Pentapeptide YP5 with function of relieving diabetic kidney injury as well as preparation method and application of pentapeptide YP5

Abstract

The invention discloses a pentapeptide YP5 with a function of relieving diabetic kidney injury as well as a preparation method and application of the pentapeptide YP5, and belongs to the technical field of biology. The pentapeptide YP5 is screened from pacific salmon protamine enzymatic hydrolysate, the amino acid sequence is YVAAP, and the pentapeptide YP5 has the function of relieving the diabetic kidney injury, can remarkably reduce the content of fasting blood-glucose, serum creatinine, urea nitrogen and urine protein of diabetic mice, has better activity and smaller toxic and side effects than a positive drug metformin, and can be used for preparing the medicine for treating diabetic kidney injury. The method can be applied to functional food for relieving diabetic kidney injury.

Description

Technical Field

[0001] The present invention relates to a small molecule peptide, a preparation method thereof and an application thereof, and particularly relates to a pentapeptide YP5 having a function of alleviating diabetic kidney injury, a preparation method thereof and an application thereof in a functional food for alleviating diabetic kidney injury, belonging to the technical field of biotechnology. Background Art

[0002] Diabetes is one of the global health crises, and its prevalence rate shows a continuous upward trend. Among them, type 2 diabetes accounts for more than 90%, and its core pathological features are insulin resistance, β-cell function defect and peripheral tissue glucose uptake disorder. Research reveals that the abnormal inactivation of incretin hormones is the key inducement for insufficient insulin secretion, and dipeptidyl peptidase-IV (DPP-IV) is the core enzyme mediating this inactivation process. Therefore, targeting and inhibiting the activity of DPP-IV has been recognized as a frontier strategy for treating type 2 diabetes.

[0003] As a glycoprotein hydrolase with a molecular weight of 110 kDa, DPP-IV has become a core target for the prevention and treatment of type 2 diabetes. Although the synthetic inhibitors such as linagliptin and sitagliptin widely used clinically at present have curative effects, they have drug adverse reactions such as an increased risk of infection and skin allergy. However, there is still little screening and excavation of natural active substances with DPP-IV inhibitory activity and capable of alleviating diabetic kidney injury. The bioactive peptides derived from food proteolysis have the advantages of high specificity, small toxic and side effects, good curative effects, and larger available drug doses, and are a good strategy for alleviating diabetic kidney injury.

[0004] Protein raw materials from different sources such as animals and plants will obtain different enzyme hydrolysates in different enzymatic hydrolysis processes. The enzyme hydrolysate is a collection of bioactive peptides with different structures, diverse sequences and different functions. The amino acid compositions and structures of the bioactive peptides having the function of alleviating diabetic kidney injury vary greatly, and there is no fixed or unified amino acid composition. The components of food protease hydrolysis products are quite complex. Even if the same protein raw material is used, different product components will be produced due to different enzymatic hydrolysis processes. Therefore, the strategy of screening bioactive peptides with DPP-IV inhibitory activity and capable of alleviating diabetic kidney injury from enzyme hydrolysates obtained from different protein sources and different preparation processes has specificity.

