A lipid-lowering peptide derived from tussah silk fibroin hydrolysate, and a preparation method and application thereof

Abstract

The application discloses a lipid-lowering peptide derived from tussah silk fibroin hydrolysate and a preparation method and application thereof, and relates to the field of food technology. The application takes tussah cocoon as raw material, prepares tussah silk fibroin hydrolysate through enzymolysis, separates and purifies the lipid-lowering peptide through ultrafiltration and glucose gel chromatography, determines the peptide sequence, and finally obtains five peptide segments with better lipid-lowering activity through virtual screening. In-vitro lipid-lowering experiments of the synthesized peptide segments show that the IC 50 value of the PL inhibition rate of GGIGGGFGRGGGYGSG is the lowest, and the lipid-lowering activity is the best. The lipid-lowering peptide obtained in the application has small side effects and is easy to absorb.

Description

Technical Field

[0001] This invention belongs to the field of food technology, specifically relating to a lipid-lowering peptide derived from tussah silk fibroin hydrolysate, its preparation method, and its application. Background Technology

[0002] The prevalence of obesity is rising globally due to high-fat diets and sedentary lifestyles. Studies have shown that inhibiting pancreatic lipase (PL) can effectively prevent the development of obesity. Orlistat is currently the most effective PL-specific inhibitor on the market. Although it works quickly, a small number of patients discontinue its use due to side effects such as diarrhea and bloating.

[0003] Tussah silkworm cocoons are rich in tussah silk fibroin, and tussah silk is mostly processed into silk products. However, during the raw silk reeling process, 35% of the cocoon silk becomes waste due to non-compliance with standards, causing significant environmental impact. The development of tussah silkworm by-products is limited, resulting in a serious waste of large amounts of tussah silk fibroin. Studies have shown that a significant characteristic of PL inhibitors is the high content of hydrophobic amino acid residues, and hydrophobic amino acids such as alanine and glycine account for more than 60% of the total amino acids in tussah silk fibroin. Therefore, tussah silk fibroin hydrolysate may also be a source of lipid-lowering peptides.

[0004] Bioactive peptides are multifunctional compounds derived from proteins, possessing various metabolic and physiological regulatory functions in the human body, and are highly safe for consumption. In summary, developing lipid-lowering peptides from tussah silk fibroin not only utilizes production waste and protects the environment, but also avoids the side effects of traditional drugs. Summary of the Invention

[0005] The purpose of this invention is to provide a lipid-lowering peptide derived from tussah silk fibroin hydrolysate and its application. This invention uses tussah silkworm cocoons as raw material to obtain a polypeptide with lipid-lowering effects, providing a new approach for the high-value processing and utilization of cocoon waste.

[0006] To achieve the above objectives, the present invention provides a lipid-lowering peptide derived from tussah silk fibroin hydrolysate, with the amino acid sequence: GGIGGGFGRGGGYGSG.

[0007] The preparation method of the lipid-lowering peptide derived from tussah silk fibroin hydrolysate includes the following steps: Preparation of regenerated tussah silk fibroin ASF: Tussah silkworm cocoons were placed in Na2CO3 solution for degumming for 30 minutes, rinsed three times with deionized water, and then air-dried to obtain tussah silk fibroin fibers. The tussah silk fibroin fibers were dissolved in Ca(NO3)2•4H2O molten solution and heated. Then, a MWCO 7000 kDa dialysis bag was placed in deionized water and dialyzed for 3 days with stirring. After centrifugation, the supernatant was collected to obtain ASF solution, which was freeze-dried to obtain solid protein powder. Hydrolysis of tussah silk fibroin: The pH of a 2 mg / mL ASF solution was adjusted with NaOH solution. The enzymatic hydrolysis temperature was set, alkaline protease was added, and the pH of the hydrolysate was maintained constant with NaOH solution. After hydrolysis, the enzyme was inactivated by boiling water bath for 10 min, cooled to room temperature, and centrifuged at 6000 rpm for 10 min. The resulting supernatant was freeze-dried for 48 h and stored at -20℃, which is ASFH. Preparation of lipid-lowering peptides from tussah silk fibroin: The enzymatic hydrolysis product was separated by ultrafiltration centrifuge tubes, and the ultrafiltration fraction with a molecular weight < 3 kDa was purified by dextran gel chromatography to obtain lipid-lowering peptides.

