Application and preparation method of marine fish protein peptide with effects of reducing uric acid and protecting kidney

Abstract

The invention discloses an application and a preparation method of a marine fish protein peptide with effects of reducing uric acid and protecting kidneys. The marine fish protein peptide VK with uric acid reduction and kidney protection activity is prepared, and the marine fish protein peptide VK can remarkably reduce the blood uric acid level in the body and improve pathological injury of the kidney. Furthermore, the preparation process is further optimized, and the content of the dipeptide VK in the zymolyte is increased. The preparation method provided by the invention is simple in process, realizes enzymolysis controlled release of the target polypeptide, and can be used in common food, food additives and functional food for preventing and treating hyperuricemia and kidney injury or health care products or medicines for relieving hyperuricemia and kidney injury.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to the application and preparation method of a marine fish protein peptide with uric acid-lowering and kidney-protective effects. Background Technology

[0002] Hyperuricemia is a metabolic disease characterized by elevated serum uric acid levels. In recent years, the global prevalence of hyperuricemia has risen sharply, reaching 25.8% in Japan, 21.0% in the United States, and 13.3% in China, with the age of onset increasing annually. Hyperuricemia has become the second leading metabolic disease globally after diabetes, posing a significant threat to human health; however, current strategies for effectively controlling the disease remain limited. Studies show that approximately 90% of primary hyperuricemia cases are caused by insufficient uric acid excretion. Elevated uric acid levels can also lead to hyperuricemic nephropathy, which in turn can cause chronic kidney disease. The kidneys, as the core site of uric acid metabolism, play a crucial role in maintaining uric acid homeostasis. Specifically, this manifests in the following three aspects: First, the transmembrane transport of uric acid in the kidneys depends on specific uric acid transport proteins on the surface of renal tubular epithelial cells. Among them, GLUT9 and URAT1 are responsible for uric acid reabsorption, while ABCG2 and OAT1 are responsible for uric acid secretion. Second, ATP produced by the mitochondria of renal tubular epithelial cells provides energy for the active transport of uric acid. Third, sodium-potassium ATPase on the basolateral side of renal tubular epithelial cells not only provides the driving force for uric acid transport proteins but also maintains intracellular osmotic pressure balance. Therefore, protecting the kidneys to promote uric acid excretion may be an effective strategy for the prevention and treatment of hyperuricemia.

[0003] Currently, clinical interventions for kidney damage and hyperuricemia primarily rely on medications (such as nonsteroidal anti-inflammatory drugs, xanthine oxidase inhibitors, and uricosuric drugs). However, long-term use of these medications can easily lead to side effects such as liver and kidney damage, gastrointestinal discomfort, and allergies. Against this backdrop, bioactive peptides, as potential therapeutic agents, have become a research hotspot due to their advantages of natural origin, relatively high safety, diverse activities, and easy absorption and metabolism. However, the number of peptides with clearly defined renal protective activity is currently limited, and enriching specific bioactive peptides from complex proteolytic hydrolysates faces numerous challenges, such as low bioactive peptide content, high separation and purification costs, and complex processes. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of marine fish protein peptides with uric acid-lowering and kidney-protecting effects.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned marine fish protein peptides with uric acid-lowering and kidney-protective effects.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects includes the following steps:

[0008] Marine fish are mixed with water, the pH is adjusted, enzymes are added for enzymatic hydrolysis, the enzymes are inactivated after hydrolysis, the mixture is centrifuged, the supernatant is collected, filtered, and the filtrate is dried to obtain marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects.

[0009] The mass ratio of marine fish to water is 1:1 to 3; preferably 1:2.

[0010] The marine fish mentioned are fish species that live in seawater; preferably skipjack tuna.

[0011] The pH adjustment is to adjust the pH to 6-8; preferably, the pH is adjusted to 7.

[0012] The enzymes include at least one of papain and aminopeptidase; preferably, papain of 1.5% marine fish protein content and aminopeptidase of 1% marine fish protein content.

[0013] The enzymatic hydrolysis conditions are 50-60℃ for 5-7 hours; preferably 55℃ for 6 hours.

[0014] The centrifugation conditions are as follows: centrifugation at 8000-9000 rpm for 10-15 minutes at 4°C.

[0015] The drying process described is freeze-drying.

[0016] A marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects was prepared according to the above preparation method.

