A special process for preparing small molecule bioactive peptides from coconut meat and their application in hypoglycemic products.

Abstract

This invention provides a method for preparing small-molecule bioactive peptides from coconut meat using a special process and their application in hypoglycemic products. Specifically, using coconut dietary fiber powder as raw material, an enzymatic hydrolysate is prepared by stepwise enzymatic hydrolysis with a combination of ultrasonic-assisted neutral and alkaline proteases. The hydrolysate is then purified by ultrafiltration and gel chromatography to obtain the active components in the 1-3 kDa molecular weight region. Subsequently, three hypoglycemic bioactive peptides with sequences of LPFPRPAGPR, FDLPAR, and ANVFNPR are obtained by liquid chromatography-tandem mass spectrometry. The obtained bioactive peptides significantly inhibit α-glucosidase activity and significantly improve glucose metabolism in insulin-resistant HepG2 cells. The preparation method of this invention uses readily available raw materials, has a simple process, and is low in cost. The obtained products have great potential as novel hypoglycemic food ingredients and have broad application prospects in hypoglycemic functional foods, health products, and special medical foods.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and functional foods, and more specifically, to a small molecule peptide with hypoglycemic activity extracted from coconut dietary fiber powder through a special process, and its application in hypoglycemic products such as functional foods. Background Technology

[0002] With changes in modern dietary structures and the accelerated pace of life, hyperglycemia and diabetes have become one of the major chronic diseases threatening public health. Type 2 diabetes mellitus (T2DM), as a serious disorder of glucose metabolism, can lead to various complications due to long-term hyperglycemia, seriously threatening patients' lives. Currently, commonly used hypoglycemic drugs in clinical practice mainly include insulin, sulfonylureas, biguanides, and alpha-glucosidase inhibitors. However, long-term use of these drugs is often accompanied by side effects (such as hypoglycemia, weight gain, gastrointestinal discomfort, etc.) and drug resistance problems. Therefore, finding safe and effective hypoglycemic active substances from natural plant resources has become an important direction for the development of functional foods and special medical foods.

[0003] Coconut, a tropical crop, contains abundant protein, dietary fiber, and various bioactive components in its meat. After coconut oil production, the remaining coconut residue is typically discarded as waste. However, this residue can be further processed to extract coconut dietary fiber powder. This powder is rich in high-quality plant protein, widely available, and inexpensive, making it a potentially high-quality raw material for preparing bioactive peptides. Currently, research on the in-depth development of coconut dietary fiber powder protein and its bioactive substances is limited, especially regarding the discovery and functional research of its hypoglycemic active peptides, which remains largely unexplored.

[0004] It is worth noting that Shanghai Ocean University, in its research on coconut fiber by-products, discovered that the enzymatic hydrolysis products contain components with a molecular weight range of 1-3 kDa that can significantly enhance glucose metabolism in insulin-resistant HepG2 cells, and these components also have an inhibitory effect on α-glucosidase activity. This discovery provides a key technical inspiration for this invention: using coconut dietary fiber powder as raw material, extracting a small molecule peptide composition with hypoglycemic activity through a specific enzymatic hydrolysis process, and using it in the development of functional foods to assist in the control of postprandial blood glucose.

[0005] Compared to single peptides, complex peptides derived from the hydrolysis of dietary proteins offer advantages in both efficacy and cost, making them more suitable for the development of hypoglycemic dietary foods. However, existing technologies for the efficient and high-purity extraction of specific hypoglycemic active peptides from coarse coconut fiber powder are still immature, resulting in high development costs for related functional foods and limiting their application prospects. Summary of the Invention

[0006] This invention addresses the aforementioned problems by providing a novel hypoglycemic small-molecule bioactive peptide extracted from coconut dietary fiber powder via a specific enzymatic hydrolysis method. The preparation method is simple and low-cost, and the resulting product exhibits a significant inhibitory effect on α-glucosidase activity. This bioactive peptide can be used as a novel hypoglycemic functional food ingredient.

[0007] The present invention adopts the following technical solution: A small molecule hypoglycemic peptide from coconut meat with α-glucosidase inhibitory activity, wherein the amino acid sequence is selected from any of the following: FDLPAR; ANVFNPR; LPFPRPAGPR.

[0008] A composition comprising at least two of the said active peptides.

[0009] According to a specific embodiment of the present invention, the composition is the F2 component obtained by enzymatic hydrolysis, separation and purification of coconut dietary fiber powder; the F2 component is a component collected by Sephadex G-25 gel chromatography during an elution period of 70-110 min.

