Rice bran protein source antagonizes gastric mucosal damage peptide and preparation method and application thereof

Abstract

The application provides a rice bran protein source antagonistic gastric mucosa damage peptide and a preparation method and application thereof, and belongs to the technical field of bioactive peptide preparation.The preparation method of the rice bran protein peptide provided by the application comprises the following steps: mixing and hydrolyzing a rice bran protein powder solution with a complex protease to obtain a hydrolysate, and sequentially performing the steps of enzyme inactivation, membrane separation and freeze-drying on the hydrolysate to obtain the rice bran protein peptide.The rice bran protein peptide prepared by the method can significantly increase the IL-10 content in the stomach of a Helicobacter pylori infected person, reduce the contents of TNF-alpha, IL-6, TLR4, MyD88 and NF-kappa B in the stomach, has a significant effect of improving gastric mucosa damage caused by Helicobacter pylori infection, and reshapes the gastric mucosa barrier function.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, and particularly relates to a rice bran protein-derived peptide that antagonizes gastric mucosal damage, its preparation method, and its application. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori , H.pyloiri Helicobacter pylori (H. pylori) is a Gram-negative bacterium with extremely high pathogenicity, classified as a Group 1 carcinogen by the World Health Organization. H. pylori is a unique pathogen that can adhere to and colonize the human stomach, releasing virulence factors that damage the gastric mucosa, causing oxidative stress damage and inducing an inflammatory response, thus disrupting the gastric mucosal barrier function. Studies have found that individuals infected with H. pylori all experience varying degrees of gastritis. Without drug intervention, long-term H. pylori infection can exacerbate gastric mucosal damage, leading to diseases such as gastric ulcers, gastric metaplasia, and even gastric cancer.

[0003] Currently, there is no effective treatment for gastric mucosal damage caused by Helicobacter pylori infection. Treatment mainly involves taking antibiotics and other medications to alleviate the damage, but these medications inevitably cause side effects and may even trigger other related diseases. Therefore, it is essential to actively conduct research on safer, side-effect-free methods to improve gastric mucosal damage caused by Helicobacter pylori infection. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing a rice bran protein source antagonistic to gastric mucosal damage peptide. The rice bran protein source antagonistic to gastric mucosal damage peptide prepared by the method of the present invention has a significant effect on improving gastric mucosal damage and gastric inflammation caused by Helicobacter pylori infection.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing rice bran protein-based peptides that antagonize gastric mucosal damage, comprising the following steps: mixing and hydrolyzing rice bran protein powder solution with a complex protease to obtain hydrolysate; subjecting the hydrolysate to enzyme inactivation, membrane separation, and freeze-drying steps in sequence to obtain rice bran protein-based peptides that antagonize gastric mucosal damage.

[0006] Preferably, the amount of the compound protease added is 1g-2g of compound protease per 100g of rice bran protein powder.

[0007] Preferably, the enzyme activity of the complex protease is 30,000 U / g.

[0008] Preferably, the concentration of the rice bran protein powder solution is 10% to 14%.

[0009] Preferably, the hydrolysis temperature is 50℃~60℃, the hydrolysis time is 1.5h~2.5h, and the hydrolysis pH value is 6.5~7.5.

[0010] Preferably, the membrane separation is nanofiltration.

[0011] The present invention also provides a rice bran protein-derived peptide for antagonizing gastric mucosal damage prepared by any of the above preparation methods.

[0012] The present invention also provides the application of the above-mentioned rice bran protein source antagonistic peptide against gastric mucosal damage in the preparation of products for preventing and treating gastric mucosal damage caused by Helicobacter pylori infection.

[0013] The present invention also provides the application of the above-mentioned rice bran protein source antagonistic peptide against gastric mucosal damage in the preparation of products for preventing and treating gastric inflammation caused by Helicobacter pylori infection.

[0014] This invention also provides the application of the above-mentioned rice bran protein source antagonistic peptide against gastric mucosal damage in the preparation of products for preventing Helicobacter pylori colonization.

