Buffalo milk protein peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene drugs, in particular to buffalo milk protein peptide as well as a preparation method and application thereof, and mainly aims at determining the optimal hydrolysis condition for extracting antidiabetic protein hydrolysate from buffalo milk. In the hydrolysis process, several milk source raw materials of buffalo milk, namely freeze-dried skimmed milk, casein, whey protein and skimmed milk, are used, then the several raw materials are subjected to enzymolysis, and pepsin, alkaline protease and papain are respectively used as three enzymes for enzymolysis; by researching the optimal enzymolysis conditions, further analyzing enzymolysis products with alpha-glucosidase inhibition and recovering the enzymolysis products, several six protein peptides with alpha-glucosidase inhibition characteristics are obtained, and through protein structure analysis, the protein peptides have the alpha-glucosidase inhibition characteristics, have a good blood sugar reduction function and can be used for preparing the blood sugar-reducing peptide with the alpha-glucosidase inhibition characteristics. And a new raw material is provided for later preparation of hypoglycemic drugs or foods.

Description

[Technical field]

[0001] The invention relates to the technical field of food and gene medicine, in particular to buffalo milk protein peptide and a preparation method and application thereof. [Background technology]

[0002] Cow's milk is one of the most nutritious beverages for human consumption. Buffalo milk production is second only to cow's milk, accounting for about 12% of global cow's milk production. In 2022, China's buffalo milk production exceeded 3 million tons, making it one of the main countries in the current buffalo milk production. Buffalo milk is an excellent source of protein, vitamins and minerals due to its unique nutritional composition, and its production is constantly increasing. Therefore, it provides more energy than cow's milk because the former has higher fat and protein content than the latter.

[0003] Over the years, many studies have found that buffalo milk extracts have value-added bioactive properties, such as antihypertensive, antioxidant, anti-inflammatory, antibacterial, anti-thrombocytopenic and anti-apoptotic properties. It has been reported that peptides extracted from buffalo whey cheese can reduce oxidative stress in intestinal epithelial cells. In addition, a decapeptide (YQEPVLGPVR) extracted from buffalo milk casein has anti-inflammatory and antioxidant properties, enzymatic hydrolyzed casein has antioxidant and antihypertensive effects, and several new peptides have been found. On the other hand, a study showed that a peptide derived from buffalo milk casein (NAVPITPTL) has an anti-osteoporotic effect on ovariectomized rats. This peptide can be used to prevent and treat postmenopausal osteoporosis, and it has also been reported that a peptide extracted from buffalo milk casein, VLPVPQK, has antioxidant and anti-apoptotic effects on serum-deficient skin fibroblasts. These studies have proven that buffalo milk and its extracts are beneficial to human health. However, there are few research reports on the antidiabetic properties of buffalo milk protein hydrolysate.

[0004] Diabetes is one of the fastest growing and most common chronic metabolic diseases. Diabetes is divided into two major categories, including type I and type II, and gestational diabetes is contracted during pregnancy. Type I diabetes is a type of autoimmune system damage in which the immune system attacks insulin-secreting cells, while type II diabetes is caused by insufficient insulin secretion or insulin dysfunction. Type II diabetes is the most common type of diabetes in patients with diabetes, with more than 90% of cases being type II diabetes. Therefore, there is an urgent need for diversified methods to prevent and treat type II diabetes. There are several commonly used methods for preventing and treating type II diabetes, such as inhibiting α-amylase, α-glucosidase, and dipeptidyl peptidase IV (DPPIV) enzymes. Although there are studies on inhibiting diabetes-related peptides in the prior art, there is no research on extracting related peptides that can inhibit α-glucosidase from buffalo milk. Therefore, in order to improve the ability to prevent and treat diabetes, it is necessary to conduct relevant extract research on nutrient-rich buffalo milk. [Summary of the invention]

[0005] In view of the above, it is necessary to study the buffalo milk hydrolysates to find out the peptides that can inhibit α-glucosidase and provide new research and development directions for the later development of new drugs or foods.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] Buffalo milk protein peptide, the amino acid sequence of the buffalo milk protein peptide is shown as SEQ ID NO.1-SEQ ID NO.6.

[0008] The present invention also includes the use of the buffalo milk protein peptide in inhibiting alpha-glucosidase.