[0010] An extract is a 1-3 kDa active component obtained from coconut dietary fiber powder through compound stepwise enzymatic hydrolysis, ultrasound-assisted separation, and ultrafiltration; the extract contains the F2 component.

[0011] A method for preparing small molecule bioactive peptides from coconut meat using a special extraction process includes the following steps: (1) Enzymatic hydrolysis of coconut dietary fiber powder: After mixing coconut dietary fiber powder with deionized water, the mixture is subjected to stepwise enzymatic hydrolysis by a combination of neutral protease and alkaline protease with ultrasonic assistance. After inactivation of enzymes, the mixture is centrifuged and the supernatant is collected to obtain the hydrolysate. (2) Separation of hypoglycemic active peptides: The enzymatic hydrolysate is separated by ultrafiltration membrane to obtain ultrafiltration components with a molecular weight of 1-3 kDa; (3) Purification of hypoglycemic active peptides: The ultrafiltration fraction was filtered through a 0.45 µm filter membrane and then separated by gel chromatography using Sephadex G-25 as the separation medium. The α-glucosidase inhibitory activity of the eluted fractions corresponding to each absorption peak was measured. The fraction with the highest α-glucosidase inhibitory activity was selected and freeze-dried to obtain fraction F2. Fraction F2 was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and three active peptides with amino acid sequences of FDLPAR, ANVFNPR and LPFPRPAGPR were separated.

[0012] In this application, "combined stepwise enzymatic hydrolysis of neutral protease and alkaline protease" means that neutral protease hydrolysis is performed first, followed by alkaline protease hydrolysis.

[0013] Furthermore, the "coconut meat dietary fiber powder" is preferably a product obtained by supercritical extraction, drying, and mechanical pulverization of coconut meat after coconut oil pressing.

[0014] Furthermore, the amount of neutral protease added is 5000~9000 U / g, the pH is adjusted to 5.5~9.5, and enzymatic hydrolysis is carried out at a temperature of 30~60℃.

[0015] Furthermore, the amount of alkaline protease added is 5000~9000 U / g, the pH is adjusted to 7.5~11.5, and enzymatic hydrolysis is carried out at a temperature of 30~60℃.

[0016] Furthermore, the total time for ultrasonic enzymatic hydrolysis is 60-180 min, the hydrolysis time for neutral protease is 30-60 min, the hydrolysis time for alkaline protease is 30-60 min, the ultrasonic frequency is 40-100 kHz, the ultrasonic power is 100-300 W, and the hydrolysis temperature is 30-60℃.

[0017] Furthermore, in step (3), the eluent for gel chromatography separation is ultrapure water, and the flow rate is 0.4 mL / min.

[0018] A small-molecule hypoglycemic active peptide from coconut meat, with the amino acid sequence FDLPAR.

[0019] A small-molecule hypoglycemic active peptide from coconut meat, with the amino acid sequence ANVFNPR.

[0020] A small-molecule hypoglycemic active peptide from coconut meat, with the amino acid sequence LPFPRPAGPR.

[0021] The application of the aforementioned coconut meat small molecule hypoglycemic active peptide, or the aforementioned composition or extract, in the preparation of hypoglycemic functional foods, health foods, and special medical foods.

[0022] The application of small-molecule hypoglycemic active peptides from coconut meat is in the fields of functional foods, health products, dietary additives, and cosmetics.

[0023] Before molecular docking of small-molecule hypoglycemic active peptides from coconut meat with α-glucosidase, the functional activity, toxicity, sensitization, hydrophobicity, and isoelectric point of the peptide sequence are predicted.

[0024] The advantages of this invention are as follows: (1) The preparation method of the present invention is simple and easy to operate. The hypoglycemic active peptide (1-3kDa ultrafiltration fraction) obtained by ultrafiltration separation from the enzymatic hydrolysate of coconut dietary fiber powder has a high yield, high purity, strong α-glucosidase inhibitory activity, and can significantly improve the glucose metabolism level of insulin-resistant HepG2 cells. Therefore, it can be used in functional foods, health food products, and special medical foods. (2) The hypoglycemic active peptides (three peptides) obtained from coconut dietary fiber powder by the present invention have strong α-glucosidase inhibitory activity, and therefore can be applied to food health products, special medical foods and drugs; (3) The hypoglycemic active peptides isolated by the present invention are small molecule peptides with a molecular weight between 700 and 1200 Da, which are easily digested and absorbed by the human body and have good prospects for development and utilization. (4) The raw materials of this invention are readily available and inexpensive, which increases the added value of coconut meat fiber and provides a foundation for the comprehensive utilization of coconut meat dietary fiber powder, and has good prospects for promotion. Attached Figure Description

[0025] Figure 1 The graph shows the inhibition rate of α-glucosidase by the enzymatic hydrolysate with different total ultrasonic hydrolysis times in Example 1.