[0005] The article "Hypoglycemic Activity and Renal Protection of Seabuckthorn Seed Protein Peptides in db / db Mice" (Shu Danyang, Xiong Jian, Liu Pengzhan, et al., Science and Technology of Food Industry, 2020, 41(21): 317-321) reported that the bioactive peptides obtained from the enzymatic hydrolysate of seabuckthorn seeds could significantly reduce the blood glucose level of diabetic mice and alleviate histopathological damage, but the identification of the active components and their sequences was not further completed; the article "Rapeseed protein-derived antioxidant peptide RAP alleviates renal fibrosis through MAPK / NF-κB signaling pathways in diabetic nephropathy" (Zhang M, Yan Z, Bu L, An C, Wang D, Liu X, Zhang J, Yang W, Deng B, Xie J, Zhang B, Drug Des Devel Ther, 2018 May 15; 12: 1255-1268) reported that the bioactive peptide YWDHNNPQIR obtained from the enzymatic hydrolysate of rapeseed had the effects of reducing urinary albumin, serum creatinine and urea nitrogen levels in a streptozotocin (STZ) and high-fat diet-induced diabetic mouse model; the article "Rapid screening and identification of novel dipeptidyl peptidase IV inhibitory peptides from buffalo milk" (Zheng Q, Luo X, Pan D, Wang Y, Suo S, Dang Y, Gao X, J Sci Food Agric, 2025 May, 105(7): 3942-3950) reported that the bioactive peptides GPFPIIV and FPQYL obtained from the enzymatic hydrolysate of buffalo milk showed excellent DPP-IV inhibitory activity in in vitro inhibition experiments and cell experiments; the article "Dipeptidyl Peptidase IV-Inhibitory Peptides Derived from Silver Carp ([[]] Hypophthalmichthys molitrix"Val.) Proteins" (Zhang Y, Chen R, Chen X, Zeng Z, Ma H, Chen S, J Agric Food Chem, 2016 Feb 3; 64(4): 831-9) reported that different enzymatic hydrolysates of silver carp showed high DPP-IV inhibitory activity, among which the neutral enzymatic hydrolysate had the highest DPP-IV inhibitory activity, and further focused on the functional peptides with the sequences of LPIIDI and APGPAGP. The article "Anti-diabetic effects of bioactive peptides: recent advances and clinical implications" (Acquah C, Dzuvor CKO, Tosh S, Agyei D, Crit Rev Food Sci Nutr, 2022; 62(8): 2158-2171) systematically summarized the preparation methods of enzymatic hydrolysates with diabetes remission function discovered in recent years and the structures of bioactive peptides isolated therefrom.

[0006] The above reports did not mention peptides with the same or similar structures as the bioactive peptides with the function of alleviating diabetic kidney injury in the present invention. In addition, if we want to modify based on the reported bioactive peptide sequences, since there are 20 common amino acids available for selection at each amino acid position, the data volume increases exponentially, which is not feasible.

[0007] Chum salmon belongs to the order Salmoniformes, family Salmonidae, and genus Oncorhynchus in biological taxonomy. It is also called Pacific salmon in China, and its scientific name is Oncorhynchus keta , which is an anadromous mid-upper layer fish, characterized by high protein, low fat, and rich in ω-3 polyunsaturated fatty acids. It is an important aquatic resource with both nutritional and ecological values. Protamine is a high-quality raw material for preparing antibacterial peptides, immunomodulatory peptides, and metabolic active peptides due to its rich content of arginine and histidine. Summary of the Invention

[0008] The object of the present invention is to provide a small molecule peptide screened from the enzymatic hydrolysate of Pacific salmon protamine, which has the function of alleviating diabetic kidney injury and can be applied to functional foods for alleviating diabetic kidney injury.

[0009] In order to achieve the above object, the present invention adopts the following technical scheme: A pentapeptide YP5 with the function of alleviating diabetic kidney injury, the amino acid sequence of the pentapeptide YP5 is YVAAP, as shown in SEQ ID NO: 4 in the sequence listing, and it has the function of alleviating diabetic kidney injury.

[0010] Application of the pentapeptide YP5 with the function of alleviating diabetic kidney injury in functional foods for alleviating diabetic kidney injury.

[0011] Preparation method of the pentapeptide YP5 with the function of alleviating diabetic kidney injury, using solid-phase synthesis method, specifically: Using Fmoc-protected amino acids as raw materials and polystyrene resin as the solid-phase carrier, solid-phase synthesis is carried out using the Fmoc solid-phase synthesis strategy.

[0012] Preparation method of the pentapeptide YP5 with the function of alleviating diabetic kidney injury, using enzymatic hydrolysis method, specifically: (1) Take Pacific salmon sperm, remove impurities, add water, heat to 65 ± 2 °C, and maintain for 12 h; (2) Fish out the sperm and make it into a slurry, put it into a reaction kettle, add water, start the stirrer and heat up to 48 °C, add papain for enzymatic hydrolysis for 2 h, continue to heat up to 57 °C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 h, then add flavor protease for enzymatic hydrolysis for 4 h, continue to heat up to 85 °C, and maintain for 30 min to obtain an enzymatic hydrolysate; (3) While cooling, add an aqueous solution of chitosan deacetate to the enzymatic hydrolysate. After flocculants appear, centrifuge to retain the supernatant and filter press to obtain a clear and transparent filtrate; (4) Adjust the pH value of the filtrate to 5.50 ± 1.50, then add coconut shell activated carbon, decolorize and deodorize at 70 ± 2 °C for 30 min, filter press again, desalt and concentrate the filtrate to obtain a concentrated solution; (5) Dry the above concentrated solution to obtain Pacific salmon sperm protein polypeptide, which contains a relatively large amount of pentapeptide YP5.

