Protein hydrolysate with gastric mucosa repair activity and application

Abstract

The invention relates to a proteolysis product with gastric mucosa repair activity and application thereof. The protein zymolyte with gastric mucosa repair activity is prepared by the following method: firstly, carrying out primary enzymolysis on mycoprotein by using a first enzyme to obtain a primary enzymolysis solution, and then carrying out secondary enzymolysis on the primary enzymolysis solution by using a second enzyme to obtain a secondary enzymolysis solution, the secondary enzymatic hydrolysate is a solution in which the protein hydrolysate with gastric mucosa repair activity is dispersed; the first enzyme comprises aspergillus oryzae protease; the second enzyme comprises aspergillus niger lipase. The proteolysis product provided by the invention has the effects of increasing the antioxidant level of gastric tissues, improving the defense factor level of gastric mucosa and reducing the level of related inflammatory factors in serum, can play a role in protecting the gastric mucosa, has gastric mucosa repairing activity, and is safe and easily available in raw materials, simple in preparation method and easy for batch production.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteolysis, and particularly relates to a proteolysate with gastric mucosa repair activity and its use. Background Art

[0002] Gastrointestinal diseases include but are not limited to digestive dysfunction, gastric ulcer, acid regurgitation, gastroesophageal reflux, chronic gastritis, etc. Commonly used drugs for treating gastrointestinal diseases, such as omeprazole, are proton pump inhibitors that can effectively inhibit gastric acid secretion and are used for gastric and duodenal ulcers, reflux esophagitis or erosive esophagitis, Zollinger-Ellison syndrome, etc.

[0003] The treatment of gastric diseases is a long process, and most drugs have side effects after long-term intake. The greatest advantage of food or substances homologous to food and medicine lies in prevention, treating pre-disease or the pre-disease state, and can also play an auxiliary role in the treatment of diseases during the onset stage.

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

[0005] In view of the deficiencies of the prior art, one of the purposes of the present application is to provide a proteolysate with gastric mucosa repair activity, which is prepared by the following method:

[0006] First, the bacterial protein is subjected to primary enzymolysis with a first enzyme to obtain a primary enzymolysis solution, and then the primary enzymolysis solution is subjected to secondary enzymolysis with a second enzyme to obtain a secondary enzymolysis solution; the secondary enzymolysis solution is a solution in which the proteolysate with gastric mucosa repair activity is dispersed;

[0007] The first enzyme includes protease produced by Aspergillus oryzae; the second enzyme includes lipase produced by Aspergillus niger.

[0008] The proteolysate provided by the present application has the effect of repairing ethanol-induced gastric mucosal damage in rats and has the effect of protecting the gastric mucosa. It is speculated that substances beneficial to gastric mucosa repair may be produced by the bacterial protein under the sequential action of specific enzymes, achieving the repair of damaged gastric mucosa.

[0009] It should be noted that protease produced by Aspergillus oryzae is the protease produced by Aspergillus oryzae, and lipase produced by Aspergillus niger is the lipase produced by Aspergillus niger. The preparation processes of these two enzymes both belong to the prior art in this field. The present application does not make specific limitations, and those skilled in the art can prepare them by themselves according to background knowledge or obtain them commercially.

[0010] Preferably, the microbial protein includes yeast protein, preferably Saccharomyces cerevisiae protein.

[0011] It should be noted that the microbial protein described in this application is the microbial cells (or microorganisms), such as yeast (exemplarily including Saccharomyces cerevisiae, etc.). After cultivation, fermentation, and centrifugation, 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 it.

[0012] Preferably, the first enzyme further includes papain and / or bromelain.

[0013] It should be noted that in the first enzyme described in this application, the protease produced by Aspergillus oryzae is the main component, usually accounting for more than 80 wt%, and other types of proteases, such as papain and / or bromelain, can be appropriately mixed, and other excipients, such as glucose, can also be appropriately added.

[0014] Preferably, the addition amount of the first enzyme for the first enzymatic hydrolysis is 0.18% - 0.25% of the mass of the microbial protein, such as 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, etc.

