Hemp seed protein source pancreatic lipase inhibitory peptide as well as preparation method and application thereof

Abstract

The invention discloses a hemp seed protein source pancrelipase inhibitory peptide as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The hemp seed protein source pancrelipase inhibitory peptide provided by the invention has an amino acid sequence as shown in SEQ ID NO. 1 (YNLPILRF). The cannabis sativa L. protein source pancreatic lipase inhibitory peptide provided by the invention is separated from cannabis sativa L. protein for the first time, is a novel cannabis sativa L. protein source pancreatic lipase inhibitory peptide, has good pancreatic lipase inhibitory activity, can be used as a natural anti-obesity agent in food, health care products and weight control related drugs, and has broad application prospects. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptide preparation, and specifically relates to a hemp kernel protein-derived pancreatic lipase inhibitory peptide and a preparation method and application thereof. Background Art

[0002] In the prevention and treatment of obesity, pancreatic lipase is a highly-regarded therapeutic target. Its main function is to hydrolyze triglycerides in the diet and limit their absorption in the gastrointestinal tract.

[0003] Currently known pancreatic lipase inhibitors include orlistat. However, the drug is associated with a variety of side effects, including allergic reactions such as urticaria, abdominal pain, dyspnea, diarrhea, nausea, vomiting, and oily bowel movements, and may cause discomfort symptoms such as swelling of the face and throat. Therefore, the development of safe and effective lipid-lowering and weight-loss drugs has become increasingly important. Studies have shown that plant-derived peptides, as part of a daily diet, are expected to have potential therapeutic effects to improve obesity symptoms and reduce side effects.

[0004] Hemp seed protein has attracted wide attention in the food industry due to its excellent nutritional value, excellent digestibility and low allergy. Studies have shown that hemp seed protein hydrolysates have a variety of biological activities, including antioxidant, anti-hypertensive, anti-thrombotic, acetylcholinesterase (AChE) inhibition, α-glucosidase inhibition, anti-cancer, anti-fatigue, and immunomodulatory. Therefore, if hemp peptide functional foods with pancreatic lipase inhibitory activity are developed through enzymatic hydrolysis, it will be of great significance to the high-value utilization of hemp kernel protein and the prevention and improvement of obesity by dietary hemp peptides. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the existing trypsin inhibitory drugs have unsatisfactory effects, and to provide a hemp kernel protein-derived pancreatic lipase inhibitory peptide and a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A hemp kernel protein-derived pancreatic lipase inhibitory peptide, the amino acid sequence of which is YNLPILRF (Tyr-Asn-Leu-Pro-lle-Leu-Arg-Phe).

[0008] A method for preparing the above-mentioned pancreatic lipase inhibitory peptide derived from hemp kernel protein, the method comprising: using papain to enzymolyze hemp kernel protein powder to obtain an enzymolyzate; after simulating gastrointestinal digestion, collecting components with a molecular weight of 1-3 kDa in the enzymolyzate, subjecting them to polysaccharide gel G-25 column chromatography separation, determining the pancreatic lipase inhibitory activity of each peak collected solution, collecting the components with the highest activity, subjecting them to LC-MS / MS identification, and chemically synthesizing them to obtain the above-mentioned hemp kernel peptide.

[0009] Furthermore, the hemp kernel protein powder is the hemp kernel protein powder obtained after supercritical extraction defatting, alkali extraction and acid precipitation.

[0010] Furthermore, in the enzymatic hydrolyzate, the liquid-to-water ratio of water to hemp kernel protein powder is 20:1 mL / g, and the amount of papain used is calculated on a protein weight basis, with 40 mg of papain added per gram of hemp kernel protein powder.

[0011] Furthermore, the enzymatic hydrolysis temperature is 50-70°C, preferably 65°C, the enzymatic hydrolysis time is greater than 120 min, preferably 180 min, and the pH is 5.5-7.5, preferably 6.0.

[0012] Furthermore, the dextran gel G-25 column chromatography, the eluent is ultrapure water; the column shape is: φ (diameter) 1.6×100 cm; the detection wavelength is 280 nm; the sample volume is 2-10 mL, preferably 5 mL, the sample concentration is 1-100 mg / mL, preferably 40 mg / mL, and the flow rate is 0.1-1 mL / min, preferably 0.5 mL / min.