[0013] The beneficial effects of the present invention are as follows: (1) The pentapeptide YP5 screened from the enzymatic hydrolysate of Pacific salmon sperm by the present invention has the function of alleviating diabetic kidney injury, can significantly reduce the contents of fasting blood glucose, serum creatinine, urea nitrogen and urinary protein in diabetic mice, and has better activity than the positive drug metformin, and can be applied to functional foods for alleviating diabetic kidney injury; (2) The pentapeptide YP5 provided by the present invention is screened from the enzymatic hydrolysate of Pacific salmon sperm, and has the advantages of small toxic and side effects and high safety in use compared with metformin, and can be taken for a long time. Description of the Drawings

[0014] Figure 1 It is a 2D schematic diagram of the binding mode of pentapeptide YP5 and DPP-IV; Figure 2 It is a graph showing the effect of pentapeptide YP5 on the body weight of diabetic mice, *** indicates P < 0.001; Figure 3 Effect diagram of pentapeptide YP5 on fasting blood glucose in diabetic mice. *** indicates P < 0.001; Figure 4 Effect diagram of pentapeptide YP5 on urinary protein in diabetic mice. ** indicates P < 0.01, *** indicates P < 0.001; Figure 5 Effect diagram of pentapeptide YP5 on serum creatinine in diabetic mice. *** indicates P < 0.001; Figure 6 Effect diagram of pentapeptide YP5 on serum urea nitrogen in diabetic mice. *** indicates P < 0.001. Detailed implementation mode

[0015] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0016] I. Preparation of protamine polypeptide from Pacific salmon Take Pacific salmon protamine, remove impurities, add an appropriate amount of pure water, heat to 65 ± 2 °C, and maintain for 12 h (to fully swell the testis tissue for easy extraction and subsequent impurity removal), and turn it over every 4 h during this period.

[0017] Fish out the protamine and beat it into pulp twice with a bone pulp machine, then put it into a reaction kettle, add pure water according to the solid-liquid ratio of 1:5, start the stirrer (rotation speed 100 rpm) and heat up to 48 °C, add papain (enzyme-substrate ratio 1.0%) and enzymolyze for 2 h, continue to heat up to 57 °C, add nuclease (enzyme-substrate ratio 0.5%), deaminase (enzyme-substrate ratio 0.3%), alkaline protease (Alcalase® 2.4L, enzyme-substrate ratio 1.0%) and neutral protease (Neutrase® 0.8L, enzyme-substrate ratio 0.5%) and enzymolyze for 3 h, then add flavor protease (Flavourzyme® 500MG, enzyme-substrate ratio 1.0%) and enzymolyze for 4 h, continue to heat up to 85 °C, and maintain for 30 min (inactivate the enzyme) to obtain an enzymolysis solution.

[0018] While cooling, add an aqueous solution of deacetylated chitosan with a concentration of 1 wt% (food grade, to promote the sedimentation of impurities) to the enzymolysis solution. After flocculants appear, centrifuge (5000 rpm, 3 min) to retain the supernatant, and perform pressure filtration with a plate and frame filter press at a pressure of 0.3 MPa to obtain a clear and transparent filtrate.

[0019] Put the filtrate into a reaction kettle, adjust the pH value of the filtrate to 5.50 ± 1.50, then add 1% of the total liquid volume of coconut shell activated carbon, decolorize and deodorize at 70 ± 2 °C for 30 min, and perform pressure filtration again with a plate and frame filter press. The obtained filtrate is desalted by a nanofiltration system, and when no salt is detected in the discharged liquid, it is transferred to a double-effect concentrator for further concentration to obtain a concentrated solution.