[0009] The present invention also includes the use of the buffalo milk protein peptide in the preparation of blood sugar lowering medicines or foods.

[0010] The present invention also includes a method for preparing the buffalo milk protein peptide, which comprises:

[0011] 1) Dissolve casein in warm distilled water and adjust the pH to 2, then preheat for 2 minutes; add enzyme solution to the preheated sample, terminate the enzymatic hydrolysis, centrifuge and filter the enzymatic hydrolyzate to obtain the supernatant to obtain the hydrolyzate;

[0012] 2) performing a hydrolysis degree determination, a molecular weight determination, and an α-glucosidase inhibition test on the hydrolyzate obtained in step 1);

[0013] 3) Using membranes with a molecular weight cutoff of 3 kDa and 10 kDa, the hydrolyzed protein with the strongest α-glucosidase inhibitory activity is fractionated by ultrafiltration, and the A (<3 kDa), B (3-10 kDa) and C (>10 kDa) fractions in the reflux liquid and the permeate are collected respectively, and the protein peptides described in claim 1 are obtained by freeze-drying them.

[0014] Furthermore, the enzyme in step 1) is pepsin.

[0015] Furthermore, in the step 1), the enzyme is added at an enzyme / substrate ratio of 1:100.

[0016] Furthermore, the enzymatic hydrolysis time in step 1) is 2 hours.

[0017] Furthermore, in the step 3), a fraction with a molecular weight cut-off of <3 kDa is recovered.

[0018] The present invention has the following beneficial effects:

[0019] 1. The present invention focuses on extracting buffalo milk protein hydrolysates with α-glucosidase inhibitory properties. The main purpose of this study is to determine the optimal hydrolysis conditions for extracting anti-diabetic protein hydrolysates from buffalo milk. Several milk-derived raw materials of buffalo milk are used in the hydrolysis process: freeze-dried skim milk, casein, whey protein and skim milk. These raw materials are then enzymatically hydrolyzed, and three enzymes for enzymatic hydrolysis are used respectively: pepsin, alkaline protease and papain; then, by studying the optimal enzymatic hydrolysis conditions, the enzymatic hydrolysis products with α-glucosidase inhibitory properties are further analyzed, and the enzymatic hydrolysis products are recovered to obtain 6 protein peptides with α-glucosidase inhibitory properties. Through protein structure analysis, we believe that the protein peptides have α-glucosidase inhibitory properties and have good blood sugar lowering function, providing new raw materials for the later preparation of blood sugar lowering drugs or foods.

Brief Description of the Drawings

[0020] Figure 1 The effect of different enzymes on the pH hydrolysis degree of buffalo milk; in the figure, different letters indicate significant differences between protein hydrolysates (p<0.05).

[0021] Figure 2-Figure 5 The effect of different enzymes on the pH molecular weight of buffalo milk; Figure 2 For skim milk, Figure 3 Freeze-dried skim milk, Figure 4 For casein, Figure 5 For whey protein.

[0022] Figure 6 Figure 2 is the α-glucosidase inhibition rate of buffalo milk pH hydrolyzed by different enzymes. In the figure, different letters indicate significant differences among the protein hydrolysates (p<0.05).

[0023] Figure 7 Figure 2 is the α-glucosidase inhibition rate of different molecular weight fractions in three groups of pH. In the figure, A is <3kDa, B is 3-10kDa, and C is >10kDa fractions. Different letters indicate significant differences among protein hydrolysates (p<0.05).

[0024] Figure 8 The figure is a percentage diagram of the amino acid content in the six identified polypeptides; in the figure, * represents hydrophobic amino acids, A-alanine, D-aspartic acid, E-glutamic acid, F-phenylalanine, G-glycine, H-histidine, I-isoleucine, K-lysine, L-leucine, M-methionine, N-asparagine, P-proline, Q-glutamine, S-serine, T-threonine, V-valine, W-tryptophan, and Y-tyrosine.

[0025] Figure 9-14 It is the molecular docking diagram of α-glucosidase and purified polypeptide; Fig. 9 The peptide is SEQ ID NO.1, Fig.10 SEQ ID NO.2, Fig.11 SEQ ID NO.3, Fig.12 SEQ ID NO.4, Fig.13 SEQ ID NO.5, Fig.14 It is SEQ ID NO.6. [Specific implementation method]

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0027] Embodiment 1:

[0028] This example is a method for analyzing the α-glucosidase inhibition of buffalo milk hydrolysate and an extraction method.