[0008] In the above preparation method, in step (1), the concentration of Na2CO3 solution is 0.5% w / v.

[0009] In the above preparation method, in step (1), the degumming is performed three times at 98 ~ 100℃.

[0010] In the above preparation method, in step (1), the heating is carried out at 110 °C for 3 h.

[0011] In the above preparation method, in step (2), the pH is 10.0.

[0012] In the above preparation method, in step (2), the enzymatic hydrolysis temperature is 45℃, the amount of alkaline protease added is 6000 U / g, and the hydrolysis time is 4h.

[0013] In the above preparation method, in step (3), the molecular weight cutoff of the ultrafiltration centrifuge tube is 3 kDa.

[0014] In the above preparation method, in step (3), the dextran gel column Sephadex G-25 is used for filtration and separation, and deionized water is used for elution at a flow rate of 0.5 mL / min.

[0015] The present invention has the following beneficial effects: The lipid-lowering peptide of this invention exhibits high PL inhibition rate, cholesterol micelle solubility inhibition rate, and bile salt binding rate, demonstrating good in vitro lipid-lowering activity. Furthermore, compared to orlistat, currently the most effective PL-specific inhibitor on the market, it has the advantages of fewer toxic side effects and better absorption.

[0016] The lipid-lowering peptide of this invention is obtained from tussah silkworm cocoons, which can make full use of the waste cocoons, protect the ecological environment, and provide a new idea for the high-value processing and utilization of waste cocoons. Attached Figure Description

[0017] Figure 1The in vitro lipid-lowering activities of different components are shown. (A) represents the properties of ASFH and its ultrafiltration fractions; (B) represents the UV absorption spectrum of the <3 kDa fraction; (C) represents the elution fraction obtained from the <3 kDa fraction using a dextran gel chromatography column; and (D) represents the in vitro lipid-lowering activities of the <3 kDa fraction and its elution fractions F1 and F2. Different lowercase letters indicate significant differences (p < 0.05).

[0018] Figure 2 The image shows the HPLC-MS / MS mass spectrum of F1.

[0019] Figure 3 IC50 of the inhibition rates of 5 peptides and orlistat on PL 50 Values. Where P1: GGIGGGFGRGDGGYGSG, P2: GWDYESYGSG, P3: GGIGGGFGRGDGGYGS, P4: GGIGGGFGRGDGG and P5: AAGSSARRGGGFY. Different lowercase letters indicate significant differences (p<0.05). Detailed Implementation

[0020] To more clearly explain the experimental procedure of this invention, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. These examples are merely illustrative and should not be considered as limiting the scope of the invention.

[0021] Preparation of regenerated tussah silk fibroin (ASF): Tussah silkworm cocoons were placed in a 0.5% (w / v) Na2CO3 solution and degummed three times at 98-100℃ for 30 minutes each time. After rinsing three times with deionized water, the cocoons were air-dried to obtain tussah silk fibroin fibers. 10 g of tussah silk fibroin fibers were dissolved in 100 mL of Ca(NO3)2•4H2O molten solution and heated at 110℃ for 3 h. Then, a dialysis bag (MWCO 7000 kDa) was placed in deionized water and dialyzed for 3 days with stirring. After centrifugation at 2000 r / min for 15 min, the supernatant was collected to obtain the ASF solution, which was then freeze-dried for 48 h to obtain solid protein powder.

[0022] Hydrolysis of tussah silk fibroin f: The pH of a 2 mg / mL ASF solution was adjusted to pH 10.0 using NaOH solution, the temperature was set to 45 ℃, the alkaline protease dosage was 6000 U / g, and the pH of the hydrolysate was maintained constant using NaOH solution. After hydrolysis for 4 h, the enzyme was inactivated by boiling water bath for 10 min, cooled to room temperature, and centrifuged at 6000 rpm for 10 min. The resulting supernatant was freeze-dried for 48 h and stored at -20 ℃, which is ASFH.

[0023] Preparation of lipid-lowering peptides from tussah silk fibroin: Ultrafiltration centrifuge tubes with molecular weight cutoffs of 3 kDa and 10 kDa were centrifuged at 4000 rpm for 30 min to obtain three enzymatic hydrolysates with different molecular weights: < 3 kDa, 3 kDa-10 kDa, and > 10 kDa. After lyophilization, the PL inhibition rate, cholesterol micelle solubility inhibition rate, and bile salt binding rate were measured.