[0020] The concentrated solution was spray-dried using a spray dryer with a feeding pressure of 18.0 MPa, a flow rate of 300 L / h, an inlet air temperature controlled at 130 °C, and an outlet air temperature controlled at 95 °C to obtain protamine polypeptide from Pacific salmon (beige powder).

[0021] II. Obtaining the sequence of the above-mentioned protamine polypeptide from Pacific salmon The protamine polypeptide from Pacific salmon obtained above was detected by LC-MS / MS, and the detection results were analyzed using mass spectrometry analysis software to obtain several polypeptide sequences.

[0022] The LC-MS / MS detection conditions were as follows: (1) In the liquid phase method: The chromatographic column was C18, 3 μm, 250 mm × 75 μm (Eksigent), phase A was water with 0.1% formic acid; phase B was acetonitrile with 0.1% formic acid, the flow rate was 300 nL / min, the injection volume was 1 μL, and the chromatographic gradient was 70 min. The specific elution gradient was: 0 - 55 min, phase A decreased uniformly from 95% to 65%; 55 - 63 min, phase A decreased uniformly from 65% to 50%; 63 - 64 min, phase A decreased uniformly from 50% to 0; 64 - 70 min, phase A was maintained at 0%. (2) In the mass spectrometry method: Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific), positive ion detection mode, the primary resolution was 120000, the AGC was set to 310, and the scanning range was 110 - 2000 m / z. The MIPS mode was peptide, the selected valence states were 1 - 6, the secondary resolution was 17500, and the isolation window was 1.6 m / z.

[0023] III. Screening active oligopeptides with a peak area ≥ 10 7 and the number of amino acids ≤ 6 From the polypeptide sequences obtained above, 11 active oligopeptides with a peak area ≥ 10 7 and the number of amino acids ≤ 6 were finally screened out, and the screening results are shown in Table 1 specifically.

[0024] Table 1 Active oligopeptide sequences in protamine polypeptide from Pacific salmon with a peak area ≥ 10 7 and the number of amino acids ≤ 6

[0025] IV. Screening active oligopeptides with strong binding ability to DPP-IV Using Discovery Studio software, the 11 bioactive oligopeptides in Table 1 were respectively subjected to molecular docking with the crystal structure of dipeptidyl peptidase-4 (DPP-IV) (PDB ID: 5Y7H). Before docking, the 2D structures of the bioactive oligopeptides were converted to 3D structures by energy minimization, and bioactive oligopeptides with strong binding ability to DPP-IV were screened out.

[0026] The docking results were expressed as docking scores (-CiE). The larger the -CiE value, the stronger the interaction between the bioactive oligopeptide and DPP-IV, and the more likely it was to exhibit the function of alleviating diabetic kidney injury.

[0027] The molecular docking results of the 11 bioactive oligopeptides with DPP-IV are shown in Table 2.

[0028] Table 2 Prediction results of the interaction between 11 bioactive oligopeptides and DPP-IV

[0029] As can be seen from Table 2, among the 11 bioactive oligopeptides, only the -CiE of YVAAP with DPP-IV exceeded 80 kcal / mol. Therefore, YVAAP (denoted as pentapeptide YP5) was selected for further prediction analysis.

[0030] After analysis, the binding mode of pentapeptide YP5 to DPP-IV was as Figure 1 shown, and the specific molecular docking situation was as follows: Six H-H bond interactions, five C-H bond interactions, and two electrostatic interactions were formed between pentapeptide YP5 and DPP-IV. Seventeen amino acid residues participated in the interaction between pentapeptide YP5 and DPP-IV, and the van der Waals force between the two was -16.09 kcal / mol.

[0031] V. Evaluating the function of pentapeptide YP5 in alleviating diabetic kidney injury 1. Solid-phase synthesis of pentapeptide YP5 Using the Fmoc solid-phase synthesis strategy, with Fmoc-protected amino acids as raw materials and polystyrene resin as the solid-phase carrier, pentapeptide YP5 (amino acid sequence: YVAAP) was solid-phase synthesized with a purity > 90%.