[0026] Figure 2 This is a graph showing the inhibition rate of α-glucosidase by different ultrafiltration components in Example 1.

[0027] Figure 3 The effect of different concentrations of 1-3 kDa ultrafiltration fractions on the cytotoxicity of HepG2 cells.

[0028] Figure 4 The effect of different concentrations of 1-3 kDa ultrafiltration fractions on glucose consumption in IR-HepG2 cells.

[0029] Figure 5 Elution curves of the gel column separation products of this invention: F1 component, F2 component, F3 component, and F4 component.

[0030] Figure 6 The graph shows the inhibition rates of α-glucosidase on the gel column separation products of this invention: F1, F2, F3, and F4 components.

[0031] Figure 7 This is a graph showing the inhibition rate of α-glucosidase by the three hypoglycemic active peptides of this invention at different concentrations. Detailed Implementation

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0033] Example 1: (1) Weigh 10 g of coconut dietary fiber powder, add 3% (w / v) deionized water and homogenize. After homogenization, add 8000 U / g neutral protease and alkaline protease sequentially for double enzymatic hydrolysis. The hydrolysis system is heated to 50℃ and reacted under ultrasonic assistance at 40 kHz and 200W. Neutral protease hydrolysis is performed first, followed by alkaline protease hydrolysis. The total ultrasonic hydrolysis time is set to 60 min, 90 min, 120 min, 150 min and 180 min, and the hydrolysis time for each step is evenly divided. After the hydrolysis is completed, the enzyme is inactivated, and then centrifuged at 8000 rpm for 15 min. The supernatant is collected for the determination of α-glucosidase inhibition rate. Figure 1 ); (2) The above-mentioned enzymatic hydrolysate was subjected to ultrafiltration separation using ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa, separating it into four components with different molecular weight ranges: a component with a molecular weight greater than 10 kDa, a component with a molecular weight between 3 kDa and 10 kDa, a component with a molecular weight between 1 kDa and 3 kDa, and a component with a molecular weight less than 1 kDa. The α-glucosidase inhibition rate was used as an indicator for evaluation. Figure 2 It can be seen that the α-glucosidase inhibition rate is best for the components with molecular weights between 1 kDa and 3 kDa; (3) Collect samples with molecular weight fractions of 1-3 kDa, filter them through a 0.45 µm filter membrane, and separate them using a Sephadex G-25 gel chromatography column. The eluent is ultrapure water, the flow rate is 0.4 mL / min, and the absorbance value is detected at 220 nm. Four peaks were separated and purified, and the in vitro α-glucosidase inhibitory activity of the components corresponding to the four peaks (F1 component, F2 component, F3 component, F4 component) was determined. The F2 component with the highest α-glucosidase inhibition rate was selected and analyzed by liquid chromatography-tandem mass spectrometry to obtain three peptides with high α-glucosidase inhibition rates (LPFPRPAGPR, FDLPAR and ANVFNPR).

[0034] (4) Collect the purified peak F2 of the elution fraction corresponding to the absorption peak, determine the amino acid sequence using LC-MS / MS, sort the predicted scores of peptide bioactivity using Peptide Ranker, and screen out peptides with a score of 0.5; predict the potential toxicity of peptide sequences using ToxinPred; predict allergenicity using AllerTOP v.2.1; and predict the physicochemical properties of peptide sequences, including hydrophobicity, net charge, and isoelectric point, using PepDraw online.

[0035] The results are shown in Table 1.

[0036] Table 1 As shown in Table 1, the three types of small-molecule hypoglycemic active peptides from coconut meat are all small-molecule peptides with a molecular weight of about 700-1200 Da. Small-molecule peptides are more easily absorbed by the human body. The tests showed that they have a good α-glucosidase inhibition rate, which indicates that the small-molecule hypoglycemic active peptides from coconut meat have good application prospects.

[0037] (5) Determination of the cytotoxicity of hypoglycemic active peptides.