[0029] 1. Materials:

[0030] Fresh buffalo milk. The milk was defatted using a milk fat separator (D9N-50, Jinshan, China). The enzymes used in the hydrolysis process, including alkaline protease, pepsin, and papain, were purchased from Sigma. α-Glucosidase was purchased from Shyuanye, China. In addition, SDS-Page materials (including precast gels, running buffer, and 5X sample buffer) were purchased from Genscript, USA.

[0031] Skimmed buffalo milk (skimmed milk for short): fresh buffalo milk is slowly poured into a milk fat separator (D9N-50, China Jinshan brand) for skimming to obtain skimmed buffalo milk.

[0032] Freeze-dried skim milk powder (freeze-dried skim milk for short): first pre-freeze the skim buffalo milk in a -80℃ refrigerator, then freeze-dry the skim milk in a freeze dryer until it is completely dry (about 48 hours).

[0033] Buffalo milk casein freeze-dried powder (abbreviated as casein): Take part of the skimmed buffalo milk, adjust the pH to 4 with acetic acid, precipitate the casein, filter with filter paper, the filtrate is the whey for standby use, and the solid is the casein. Rinse the casein with pure water at least 4 times to remove the residual acetic acid, dissolve the precipitated casein in water again, adjust the pH to 7, put it in a -80℃ refrigerator for pre-freezing, and then transfer it to a freeze dryer for freeze drying.

[0034] Buffalo milk whey protein freeze-dried powder (abbreviated as whey protein): adjust the pH of the filtered whey to 7, place it in a -80℃ refrigerator for pre-freezing, and then transfer it to a freeze dryer for freeze drying until it is completely dry.

[0035] 2. Methods:

[0036] 1. Hydrolysis process: Freeze-dried skim milk, casein and whey protein groups were dissolved in warm water (10% w / v), while the skim milk group was directly used for hydrolysis. First, HCl and NaOH were used to adjust the four groups of substrate solutions to pH 2 (pepsin), pH 7 (papain) and pH 8.5 (alkaline protease), respectively, and then preheated to 60°C for 2 min, and then the substrate solution was placed in water preheated to 60°C for 2 min, and the corresponding enzyme was added to the substrate solution at an enzyme / substrate ratio (w / v) of 1:100 to obtain 12 groups of enzymatic hydrolysates. At the same time, without adding enzymes, the substrate solution was adjusted to the corresponding pH value to obtain 12 control groups. The hydrolysis was continued at 60°C for 2h and 4h, respectively. At the end, the hydrolysis solution was heated in a 100°C water bath for 15min, and then immersed in ice water and immediately cooled to room temperature, thereby terminating the hydrolysis. The pH value of the hydrolyzed solution was adjusted to 7, centrifuged at 10000xg (Heraeus Biofuge Stratos, Thermoscientific, USA) for 15 min (4°C), the supernatant was filtered, freeze-dried (Alpha1-2LDplus, ChristSigma, Germany), and 48 groups of hydrolyzed solutions (abbreviated as PH) were obtained and stored at -80°C for use. Among them, the enzymatic activity of pepsin was 250 units / mg, the enzymatic activity of papain was 1.5-10 units / mg, and the enzymatic activity of alkaline protease was 2.4 units / g.

[0037] 2. Chemical composition determination of buffalo fresh milk and skim milk: The chemical composition of buffalo fresh milk and skim milk was determined using a milk analyzer F120 (Foss, Denmark). The chemical composition, including the content of protein, fat, lactose, total solids (TS) and non-fat milk solids (SNF), is shown in Table 1.