[0015] Preferably, the addition amount of the second enzyme for the second enzymatic hydrolysis is 0.28% - 0.35% of the mass of the microbial protein, such as 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, etc.

[0016] Preferably, the mass ratio of the first enzyme to the second enzyme is 2:2.3 - 2:3.2, such as 2:2.4, 2:2.5, 2:2.6, 2:2.7, 2:2.8, 2:2.9, 2:3.0, 2:3.1, etc.

[0017] Preferably, the time for the first enzymatic hydrolysis is 0.4 - 0.8 h (such as 0.5 h, 0.6 h, 0.7 h, etc.), and the temperature for the first enzymatic hydrolysis is 48 - 55 °C (such as 49 °C, 50 °C, 51 °C, 53 °C, etc.).

[0018] Preferably, the time for the second enzymatic hydrolysis is 0.9 - 1.2 h (such as 1.0 h, 1.1 h, etc.), and the temperature for the first enzymatic hydrolysis is 35 - 50 °C (such as 36 °C, 38 °C, 42 °C, 45 °C, 48 °C, etc.).

[0019] Preferably, the microbial protein is dispersed in water at a mass ratio of 1:12 - 1:20 (such as 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, etc.) to form a microbial protein dispersion, and then the enzymatic hydrolysis of the microbial protein is carried out.

[0020] The proteolysate with gastric mucosa repair activity described in this application is a mixture obtained through a specific enzymatic hydrolysis process. First, the microbial protein (such as yeast protein) is enzymatically hydrolyzed once using the first enzyme (including protease produced by Aspergillus oryzae) to obtain a primary enzymatic hydrolysate, and then the primary enzymatic hydrolysate is enzymatically hydrolyzed a second time using the second enzyme (including lipase produced by Aspergillus niger) to obtain the product. It should be noted that the specific selection of the type, ratio, enzymatic hydrolysis time, enzymatic hydrolysis temperature, and the state of the microbial protein in the enzymes described in this application can improve the repair effect on damaged gastric mucosa. However, compared with the scheme where the protease described in this application is not used, the scheme without specific selection (type, ratio, enzymatic hydrolysis time, enzymatic hydrolysis temperature, and the state of the microbial protein) also has the effect of repairing damaged gastric mucosa, but it has no advantage compared with the scheme with specific selection (type, ratio, enzymatic hydrolysis time, enzymatic hydrolysis temperature, and the state of the microbial protein).

[0021] Preferably, the water is removed from the secondary enzymatic hydrolysate to obtain a powdered proteolysate.

[0022] Preferably, the method of removing water includes freeze-drying or spray-drying.

[0023] This application does not specifically limit the conditions of freeze-drying and spray-drying, and only gives the following exemplary examples:

[0024] Exemplarily, the vacuum degree of freeze-drying is 10 Pa to 30 Pa, the temperature is -80 °C to -100 °C, and the time is 3 to 6 days.

[0025] Exemplarily, the inlet temperature of spray-drying is ≥150 °C, the outlet temperature is 50 - 80 °C, the spraying speed is 2% - 6%, and the number of gas jetting times is 7 - 10 times / min.

[0026] As an optional method for preparing the proteolysate with gastric mucosa repair activity, it includes the following steps:

[0027] (1) Disperse the microbial protein in water to obtain a microbial protein dispersion;

[0028] (2) Add the first enzyme to the microbial protein dispersion for primary enzymatic hydrolysis to obtain a primary enzymatic hydrolysate; the first enzyme includes protease produced by Aspergillus oryzae;

[0029] (3) Add the second enzyme to the primary enzymatic hydrolysate for secondary enzymatic hydrolysis to obtain a secondary enzymatic hydrolysate, which is a solution containing the proteolysate with gastric mucosa repair activity; the second enzyme includes lipase produced by Aspergillus niger;

[0030] Optionally, (4) Dry the secondary enzymatic hydrolysate to remove water to obtain a powder of the proteolysate with gastric mucosa repair activity.

[0031] A second object of the present application is to provide a use of a proteolysate having gastric mucosa repair activity as described in the first object, and the proteolysate is used as any one or a combination of at least two of health product additives, health food additives or drugs.