[0017] The above-mentioned marine fish enzymatic hydrolysates or dipeptides VK, which have the effects of lowering uric acid and protecting the kidneys, are used in the preparation of drugs for the treatment / prevention of kidney diseases.

[0018] The above-mentioned marine fish enzymatic hydrolysates or dipeptides VK, which have the effects of lowering uric acid and protecting the kidneys, are used in the preparation of uric acid-lowering drugs.

[0019] The above-mentioned marine fish enzymatic hydrolysates or dipeptides VK, which have the effects of lowering uric acid and protecting the kidneys, are used in the preparation of food or health products with the effects of lowering uric acid or protecting the kidneys.

[0020] The amino acid sequence of the dipeptide VK is Val-Lys.

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] (1) The present invention prepared marine fish protein peptide VK with uric acid reduction and kidney protection effects, which can significantly reduce the blood uric acid level in rats, promote uric acid excretion, and improve kidney pathological damage.

[0023] (2) The preparation method provided by the present invention further improves the content of marine fish protein peptide VK with uric acid reduction and kidney protection effects. The process is simple and realizes the enzymatic hydrolysis and controlled release of the target polypeptide. The entire process can meet the food grade requirements and can be applied to ordinary foods, food additives, functional foods or health products or drugs that can relieve hyperuricemia and kidney damage for the prevention and treatment of hyperuricemia and kidney damage. Attached Figure Description

[0024] Figure 1 The content of VK in the enzymatic hydrolysates obtained in the examples and comparative examples.

[0025] Figure 2 The graphs show the changes in uric acid levels and kidney function in rats after VK intervention. In the graphs, A shows the changes in serum uric acid levels, B shows the changes in serum creatinine levels, C shows the changes in urinary creatinine levels, and D shows the changes in urinary uric acid levels.

[0026] Figure 3 The graph shows the kidney damage in rats in each group after VK intervention. A represents the renal collagen volume fraction, and B and C represent the changes in the levels of kidney damage markers KIM-1, IL-18, NAG, and Cys-C, respectively.

[0027] Figure 4 This is a microscopic image of a kidney tissue section from Example 9. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0029] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0030] The enzymes used in the following examples include papain (Pompeo) and aminopeptidase (Amano, Japan).

[0031] Example 1

[0032] (1) Mix the skipjack tuna with deionized water at a ratio of 1:2 (w / v);

[0033] (2) Adjust the pH of the solution to 7, add papain and aminopeptidase to it for enzymatic hydrolysis, the amount added is 1.5% and 1% of the protein content of bonito meat (based on the protein content of 24%), the enzymatic hydrolysis temperature is 55℃, the enzymatic hydrolysis is carried out for 6 hours, and the enzyme is inactivated by boiling water at 100℃ for 15 minutes to obtain the enzymatic hydrolysate.

[0034] (3) Cool to room temperature, then centrifuge at 8000 rpm for 10 min at 4℃, take the supernatant, filter and obtain marine fish enzymatic hydrolysate.

[0035] (4) The filtered enzymatic hydrolysate was freeze-dried to obtain marine fish uric acid-lowering peptides with kidney protection function.

[0036] Example 2

[0037] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0038] In step (2), the amount of papain and aminopeptidase added is 1.5% and 0.5% of the protein content of bonito meat, respectively.

[0039] Example 3

[0040] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0041] In step (2), the amount of papain and aminopeptidase added is 1.5% and 0.25% of the protein content of bonito meat, respectively.

[0042] Example 4

[0043] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0044] In step (2), the amount of papain and aminopeptidase added is 1.5% and 0.1% of the protein content of bonito meat, respectively.

[0045] Example 5

[0046] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0047] In step (2), the amount of papain and aminopeptidase added is 1% and 1% of the protein content of bonito flesh, respectively.

[0048] Example 6

[0049] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0050] In step (2), the amount of papain and aminopeptidase added is 1% and 0.25% of the protein content of bonito meat, respectively.

[0051] Example 7

[0052] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0053] In step (2), the amount of papain and aminopeptidase added is 1% and 0.1% of the protein content of bonito meat, respectively.

[0054] Comparative Example 1

[0055] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0056] In step (2), papain is added at a rate of 2% of the bonito meat protein content.

[0057] Comparative Example 2

[0058] Marine fish peptides were prepared according to the method of Example 1, with the only difference being:

[0059] In step (2), papain and aminopeptidase are added, with the amounts being 0.5% and 0.5% of the bonito meat protein content, respectively.