[0038] Table 1 Chemical composition of buffalo fresh milk and skim milk

[0039]

[0040] 3. Determination of hydrolysis degree: The degree of hydrolysis of the hydrolyzate was determined by the o-phthalaldehyde (OPA) assay. The OPA solution was prepared with 3.81 g sodium tetraborate, 0.1 g sodium dodecyl sulfate (SDS), 0.088 g dithiothreitol (DTT) and 0.08 g OPA (in 2 mL ethanol) to a final volume of 100 mL. 150 μL of OPA solution was added to 20 μL of sample (10 mg / mL). The mixture was then incubated at 30 °C for 2 min, and the absorbance Abs was read at a wavelength of 340 nm using a spectrophotometer (Agilent Biotek Epoch 2, USA). Serine was used as the standard reference and the same method was used. The percentage of hydrolysis was calculated as follows:

[0041]

[0042] In the formula, α is 1.039, β is 0.383, and h tot It is 8.2 mol / g.

[0043] 4. Molecular weight determination: The molecular weight of each group of PH was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the following method. SDS-PAGE electrophoresis used a 4-20% separation gel, and the prepared sample concentration was 5 mg / mL, of which 10 μL of sample was added to the SDS loading buffer and then boiled for 5 minutes. The protein ladder of 6.5-240 kDa was loaded onto the gel, and then the sample was loaded. The voltage was set to 70 V for 90 minutes. The gel was stained with Coomassie blue solution and then eluted twice with methanol: water: acetic acid: distilled water (5:3:1:1), each for 30 minutes.

[0044] 5. Determination of α-glucosidase inhibition rate: Add 50 μL α-glucosidase (1U, 0.1 mol / L PBS, pH 6.8) to 50 μL PH, and then incubate at 37 ° C for 10 min. Then add 50 μL 4-nitrobenzene α-D-pyranoglucoside (PNPG) to the mixture and incubate at 37 ° C for 20 min. Add 100 μL 1 mol / L sodium carbonate (Na2CO3) to terminate the enzyme activity. Use a spectrophotometer (Agilent Biotek Epoch2, USA) to read the absorbance Abs at a wavelength of 405 nm. The standard used in the test is acarbose. Parallel 3 times. The inhibition rate is calculated according to the following formula:

[0045]

[0046] 6. Fractionation of protein hydrolysate: Using membranes with a molecular weight cutoff (MWCO) of 3 kDa and 10 kDa, PH with the strongest α-glucosidase inhibitory activity was separated and graded by ultrafiltration. 10 mL of PH with a concentration of 10 mg / mL was placed in a molecular weight cutoff (MWCO) centrifuge tube (Vivaspin, Sartorius, Germany), and then centrifuged at 2701 g (Heraeus Biofuge Stratos, Thermo scientific, USA) for 30 min at 4°C. The A (<3 kDa), B (3-10 kDa) and C (>10 kDa) fractions in the reflux and permeate were collected, respectively, and freeze-dried (Alpha1-2 LDplus, ChristSigma, Germany) for determination of α-glucosidase inhibition rate.

[0047] 7. Identification of peptides using liquid chromatography-mass spectrometry (LC-MS / MS): The peptide sequences were identified using a Vanquish Neo ultra-high performance liquid chromatography (UHPLC) system and an Orbitrap Astral mass spectrometer (Thermo Scientific, USA). The enzymatic hydrolyzed sample was ultrafiltered with 8 mol / L urea solution and then desalted using a C18 filter cartridge. Chromatographic separation was performed using a PepMap Neo (5 μm) C18 column (300 μmX5 mm, gradient elution). The column was equilibrated with 96% mobile phase A (0.1% formic acid aqueous solution). The elution gradient of mobile phase B (0.1% acetonitrile-formic acid-80% acetonitrile) was set as follows: linear gradient 4-10%, 1.2 min; 10-28%, 10 min; 28-45%, 2.3 min; 99%, 1.5 min. Peptides were separated using DDA mass spectrometry analysis using an Orbitrap Astral mass spectrometer (Thermo Scientific). The analysis time was 15 min, the electrospray voltage was 2.2 kV, the detection mode was positive ion, and the precursor ion scanning range was 380-980 m / z.

[0048] 8. Molecular docking of peptides: AutodockVina was used to dock the six peptides identified by LCMS / MS with α-glucosidase, the crystal structure of which was from the RCSB protein database, and the two-dimensional peptide sequence was from ChemBio3DUltra14.0. The water molecules in the protein molecules were deleted, and hydrogen bonds and Kollman charges were added. The grid frame was set to center_x=-21.672, center_y=-21.563, center_z=9.708, and the dimensions were size_x=78, size_y=68, size_z=104. The docking complex of the protein receptor and peptide sequence with α-glucosidase inhibition was analyzed using PyMOL, and the hydrophobic interactions and salt bridges between the ligand and the protein were analyzed using LigPlot+Version v.2.2.9 software.