[0032] Preferably, the health products include health products with an auxiliary protective function for the gastric mucosa.

[0033] Preferably, the dosage of the proteolysate having gastric mucosa repair activity is 200-300 mg / kg / day, such as 220 mg / kg / day, 240 mg / kg / day, 260 mg / kg / day, 280 mg / kg / day, 290 mg / kg / day, etc.

[0034] A third object of the present application is to provide a health product with an auxiliary protective function for the gastric mucosa, and the health product includes the proteolysate having gastric mucosa repair activity as described in the first object.

[0035] A fourth object of the present application is to provide a complex having gastric mucosa repair activity, including the proteolysate having gastric mucosa repair activity as described in any one of claims 1-6, hawthorn powder, yam powder, Poria powder and tangerine peel;

[0036] Preferably, the complex may further include maltodextrin;

[0037] Preferably, the complex includes the following components by weight:

[0038]

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) The proteolysate provided by the present application has the effects of increasing the antioxidant level of gastric tissue, improving the level of gastric mucosa defense factors, and reducing the level of related inflammatory factors in serum, can play a role in protecting the gastric mucosa, has gastric mucosa repair activity, the raw materials are safe and easy to obtain, and the preparation method is simple and easy for mass production.

[0041] (2) The proteolysate provided by the present application is derived from food, has gastric mucosa repair activity, and can be used as a health product with an auxiliary protective function for the gastric mucosa. Description of the Drawings

[0042] Figure 1 Photographs of macroscopic observation of gastric tissue damage in rats of the blank control group, model group, positive control group, experimental group A and experimental groups 1-12 are given;

[0043] Figure 2 Photographs of macroscopic observation of gastric tissue damage in rats of comparative experimental groups 1-5 are given. Detailed Embodiments

[0044] The technical solution of the present invention will be further explained below in conjunction with specific embodiments. It should be noted that the specific embodiments are only a specific implementation and explanation of the essence of the technical solution of the present invention, and should not be construed as a limitation on the protection scope of the present invention.

[0045] 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.

[0046] Example 1

[0047] A protease hydrolyzate with gastric mucosa repair activity is prepared by the following method:

[0048] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to a solid-liquid ratio of 1:15, and stir evenly to obtain a cell protein dispersion;

[0049] (2) Heat the cell protein dispersion to 50 °C, add protease produced by Aspergillus oryzae accounting for 0.20% of the mass of the cell protein, and enzymatically hydrolyze for 0.5 h to obtain a primary hydrolyzate;

[0050] (3) Maintain the primary hydrolyzate at 40 °C, add lipase produced by Aspergillus niger accounting for 0.30% of the mass of the cell protein, and enzymatically hydrolyze for 1 h. After the enzymatic hydrolysis is completed, quickly heat to 90 °C and maintain for 15 min to inactivate the enzyme activity, obtaining a secondary hydrolyzate;

[0051] (4) Freeze-dry the secondary hydrolyzate at a vacuum of 20 Pa and a temperature of -90 °C for 4 days to remove water, obtaining a protease hydrolyzate powder with gastric mucosa repair activity.

[0052] Example 2

[0053] A protease hydrolyzate with gastric mucosa repair activity is prepared by the following method:

[0054] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to a solid-liquid ratio of 1:12, and stir evenly to obtain a cell protein dispersion;

[0055] (2) Heat the cell protein dispersion to 55 °C, add protease produced by Aspergillus oryzae accounting for 0.18% of the mass of the cell protein, and enzymatically hydrolyze for 0.8 h to obtain a primary hydrolyzate;

[0056] (3) Maintain the primary hydrolyzate at 50 °C, add lipase produced by Aspergillus niger accounting for 0.28% of the mass of the cell protein, and enzymatically hydrolyze for 0.9 h. After the enzymatic hydrolysis is completed, quickly heat to 90 °C and maintain for 15 min to inactivate the enzyme activity, obtaining a secondary hydrolyzate;

[0057] (4) Freeze-dry the secondary enzymolysis solution for 4 days at a vacuum degree of 20 Pa and a temperature of -90 °C to remove moisture, obtaining a protease hydrolyzate powder with gastric mucosa repair activity.