[0024] Determination of PL inhibition rate A PL solution with a concentration of 4 mg / mL was prepared in a buffer solution (13 mM Tris-HCl, 1.3 mM CaCl2 and 150 mM NaCl, pH 8.5). 50 μL of PL solution and 50 μL of tussah silk fibroin lipid-lowering peptide solution (2 mg / mL) were incubated at 37°C for 10 min. Then, 50 μL of 2 mg / mL p-nitrophenylbutyric acid (p-NPB) ethanol solution was added, and the mixture was incubated at 37°C for 20 min. The buffer solution was used instead of the tussah silk fibroin lipid-lowering peptide solution as a negative control. An enzyme-free reaction mixture was used as a blank control, and orlistat as a positive control. The absorbance of p-nitrobenzene produced by each sample at 405 nm was recorded. The inhibition rate of PL was calculated using the following formula:

[0025] Wherein, A1—absorbance containing tussah silk fibroin lipid-lowering peptide and PL; A2—absorbance containing only the sample, without PL; A3—absorbance containing only PL; A4—absorbance without tussah silk fibroin lipid-lowering peptide solution and PL.

[0026] Determination of cholesterol micelle solubility inhibition rate Artificial cholesterol micelles consisted of 10 mM sodium taurocholate, 1 mM oleic acid, 132 mM NaCl, 0.4 mM cholesterol, and 15 mM sodium phosphate buffer (pH 7.4). Tussah silk fibroin lipid-lowering peptides were dissolved in the micelle solution to a final concentration of 0.5 mg / mL and incubated at 37°C for 24 h. After centrifugation for 30 min, the absorbance of the supernatant was measured at 560 nm, and the cholesterol micelle solubility inhibition rate was calculated using the following formula:

[0027] In the formula, A0 is the absorbance value of the blank group (using distilled water instead of tussah silk fibroin lipid-lowering peptide), and A1 is the absorbance value of tussah silk fibroin lipid-lowering peptide.

[0028] Determination of bile salt binding rate Mix 1 mL of tussah silk fibroin lipid-lowering peptide with 4 mL of bile salt solution (0.3 mmol / L sodium cholate, sodium glycinecholate, sodium taurocholate), incubate at 37℃ for 2 h, centrifuge at 3200 g for 20 min, aspirate the supernatant, add 3.75 mL of 60% sulfuric acid, store at 70℃ for 30 min, and measure the absorbance at 387 nm. Calculate the bile salt binding rate using the following formula:

[0029] A0 - Absorbance value of the blank group, A1 - Absorbance value of the sample group.

[0030] like Figure 1 As shown in (A): at the same concentration of 2 mg / mL, the PL inhibition rate, cholesterol micelle solubility inhibition rate, and bile salt binding rate of the < 3 kDa fraction were 69.61±1.43%, 93.06±0.26%, and 80.79±1.08%, respectively, all of which were significantly higher than those of ASFH, >10 kDa, and 3-10 kDa fractions (p<0.05). Therefore, the ultrafiltration fraction with a molecular weight of < 3 kDa was selected.

[0031] A sample of <3kDa component with a concentration of 2 mg / mL was prepared. Blank calibration was performed using deionized water. The scanning speed was 4000 nm / m, and ultraviolet spectroscopy was performed in the range of 190-500 nm, as shown in Figure (B).

[0032] Fractions with a molecular weight <3kDa were purified by dextran gel chromatography to obtain more precise lipid-lowering components. The specific process is as follows: The activated dextran gel column (Sephadex G-25) was slowly packed into a 2.0 × 40 cm glass chromatography column, leaving a margin of approximately 5 cm from the top. Deionized water was used to equilibrate for 3-5 column volumes at a flow rate of 1.0 mL / min. A 20 mg / mL solution of the active component was prepared and filtered through a 0.45 μm filter to remove impurities. 2 mL of sample was loaded, and elution was performed at a flow rate of 0.5 mL / min. 3 mL of eluent was collected per tube, and the absorbance of each tube was measured at 220 nm to plot the elution curve. The two collected peak components, F1 and F2, were freeze-dried and the PL inhibition rate, cholesterol micelle solubility inhibition rate, and bile salt binding rate were determined according to methods ①, ②, and ③ in Example 1, respectively.

[0033] like Figure 1 As shown in (C), the ultrafiltration fraction with a molecular weight < 3 kDa was separated into two peak components, F1 and F2, after being separated by Sephadex G-15 gel filtration.