[0049] 9. Statistical analysis: All experiments were repeated three times and expressed as mean ± standard deviation. SPSS16.0 software was used for statistical analysis, and the confidence level was α≤0.05. One-way ANOVA was used for the data of hydrolysis degree and α-glucosidase inhibition rate. Tukey's one-way ANOVA was used for mean comparison.

[0050] 4. Results and Discussion

[0051] 1. Hydrolysis degree: The hydrolysis degree of 48 groups of PH was determined using the o-phthalaldehyde (OPA) method. The hydrolysis degree of PH in the skim milk and casein groups was higher (68-100%), while the hydrolysis degree of PH in the whey protein group was lower (6-62%). In addition, the hydrolysis degree of PH in the enzymatic hydrolysis group was higher than that in the control group (e.g. Figure 1 As shown). The hydrolysis degree of casein PH using the enzymatic group was the highest (92-100%), followed by skim milk PH (77-100%), freeze-dried skim milk PH (75-98%), and whey protein PH (18-62%). This shows that the three enzymes have a better hydrolysis effect on casein than whey protein. Casein is more easily hydrolyzed by enzymes due to its open and disordered structure, while whey protein has a lower degree of hydrolysis due to its globular structure. In addition, the results also show that the hydrolysis degree of papain group PH is the highest, followed by alkaline protease and pepsin, especially for the casein group. This shows that as the degree of hydrolysis increases, more amino groups in the parent protein are released or hydrolyzed. In general, the degree of hydrolysis of the sample is greatly affected by the specificity of the enzyme, the enzyme reaction rate, and the substrate affinity. Pepsin is an aspartic protease that cleaves peptide bonds adjacent to phenylalanine, tyrosine, and tryptophan, as well as the carboxyl groups of glutamic acid and leucine. However, this enzyme does not cleave linkages in valine, glycine, and alanine, thereby preserving disulfide groups in the extracted proteins. Papain is a cysteine ​​protease that hydrolyzes peptide bonds in amino acids next to lysine, arginine, and phenylalanine as well as several hydrophobic regions, including tyrosine, valine, alanine, leucine, isoleucine, and tryptophan. Alkaline protease is a serine protease that cleaves the carboxyl side of hydrophobic amino acid residues. This enzyme also hydrolyzes protein bonds involving aromatic amino acids. Therefore, the enzyme hydrolyzes peptide bonds more extensively according to its specificity compared to the control group where only pH was adjusted for hydrolysis. However, the degree of hydrolysis value of pH should be controlled because it affects the amino acid sequence, the number of charged groups, hydrophobicity, polarity, and molecular weight (size) of the protein hydrolysate, which in turn affects the biological activity of the hydrolysate.

[0052] 2. Comparative analysis of the effect of different enzymes on the molecular weight distribution of buffalo milk pH: The molecular weight of protein hydrolysates was determined by SDS-PAGE electrophoresis. Figure 2-Figure 5 The molecular weight distribution of hydrolyzed proteins extracted from buffalo milk and their components is shown. Figure 2 , Figure 3 and Figure 4 The protein hydrolysates extracted by pH-dependent hydrolysis showed distinct bands around 13 kDa, 18 kDa, 25 kDa, 31 kDa, and >58 kDa. These bands appeared similar to those of the unhydrolyzed samples. However, except for the papain-free skim milk pH group, which had a distinct band around 13 kDa, see Figure 3, no obvious bands were found in other enzymatic hydrolysis groups (all faded). The lack of bands after enzymatic hydrolysis is consistent with the literature, because after the hydrolysis process, the bands related to bovine milk casein, especially the 25 kDa band, disappeared significantly.