[0058] Example 3

[0059] A protease hydrolyzate with gastric mucosa repair activity is prepared by the following method:

[0060] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to the reaction kettle, add potable water according to a material-liquid ratio of 1:20, and stir evenly to obtain a cell protein dispersion;

[0061] (2) Heat the cell protein dispersion to 48 °C, add protease produced by Aspergillus oryzae accounting for 0.25% of the mass of the cell protein, and enzymolyze for 0.8 h to obtain a primary enzymolysis solution;

[0062] (3) Maintain the primary enzymolysis solution at 35 °C, add lipase produced by Aspergillus niger accounting for 0.35% of the mass of the cell protein, enzymolyze for 1.2 h, and quickly heat to 90 °C after the enzymolysis ends, and maintain for 15 min to inactivate the enzyme, obtaining a secondary enzymolysis solution;

[0063] (4) Freeze-dry the secondary enzymolysis solution for 4 days at a vacuum degree of 20 Pa and a temperature of -90 °C to remove moisture, obtaining a protease hydrolyzate powder with gastric mucosa repair activity.

[0064] Examples 4 - 5

[0065] The difference from Example 1 is only that the addition amount of lipase produced by Aspergillus niger in step (3) is 0.32% (Example 4), 0.23% (Example 5) of the mass of the cell protein.

[0066] Example 6

[0067] The difference from Example 1 is only that the protease produced by Aspergillus oryzae in step (2) is equivalently replaced with a composite enzyme which is a mixture of protease produced by Aspergillus oryzae and papain with a mass ratio of 9:1.

[0068] Example 7

[0069] The difference from Example 1 is only that the protease produced by Aspergillus oryzae in step (2) is equivalently replaced with a composite enzyme which is a mixture of protease produced by Aspergillus oryzae, bromelain and papain with a mass ratio of 9:0.5:0.5.

[0070] Example 8

[0071] The difference from Example 1 is only that the addition amount of protease produced by Aspergillus oryzae is adjusted to 0.15% of the mass of the thallus protein, and the addition amount of lipase produced by Aspergillus niger is adjusted to 0.28% of the mass of the thallus protein.

[0072] Example 9

[0073] The difference from Example 1 is only that the addition amount of protease produced by Aspergillus oryzae is adjusted to 0.28% of the mass of the thallus protein, and the addition amount of lipase produced by Aspergillus niger is adjusted to 0.25% of the mass of the thallus protein.

[0074] Example 10

[0075] The difference from Example 1 is only that the enzymolysis time in step (2) is 0.2 h, and the enzymolysis time in step (3) is 1.5 h.

[0076] Example 11

[0077] The difference from Example 1 is only that the enzymolysis time in step (2) is 1.5 h, and the enzymolysis time in step (3) is 0.5 h.

[0078] Example 12

[0079] The difference from Example 1 is only in step (4): the secondary enzymolysis solution is spray-dried (the inlet temperature of the spray dryer is 155 °C, the outlet temperature is 60 °C, the spraying speed is 4%, and the number of gas jetting times is 8 times / min) to remove moisture, and a protease hydrolysate powder with gastric mucosa repair activity is obtained.

[0080] Comparative Example 1

[0081] The difference from Example 1 is only that step (3) is not carried out, specifically as follows:

[0082] A protease hydrolysate with gastric mucosa repair activity is prepared by the following method:

[0083] (1) Add Saccharomyces cerevisiae protein to the reaction kettle, add potable water according to a material-liquid ratio of 1:15, and stir evenly to obtain a thallus protein dispersion;

[0084] (2) Heat the thallus protein dispersion to 50 °C, add protease produced by Aspergillus oryzae at 0.20% of the mass of the thallus protein, and carry out enzymolysis for 0.5 h to obtain an enzymolysis solution;

[0085] (3) Carry out freeze-drying of the enzymolysis solution at a vacuum degree of 20 Pa and a temperature of -90 °C for 4 days to remove moisture, and obtain a protease hydrolysate powder with gastric mucosa repair activity.