[0015] The beneficial effects of this invention are: This invention utilizes a complex protease to hydrolyze rice bran protein powder, obtaining rice bran protein-derived peptides that antagonize gastric mucosal damage, thus achieving high-value utilization of rice bran protein. The rice bran protein-derived peptides antagonizing gastric mucosal damage prepared using this method can significantly increase the IL-10 content in the stomach of Helicobacter pylori-infected individuals and decrease the content of TNF-α, IL-6, TLR4, MyD88, and NF-κB in the stomach, demonstrating a significant effect in improving gastric mucosal damage induced by Helicobacter pylori infection and reshaping the gastric mucosal barrier function. Attached Figure Description

[0016] Figure 1 The effect of rice bran protein peptides on the IL-6 content in the stomach of mice infected with Helicobacter pylori.

[0017] Figure 2 This study investigated the effect of rice bran protein peptides on the TNF-α content in the stomach of mice infected with Helicobacter pylori.

[0018] Figure 3 The effect of rice bran protein peptides on the IL-10 content in the stomach of mice infected with Helicobacter pylori.

[0019] Figure 4 The effect of rice bran protein peptides on the TLR4 content in the stomach of mice infected with Helicobacter pylori.

[0020] Figure 5 The effect of rice bran protein peptides on the MyD88 content in the stomach of mice infected with Helicobacter pylori.

[0021] Figure 6 The effect of rice bran protein peptides on the NF-κB content in the stomach of mice infected with Helicobacter pylori. Detailed Implementation

[0022] This invention provides a method for preparing rice bran protein-based peptides that antagonize gastric mucosal damage, comprising the following steps: mixing and hydrolyzing rice bran protein powder solution with a complex protease to obtain hydrolysate; subjecting the hydrolysate to enzyme inactivation, membrane separation, and freeze-drying steps in sequence to obtain rice bran protein-based peptides that antagonize gastric mucosal damage.

[0023] In this invention, the rice bran protein source antagonistic peptide against gastric mucosal damage is simply referred to as rice bran protein peptide. The rice bran protein powder used in this invention is preferably purchased from Shaanxi Panier Co., Ltd., and the concentration of the rice bran protein powder solution is preferably 10%~14% (w / v), more preferably 11%~13% (w / v), where w / v represents the mass ratio of rice bran protein powder to the volume ratio of the solution. This invention does not specifically limit the source of the complex protease, and the enzyme activity of the complex protease is preferably 30000 U / g. In this invention, the preferred amount of complex protease added is 1g-2g of complex protease per 100g of rice bran protein powder.

[0024] In this invention, the hydrolysis temperature is preferably 50℃~60℃, more preferably 54℃~58℃; the hydrolysis time is preferably 1.5h~2.5h, more preferably 1.7h~2.1h; and the hydrolysis pH is preferably 6.5~7.5, more preferably 6.7~7.2. In this invention, the enzyme inactivation temperature is preferably 90℃~110℃, more preferably 95℃; and the enzyme inactivation time is preferably 5min~15min, more preferably 10min. After enzyme inactivation, the supernatant is subjected to membrane separation. In this invention, the membrane separation method is preferably nanofiltration. This invention does not specifically limit the nanofiltration and freeze-drying methods; conventional nanofiltration and freeze-drying methods in the art can be used.

[0025] The present invention also provides a rice bran protein peptide prepared by any of the above-described preparation methods.

[0026] The present invention also provides the application of the above-mentioned rice bran protein peptide in the preparation of products for preventing and treating gastric mucosal damage or gastric inflammation caused by Helicobacter pylori infection.

[0027] The present invention also provides the application of the above-mentioned rice bran protein peptide in the preparation of products for preventing Helicobacter pylori colonization.

[0028] In this invention, the product preferably includes a drug.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1 100 mL of a 12% (w / v) rice bran protein powder solution (purchased from Shaanxi Panier Co., Ltd.) (solvent: water) was mixed with a complex protease (enzyme activity of 30,000 U / g) (purchased from Novozymes, Denmark). The amount of complex protease added was calculated based on a ratio of 1.5 g of complex protease per 100 g of rice bran protein powder. Hydrolysis was carried out at pH 7.0 and 56℃ for 2 h to obtain hydrolysate. The hydrolysate was heated to 95℃ and held for 10 min to inactivate the enzyme. After inactivation, the hydrolysate was cooled. The cooled hydrolysate was centrifuged at 4000 r / min for 10 min, and the supernatant was subjected to nanofiltration desalting to remove sodium ions. Then, it was freeze-dried to obtain rice bran protein peptides.

