A polypeptide from adductor muscle of oyster with fat-reducing efficacy, and preparation method and application thereof

Abstract

The present application relates to the technical field of polypeptide, disclose a kind of oyster adductor muscle polypeptide with the effect of reducing fat and its preparation method and application, for the development of oyster protein resources, disclose the preparation method and application of oyster adductor muscle polypeptide with the effect of reducing fat, the oyster polypeptide is short peptide or the mixture of at least two short peptides;The short peptide is the mixture of DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP, QMFPYMPN;The oyster polypeptide is first hydrolyzed by pepsin from oyster adductor muscle, then hydrolyzed by trypsin and alkaline protease double enzyme, then it is centrifuged and filtered and retained 500-1500Da molecule;Then again by separation and purification component is freeze-dried to obtain the polypeptide powder with the effect of reducing fat.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, specifically to an oyster adductor muscle polypeptide with fat-reducing effects, its preparation method, and its application. Background Technology

[0002] Hyperlipidemia is a chronic disease characterized by abnormally high levels of lipids in the blood, closely associated with fatty liver, diabetes, and cardiovascular diseases, posing a significant threat to human health. Recent studies have shown that polypeptides possess lipid-lowering, blood sugar-lowering, anti-tumor, antioxidant, anti-inflammatory, immunomodulatory, and liver and kidney protective effects. Oysters belong to the class Bivalvia and the family Ostreidae of mollusks. Oysters are rich in protein, which is a high-quality aquatic protein. Compared to oyster protein, oyster peptides have superior physicochemical properties: higher activity, exerting unique physiological effects even in trace amounts and low concentrations; small size for easy absorption; low osmotic pressure; and the fact that oyster peptide intake does not cause diarrhea. Currently, there is limited attention paid to the application of hydrolyzed oyster adductor muscle protein peptides for lipid-lowering functions. This invention has discovered hydrolyzed peptides from this part, obtained through cooking, digestion with pepsin, and then secondary digestion with trypsin and alkaline protease, which exhibit inhibitory activity against pancreatic lipase and lipid-lowering effects in mice on a high-fat diet. Summary of the Invention

[0003] To develop oyster protein resources, the purpose of this invention is to provide an oyster adductor muscle polypeptide with fat-reducing effects, its preparation method, and its application, namely, to provide an oyster polypeptide with fat-reducing effects, and polypeptide powder containing oyster polypeptide; and a method for purifying and preparing the oyster polypeptide and polypeptide powder.

[0004] This invention provides the following technical solution:

[0005] Oyster polypeptides with fat-reducing effects, wherein the oyster polypeptides are short peptides or a mixture of at least two short peptides;

[0006] The polypeptide is a mixture of DIFMAWP, GSSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVALCFMYLMY, GPMMRGP, and QMFPYMPN;

[0007] The amino acid sequences of each short peptide are as follows:

[0008] DIFMAWP:Asp-Ile-Phe-Met-Ala-Trp-Pro;

[0009] GSWSCDGCL: Gly-Ser-Trp-Ser-Cys-Asp-Gly-Cys-Leu;

[0010] GPMGPRM: Gly-Pro-Met-Gly-Pro-Arg-Met;

[0011] FSMPPQMFPY:Phe-Ser-Met-Pro-Pro-Gln-Met-Phe-Pro-Tyr;

[0012] AVLCFMYLMY: Ala-Val-Leu-Cys-Phe-Met-Tyr-Leu-Met-Tyr;

[0013] GPMMRGP: Gly-Pro-Met-Met-Arg-Gly-Pro;

[0014] QMFPYMPN: Gln-Met-Phe-Pro-Tyr-Met-Pro-Asn.

[0015] The preparation method of the above-mentioned oyster polypeptide includes the following steps:

[0016] (1) After removing the shell from the oyster, take the adductor muscle tissue and heat it at 100℃ for 5 minutes.

[0017] (2) Pepsin was added to the oyster homogenate and hydrolyzed under acidic conditions, followed by hydrolysis under alkaline conditions by trypsin and alkaline protease.

[0018] (3) Add the enzymatic hydrolysate after step (2) to a total volume of 75% ethanol solution, precipitate at 4°C for 24 hours, and then centrifuge to obtain the supernatant.