[0053] On the other hand, the papain casein pH group showed bands around 18 kDa, 25 kDa, and 31 kDa, see Figure 4 Most of the bands between 15 and 31 kDa are caseins, of which κ-casein is about 19 kDa, β-casein is 24 kDa, and α-casein is 25 kDa, indicating that the hydrolysis of this group is not complete. The whey protein pH 8.5 control group has similar bands to the unhydrolyzed group, with 5 bands between 13 kDa and 18 kDa, a faint line between 25 and 31 kDa, and a band greater than 58 kDa, representing lactoferrin, see Figure 5 . In contrast, in the whey protein control group pH2 and pH7, there were only two obvious bands of about 13kDa and 18kDa, corresponding to α-lactalbumin and β-lactoglobulin, respectively. The disappearance of the bands after hydrolysis may be related to the destruction and denaturation of disulfide bonds in whey protein. In the whey protein hydrolysis group pH, except for papain, which had an obvious band at about 13kDa, there were no bands in other groups. This reflects the specificity of the enzyme's action on the structure of α-lactalbumin, because the cleavage site of α-lactalbumin is deeply buried in its barrel structure. This is consistent with the result that the degree of hydrolysis of casein hydrolysate is higher than that of whey hydrolysate, because casein has a more flexible random structure and is more easily hydrolyzed by enzymes.

[0054] 3. Comparative analysis of the α-glucosidase inhibition rate of different enzymes on buffalo milk PH: The hypoglycemic properties of 48 groups of PH were evaluated using the in vitro α-glucosidase inhibition rate. The results showed that only the enzymatic group PH had α-glucosidase inhibitory activity at a sample concentration of 10 mg / mL. The unhydrolyzed skim milk and the control group that only adjusted the pH without adding enzymes could not inhibit α-glucosidase even at high concentrations. Some scholars have also made similar findings that unhydrolyzed milk protein has no inhibitory effect on α-glucosidase. This shows that pH cannot effectively hydrolyze protein to produce bioactive peptides. In addition, pH will also change the ionic charge of the protein and the binding affinity of the protein to α-glucosidase.

[0055] The pH of casein hydrolyzed group had the strongest inhibitory activity on α-glucosidase, while the pH of whey hydrolyzed group had the weakest inhibitory activity. The pH of pepsin had the strongest inhibitory effect on α-glucosidase, while the pH of alkaline protease and papain had weaker inhibitory effects. Figure 6The results showed that the inhibition rate of pepsin on α-glucosidase in 2h freeze-dried skim milk pH and 2h and 4h casein pH exceeded 50%, 55.4%, 57.1% and 55.9% respectively, which was higher than that in the pH group (2.2-43.4%). The inhibition of casein pH was stronger, which may be due to the release of peptides with stronger binding affinity to the enzyme. The pepsin pH was more effective than the A1 and A2 bovine milk casein pH, which were reported to have an inhibition rate of only 12.5-26.9% for A1 and A2 bovine milk casein hydrolysates.

[0056] The three groups of pH with the best inhibitory activity on α-glucosidase were selected and their half-maximal inhibition values ​​(IC 50 ) and compared with the positive control group acarbose. IC of pepsin at 2h and 4h casein pH 50 The value is 5.3-5.6 mg / mL, which is significantly lower than the IC value of freeze-dried skim milk pH. 50 Compared with the standard drug acarbose, the IC values ​​of the three PH groups were 50 The higher values ​​may be due to the presence of both bioactive and non-bioactive peptides in the pH. However, the efficacy of pepsin-digested casein pH was comparable to that of fermented cheese spreads, which had an IC 50 The value was 5.3 mg / mL. These results emphasize the specificity of enzymatic hydrolysis in releasing bioactive peptides from casein, while whey hydrolysates showed minimal activity due to limited release of inhibitory peptides. Previous studies have reported that α-glucosidase inhibitory activity in whey is mainly associated with β-lactoglobulin, followed by α-lactalbumin, while lactoferrin has negligible activity.

[0057] In conclusion, the pepsin-supplemented PHs, especially casein PH, had the highest α-glucosidase inhibitory activity. Based on these results, we selected three pepsin PHs, namely freeze-dried skim milk PH (2h), casein PH (2h), and casein PH (4h), and further fractionated them by ultrafiltration to isolate and identify their bioactive peptides.

[0058] Table 2 Half-maximal inhibition values ​​IC of standard drugs and three preferred groups of PH 50

[0059]

[0060] Note: Means with different letters indicate significant differences (P<0.05).