[0038] Cell viability was detected using the MTT assay with tetramethylazazole salts. HepG2 cells were passaged to the logarithmic growth phase, digested with trypsin, and then inoculated with 1×10⁻⁶ cells. 4 Cells were seeded at a density of 200 μL / well in 96-well plates and cultured adherently for 12 h. Then, the plates were starved for 2 h in serum-free medium, followed by incubation for 24 h with gradient concentrations of 1-3 kDa ultrafiltration fraction. 20 μL of MTT (5 mg / mL) was added to each well, and the plates were incubated at 37°C in the dark for 4 h. The supernatant was discarded, and 150 μL of DMSO was added and the plates were shaken for 10 min to dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated using Equation 2.

[0039] Cell viability (%) = 00% formula (2) A0 is the reagent blank group. A1 is the sample group. A2 is a blank group. (6) Effect of hypoglycemic active peptides on glucose consumption in insulin-resistant HepG2 cell model.

[0040] An insulin-resistant HepG2 cell model (IR-HepG2) was established using insulin induction to determine the optimal insulin concentration and duration. The effects of the aforementioned 1-3 kDa ultrafiltration fractions at different concentrations on glucose consumption in IR-HepG2 cells were investigated. A normal control group (NC group), an insulin-resistant model group (M0 group), and a metformin-positive drug group (Drug group) were used as controls.

[0041] (7) Molecular docking of hypoglycemic active peptides with α-glucosidase The three hypoglycemic active peptides mentioned above were selected to study the molecular docking results with α-glucosidase. The crystal structure of α-glucosidase (PDB ID: 3WY1) was obtained from the PDB database. AutoDock Vina was used for docking, and the docking conformation with the lowest docking energy was selected. Pymol software was used for visualization analysis to obtain the interaction points, and Ligplot software was used to analyze the interaction forces.

[0042] The method for determining the inhibition rate of the hypoglycemic active peptide against α-glucosidase in the examples is as follows: Mix 30 μL of sample solution with 100 μL of 0.1 U / mL α-glucosidase solution and incubate at 37℃ for 10 min. Then add 30 μL of 2.5 mmol / L PNPG solution and react at 37℃ for 20 min. Finally, add 100 μL of 0.2 mol / L sodium carbonate solution and incubate at 37℃ for 10 min to terminate the reaction. Measure the absorbance at 405 nm using a microplate reader. Calculate the α-glucosidase inhibition rate according to Formula 1: Inhibition rate (%) = (1 - (As - Ab) / Ac) × 100% Formula (1) As: Absorbance measured from the α-glucosidase solution of the sample solution. Ab: The absorbance measured using an equal volume of PBS solution at pH 6.8 instead of α-glucosidase solution. Ac: The absorbance measured was obtained by replacing the sample solution with an equal volume of PBS solution at pH 6.8. Depend on Figure 1 It can be seen that the α-glucosidase inhibition rate of the coconut meat fiber hypoglycemic active peptide enzymatic hydrolysate varies with the ultrasonic enzymatic hydrolysis time, ranging from 40% to 70.62%. As shown in the figure, the optimal ultrasonic enzymatic hydrolysis time in this invention is 120 min.

[0043] Depend on Figure 2 It can be seen that the α-glucosidase inhibition rate is the best in the ultrasound-assisted complex enzymatic hydrolysis system with ultrafiltration fractions of 1-3 kDa molecular weight, with an inhibition rate as high as 74.49%.

[0044] Depend on Figure 3 It can be seen that when the treatment time is 24 h and the amount of 1-3 kDa ultrafiltration component added is 0.1, 1, 10, 100, and 1000 μg / mL, the survival rate of HepG2 cells is higher than 95%, indicating that the 1-3 kDa ultrafiltration component has no cytotoxic effect.

[0045] Depend on Figure 4 It was found that after intervention with different concentrations of 1-3 kDa ultrafiltration fraction, the glucose consumption of IR-HepG2 cells in all groups was significantly increased, and the increase in glucose consumption was dose-dependent. The glucose metabolism recovery effect was particularly outstanding in the 250 μg / mL treatment group, reaching 95.4% of the improvement effect of the drug group. This indicates that the 1-3 kDa ultrafiltration fraction has a significant hypoglycemic effect and contains highly active hypoglycemic peptides.

[0046] Depend on Figure 5It can be seen that the four elution fractions obtained by gel column separation have four peaks appearing sequentially as follows: F1 fraction 30-70 min, F2 fraction 70-110 min, F3 fraction 110-130 min, and F4 fraction 130-150 min.