[0013] An application of the above-mentioned hemp kernel protein-derived pancreatic lipase inhibitory peptide in the preparation of a product with pancreatic lipase inhibitory activity, wherein the product includes food, medicine or health care product.

[0014] The beneficial effects of the present invention compared to the prior art are as follows: the present invention is the first to separate and identify a hemp kernel peptide with an amino acid sequence of YNLPILRF from hemp kernel protein hydrolysate, which has good pancreatic lipase inhibitory activity. The hemp kernel peptide provided by the present invention can be used to prepare foods and medicines with pancreatic lipase inhibitory function, providing new ideas for the prevention and treatment of overweight and obese people, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of hydrolysis time on the degree of hydrolysis (DH) and pancreatic lipase (PL) inhibition rate of hemp seed protein hydrolysate (HPH). Letters a–d indicate significant differences between sample means (p<0.05).

[0016] Figure 2 PL inhibition rates of HPH, HPH-SGD and U1-U4 at 10 mg / mL. Letters a–d indicate significant differences between sample means (p<0.05).

[0017] Figure 3 This is a diagram showing the changes in the molecular weight distribution of HPH-SGD before and after ultrafiltration treatment.

[0018] Figure 4This is a diagram showing the results of protein purification of U3 via Sephadex G-25.

[0019] Figure 5 PL inhibition rates of F1-F4 at 10 mg / mL. Letters a–c indicate significant differences between sample means (p<0.05).

[0020] Figure 6 This is the identification result diagram of hemp peptide YNLPILRF.

[0021] Figure 7 This is the Lineweaver-Burk plot between hemp peptide YNLPILRF and pancreatic lipase.

[0022] Figure 8 Flow chart of the test of the present invention. DETAILED DESCRIPTION

[0023] The technical scheme provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. In the following embodiments, unless otherwise specified, all are conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0024] Example 1

[0025] A hemp bean peptide having pancreatic lipase inhibitory activity, the specific preparation method is as follows:

[0026] 1. Preparation of Hemp Protein Isolate

[0027] Hemp protein isolate (HPI) is obtained by alkali-soluble acid precipitation technology. 5 grams of hemp protein powder (HPM) defatted by supercritical extraction was dispersed in 100 ml of distilled water, and the pH value of the mixture was adjusted to 10.0 using 2 mol / L sodium hydroxide, and stirred at room temperature for 2 hours. Subsequently, centrifugation was carried out at 8000 rpm for 30 minutes at 4°C. After collecting the supernatant, its pH value was adjusted to 5.0 by adding 2 mol / L hydrochloric acid. After centrifugation, the precipitate was collected and its pH value was adjusted to 7.0. Finally, the protein content was quantitatively analyzed using the Dumas combustion method and a protein conversion factor of 5.7, and the protein purity was 94.36%.

[0028] 2. Preparation of hemp seed enzymatic hydrolysate and simulated gastrointestinal digestion

[0029] 4% (w / w, pH 6.0) of papain was dissolved in a 50 mg / mL aqueous solution of HPI and stirred continuously at 65°C for 1-5 hours to obtain hemp protein isolate hydrolysate (HPH). In order to evaluate the digestive enzyme stability of pancreatic lipase inhibitory peptides, the samples were subjected to simulated gastrointestinal digestion experiments after the hydrolysis was completed. At this stage, 2% (w / w, pH 2.0) of pepsin was added to promote hydrolysis. After reacting for 2 hours at 37°C, the reaction was terminated by placing the mixture in boiling water. Subsequently, 2% (w / w, pH 7.5) of trypsin was added to continue hydrolysis at 37°C for 2 hours, followed by centrifugation. The supernatant obtained from the process is called HPH-SGID. The protein purity was quantitatively analyzed by the BCA kit, and the result was 94.36%.