[0060] Example 8 Peptide Identification and Quantification

[0061] An active polypeptide VK was identified from the enzymatic hydrolysate prepared in Example 1 through separation and screening. This experiment used liquid chromatography-tandem mass spectrometry (LC-MS / MS) to identify and quantify the VK peptide in marine fish enzymatic hydrolysates prepared by the aforementioned different methods. The specific procedures are as follows:

[0062] (1) The lyophilized enzymatic hydrolysate was diluted with 0.1% formic acid to prepare a solution with a final concentration of 0.5 mg / mL (protein content). The solution was centrifuged at 10000×g for 10 min at 4 °C, and the supernatant was collected and filtered through a 0.22 μm filter membrane. Peptide sequences were determined using ultra-high performance liquid chromatography (UPLC) tandem quadrupole time-of-flight (Q-TOF) mass spectrometry. Electrospray ionization (EIC) was used for mass spectrometry sampling in positive ion mode. The mass spectrometry acquisition range was 50–1500 m / z, and the acquisition rate was 5 Hz. The nebulizer gas pressure was 1.5 bar, the nitrogen drying gas flow rate was 8 L / min, and the nitrogen drying gas temperature was 200 °C. Analysis was performed using a Waters ACQUITYUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The injection volume was 2 μL, and the elution rate was 0.20 mL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 100% acetonitrile. The gradient conditions were as follows: 0-2 min, 100% A; 2-10 min, 100%-60% A; 10-12 min, 60%-20% A; 12-14 min, 20% A; 14-14.5 min, 20%-10% A; 14.5-18 min, 100% A.

[0063] (2) The obtained MS / MS chromatograms were analyzed using Data Analysis 4.4 (Bruker Daltonics), and the peptide content was absolutely quantified by extracting the peak area of ​​ion chromatography.

[0064] Experimental results are as follows Figure 1 As shown, the VK content was increased in Examples 1-7 compared to Comparative Examples 1-2.

[0065] Example 9: Evaluation of in vivo renal protective and uric acid-lowering activities

[0066] To further verify the activity of the active peptide VK, an animal model was designed to verify its specific effects. The pure peptide VK was prepared by solid-phase synthesis.

[0067] 9.1 Animal husbandry

[0068] Six- to eight-week-old SD rats were acclimatized for one week and then randomly divided into four groups (n=8): control group (NC), model group (MC), positive control group (BenZ), and VK group.

[0069] Potassium oxonate and adenine were dispersed in a 0.5% (w / w) sodium carboxymethyl cellulose solution used for tube feeding. The control group was administered 0.5% sodium carboxymethyl cellulose solution by gavage every morning, while the other groups were administered potassium oxonate (1.5 g / kg) and adenine (50 mg / kg) by gavage for 7 consecutive days to induce a hyperuricemic kidney injury model.

[0070] After the hyperuricemia-induced kidney injury model was successfully established, the experiment officially began. For the next 28 days, rats in the model group, BenZ group, and VK group were administered potassium oxonate (1.5 g / kg) and adenine (50 mg / kg) by gavage in the morning. In the afternoon, rats in the model group received only saline. The BenZ group was given benzbromarone (10 mg / kg), the VK group was given dipeptide VK (40 mg / kg), and the NC group was administered saline by gavage.

[0071] 9.2 Sample Collection and Processing

[0072] During the experiment, orbital blood was collected from rats weekly, and the supernatant was collected by centrifugation.

[0073] Two days before euthanasia, rats were placed in metabolic cages to collect urine for 24 hours. Urine and serum samples were obtained for comprehensive evaluation. After euthanasia, kidney tissue was taken for detailed histopathological examination and biochemical analysis.

[0074] 9.3 Detection of serum uric acid, serum creatinine, urine creatinine, and urine uric acid levels.

[0075] Commercially available kits were used to detect the levels of serum uric acid, serum creatinine, urine creatinine, and urine uric acid.

[0076] Experimental results are as follows Figure 2 As shown, where, Figure 2 The figure shows the changes in serum uric acid levels in each group of rats. Compared with the control group, the serum uric acid level in the model group increased significantly. After intervention with dipeptide VK, the serum uric acid level decreased significantly by 20.92% (p<0.05).