[0047] Depend on Figure 6 It was found that the four gel components exhibited α-glucosidase inhibition rates ranging from 50% to 76.57%, with component F2 showing the highest inhibition rate. Therefore, component F2 was selected for mass spectrometry identification of its peptide sequence composition.

[0048] Depend on Figure 7 It can be seen that the α-glucosidase inhibition rate of the three hypoglycemic active peptides increases with increasing concentration, and the order of α-glucosidase inhibition activity is FDLPAR>ANVFNPR>LPFPRPAGPR.

[0049] The molecular docking results between the hypoglycemic active peptide and α-glucosidase are as follows: FDLPAR forms hydrogen bonds with amino acid residues Tyr389, Gly399, Asp333, and Ala378 in the active site of α-glucosidase, and forms hydrophobic interactions with 17 surrounding amino acid residues (Pro230, Leu227, Val334, Met302, and Ala229). LPFPRPAGPR forms hydrogen bonds with Leu227, Arg340, Thr339, and Glu377 in the active site of α-glucosidase, and forms hydrophobic interactions with 18 surrounding amino acid residues, such as Asp333, Lys398, Pro230, and Tyr389. ANVFNPR forms hydrogen bonds with three amino acid residues (Lys225, Gly399, and Glu377) in the active site of α-glucosidase, and forms hydrophobic interactions with 16 surrounding amino acid residues (such as Asp401, Phe397, Ala229, and Val334). Therefore, the inhibitory effect of these three polypeptides on α-glucosidase is due to their binding to the active site on α-glucosidase, thereby blocking the binding of α-glucosidase to the substrate or destroying its catalytic activity.

Claims

1. A method for preparing small molecule bioactive peptides from coconut meat using a special extraction process, characterized in that, Includes the following steps: (1) Enzymatic hydrolysis of coconut dietary fiber powder: After mixing coconut dietary fiber powder with deionized water, the mixture is subjected to stepwise enzymatic hydrolysis by a combination of neutral protease and alkaline protease with ultrasonic assistance. After inactivation of enzymes, the mixture is centrifuged and the supernatant is collected to obtain the hydrolysate. (2) Separation of hypoglycemic active peptides: The enzymatic hydrolysate is separated by ultrafiltration membrane to obtain ultrafiltration components with a molecular weight of 1-3 kDa; (3) Purification of hypoglycemic active peptides: After the ultrafiltration fraction was filtered through a 0.45 µm filter membrane, it was separated by gel chromatography using Sephadex G-25 as the separation medium. The α-glucosidase inhibitory activity of the eluted fraction corresponding to each absorption peak was determined. The fraction with the highest α-glucosidase inhibitory activity was selected and freeze-dried to obtain the F2 fraction. The F2 fraction was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and active peptides with amino acid sequences of FDLPAR, ANVFNPR and LPFPRPAGPR were isolated.

2. The method for preparing small molecule bioactive peptides from coconut meat according to claim 1, characterized in that: The coconut meat dietary fiber powder is preferably a product obtained by supercritical extraction, drying, and mechanical pulverization of coconut meat after pressing coconut oil.

3. The method for preparing small molecule bioactive peptides from coconut meat according to claim 1, characterized in that: In step (1), the amount of neutral protease added is 5000~9000 U / g, the pH is adjusted to 5.5~9.5, and enzymatic hydrolysis is performed at a temperature of 30~60℃.

4. The method for preparing small molecule bioactive peptides from coconut meat according to claim 1, characterized in that: In step (1), the amount of alkaline protease added is 5000~9000 U / g, the pH is adjusted to 7.5~11.5, and enzymatic hydrolysis is carried out at a temperature of 30~60℃.

5. The method for preparing small molecule bioactive peptides from coconut meat according to claim 1, characterized in that: In step (1), the total time for ultrasonic enzymatic hydrolysis is 60-180 min, the time for hydrolysis of neutral protease is 30-60 min, the time for hydrolysis of alkaline protease is 30-60 min, the ultrasonic frequency is 40-100 kHz, the ultrasonic power is 100-300 W, and the hydrolysis temperature is 30-60 ℃.

6. The method for preparing coconut meat small molecule bioactive peptides according to claim 1, characterized in that: In step (3), the eluent for gel chromatography separation is ultrapure water, and the flow rate is 0.4 mL / min.

7. The small molecule active peptide or a composition containing the small molecule active peptide prepared by the method of any one of claims 1–6, and its application in the preparation of hypoglycemic functional foods, health foods, and special medical foods.

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