[0030] 3. Determination of degree of hydrolysis (DH) and pancreatic lipase inhibitory activity

[0031] 20 μL of L-serine or HPH was mixed with 150 μL of OPA and incubated for 2 minutes. Subsequently, the optical density was detected at 340 nm using a microplate reader (Molecular Devices, USA), with L-serine (0-100 μg / mL) as the standard. 100 mg HPI was hydrolyzed in 6 mol / L HCl at 115 °C for 22 hours, and the total amino acid content was calculated. The following formula was then used to calculate the degree of hydrolysis DH value of HPI after different enzymatic hydrolysis times:

[0032] DH(%)=(OD sample -OD blank) / (OD total -OD blank )×100

[0033] Among them, OD sample OD blank and OD total The optical density of the sample after enzymatic hydrolysis, the optical density of the sample before enzymatic hydrolysis, and the optical density of the sample after complete hydrolysis are defined as follows. Figure 1 shown.

[0034] 4. The pancreatic lipase inhibition experiment was performed using p-NPB as a substrate. Specifically, 50 μL of sample was mixed with 25 μL of pancreatic lipase at a concentration of 1.25 mg / mL and incubated at 37°C for 15 minutes. Subsequently, 25 μL of 5 mM p-NPB solution was added and diluted to a final volume of 150 μL. After another 15 min of incubation, the optical density of the resulting solution was measured at 405 nm. The sample refers to HPH or HPH-SGID, and subsequent U1~U4, F1~F4 can also be used for activity determination using this method. The PL inhibition rate is calculated by the following formula:

[0035] PL inhibition rate (%) = [1-(CD) / (AB)] × 100

[0036] Among them, A represents the optical density value when there is enzyme solution but no sample is added; B represents the optical density value when there is no enzyme solution and sample; C represents the optical density value when there is enzyme solution and sample; D represents the optical density value when there is only sample but no enzyme solution.

[0037] The results showed that although the DH value reached a maximum of 21.75% ± 0.34% at 4 h, the papain hydrolysate had the highest pancreatic lipase inhibitory activity at 3 h, with a value of 71.00% ± 4.12%. Therefore, the subsequent hydrolysis time was determined to be 3 h. At a concentration of 10 mg protein / mL, the pancreatic lipase inhibition rate exposed to gastrointestinal conditions increased from 22.51 ± 0.57% to 38.51% ± 5.23% (p < 0.05). The pancreatic lipase inhibitory activity of HPH and HPH-SGID was shown in Figure 2. Figure 1 shown.

[0038] 5. Ultrafiltration and molecular weight distribution

[0039] The concentration of HPI-SGID was adjusted to 20 mg / mL and separated by 5 kDa, 3 kDa and 1 kDa molecular weight cutoff membranes, respectively. The individual fractions were named U1 (>5 kDa), U2 (3-5 kDa), U3 (1-3 kDa) and U4 (<1 kDa). High performance liquid chromatography (HPLC) was performed using an Agilent 1260 Infinity II system (USA), using a TSKgel G2000 SWXL column, and 10 μL of the test sample was transferred to the HPLC equipment and eluted with ultrapure water containing 45% (v / v) acetonitrile and 0.1% (v / v) trifluoroacetic acid. The elution rate was maintained at 0.5 mL / min, commercial standards were used as external standards, and the detection wavelength was set to 220 nm. According to the standard curve (y = -0.2402x + 7.065, R 2 =0.9823) where X represents the retention time.

[0040] The results are as follows Figure 2 The results showed that at a concentration of 10 mg protein / mL, the component with a molecular weight of 1-3 kDa (U3) had the highest phospholipase inhibitory activity (51.88% ± 1.86%), which was significantly higher than that of HPH-SGID. Figure 3 shown.

[0041] 6. Gel filtration chromatography G-25 separation

[0042] 5 mL of 40 mg / mL U3 was then further separated using a Sephadex G-25 gel filtration column (16×100 mm). The sample was eluted with ultrapure water at a flow rate of 0.5 mL / min. The sample was detected by UV detector (Cytiva The absorbance was monitored at 280 nm and the samples were collected using an automatic fraction collector. Figure 4 As shown, a total of 4 peak fractions (F1 to F4) were collected and freeze-dried for subsequent experiments.

[0043] The results are as follows Figure 5 The results showed that at a concentration of 10 mg protein / mL, the F3 fraction had the highest pancreatic lipase inhibition rate of 81.62%±2.86%.