[0033] Example 2 100 mL of a 10% (w / v) rice bran protein powder solution (purchased from Shaanxi Panier Co., Ltd.) was mixed with a complex protease (enzyme activity of 30,000 U / g) (purchased from Novozymes, Denmark). The amount of complex protease added was calculated based on a ratio of 1.0 g of complex protease per 100 g of rice bran protein powder. Hydrolysis was carried out for 1.5 h at pH 6.5 and 50 °C to obtain hydrolysate. The hydrolysate was heated to 90 °C and held for 5 min to inactivate the enzyme. After inactivation, the hydrolysate was cooled. The cooled hydrolysate was centrifuged at 3000 r / min for 15 min, and the supernatant was desalted by nanofiltration to remove sodium ions. Then, it was freeze-dried to obtain rice bran protein peptides.

[0034] Example 3 100 mL of a 14% (w / v) rice bran protein powder solution (purchased from Shaanxi Panier Co., Ltd.) was mixed with a complex protease (enzyme activity of 30,000 U / g) (purchased from Novozymes, Denmark). The amount of complex protease added was calculated based on a ratio of 2.0 g of complex protease per 100 g of rice bran protein powder. Hydrolysis was carried out at pH 7.5 and 60℃ for 2.5 h to obtain hydrolysate. The hydrolysate was heated to 110℃ and held for 15 min to inactivate the enzyme. After inactivation, the hydrolysate was cooled. The cooled hydrolysate was centrifuged at 5000 r / min for 5 min, and the supernatant was desalted by nanofiltration to remove sodium ions. Then, it was freeze-dried to obtain rice bran protein peptides.

[0035] Example 4 The rice bran protein peptides obtained in Example 1 were tested in Kunming mice. Forty-eight male Kunming mice were divided into six groups of eight each, as follows: ① Control group (normal group): administered normal saline by gavage for 5 weeks; ② Model group: Administered normal saline by gavage for 1-2 weeks, and normal saline + normal saline by gavage for 3-4 weeks. H. pylori Bacterial solution, administered via gavage with physiological saline for 5 weeks; ③ 125 mg / kg rice bran protein peptide group (125 mg / kg rice bran peptide group): Rice bran protein peptide was administered by gavage for 1-2 weeks, and rice bran protein peptide + 125 mg / kg rice bran peptide was administered by gavage for 3-4 weeks. H. pylori Bacterial solution, administered via gavage with rice bran protein peptides for 5 weeks; ④ 250 mg / kg rice bran protein peptide group (250 mg / kg rice bran peptide group): Rice bran protein peptide was administered by gavage for 1-2 weeks, and rice bran protein peptide + 250 mg / kg rice bran peptide was administered by gavage for 3-4 weeks. H. pylori Bacterial solution, administered via gavage with rice bran protein peptides for 5 weeks; ⑤ 500 mg / kg rice bran protein peptide group (500 mg / kg rice bran peptide group): Rice bran protein peptide was administered by gavage for 1-2 weeks, and rice bran protein peptide + 500 mg / kg rice bran peptide was administered by gavage for 3-4 weeks. H. pylori Bacterial solution, administered via gavage with rice bran protein peptides for 5 weeks; Note: Fasting for 12 hours before each gavage administration of bacterial solution, and fasting and abstaining from water for 4 hours after gavage administration; administer bacterial solution by gavage every other day, for a total of 5 times.

[0036] After the last gavage, mice were fasted for 12 hours. Gastric tissue homogenates were then collected and relevant indicators were analyzed, as follows: (1) The contents of IL-10, TNF-α, IL-6, TLR4, MyD88 and NF-κB in the homogenate were determined by the corresponding kits.

[0037] The results are as follows Figure 1-6 As shown. By Figures 1-3 It can be seen that, compared with the normal group (control group), the levels of inflammatory factors IL-6 and TNF-α in the model group were significantly increased. P＜0.05 ), while the level of the inflammatory suppressor IL-10 was significantly reduced ( P＜0.05 ).show H. pylori Infection led to abnormal secretion of inflammatory factors related to gastric mucosal damage in mice, resulting in a significant inflammatory response in the gastric mucosa.

