Lipid-lowering peptide derived from tussah silk fibroin hydrolysate as well as preparation method and application of lipid-lowering peptide

Abstract

The invention discloses lipid-lowering peptide derived from tussah silk fibroin hydrolysate as well as a preparation method and application of the lipid-lowering peptide, and relates to the technical field of food. Tussah cocoons are used as raw materials, tussah silk fibroin protein hydrolysate is prepared through enzymolysis, lipid-lowering peptide is separated and purified through ultrafiltration and glucose gel chromatography, the peptide sequence of the lipid-lowering peptide is measured, and five peptide fragments with good lipid-lowering activity are finally obtained through virtual screening. The in-vitro lipid-lowering experiment of the synthetic peptide fragment shows that the IC50 value of the GGIGGFFGRGGGYGSG on the PL inhibition ratio is the lowest, and the lipid-lowering activity is the best. The obtained lipid-lowering peptide is small in side effect and easy to absorb, and has certain significance in development and application of tussah silk fibroin functional food.

Description

Technical Field

[0001] The invention belongs to the field of food technology, and particularly relates to a lipid-lowering peptide derived from tussah silk fibroin hydrolysate and a preparation method and application thereof. Background Art

[0002] The prevalence of obesity is on the rise worldwide due to high-fat diets and long periods of sedentary life. 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 stop taking it due to side effects such as diarrhea and flatulence.

[0003] Tussah cocoons are rich in tussah silk fibroin, and tussah silk is mostly processed into silk products. However, in the process of raw silk reeling, 35% of the cocoon silk does not meet the standards and becomes waste cocoon waste, which has a significant impact on the ecological environment. The development of tussah by-products is limited, and a large amount of tussah silk fibroin is seriously wasted. Studies have shown that the notable feature of PL inhibitors is the high content of hydrophobic amino acid residues, and the content of hydrophobic amino acids such as alanine and glycine in tussah silk fibroin accounts for more than 60% of the total amino acids. Therefore, tussah silk fibroin hydrolyzate may also be a source of lipid-lowering peptides.

[0004] Bioactive peptides are multifunctional compounds derived from proteins, which have a variety of metabolic and physiological regulatory effects on the human body and are highly safe for consumption. In summary, the development of tussah silk fibroin lipid-lowering peptides can not only utilize production waste and protect the ecology, but also avoid the side effects of traditional drugs and be applied to functional foods. Summary of the invention

[0005] The purpose of the present invention is to provide a lipid-lowering peptide derived from tussah silk protein hydrolysate and its application. The present invention uses tussah cocoons as raw materials to obtain polypeptides with lipid-lowering effects, providing new ideas for the high-value processing and utilization of waste cocoon waste, and also expanding resources for the development of natural lipid-lowering functional products.

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

[0007] The method for preparing the lipid-lowering peptide derived from tussah silk fibroin hydrolysate comprises the following steps:

[0008] (1) Preparation of regenerated tussah silk fibroin ASF: tussah cocoons were placed in a Na2CO3 solution for degumming for 30 minutes, rinsed with deionized water three times and air-dried to obtain tussah silk fibroin fibers. The tussah silk fibroin fibers were dissolved in a Ca(NO3)2·4H2O molten solution and heated. A MWCO 7000 kDa dialysis bag was placed in deionized water and stirred for 3 days. After centrifugation, the supernatant was collected to obtain an ASF solution, which was freeze-dried to obtain a solid protein powder.

[0009] (2) Hydrolysis of tussah silk fibroin: Use NaOH solution to adjust the pH of 2 mg / mL ASF solution, set the enzymatic temperature, add alkaline protease, and use NaOH solution to maintain the pH value of the hydrolyzate constant. After hydrolysis, inactivate the enzyme in a boiling water bath for 10 min, cool to room temperature, and centrifuge at 6000 rpm for 10 min. The obtained supernatant is freeze-dried for 48 h and stored at -20 °C, which is ASFH.

[0010] (3) Preparation of tussah silk fibroin lipid-lowering peptide: The enzymatic hydrolysis product is separated by ultrafiltration centrifuge tube, and the ultrafiltration component with a molecular weight <3 kDa is purified by dextran gel chromatography to obtain the lipid-lowering peptide.