[0044] 7. Identification of peptide sequences

[0045] F3 was separated on a 0.15 × 150 mm AcciaimPepMap RP-C18 column connected to an Easy-nLC 1200 (Thermo Fisher Scientific, USA) system with a loading volume of 3 μL. The column was equilibrated with 95% mobile phase A (0.1% formic acid). Mobile phase B consisted of 84% acetonitrile and 0.1% formic acid. The elution flow rate was set at 300 nL / min, and the gradient was set as follows: 0-50 min, 4-50% B; 50-54 min, 50-100% B; 54-60 min maintained at 100% B.

[0046] The separated peptides were analyzed on a Q-Exactive HF-X (Thermo Fisher Scientific, USA) using a data-dependent Top 20 method. Mass spectrometry data were collected in the m / z range of 350 to 1500 in electrospray positive ionization mode. The MS resolution was set to 60,000 (m / z 200) and the automatic gain value (AGC) was 3 × 10 6 In the MS / MS analysis, the resolution was set to 15000 (m / z 200) and the AGC value was 1×10 5 .

[0047] The data were identified using MaxQuantVer.1.5.5.1 in combination with the UniProtkb_Cannabis_stiva_76512_2024_06_13 database.

[0048] The results showed that among the 18 peptides identified with a relative abundance greater than 1%, YNLPILRF (e.g. Figure 6 The sequence shown) showed the strongest inhibitory activity.

[0049] 8. IC of peptides inhibiting pancreatic lipase 50 Determination of value

[0050] IC 50 The values ​​(concentration required to reduce pancreatic lipase activity by 50%) were determined by nonlinear regression analysis using GraphPad Prism 10.1.2 software. At the same time, orlistat was used as a positive control. The inhibition type was obtained by Lineweaver-Burk plot. The substrate concentration was adjusted to 1, 2, 3, 4 and 5 mM, while the synthetic peptide concentration was varied to 0, 0.1 and 0.2 mg / mL.

[0051] The results are as follows Figure 7 It was shown that YNLPILRF inhibited pancreatic lipase by IC 50 The value is 0.377±0.041mM.

Claims

1. A pancreatic lipase inhibitory peptide derived from hemp kernel protein, characterized in that: The amino acid sequence of the hemp kernel protein-derived pancreatic lipase inhibitory peptide is YNLPILRF.

2. A method for preparing the hemp kernel protein-derived pancreatic lipase inhibitory peptide according to claim 1, characterized in that: The method comprises the following steps: using papain to enzymolyze hemp kernel protein powder to obtain an enzymolyzate; after simulating gastrointestinal digestion, collecting components with a molecular weight of 1-3 kDa in the enzymolyzate, performing polysaccharide gel G-25 column chromatography separation, determining the pancreatic lipase inhibitory activity of each peak collected solution, and collecting the component with the highest activity to obtain the hemp kernel protein-derived pancreatic lipase inhibitory peptide.

3. The method for preparing a pancreatic lipase inhibitory peptide derived from hemp kernel protein according to claim 1, characterized in that: The hemp kernel protein powder is the hemp kernel protein powder obtained after supercritical extraction defatting, alkali extraction and acid precipitation.

4. The method for preparing a pancreatic lipase inhibitory peptide derived from hemp kernel protein according to claim 1, characterized in that: In the enzymatic hydrolysate, the liquid-to-water ratio of water to hemp kernel protein powder is 20:1 mL / g, and the amount of papain used is based on protein weight, with 40 mg of papain added per gram of hemp kernel protein powder.

5. The method for preparing a pancreatic lipase inhibitory peptide derived from hemp kernel protein according to claim 1, characterized in that: The enzymatic hydrolysis temperature is 50-70° C., the enzymatic hydrolysis time is greater than 120 min, and the pH value is 5.5-7.

5.

6. The method for preparing a pancreatic lipase inhibitory peptide derived from hemp kernel protein according to claim 1, characterized in that: The dextran gel G-25 column chromatography has an eluent of ultrapure water; a column shape of φ (diameter) 1.6×100 cm; a detection wavelength of 280 nm; a sample volume of 2-10 mL, a sample concentration of 1-100 mg / mL, and a flow rate of 0.1-1 mL / min.

7. Use of the hemp kernel protein-derived pancreatic lipase inhibitory peptide according to claim 1 in the preparation of a product with pancreatic lipase inhibitory activity, characterized in that: The products include food, medicine or health products.

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