[0038] H. pyloriInfection can cause gastric mucosal damage, leading to the secretion of inflammatory factors such as IL-6 and TNF-α. Both IL-6 and TNF-α are cytokines involved in human immune regulation and mediating inflammatory responses. They can activate and recruit inflammatory cells, causing infiltration and exacerbating the inflammatory response, playing an important role in gastric mucosal lesions. These inflammatory factors can also reduce gastric acid secretion, further aggravating gastric mucosal damage. Compared with the model group, a medium dose of rice bran protein peptides can significantly reduce IL-6 levels (…). P＜ 0.05 All doses of rice bran protein peptides significantly reduced TNF-α levels compared to the model group. P＜0.05 Among the various groups, the high-dose group showed the best effect from rice bran protein peptides. The results indicate that intervention with rice bran protein peptides can effectively alleviate gastric mucosal damage induced by H. pylori infection.

[0039] IL-10 is an important immunomodulatory factor; IL-10 can inhibit... H. pylori The synthesis and activity of inflammatory factors during infection synergistically interact with anti-inflammatory mediators to exert an anti-inflammatory effect. Compared with the model group, the IL-10 content in the high-dose rice bran protein peptide group was significantly increased ( P＜0.05 This indicates that high doses of rice bran protein peptides can promote... H. pylori Infecting mice with IL-10 significantly improved the gastric mucosal injury response by increasing gastric IL-10 secretion.

[0040] from Figures 4-6 It can be seen that, compared with the normal group (control group), the levels of the three key factors of the NF-κB signaling pathway in the stomach, TLR4, MyD88 and NF-κB, were significantly increased in the model group. P＜0.05 ),show H. pylori Excessive activation of the NF-κB signaling pathway by infection leads to and exacerbates gastric mucosal damage.

[0041] H. pylori Infection led to an increase in the expression of TLR4 receptors on the surface of gastric mucosal epithelial cells. Compared with the model group, different doses of rice bran protein peptides significantly reduced the content of TLR4 in the stomach. P＜0.05 Medium doses of rice bran peptides have shown a high ability to inhibit TLR4 receptor expression.

[0042] Increased TLR4 receptor expression induces an increase in MyD88, and the two combine to form the TLR4 / MyD88 complex. Compared with the model group, the MyD88 content in the low-dose rice bran peptide group was decreased, but not significantly. P＞0.05 Medium and high doses of rice bran protein peptides significantly reduced MyD88 content. P＜0.05 The high-dose rice bran protein peptide group had the lowest MyD88 content.

[0043] The TLR4 / MyD88 complex, through a signaling cascade, degrades the NF-κB inhibitor IκB, freeing NF-κB and allowing it to enter the cell nucleus to regulate the transcription and expression of inflammatory cytokines and chemokines. Compared with the model group, different doses of rice bran protein peptides significantly reduced the NF-κB content in the stomach. P＜0.05 Furthermore, this effect is dose-dependent. Decreased NF-κB expression leads to a corresponding decrease in the expression of inflammatory factors and chemokines, thus mitigating the effects of [the condition]. H. pylori Infection-induced gastric mucosal damage is of great significance.

[0044] In summary, the results show that the intervention of rice bran protein peptides prepared by the method of the present invention can significantly reduce the content of gastric inflammatory factors and increase the content of inflammatory inhibitory factors, thereby alleviating the inflammatory response of gastric mucosal damage. In addition, the rice bran protein peptides prepared by the present invention can significantly improve gastric mucosal damage caused by Helicobacter pylori infection.

[0045] (2) Analysis of Helicobacter pylori content in mouse gastric mucosa A certain mass of mouse gastric mucosa was taken and homogenized with PBS solution at a ratio of 1:9. The homogenate was then diluted to a series of concentrations and plated. The concentration of the homogenate in the mouse mucosa was calculated. H. pylori Content. The colonization level of Helicobacter pylori in the stomach of mice was detected. The results are shown in Table 1.

[0046] Table 1 Gastric mucosa of mice in each experimental group H. pylori Planting quantity

[0047] Note: Each lowercase letter represents... P＜0.05 .

