratios 1 - 2 was not improved.

[0117] (2b) Results of gastric mucosa injury

[0118] For the rat gastric tissues intragastrically administered with different samples, the bleeding points and bleeding zones were counted according to the steps for measuring rat gastric mucosa injury, the injury inhibition rate was calculated, and the results were recorded in Table 3.

[0119] Table 3

[0120] sample injury incidence injury inhibition rate sample injury incidence injury inhibition rate blank control group — — experimental group 3 100％ 58.60％ model group 100％ — experimental group 4 100％ 50.00％ positive control group 100％ 32.0％ experimental group 5 100％ 45.73％ experimental group A 100％ 2.89％ experimental group 6 100％ 46.89％ experimental group 1 100％ 61.33％ comparative experimental group 1 100％ ＜0 experimental group 2 100％ 55.47％ comparative experimental group 2 100％ 10.22％

[0121] Note: In Table 3, an injury inhibition rate < 0 means that the gastric tissue injury after intragastric administration was more severe than that in the model group.

[0122] As can be seen from Table 3, the injury inhibition rate of the gastric tissues of the rats in the positive control group fed with omeprazole was 32.0%. The injury inhibition rates of the proteolysates with gastric mucosa repair activity provided in this application were all ≥ 45%, and the best one could reach 61.33%. Moreover, they were significantly higher than the injury inhibition rates of the gastric tissues in the comparative experimental group. This indicates that the proteolysates provided in this application have an effect on improving and protecting gastric mucosa injury that is close to that of omeprazole, and the effect is significantly higher than the injury inhibition rates of the gastric tissues of the single yeast protein (Experimental Group A), the proteolysate enzymatically hydrolyzed with bromelain, and the proteolysates enzymatically hydrolyzed with flavorzyme and lipase.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A proteolytic product with gastric mucosa protection function, characterized in that, The protease hydrolyzate is obtained by enzymatically hydrolyzing the thallus protein dispersion with papain to obtain a solution in which the protease hydrolyzate with gastric mucosa protection effect is dispersed.

2. The proteolytic product according to claim 1, characterized in that, The thallus protein includes yeast protein, preferably Saccharomyces cerevisiae protein. Preferably, the concentration of the thallus protein dispersion is 5.8-6.7%.

3. The proteolytic product according to claim 1 or 2, characterized in that, The molecular weight of the papain is 23-25 kDa. Preferably, the activity of the papain is 150,000-250,000 u / g. Preferably, the addition amount of the papain is 0.28%-0.32% of the mass of the thallus protein.

4. The protease hydrolyzate according to any one of claims 1 to 3, characterized in that, The temperature of the enzymatic hydrolysis is 50-55 °C, and the enzymatic hydrolysis time is 1.2-1.8 h.

5. The protease hydrolyzate according to any one of claims 1 to 4, characterized in that, The protease hydrolyzate contains any one or at least two combinations of the following peptide chains: LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, or TGVIKPGMVVT.

6. The protease hydrolyzate according to any one of claims 1 to 5, characterized in that, The solution in which the protease hydrolyzate with gastric mucosa protection effect is dispersed is dried to obtain a protease hydrolyzate powder with gastric mucosa protection effect. Preferably, the drying temperature ≤ 90 °C. Preferably, the drying includes any one or at least two combinations of freeze-drying, drying, and air-drying.

7. A small molecule proteolysate with gastric mucosa protective effect, characterized in that, It is prepared by the following method: Obtain a solution in which the protease hydrolyzate with gastric mucosa protection effect described in any one of claims 1-6 is dispersed, and then intercept the component with a molecular weight ≤ 5 kDa to obtain a solution in which the small molecule protease hydrolyzate with gastric mucosa protection effect is dispersed; the solution in which the protease hydrolyzate with gastric mucosa protection effect described in any one of claims 1-6 is dispersed includes the solution in which the protease hydrolyzate with gastric mucosa protection effect described in any one of claims 1-5 is dispersed, or the re-dissolved solution of the small molecule protease hydrolyzate with gastric mucosa protection effect described in claim 6.

8. The small molecule protease hydrolyzate according to claim 7, characterized in that, The solution in which the small molecule protease hydrolyzate with gastric mucosa protection effect is dispersed is dried to obtain a protease hydrolyzate powder with gastric mucosa protection effect. Preferably, the drying temperature ≤ 90 °C. Preferably, the drying includes any one or at least two combinations of freeze-drying, spray-drying, drying, and air-drying.

9. The small molecule protease hydrolyzate according to claim 7 or 8, characterized in that, The protease hydrolyzate contains any one or at least two combinations of the following peptide chains: LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, or TGVIKPGMVVT. Preferably, the sum of the relative mass contents of LPLQDVYKIG, LPTFGAKDYETHRVA, RVETGVIKPGMVVT, and TGVIKPGMVVT in the protease hydrolyzate ≥ 6%.

10. Use of a proteolysate having a gastric mucosa protective effect as described in any one of claims 1 to 6 or a small molecule proteolysate having a gastric mucosa protective effect as described in any one of claims 7 to 9, characterized in that The protease hydrolyzate or small molecule hydrolyzate is used as any one or at least two combinations of a health product additive, a health food additive, or a medicine. Preferably, the health product includes a health product with an auxiliary protection function for the gastric mucosa.

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