[0034] likeFigure 1 As shown in (D), the positive control orlistat exhibited the highest PL inhibition rate, cholesterol micelle solubility inhibition rate, and bile salt binding rate, showing significant differences compared to other experimental groups (p<0.05). The PL inhibition rate, cholesterol micelle solubility inhibition rate, and bile salt binding rate of fraction F1 were 83.30±2.10%, 93.58±0.64%, and 82.87±0.38%, respectively, significantly higher than those of fractions F2 and <3 kDa (p<0.05), indicating that fraction F1 demonstrated the best in vitro lipid-lowering activity. Therefore, the amino acid sequence of fraction F1 was analyzed.

[0035] Mass spectrometry identification and virtual screening: such as Figure 2 As shown, component F1 was identified by HPLC-MS / MS, yielding a total of 921 amino acid sequences. To improve reliability, relevant literature was reviewed and, based on the actual situation of this invention, five peptides were screened after considering -10lgP ≥ 40, Peptideranker score > 0.6, molecular weight < 1500 Da, and good water solubility assessment: GGIGGGFGRGDGGY, GGIGGGFGRGDGGYGSG, AAGSSARRGGGFY, GGIGGGFGRGDGGYGS, and GWDYESYGSG. The predicted water solubility and isoelectric point of the 42 peptides are shown in Table 1. Comparison with current data in the bioactive peptide database (BIOPEP-UWM) revealed no identical sequences; therefore, this may be a novel lipid-lowering peptide.

[0036] Table 1. Predicted water solubility and isoelectric point of 42 peptides GSGAGGIGGGF 0.87 53.69 835.38 poor 3.7 AGSGAGGIGGGF 0.86 63.49 906.42 poor 3.77 GSGAGGIGGGFGRGDGGY 0.84 45.32 1497.66 poor 6.59 GWGDGGYG 0.84 40.15 767.29 poor 0.68 SGAGGIGGGF 0.84 63.52 778.36 poor 3.43 GGGYGWGDGGYG 0.84 43.24 1101.42 poor 0.68 GAGGIGGGF 0.83 43.3 691.33 poor 3.7 AAGSGAGGIGGGF 0.83 65 977.46 poor 3.77 GGSYGWGDGGYG 0.82 44.32 1131.43 poor 0.68 GGAGGSYGWGDGGY 0.82 49.16 1259.48 poor 0.74 GGSGGSYGWGDGGY 0.81 48.82 1275.48 poor 0.74 GSGAGGVGGGYGW 0.80 45.35 1080.46 poor 3.54 GGGYGWGDGGY 0.80 46.99 1044.39 poor 0.74 AAAAGSGAGGIGGGF 0.80 55.67 1119.53 poor 3.77 GAGGSYGWGDGGY 0.79 56.34 1202.46 poor 0.74 GGYGWGDGGYG 0.79 41.46 1044.39 poor 0.68 AAAGSGAGGIGGGF 0.79 61.29 1048.49 poor 3.77 GGSGGGYGWGDGGY 0.79 51.61 1245.47 poor 0.74 SGGGYGWGDGGY 0.78 50.62 1131.43 poor 0.74 GSGGGYGWGDGGY 0.78 40.17 1188.45 poor 0.74 GAGGSGGSYGWGDGGY 0.78 42.39 1403.54 poor 0.74 GGAGGGYGWGDGGY 0.78 52.45 1229.47 poor 0.74 GGSYGWGDGGY 0.78 45.3 1074.40 poor 0.74 SGGSYGWGDGGY 0.77 56.32 1161.44 poor 0.74 GGIGGGFGRGDGGY 0.74 40.78 1225.55 good 6.59 AGGGYGWGDGGY 0.74 44.08 1115.43 poor 0.74 GGYGWGDGGY 0.73 46.32 987.37 poor 0.74 SGGSYGWDY 0.73 51.56 990.37 poor 0.74 GAGGGYGWGDGGY 0.73 42.9 1172.45 poor 0.74 GSYGWGDGGY 0.71 40.47 1017.38 poor 0.74 GGIGGGFGRGDGGYGSG 0.71 46.92 1426.62 good 6.59 GGSGGSYGWDY 0.70 42.06 1104.41 poor 0.74 GGVGGGYGWGDGGY 0.70 47.97 1257.51 poor 0.74 GYGWGDGGY 0.70 40.66 930.35 poor 0.74 AAGSSARRGGGFY 0.69 48.01 1255.61 good 11.02 SYGWGDGGY 0.68 41.48 960.36 poor 0.74 GRSGGSYGWGDGGY 0.66 48.67 1374.56 poor 6.54 YGWGDGGY 0.65 48.08 873.33 poor 0.74 GGIGGGFGRGDGGYGS 0.65 52.12 1369.60 good 6.59 SGAGGAGGYGGY 0.62 41.83 972.39 poor 3.37 GWGDGGYGSGS 0.61 43.32 998.37 poor 0.79 GWDYESYGSG 0.61 40.88 1119.41 good 0.62