[0048] As shown in Table 1, except for the control group, the gastric mucosa of mice in each experimental group showed [significant abnormalities / conditions]. H. pylori It was present in the gastric mucosa of the model group mice. H. pylori The planting density was 3.11 × 10⁶. 9 The cfu / g gastric tissue concentration was significantly higher than that in the control group. P＜0.05 ),show H. pylori Successful colonization of the mouse gastric mucosa, indicating successful model establishment.

[0049] Compared with the model group, the gastric mucosa of mice in the low-dose group (125 mg / kg rice bran protein peptide group) showed better results. H. pylori The planting density was slightly lower, but there was no significant difference. P＞0.05 Gastric mucosa of mice in the medium-dose group (250 mg / kg rice bran protein peptide group) and the high-dose group (500 mg / kg rice bran protein peptide group) H. pylori The planting density was significantly lower than that of the model group ( P＜0.05 The results showed that with the increase of rice bran protein peptide dosage, the gastric mucosa of mice... H. pylori The colonization rate gradually decreased, demonstrating a dose-dependent effect. This further illustrates that the rice bran protein peptides prepared by the method of this invention can reduce the colonization rate of Helicobacter pylori in the gastric mucosa.

[0050] The rice bran protein peptides obtained in Examples 2 and 3 were used in Kunming mouse experiments, with the specific steps being the same as in Example 4. The results were similar to those in Example 4, both indicating that the intervention of the rice bran protein peptides prepared by the method of the present invention can significantly reduce the content of gastric inflammatory factors and increase the content of inflammatory inhibitory factors, thereby alleviating the inflammatory response of gastric mucosal damage. It can significantly improve gastric mucosal damage caused by Helicobacter pylori infection and reduce the colonization of Helicobacter pylori in the gastric mucosa.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a rice bran protein-derived peptide antagonizing gastric mucosal damage in the preparation of products for preventing and treating gastric mucosal damage caused by Helicobacter pylori infection, characterized in that, The preparation method of the rice bran protein source antagonistic peptide against gastric mucosal damage includes the following steps: mixing and hydrolyzing rice bran protein powder solution with a complex protease to obtain hydrolysate; subjecting the hydrolysate to enzyme inactivation, membrane separation, and freeze-drying steps in sequence to obtain the rice bran protein source antagonistic peptide against gastric mucosal damage; the membrane separation is nanofiltration; the product is a drug.

2. The application of a rice bran protein-derived peptide antagonizing gastric mucosal damage in the preparation of products for preventing and treating gastric inflammation caused by Helicobacter pylori infection, characterized in that... The preparation method of the rice bran protein source antagonistic peptide against gastric mucosal damage includes the following steps: mixing and hydrolyzing rice bran protein powder solution with a complex protease to obtain hydrolysate; subjecting the hydrolysate to enzyme inactivation, membrane separation, and freeze-drying steps in sequence to obtain the rice bran protein source antagonistic peptide against gastric mucosal damage; the membrane separation is nanofiltration; the product is a drug.

3. The application of a rice bran protein-derived peptide that antagonizes gastric mucosal damage in the preparation of products for preventing Helicobacter pylori colonization, characterized in that, The preparation method of the rice bran protein source antagonistic peptide against gastric mucosal damage includes the following steps: mixing and hydrolyzing rice bran protein powder solution with a complex protease to obtain hydrolysate; subjecting the hydrolysate to enzyme inactivation, membrane separation, and freeze-drying steps in sequence to obtain the rice bran protein source antagonistic peptide against gastric mucosal damage; the membrane separation is nanofiltration; the product is a drug.

4. The application according to any one of claims 1 to 3, characterized in that, The amount of the compound protease added is 1g-2g per 100g of rice bran protein powder.

5. The application according to any one of claims 1 to 3, characterized in that, The enzyme activity of the complex protease is 30,000 U / g.

6. The application according to any one of claims 1 to 3, characterized in that, The concentration of the rice bran protein powder solution is 10%~14%; the concentration is the mass ratio of the rice bran protein powder to the volume ratio of the solution.

7. The application according to any one of claims 1 to 3, characterized in that, The hydrolysis temperature is 50℃~60℃, the hydrolysis time is 1.5h~2.5h, and the hydrolysis pH value is 6.5~7.5.

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