[0061] 4. Effect of different molecular weight fractions on α-glucosidase inhibition: 3 and 10 kDa molecular weight cutoff (MWCO) membranes were used to fractionate the three groups of pepsin PH to obtain three fractions: fraction A (<3 kDa), fraction B (3-10 kDa) and fraction C (>10 kDa). The results showed that the fractions with lower molecular weight had significantly higher α-glucosidase inhibition activity, among which casein PH fraction A had the highest inhibition rate, which was comparable to casein PH fraction B (IC 50 =5.9 mg / mL) and freeze-dried skim milk fraction B (IC 50 =5.3 mg / mL), casein pH fraction A had the highest inhibition rate (43.6-47.4%) ( Figure 7 ), IC 50 The lowest value was 4.9 mg / mL (Table 3). Peptides smaller than 2 kDa showed greater potency than larger peptides, with IC 50 The values ​​ranged between 470 and 543 M. Low molecular weight peptides showed stronger inhibition against α-glucosidase, thanks to their exposed amino acid residues, which allow for better interaction with the active site of the enzyme. The composition, molecular weight and chain length of these peptides play a crucial role in their inhibitory potential.

[0062] Table 3 Half maximum inhibition values ​​(IC 50 )

[0063]

[0064] Note: A is <3kDa, B is 3-10kDa, and C is >10kDa fraction; different letters indicate significant differences in the same column (p<0.05)

[0065] 5. Peptide identification: Six peptides with molecular weights between 1.3 and 1.8 kDa were identified from the pepsin-digested casein PH fraction A. These oligopeptides include DELQDKIHPFAQTQS, IVSVEPTSTPTTE, AEEQLHSMKEGIHA, IVSVEPTSTPITE, TVAGGAWTYNTTSAVTVK, and VYPFPGPIPNSL. Previous studies have shown that antidiabetic peptides are usually oligopeptides (<2 kDa) because of their small size, which can improve bioavailability and stability during gastrointestinal digestion.

[0066] These peptides are rich in hydrophobic and aliphatic amino acids, including leucine (L), isoleucine (I), proline (P), methionine (M), and alanine (A), which are known to enhance their interaction with α-glucosidase. Hydrophobic amino acids account for 44.7% of the amino acid composition, with the most prominent contributions from P (10.6%), A (7.1%), and I (7.1%). Figure 8 ). In addition, several amino acids involved in insulin regulation and secretion, as well as inhibition of α-glucosidase, were also present in large quantities. These amino acids include glutamate (E), arginine (R), serine (S), threonine (T), and valine (V). Notably, the identified peptides contained a considerable proportion of T (15.4%), as well as other key amino acids E, S, and V (9.4%). In addition, these peptides also contained 2.4-4.7% of tyrosine (Y), glycine (G), aspartic acid (D), histidine (H), and lysine (K), all of which contribute to the inhibition of α-glucosidase. The potency of these peptides depends primarily on the enzyme used for hydrolysis, as the specificity of the enzyme determines the cleavage site and fragment size, ultimately affecting its biological activity.

[0067] 6. Molecular docking and interaction: Molecular docking studies evaluated the binding interactions of the six identified peptides with the active site of α-glucosidase (PDB code: 4J5T) ( Figure 9-14 ). The binding affinities of these peptides ranged from -7.7 to -12.2 kcal / mol, with IVSVEPTSTPTTE having the strongest binding affinity (-12.2 kcal / mol) (Table 4). These peptides had stronger binding affinities than the standard drug acarbose (-7.6 kcal / mol), indicating that they have comparable enzyme inhibition potential because they bind to the protein ligand of α-glucosidase more stably than acarbose. These peptides interact with α-glucosidase mainly through hydrogen bonding and hydrophobic interactions. Common hydrogen bonding sites include Arg428, Glu470, Ser466, and Tyr709, while hydrophobic interactions occur at residues such as Asp392, Gly556, Ile469, and Phe385 (Table 5). These interactions are consistent with the catalytic domain previously reported as binding residues for α-glucosidase, which are important for enzyme inhibition, preventing the formation of the enzyme-substrate complex, thereby inhibiting the glycosylation process. These findings suggest that the six peptides identified from buffalo milk casein PH are potent inhibitors of α-glucosidase and have strong potential for antidiabetic applications. The identified peptides, especially those with smaller molecular weight, are able to target the active site of the enzyme with high affinity, show good biological activity, and may provide another approach for controlling type 2 diabetes.