[0037] The peptides were synthesized by Anhui Guoping Pharmaceutical Co., Ltd., and the purity of the obtained peptides reached over 95%. The PL inhibition rate was determined using the method in heading ① of Example 1.

[0038] like Figure 3 It can be seen that the IC50 of the P1 peptide is... 50 The lowest value was 0.76 ± 0.02 mg / mL, while the IC50 values ​​for other peptides were... 50 The concentrations of the five peptides, from lowest to highest, were P2 (0.83±0.08 mg / mL), P4 (0.96±0.03 mg / mL), P3 (1.13±0.04 mg / mL), and P5 (1.65±0.05 mg / mL). The IC50 values ​​for the five peptides were... 50 There is a significant difference.

[0039] In summary, the lipid-lowering peptide GGIGGGFGRGDGGYGSG of this invention exhibits the best in vitro lipid-lowering activity, with an IC50 value of [missing value]. 50Although its value is slightly higher than that of orlistat, it has the advantages of fewer side effects and easier absorption compared to this synthetic drug.

Claims

1. A lipid-lowering peptide derived from tussah silk fibroin hydrolysate, characterized in that, The amino acid sequence is: GGIGGGFGRGDGGYGSG.

2. The method for preparing the lipid-lowering peptide derived from tussah silk fibroin hydrolysate as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of regenerated tussah silk fibroin ASF: Tussah silkworm cocoons were placed in Na2CO3 solution for degumming for 30 minutes, rinsed 3 times with deionized water and air-dried to obtain tussah silk fibroin fiber. The tussah silk fibroin fiber was dissolved in Ca(NO3)2•4H2O molten solution and heated. Then, a MWCO 7000 kDa dialysis bag was placed in deionized water and stirred for dialysis for 3 days. After centrifugation, the supernatant was taken to obtain ASF solution, which was freeze-dried to obtain solid protein powder. (2) Hydrolysis of tussah silk fibroin: The pH of 2 mg / mL ASF solution was adjusted with NaOH solution, the enzymatic hydrolysis temperature was set, alkaline protease was added, and the pH of the hydrolysate was kept constant with NaOH solution. After hydrolysis, the enzyme was inactivated by boiling water bath for 10 min, cooled to room temperature, centrifuged at 6000 rpm for 10 min, and the supernatant was freeze-dried for 48 h and stored at -20℃, which is ASFH. (3) Preparation of lipid-lowering peptides from tussah silk fibroin: The enzymatic hydrolysis product was separated by ultrafiltration centrifuge tubes, and the ultrafiltration fraction with a molecular weight < 3 kDa was purified by dextran gel chromatography to obtain lipid-lowering peptides.

3. The preparation method according to claim 2, characterized in that, In step (1), the concentration of the Na2CO3 solution is 0.5% w / v.

4. The preparation method according to claim 2, characterized in that, In step (1), the degumming is performed three times at 98-100°C.

5. The preparation method according to claim 2, characterized in that, In step (1), the heating is performed at 110 °C for 3 h.

6. The preparation method according to claim 2, characterized in that, In step (2), the pH is 10.

0.

7. The preparation method according to claim 6, characterized in that, In step (2), the enzymatic hydrolysis temperature is 45℃, the amount of alkaline protease added is 6000 U / g, and the hydrolysis time is 4h.

8. The preparation method according to claim 2, characterized in that, In step (3), the molecular weight cutoff of the ultrafiltration centrifuge tube is 3 kDa.

9. The preparation method according to claim 8, characterized in that, In step (3), the dextran gel column Sephadex G-25 was used for filtration and separation, and deionized water was used for elution at a flow rate of 0.5 mL / min.

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