[0086] Comparative Example 2

[0087] The difference from Example 1 is only that step (2) is not carried out, specifically as follows:

[0088] A protease hydrolyzate with gastric mucosa repair activity is prepared by the following method:

[0089] (1) Add brewer's yeast protein (yeast protein F80 commercially purchased from Angel Yeast) to a reaction kettle, add potable water according to a solid-liquid ratio of 1:12, and stir evenly to obtain a cell protein dispersion;

[0090] (2) Heat the cell protein dispersion to 50 °C, add lipase produced by Aspergillus niger accounting for 0.28% of the mass of the cell protein, and carry out enzymatic hydrolysis for 0.9 h. After the enzymatic hydrolysis is completed, quickly heat it to 90 °C and maintain it for 15 min to inactivate the enzyme, obtaining an enzymatic hydrolysate;

[0091] (4) Freeze-dry the enzymatic hydrolysate at a vacuum degree of 20 Pa and a temperature of -90 °C for 4 days to remove moisture, obtaining a protease hydrolyzate powder with gastric mucosa repair activity.

[0092] Comparative Examples 3 to 5

[0093] The differences from Example 1 are only that the brewer's yeast protein is replaced with chicken protein (commercially purchased, boiled and freeze-dried) (Comparative Example 3), whey protein (Fonterra) (Comparative Example 4), and soy protein (Shansong Biology) (Comparative Example 5).

[0094] Performance test: Gastric mucosa repair activity test

[0095] Refer to the evaluation method for the auxiliary protection function of gastric mucosa damage in the notice of the State Food and Drug Administration of China

[2012] No. 107 to conduct the gastric mucosa repair activity test. The test is as follows:

[0096] Step (1) Animal experiment for gastric mucosa injury:

[0097] (1) Animal feeding conditions and ethics

[0098] 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 Vital River Laboratory Animal Technology Co., Ltd. (approval number: PA23070601). Rats (Wistar, male, 6 weeks old) were housed in IVC (independent ventilation system) cages in an SPF-class animal room. The breeding temperature was 20-26°C, the humidity was 40-70%, and the light cycle was 12 hours of light alternating with 12 hours of darkness. After adaptive feeding, the rats began the formal gavage experiment. During the experiment, the rats had free access to food and water. After the breeding 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 yeast protein), experimental group 1 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 1), experimental group 2 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 2), experimental group 3 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 3), experimental group 4 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 4), experimental group 5 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 5), experimental group 6 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 6), experimental group 7 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 7), experimental group 8 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 8), experimental group 9 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 9), experimental group 10 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 10), experimental group 11 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 11), experimental group 12 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Example 12), comparative experimental group 1 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Comparative Example 1), comparative experimental group 2 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Comparative Example 2), comparative experimental group 3 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Comparative Example 3), comparative experimental group 4 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Comparative Example 4), comparative experimental group 5 (gavaged with the aqueous reconstituted solution of the protease hydrolyzate of Comparative Example 5); there were 8 rats in each group, and the results in Tables 2-4 below were the numerical averages of 8 rats.

[0099] (2) Rat gavage experiment

[0100] Research method: Based on the evaluation method for the auxiliary protection function of gastric mucosal injury in the document of the State Food and Drug Administration of China

[2012] No. 107, and further modified on this basis, it was proved the beneficial effect of yeast protease hydrolyzate in gastric mucosal protection.

[0101] After 7 days of adaptive feeding, the rats began to be administered with drug intervention for 14 consecutive days. Specifically, the blank control group and the model group were gavaged with normal saline once a day; the positive control group was gavaged with normal saline once a day for the first 12 days, and omeprazole (140 mg / kg w) was gavaged once a day on the 13th and 14th days; the experimental group A was gavaged with a water solution of yeast protein F80 (1500 mg / kg) every day; the experimental groups 1 to 12 were gavaged with a water solution of the proteolytic product obtained in the corresponding embodiment (1500 mg / kg in terms of proteolytic product) every day; the comparative experimental groups 1 to 5 were gavaged with a water solution of the proteolytic product obtained in the corresponding comparative example (1500 mg / kg in terms of proteolytic product) every day. After fasting for 24 h on the 15th day (without water withdrawal), on the 16th day, rats in other groups (model group, positive control group, experimental group 1 to experimental group 12, comparative experimental groups 1 to 5) except the blank control group were gavaged with 1 mL of anhydrous ethanol to induce acute gastric mucosal injury. After gavage with anhydrous ethanol, the analgesic meloxicam (dose 1 to 2 mg / kg) was subcutaneously injected, and the rats were anesthetized by isoflurane breathing 1 hour later.