[0068] Table 4 Peptide sequence information and binding affinity to the active site of α-glucosidase (PDB code: 4J5T) identified by LC-MS / MS

[0069]

[0070]

[0071] Note: Note: D = aspartic acid, H = histidine, I = isoleucine, M = methionine, S = serine, V = valine, A = alanine, G = glycine, L = leucine, P = proline, T = threonine, F = phenylalanine, Y = tyrosine, W = tryptophan, N = asparagine, E = glutamic acid, Q = glutamine, K = lysine.

[0072] Table 5 Interaction of peptide sequences with the active site of α-glucosidase (PDB code: 4J5T) through hydrogen bonding and hydrophobic interactions

[0073]

[0074] In summary, this application optimizes several buffalo milk raw materials: freeze-dried skim milk, casein, whey protein and skim milk, and uses a combination of pepsin, alkaline protease and papain for enzymatic hydrolysis to obtain a hydrolyzate with a high degree of hydrolysis (68-100%) and significant biological activity. By studying the hydrolyzate, it was found that casein PH has the potential to be a valuable source of α-glucosidase inhibitors with anti-diabetic properties. Casein PH enzymatically hydrolyzed with pepsin has the strongest inhibitory activity on α-glucosidase, with an inhibition level of 55.9-57.1%. After fractionating these PHs, it was found that the smaller peptides (<3kDa) in fraction A were the most effective, and IC 50 Values ​​as low as 4.9 mg / mL. Six novel peptides were identified from the pepsin-digested casein PH fraction A: DELQDKIHPFAQTQS, IVSVEPTSTPTTE, AEEQLHSMKEGIHA, IVSVEPTSTPITE, TVAGGAWTYNTTSAVTVK, and VYPFPGPIPNSL. These peptides have a high content of hydrophobic amino acids, which contributes to their strong binding to the active site of α-glucosidase. Molecular docking studies showed that these peptides interact with the enzyme mainly through hydrogen bonds and hydrophobic interactions, resulting in a strong binding affinity (-7.7 to -12.2 kcal / mol). The study showed that this interaction inhibits the enzymatic activity of α-glucosidase by disrupting the formation of the enzyme-substrate complex. These findings highlight the therapeutic potential of buffalo milk casein PH and its derived peptides as effective natural inhibitors of α-glucosidase. This study highlights the specificity of enzymatic hydrolysis in generating bioactive peptides with enhanced anti-diabetic properties.

[0075] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Buffalo milk protein peptide, characterized in that The amino acid sequences of the buffalo milk protein peptides are shown in SEQ ID NO.1-SEQ ID NO.

6.

2. Use of the buffalo milk protein peptide as claimed in claim 1 in inhibiting α-glucosidase.

3. Use of the buffalo milk protein peptide according to claim 1 in the preparation of hypoglycemic drugs or foods.

4. A method for preparing the buffalo milk protein peptide according to claim 1, characterized in that: The method is: 1) Dissolve casein in warm distilled water and adjust the pH to 2, then preheat for 2 minutes; add enzyme solution to the preheated sample, terminate the enzymatic hydrolysis, centrifuge and filter the enzymatic hydrolyzate to obtain the supernatant to obtain the hydrolyzate; 2) performing a hydrolysis degree determination, a molecular weight determination, and an α-glucosidase inhibition test on the hydrolyzate obtained in step 1); 3) Using membranes with a molecular weight cutoff of 3 kDa and 10 kDa, the hydrolyzed protein with the strongest α-glucosidase inhibitory activity is fractionated by ultrafiltration, and the A (<3 kDa), B (3-10 kDa) and C (>10 kDa) fractions in the reflux liquid and the permeate are collected respectively, and the protein peptides described in claim 1 are obtained by freeze-drying them.

5. The method according to claim 4, characterized in that The enzyme in step 1) is pepsin.

6. The method according to claim 4, characterized in that The enzyme addition ratio in step 1) is an enzyme / substrate ratio of 1:

100.

7. The method according to claim 4, characterized in that The enzymatic hydrolysis time of the protease in step 1) is 2 hours.

8. The method according to claim 4, characterized in that In the step 3), a fraction with a molecular weight cut-off of <3 kDa is recovered.

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