[0077] Figure 2 Tables B and C show the changes in serum and urinary creatinine levels in rats. Compared with the control group, the model group showed a significant increase in serum creatinine and a significant decrease in urinary creatinine (p<0.05), reflecting impaired kidney function in rats. Dipeptide VK intervention significantly decreased serum creatinine levels in rats (p<0.05), while urinary creatinine levels showed an upward trend; however, this change did not reach a significant level (p>0.05).

[0078] Figure 2 D represents the change in uric acid in urine. Compared with the model group, the uric acid level in the VK group was increased, but it did not reach the statistical significance level (p>0.05).

[0079] The results above indicate that dipeptide VK treatment can maintain normal kidney function and reduce serum uric acid levels in rats.

[0080] 9.4 Detection of renal collagen volume fraction and levels of damage markers KIM-1, IL-18, NAG, and Cys-C

[0081] Kidney tissue was fixed in 4% paraformaldehyde, then routinely dehydrated with ethanol, embedded in paraffin, and sectioned at a thickness of 4 μm. After dewaxing and rehydration, the sections were stained with hematoxylin-eosin (H&E) or Masson's trichrome. Pathological changes in the kidney tissue were observed under a 200x optical microscope. Semi-quantitative analysis was performed using ImageJ to quantitatively measure the collagen volume fraction in the renal interstitium. Collagen volume fraction (%) = collagen area / total tissue area * 100%. Simultaneously, the levels of kidney injury markers KIM-1, IL-18, NAG, and Cys-C were measured using ELISA kits.

[0082] Experimental results are as follows Figures 3-4 As shown, where, Figure 3 In the figure, A represents the collagen volume fraction, which reflects the presence of collagen deposition and renal interstitial fibrosis in rat kidneys. The collagen volume fraction in the kidneys of rats in the model group was significantly increased (p<0.05); however, after intervention with dipeptide VK, the collagen volume fraction decreased by 11.70%. Figure 3 Figures B through E show the changes in the levels of kidney injury markers. Compared with the control group, the levels of kidney injury markers KIM-1, IL-18, NAG, and Cys-C in the model group rats were significantly increased (p<0.05), while the levels of the above kidney injury markers decreased by 32.35%, 23.67%, 28.79%, and 35.60%, respectively, after intervention with dipeptide VK.

[0083] The results above indicate that dipeptide VK treatment can effectively improve kidney damage in rats and has potential applications in the preparation of drugs for treating related diseases.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects, characterized in that... The steps include the following: Marine fish are mixed with water, the pH is adjusted, enzymes are added for enzymatic hydrolysis, the enzymes are inactivated after hydrolysis, the mixture is centrifuged, the supernatant is collected, filtered, and the filtrate is dried to obtain marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects.

2. The method for preparing marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects according to claim 1, characterized in that: The marine fish mentioned are species of fish that live in seawater; The mass ratio of marine fish to water is 1:1 to 3; The pH adjustment mentioned refers to adjusting the pH to 6-8.

3. The method for preparing marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects according to claim 1, characterized in that: The enzymes include at least one of papain and aminopeptidase, wherein the amount of papain added is 1%-1.5% of the marine fish protein content, and the amount of aminopeptidase added is 0.1%-1% of the marine fish protein content.

4. The method for preparing marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects according to claim 1, characterized in that: The enzymatic hydrolysis conditions are 50–60°C for 5–7 hours.

5. The method for preparing marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects according to claim 1, characterized in that: The centrifugation conditions are as follows: centrifugation at 8000-9000 rpm for 10-15 min at 4℃; The drying process described is freeze-drying.

6. A marine fish enzymatic hydrolysate with uric acid-lowering and kidney-protective effects, prepared according to any one of claims 1 to 5.

7. The use of the marine fish enzymatic hydrolysate or dipeptide VK with uric acid-lowering and kidney-protective effects as described in claim 6 in the preparation of drugs for the treatment / prevention of kidney diseases.

8. The use of the marine fish enzymatic hydrolysate or dipeptide VK with uric acid-lowering and kidney-protective effects as described in claim 6 in the preparation of uric acid-lowering drugs.

9. The use of the marine fish enzymatic hydrolysate or dipeptide VK with uric acid-lowering and kidney-protective effects as described in claim 6 in the preparation of food or health products with uric acid-lowering or kidney-protective effects.

10. The application according to any one of claims 7 to 9, characterized in that: The amino acid sequence of the dipeptide VK is Val-Lys.

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