[0011] In the above preparation method, in step (1), the concentration of the Na2CO3 solution is 0.5% w / v.

[0012] In the above preparation method, in step (1), the degumming is performed at 98-100° C. for 3 times.

[0013] In the above preparation method, in step (1), the heating is performed at 110° C. for 3 hours.

[0014] In the above preparation method, in step (2), the pH is 10.0.

[0015] In the above preparation method, in step (2), the enzymatic hydrolysis temperature is 45°C, the amount of alkaline protease added is 6000U / g, and the hydrolysis time is 4h.

[0016] In the above preparation method, in step (3), the molecular weight cutoff of the ultrafiltration centrifuge tube is 3kDa.

[0017] In the above preparation method, in step (3), Sephadex G-25 column is used for filtration and separation, and deionized water is used for elution, and the elution flow rate is 0.5 mL / min.

[0018] The application of the lipid-lowering peptide derived from tussah silk fibroin hydrolysate in the preparation of natural functional food with lipid-lowering effect.

[0019] The present invention has the following beneficial effects:

[0020] 1. The lipid-lowering peptide of the present invention has high PL inhibition rate, cholesterol micelle solubility inhibition rate and bile salt binding rate, showing good in vitro lipid-lowering activity, and can be applied to natural functional foods. At the same time, compared with orlistat, the most effective PL-specific inhibitor currently on the market, it has the advantages of less toxic side effects and better absorption.

[0021] 2. The lipid-lowering peptide of the present invention is obtained from tussah cocoons as the raw material for preparation, which can make full use of the waste cocoon materials, protect the ecological environment, provide new ideas for the high-value processing and utilization of the waste cocoon materials, and also expand resources for the development of natural lipid-lowering functional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The in vitro lipid-lowering activity of different fractions. (A) is the properties of ASFH and its ultrafiltration fractions; (B) is the UV absorption spectrum of the <3kDa fraction; (C) is the elution fraction obtained from the <3kDa fraction using a dextran gel chromatography column; (D) is the in vitro lipid-lowering activity of <3kDa and its elution fractions F1 and F2. Different lowercase letters indicate significant differences (p<0.05).

[0023] Figure 2 This is the HPLC-MS / MS mass spectrum of F1.

[0024] Figure 3 The IC of PL inhibition rate of five peptides and orlistat 50 Among them, P1: GGIGGGFGRGDGGYGSG, P2: GWDYESYGSG, P3: GGIGGGFGRGDGGYGS, P4: GGIGGGFGRGDGG and P5: AAGSSARRGGGFY. Different lowercase letters indicate significant differences (p<0.05). DETAILED DESCRIPTION

[0025] In order to explain the experimental operation process of the present invention more clearly, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. These examples are only for explaining the present invention and cannot be considered to limit the scope of the present invention.

[0026] Example 1 Preparation of lipid-lowering peptides from tussah silk fibroin hydrolysate

[0027] 1) Preparation of regenerated tussah silk fibroin (ASF): tussah cocoons were placed in a 0.5% (w / v) Na2CO3 solution, degummed 3 times at 98-100°C for 30 minutes, rinsed 3 times with deionized water and 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, heated at 110°C for 3 hours, and then placed in a dialysis bag (MWCO7000kDa) in deionized water for 3 days of stirring. After centrifugation at 2000r / min for 15 minutes, the supernatant was taken to obtain the ASF solution, which was freeze-dried for 48 hours to obtain solid protein powder.

[0028] 2) Hydrolysis of tussah silk fibroin f: The pH of 2 mg / mL ASF solution was adjusted to pH 10.0 with NaOH solution, the temperature was set to 45°C, the amount of alkaline protease added was 6000 U / g, and the pH value of the hydrolyzate was maintained constant with NaOH solution. After hydrolysis for 4 hours, the enzyme was inactivated in a boiling water bath for 10 minutes, cooled to room temperature, and centrifuged at 6000 rpm for 10 minutes. The obtained supernatant was freeze-dried for 48 hours and stored at -20°C, which was ASFH.