[0032] 2. Animal experiment procedure Male C57BL / 6J mice at 4 - 6 weeks of age were randomly divided into 4 groups after 1 week of adaptive feeding, namely: control group, model group, positive drug group, and pentapeptide YP5 group.

[0033] The control group was fed with normal feed, and the other groups were fed with high-sugar and high-fat feed for 4 weeks. Starting from the 5th week, after fasting for 8 hours every day, the control group was intraperitoneally injected with citric acid-sodium citrate buffer (200 μL), and the other groups were intraperitoneally injected with streptozotocin (STZ, 50 mg / kg) solution for 5 consecutive days. On the 4th day of the 5th week, the fasting blood glucose of mice in each group was measured. If the fasting blood glucose ≥ 16.7 mM, it was considered that the diabetic mouse model was successfully established. During the experiment, the mice had free access to water. After the modeling was completed, the body weight of the mice (W0) was weighed and recorded.

[0034] For the mice with successful modeling, the positive drug group was intragastrically administered 200 mg / kg metformin every day, and the pentapeptide YP5 group was intragastrically administered 100 mg / kg of solid-phase synthesized pentapeptide YP5 (purity > 90%) every day. The control group and the model group were intragastrically administered normal saline (200 μL) every day. The control group was fed with normal feed, and the other groups continued to be fed with high-sugar and high-fat feed. During the experiment, the mice had free access to water for 4 weeks. At the end of the experiment, the mice were sacrificed, and their body weight (W t ) was weighed and recorded.

[0035] 3. Sample Collection and Processing Plasma: After the last administration, the mice were fasted for 12 hours, and blood was collected from the orbital venous plexus to collect the 24-hour plasma of the mice. The fasting blood glucose of the mice was detected using a blood glucose meter.

[0036] Urine: After the last administration, the mice were fasted for 12 hours, and the 24-hour urine of the mice was collected. The supernatant was obtained by centrifugation, and the 24-hour urinary protein content in the urine was detected using a kit.

[0037] Serum: The whole blood of the mice was collected by eye puncture, placed in a refrigerator at 4°C for 3 hours, then centrifuged at 2000 rpm for 15 minutes. The upper-layer serum was aspirated with a pipette, aliquoted, and stored in a refrigerator at -80°C for the determination of urea nitrogen and creatinine contents in the serum.

[0038] The statistical results of the body weight gain (W t - W0) of mice in each group are shown in Figure 2 . It can be seen from Figure 2 that compared with the body weight gain of the control group (9.98 ± 0.88 g), the body weight gain of the model group (3.32 ± 1.07 g) was significantly reduced (p < 0.001); compared with the body weight gain of the model group (3.32 ± 1.07 g), the body weight gain of the positive drug group (5.5 ± 0.6 g) was significantly increased (p < 0.001), and the body weight gain of the pentapeptide YP5 group (8.53 ± 0.62 g) was also significantly increased (p < 0.001), which was about 1.57 times higher than that of the model group.

[0039] The statistical results of the fasting blood glucose concentration of mice in each group are shown in Figure 3 . It can be seen from Figure 3It was found that: compared with the fasting blood glucose concentration of the control group (5.45 ± 0.3 mM), the fasting blood glucose concentration of the model group (25.3 ± 1.3 mM) increased significantly (p < 0.001); compared with the fasting blood glucose concentration of the model group (25.3 ± 1.3 mM), the fasting blood glucose concentration of the positive drug group (14.5 ± 0.7 mM) decreased significantly (p < 0.001), and the fasting blood glucose concentration of the pentapeptide YP5 group (13.6 ± 0.78 mM) also decreased significantly (p < 0.001).

[0040] The statistical results of the urinary protein content of each group of mice are shown in Figure 4 . From Figure 4 It was found that: compared with the urinary protein content of the control group (1.3 ± 0.18 mg / 24 h), the urinary protein content of the model group (17.1 ± 0.8 mg / 24 h) increased significantly (p < 0.001); compared with the urinary protein content of the model group (17.1 ± 0.8 mg / 24 h), the urinary protein content of the positive drug group (16.0 ± 0.5 mg / 24 h) decreased significantly (p < 0.01), and the urinary protein content of the pentapeptide YP5 group (8.04 ± 0.6 mg / 24 h) also decreased significantly (p < 0.001).