[0102] (3) Collection of rat gastric tissue

[0103] After euthanasia, the rats were cut open in the abdominal cavity to expose the complete stomach, the pylorus was ligated, the esophagus and duodenum were cut off at both ends of the ligature, and the whole stomach was removed. After 10 minutes, it was cut open along the greater curvature of the stomach, the gastric mucosal contents were cleaned with pre-cooled saline and then dried with filter paper, the inner wall of the stomach was turned out to observe the damage of the gastric mucosa, and the bleeding points and bleeding bands, the length and width of the damage were measured at the same time, and the gastric mucosal damage pictures were taken under a stereo microscope to observe the gastric mucosal damage. The gastric tissue was then fixed with paraformaldehyde for H&E section staining.

[0104] (4) Assay of gastric mucosal injury in rats

[0105] Use a vernier caliper to measure the bleeding points of gastric tissue and the length and width of the bleeding band under a stereo microscope. The judgment method of bleeding points and bleeding bands is as follows: ① Point bleeding: the bleeding erosion points are smaller than 1mm, and the number is calculated; ② Strip bleeding: the length and width of the bleeding band are greater than 1mm and need to be measured. Because the severity of the injury represented by the width is much greater than the length, double points are assigned. The scoring criteria are shown in Table 1. The degree of gastric mucosal injury in rats in each experimental group is expressed by the injury incidence rate (%), injury score index and injury inhibition rate (the results are shown in Table 3), and the corresponding calculation formulas are shown in (1)(2)(3):

[0106] Injury incidence (%) = number of rats with bleeding or ulcers in a group / number of rats in the group × 100% (1)

[0107] Injury score index = total injury score of each group / number of animals in each group (2)

[0108] Inhibitory rate of injury (%) = (a - b) / a × 100% (3)

[0109] In formula (3), a and b are the injury scores of the model group and the experimental group, respectively.

[0110] According to the above formula, using Table 1 as the scoring standard, the injury data in Table 2 were calculated through the measured gastric mucosal bleeding results of rats in each group.

[0111] Table 1 Macroscopic Observation Scoring Standard for Acute Alcohol-induced Gastric Mucosal Injury

[0112]

[0113] Step (2) Observation of the histopathological results of gastric mucosal tissue by H&E staining of rat gastric tissue

[0114] (a) Dewax the paraffin section to water: sequentially place the section in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water.

[0115] (b) Hematoxylin staining: Immerse the section in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with differentiating solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0116] (c) Eosin staining: Immerse the section in gradient alcohols of 85% and 95% for dehydration for 5 min each, and stain in eosin staining solution for 5 min.

[0117] (d) Dehydration and mounting: Sequentially place the section in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and mount with neutral balsam.

[0118] Step (3) Experimental results of gastric mucosal injury and recovery in rats intragastrically administered with yeast protease solution

[0119] (a) Macroscopic observation results of gastric mucosal injury

[0120] The macroscopic observation results of gastric tissue injury in rats intragastrically administered with different samples under a stereomicroscope are as Figure 1 ( Figure 1 Macroscopic observation photos of gastric tissue injury in rats in the blank control group, model group, positive control group, experimental group A, and experimental groups 1 - 12 are given Figure 2Pictures of macroscopic observation of gastric tissue damage in rats of comparative experimental groups 1-5 are shown. The surface of the gastric mucosa tissue of rats in the blank control group is smooth, the folds are complete and regular in direction, and the color is light red. After ethanol induction, the gastric mucosa of rats in the model group shows severe bleeding, erosion, and the color turns red. Compared with the model group and comparative experimental groups 1-5, although rats in the positive control group and experimental groups 1-12 are induced by ethanol, the hemorrhagic injury is significantly reduced, which plays a protective role in gastric mucosa injury. However, the gastric mucosa injury of rats intragastrically administered with the protease hydrolysate complex solutions of Comparative Ratios 1-5 has not been improved, and even the bleeding and ulcers in Comparative Ratios 1 and 2 are more severe.