[0029] 3) Preparation of tussah silk fibroin lipid-lowering peptide: Ultrafiltration centrifuge tubes with a cut-off of 3 kDa and 10 kDa were used for centrifugation at 4000 rpm for 30 min to obtain three different molecular weights of enzymatic hydrolysates of <3 kDa, 3 kDa-10 kDa and >10 kDa. After freeze-drying, the PL inhibition rate, cholesterol micelle solubility inhibition rate and bile salt binding rate were determined.

[0030] ① Determination of PL inhibition rate

[0031] 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, and then 50 μL of 2 mg / mL p-nitrophenylbutyric acid (p-NPB) ethanol solution was added and incubated at 37°C for 20 min. The buffer solution was used to replace the tussah silk fibroin lipid-lowering peptide solution as a negative control. The enzyme-free reaction mixture was used as a blank control, and orlistat was used 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 as follows:

[0032]

[0033] Among them, A1—contains the absorbance of tussah silk fibroin lipid-lowering peptide and PL; A2—contains the absorbance of the sample only, without the absorbance of PL; A3—contains the absorbance of PL only; A4—contains the absorbance of neither tussah silk fibroin lipid-lowering peptide solution nor PL.

[0034] ② Determination of cholesterol micelle solubility inhibition rate

[0035] Artificial cholesterol micelles include 10mM sodium taurocholate, 1mM oleic acid, 132mM NaCl, 0.4mM cholesterol and 15mM sodium phosphate buffer (pH 7.4). The tussah silk fibroin lipid-lowering peptide was dissolved in the micelle solution to a final concentration of 0.5mg / mL and incubated at 37°C for 24h. After centrifugation for 30min, the absorbance of the supernatant was measured at 560nm, and the cholesterol micelle solubility inhibition rate was calculated as follows:

[0036]

[0037] Wherein, A0 is the absorbance value of the blank group (distilled water is used instead of tussah silk fibroin lipid-lowering peptide), and A1 is the absorbance value of tussah silk fibroin lipid-lowering peptide.

[0038] ③ Determination of bile salt binding rate

[0039] Mix 1 mL of tussah silk fibroin lipid-lowering peptide with 4 mL of bile salt solution (0.3 mmol / L sodium cholate, sodium glycine cholate, sodium taurocholate), incubate at 37°C for 2 h, centrifuge at 3200 g for 20 min, aspirate the supernatant, add 3.75 mL of 60% sulfuric acid, store at 70°C for 30 min, and measure the absorbance at 387 nm. Calculate the bile salt binding rate using the following formula:

[0040]

[0041] A0-absorbance value of blank group, A1-absorbance value of sample group.

[0042] 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 <3kDa component were 69.61±1.43%, 93.06±0.26% and 80.79±1.08%, respectively, which were significantly higher than those of ASFH, >10kDa and 3-10kDa components (p<0.05), so the ultrafiltration component with a molecular weight of <3kDa was selected.

[0043] A 2 mg / mL sample of the <3 kDa component was prepared, and blank calibration was completed with deionized water. The scanning speed was 4000 nm / m, and the UV spectrum was scanned in the range of 190-500 nm, as shown in Figure (B).

[0044] The components with molecular weight less than 3k Da were purified by dextran gel chromatography to obtain more accurate lipid-lowering components. The specific process is as follows:

[0045] Slowly fill the activated Sephadex G-25 dextran gel column into a 2.0×40cm glass chromatography column until it is about 5cm from the top of the column, and balance it with deionized water at a rate of 1.0mL / min for 3-5 column volumes. Prepare a solution of active components with a concentration of 20mg / ml, and remove impurities through a 0.45μm filter membrane. Load 2mL of sample and elute at a flow rate of 0.5mL / min. Collect the eluate, 3mL per tube, detect the absorbance of each tube at 220nm, and draw an elution curve. The two collected peak components F1 and F2 are freeze-dried, and the PL inhibition rate, cholesterol micelle solubility inhibition rate and bile salt binding rate are determined according to the methods in titles ①, ②, and ③ of Example 1, respectively.

[0046] like Figure 1 As shown in (C), the ultrafiltration fraction with a molecular weight < 3 kDa was separated into two peak fractions, F1 and F2, after being separated by Sephadex G-15 gel filtration.