[0041] The statistical results of the serum creatinine content of each group of mice are shown in Figure 5 . From Figure 5 It was found that: compared with the serum creatinine content of the control group (42.17 ± 1.19 μM), the serum creatinine content of the model group (135.8 ± 4 μM) increased significantly (p < 0.001); compared with the serum creatinine content of the model group (135.8 ± 4 μM), the serum creatinine content of the positive drug group (125.3 ± 1.9 μM) decreased significantly (p < 0.001), and the serum creatinine content of the pentapeptide YP5 group (85.99 ± 1.68 μM) also decreased significantly (p < 0.001).

[0042] The statistical results of the serum urea nitrogen content of each group of mice are shown in Figure 6 . From Figure 6 It was found that: compared with the serum urea nitrogen content of the control group (5.59 ± 0.26 mM), the serum urea nitrogen content of the model group (21.2 ± 0.80 mM) increased significantly (p < 0.001); compared with the serum urea nitrogen content of the model group (21.2 ± 0.80 mM), the serum urea nitrogen content of the positive drug group (19.15 ± 0.56 mM) decreased significantly (p < 0.001), and the serum urea nitrogen content of the pentapeptide YP5 group (12.05 ± 0.34 mM) also decreased significantly (p < 0.001).

[0043] In summary, the bioactive oligopeptide YVAAP (pentapeptide YP5) screened from the enzymatic hydrolysate of Pacific salmon sperm can significantly reduce the fasting blood glucose, serum creatinine, blood urea nitrogen and urinary protein levels in diabetic mice, and has the function of alleviating diabetic kidney injury. It has better activity and less toxic and side effects than the positive drug metformin, and can be used in functional foods for alleviating diabetic kidney injury.

[0044] It should be noted that the above examples are only illustrations for clearly explaining the present invention, and are not limitations on the implementation modes of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all implementation modes here. Any obvious changes or modifications derived from the technical solution of the present invention still fall within the protection scope of the present invention.

Claims

1. A pentapeptide YP5 with the function of alleviating diabetic kidney injury, characterized in that, The amino acid sequence of the pentapeptide YP5 is YVAAP, as shown in SEQ ID NO: 4 in the sequence listing, and it has the function of alleviating diabetic kidney injury.

2. Use of the pentapeptide YP5 with the function of alleviating diabetic kidney injury described in claim 1 in a functional food for alleviating diabetic kidney injury.

3. The method for preparing the pentapeptide YP5 having the function of alleviating diabetic kidney damage according to claim 1, characterized in that: Using the solid-phase synthesis method, specifically: Using Fmoc-protected amino acids as raw materials and polystyrene resin as the solid-phase carrier, solid-phase synthesis is carried out using the Fmoc solid-phase synthesis strategy.

4. The preparation method of pentapeptide YP5 with the function of alleviating diabetic kidney injury according to claim 1, characterized in that, Using the enzymatic hydrolysis method, specifically: (1) Take Pacific salmon sperm, remove impurities, add water, heat to 65 ± 2 °C, and maintain for 12 h; (2) Fish out the sperm and make it into a pulp, put it into a reaction kettle, add water, start the stirrer and raise the temperature to 48 °C, add papain for enzymatic hydrolysis for 2 h, continue to raise the temperature to 57 °C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 h, then add flavor protease for enzymatic hydrolysis for 4 h, continue to raise the temperature to 85 °C, and maintain for 30 min to obtain an enzymatic hydrolysate; (3) While cooling, add an aqueous solution of chitosan to the enzymatic hydrolysate. After floccules appear, centrifuge to retain the supernatant and filter press to obtain a clear and transparent filtrate; (4) Adjust the pH value of the filtrate to 5.50 ± 1.50, then add coconut shell activated carbon, decolorize and deodorize at 70 ± 2 °C for 30 min, filter press again, and desalt and concentrate the filtrate to obtain a concentrated solution; (5) Dry the above concentrated solution to obtain Pacific salmon protamine polypeptide, which contains a relatively large amount of pentapeptide YP5.

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