[0121] (b) Results of gastric mucosa injury

[0122] The bleeding points and bleeding bands of the gastric tissues of rats intragastrically administered with different samples were counted according to the steps for measuring gastric mucosa injury in rats, the injury inhibition rate was calculated, and the results were recorded in Table 2.

[0123] Table 2 Injury inhibition rates of gastric tissues of rats intragastrically administered with different samples

[0124]

[0125]

[0126] Note: In Table 2, an injury inhibition rate < 0 means that the gastric tissue injury after intragastric administration is more severe than that in the model group.

[0127] As can be seen from Table 2, the injury inhibition rate of the gastric tissue of rats in the positive control group fed with omeprazole is 38.2%. The injury inhibition rates of the protease hydrolysates with gastric mucosa repair activity provided by this application are all ≥ 17%, and the best can reach 26.68%. Moreover, it is significantly higher than the injury inhibition rate of the gastric tissue in the comparative experimental group, indicating that the protease hydrolysate provided by this application has an effect on improving and protecting gastric mucosa injury that is close to that of omeprazole, and the effect is significantly higher than that of single yeast protein (Experimental Group A), chicken protein, whey protein, and soy protein, as well as the injury inhibition rates of gastric tissue using protease produced by Aspergillus oryzae and lipase produced by Aspergillus niger alone.

[0128] (c) Determination of gastric tissue oxidative stress level and gastric mucosa defense factor level

[0129] To further reflect the degree of gastric tissue recovery, the oxidative stress levels and gastric mucosal defense factors of the gastric tissues of rats (blank control group, model group, positive group, experimental group A, experimental group 1, comparative experimental group 1, and comparative experimental group 2) intragastrically administered with different samples were measured. The expression levels of SOD (superoxide dismutase), GSH-Px (glutathione peroxidase), MDA (malondialdehyde), EGF (epidermal growth factor in gastric tissue), and NO (nitric oxide) in the gastric tissues of rats were tested. The test method was to use the corresponding enzyme-linked immunosorbent assay (ELISA) kit. Among them, the higher the SOD, the smaller the gastric injury; the higher the GSH-Px, the smaller the gastric injury; the lower the MDA, the smaller the gastric injury; the higher the EGF, the smaller the gastric injury; the higher the NO, the smaller the gastric injury. The test results are shown in Table 3:

[0130] Table 3 Measurement results of oxidative stress levels and gastric mucosal defense factor levels in gastric tissues

[0131]

[0132]

[0133] As can be seen from Table 3, the proteolysate with gastric mucosal repair activity provided by this application exerts a repair effect by increasing the contents of SOD, GSH-Px, EGF, and NO and decreasing the content of MDA.

[0134] (d) Determination of inflammatory cytokines in rat serum

[0135] To further reflect the degree of gastric tissue injury recovery, IL-6 (interleukin-6), IL-8 (interleukin-8), and TNF-α (tumor necrosis factor-α) in the sera of rats (blank control group, model group, positive group, experimental group A, experimental group 1, comparative experimental group 1, and comparative experimental group 2) intragastrically administered with different samples were measured. The lower the IL-6, the smaller the gastric injury; the lower the IL-8, the smaller the gastric injury; the lower the TNF-α, the smaller the gastric injury. The measurement method was enzyme-linked immunosorbent assay, and the measurement results are shown in Table 4.