[0047] like Figure 1 As shown in (D), the positive control orlistat had the highest PL inhibition rate, cholesterol micelle solubility inhibition rate and bile salt binding rate, which were significantly different from those of other experimental groups (p<0.05). The PL inhibition rate, cholesterol micelle solubility inhibition rate and bile salt binding rate of the F1 fraction were 83.30±2.10%, 93.58±0.64% and 82.87±0.38%, respectively, which were significantly higher than those of the F2 and <3kDa fractions (p<0.05). F1 exhibited the best in vitro lipid-lowering activity. Therefore, the amino acid sequence of the F1 fraction was analyzed.

[0048] 1) Mass spectrometry identification and virtual screening: Figure 2 As shown, HPLC-MS / MS was used to identify the component F1 by mass spectrometry, and a total of 921 amino acid sequences were obtained. In order to improve the credibility, the relevant literature was consulted and combined with the actual practice of the present invention. After -10lgP≥40, Peptideranker score>0.6, <1500Da molecular weight screening and good water solubility evaluation, a total of 5 peptides were screened out, namely GGIGGGFGRGDGGY, GGIGGGFGRGDGGYGSG, AAGSSARRGGGFY, GGIGGGFGRGDGGYGS and GWDYESYGSG. The prediction of water solubility and isoelectric point of 42 peptides is shown in Table 1. By comparing with the current data in the active peptide database (BIOPEP-UWM), no sequence identical to the above was found, so this may be a new lipid-lowering peptide.

[0049] Table 1 Prediction of water solubility and isoelectric point of 42 peptides

[0050]

[0051]

[0052] Example 2 Activity Verification of Synthetic Peptides

[0053] Anhui Guoping Pharmaceutical Co., Ltd. was commissioned to synthesize the peptide segment, and the purity of the obtained peptide segment reached more than 95%. The PL inhibition rate was determined using the method described in title ① in Example 1.

[0054] like Figure 3 It can be seen that the IC of P1 peptide 50 The lowest value is 0.76±0.02mg / mL. The IC 50 From low to high, they are P2 (0.83±0.08mg / mL), P4 (0.96±0.03mg / mL), P3 (1.13±0.04mg / mL) and P5 (1.65±0.05mg / mL). The IC 50 There is a significant difference.

[0055] In summary, the lipid-lowering peptide GGIGGGFGRGDGGYGSG of the present invention has the best lipid-lowering activity in vitro, and its IC 50 Although the value is slightly higher than that of orlistat, compared with this synthetic drug, it has the advantages of better side effects and easier absorption, and can be used to produce natural functional foods with lipid-lowering effects.

Claims

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

2. The method for preparing a lipid-lowering peptide derived from tussah silk fibroin hydrolysate according to claim 1, characterized in that: The following steps are involved: (1) Preparation of regenerated tussah silk fibroin ASF: tussah cocoons were placed in a Na2CO3 solution for degumming for 30 minutes, rinsed with deionized water three times and air-dried to obtain tussah silk fibroin fibers. The tussah silk fibroin fibers were dissolved in a Ca(NO3)2·4H2O molten solution and heated. A MWCO 7000 kDa dialysis bag was placed in deionized water and stirred for 3 days. After centrifugation, the supernatant was collected to obtain an ASF solution, which was freeze-dried to obtain a solid protein powder. (2) Hydrolysis of tussah silk fibroin: Use NaOH solution to adjust the pH of 2 mg / mL ASF solution, set the enzymatic temperature, add alkaline protease, and use NaOH solution to maintain the pH value of the hydrolyzate constant. After hydrolysis, inactivate the enzyme in a boiling water bath for 10 min, cool to room temperature, and centrifuge at 6000 rpm for 10 min. The obtained supernatant is freeze-dried for 48 h and stored at -20 °C, which is ASFH. (3) Preparation of tussah silk fibroin lipid-lowering peptide: The enzymatic hydrolysis product is separated by ultrafiltration centrifuge tube, and the ultrafiltration component with a molecular weight <3 kDa is purified by dextran gel chromatography to obtain the lipid-lowering peptide.

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 at 98-100° C. for 3 times.

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° C., the amount of alkaline protease added is 6000 U / g, and the hydrolysis time is 4 h.

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

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

10. Use of the lipid-lowering peptide derived from tussah silk fibroin hydrolysate according to claim 1 in the preparation of natural functional foods with lipid-lowering effects.

Images