[0136] Table 4 Measurement results of inflammatory cytokines

[0137] sample TNF-α, pg / mL IL-6, pg / mL IL-8, pg / mL blank control group 89.94 32.05 58.96 model group 130.54 49.77 95.27 positive group 94.71 39.06 76.15 experimental group A 110.75 42.30 80.99 experimental group 1 96.27 43.19 71.57 comparative experimental group 1 123.78 45.63 79.25 comparative experimental group 2 119.02 44.98 79.67

[0138] As can be seen from Table 4, the proteolysate with gastric mucosal repair activity provided by this application exerts a repair effect by decreasing the contents of the inflammatory factors TNF-α, IL-6, and IL-8 in the serum.

[0139] It can be seen from the test results that the yeast proteolysate provided by the present invention can increase the antioxidant level of gastric tissue (increase SOD and GSH-Px; decrease MDA), improve the level of gastric mucosal defense factors (increase EGF, NO), and decrease the level of related inflammatory factors in serum (TNF-α, IL-6, IL-8), and has the effect of protecting gastric mucosa, and the effect is close to that of omeprazole.

[0140] Application Example 1

[0141] A complex with the effect of repairing gastric mucosa, comprising the following components by weight parts:

[0142]

[0143] 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 enzyme having gastric mucosal repairing activity, characterized in that: The protein hydrolysate is prepared by the following method: Firstly, a bacterial protein is enzymatically hydrolyzed once with a first enzyme to obtain a primary enzymatic hydrolyzate, and then the primary enzymatic hydrolyzate is enzymatically hydrolyzed twice with a second enzyme to obtain a secondary enzymatic hydrolyzate; the secondary enzymatic hydrolyzate is a solution in which the proteolytic product having gastric mucosa repairing activity is dispersed; The first enzyme includes protease produced by Aspergillus oryzae; and the second enzyme includes lipase produced by Aspergillus niger.

2. The protein hydrolysate according to claim 1, wherein The bacterial protein includes yeast protein, preferably includes Saccharomyces cerevisiae protein; Preferably, the first enzyme further comprises papain and / or bromelain.

3. The protein hydrolysate according to claim 1 or 2, characterized in that The amount of the first enzyme added in the primary enzymatic hydrolysis is 0.18% to 0.25% of the bacterial protein mass; Preferably, the amount of the second enzyme added in the secondary enzymolysis is 0.28% to 0.35% of the bacterial protein mass; Preferably, the mass ratio of the first enzyme to the second enzyme is 2:2.3 to 2:3.

2.

4. The protein hydrolysate according to any one of claims 1 to 3, characterized in that: The time of the first enzymatic hydrolysis is 0.4 to 0.8 h, and the temperature of the first enzymatic hydrolysis is 48 to 55° C. Preferably, the time of the secondary enzymolysis is 0.9 to 1.2 h, and the temperature of the primary enzymolysis is 35 to 50°C.

5. The protein hydrolysate according to any one of claims 1 to 4, characterized in that: The bacterial protein is dispersed in water at a mass ratio of 1:12 to 1:20 to form a bacterial protein dispersion, and then the bacterial protein is enzymatically hydrolyzed.

6. The protein hydrolysate according to any one of claims 1 to 5, characterized in that: The secondary enzymatic hydrolysate is dehydrated to obtain a powdery enzymatic hydrolysate; Preferably, the method of removing moisture includes freeze drying or spray drying.

7. Use of the proteolytic enzyme hydrolysate having gastric mucosal repairing activity as claimed in any one of claims 1 to 6, characterized in that: The protein hydrolysate is used as any one of a health product additive, a health food additive or a medicine, or a combination of at least two of them; Preferably, the health care products include health care products that have auxiliary protective functions for gastric mucosa.

8. The use according to claim 7, characterized in that The usage amount of the proteolytic enzyme with gastric mucosal repairing activity is 200-300 mg / kg / day.

9. A health product with auxiliary protective function for gastric mucosa, characterized in that: The health product comprises the proteolysate with gastric mucosa repairing activity as claimed in any one of claims 1 to 6.

10. A compound having gastric mucosal repair activity, characterized in that: The complex comprises the proteolytic product with gastric mucosal repair activity as claimed in any one of claims 1 to 6, hawthorn powder, yam powder, tuckahoe powder and tangerine peel; Preferably, the complex may further include maltodextrin; Preferably, the composite comprises the following components by weight:

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