[0019] (4) Filter the supernatant obtained in step (3) and retain molecules with a molecular weight of 500 to 1500 Da;

[0020] (5) Separation and purification by gel column chromatography: The above-mentioned truncated enzymatic hydrolysate was dissolved in a polypeptide solution with a concentration of 200 mg / mL, centrifuged at 4℃ and 10000 r / min for 20 min, and the supernatant was added to a Sephadex LH-20 dextran gel chromatography column at a volume ratio of 1 mL: 25 mL. The column was eluted with ultrapure water, and the absorbance was detected at a wavelength of 214 nm and the absorbance curve was plotted. The eluted components were collected and lyophilized into powder.

[0021] (6) Based on the oral lipid-lowering effect on the hyperlipidemic mouse model, component 2 in step (5) was determined to be the polypeptide component with the best lipid-lowering effect.

[0022] As a preferred embodiment of the method of the present invention, in step (2), the pH of the homogenate is maintained at 1 to 3, the hydrolysis time is 2 to 4 hours, the hydrolysis temperature is 35 to 38°C, the amount of pepsin added is 2 to 4%, and the activity of pepsin is 300 to 500 U / mg.

[0023] As a preferred embodiment of the method of the present invention, in step (2), when adding trypsin and alkaline protease for enzymatic hydrolysis, the pH of the homogenate is maintained at 7.0-8.6, the hydrolysis time is 1-2 hours, the hydrolysis temperature is 35-38℃, the amount of trypsin added is 1-3%, the amount of alkaline protease added is 2-4%, the activity of trypsin is 8000-12000U / g, and the activity of alkaline protease is 30000-50000U / g.

[0024] Peptide powder containing the above-mentioned oyster polypeptides.

[0025] Further preferably, the method for preparing the above-mentioned polypeptide powder includes the following steps:

[0026] (1) After removing the shell from the oyster, take the adductor muscle tissue and heat it at 100℃ for 5 minutes.

[0027] (2) Pepsin was added to the oyster homogenate and hydrolyzed under acidic conditions, followed by hydrolysis under alkaline conditions by trypsin and alkaline protease.

[0028] (3) Add the enzymatic hydrolysate after step (2) to an 80% ethanol solution, precipitate at 4°C for 24 hours, and then centrifuge to obtain the supernatant.

[0029] (4) Filter the supernatant obtained in step (3) and retain molecules with a molecular weight of 500 to 1500 Da;

[0030] (5) Separation and purification by gel column chromatography: The above-mentioned truncated enzymatic hydrolysate was dissolved in a polypeptide solution with a concentration of 200 mg / mL, centrifuged at 4℃ and 10000 r / min for 20 min, and the supernatant was added to a Sephadex LH-20 dextran gel chromatography column at a volume ratio of 1 mL: 25 mL. The column was eluted with ultrapure water, and the absorbance was detected at a wavelength of 214 nm and the absorbance curve was plotted. The eluted components were collected and lyophilized into powder.

[0031] (6) Based on the oral lipid-lowering effect on the hyperlipidemic mouse model, component 2 in step (5) was determined to be the polypeptide component with the best lipid-lowering effect.

[0032] As a preferred embodiment of the method of the present invention, in step (2), the pH of the homogenate is maintained at 1 to 3, the hydrolysis time is 2 to 4 hours, the hydrolysis temperature is 35 to 38°C, the amount of pepsin added is 2 to 4%, and the activity of pepsin is 300 to 500 U / mg.

[0033] As a preferred embodiment of the method of the present invention, in step (2), when adding trypsin and alkaline protease for enzymatic hydrolysis, the pH of the homogenate is maintained at 7.0-8.6, the hydrolysis time is 1-2 hours, the hydrolysis temperature is 35-38℃, the amount of trypsin added is 1-3%, the amount of alkaline protease added is 2-4%, the activity of trypsin is 8000-12000U / g, and the activity of alkaline protease is 30000-50000U / g.

[0034] The above-mentioned oyster polypeptides or polypeptide powders are used in the preparation of oral liquids with fat-reducing effects.

[0035] The beneficial effects of this invention are as follows:

[0036] I. The polypeptide components obtained by hydrolysis, ultrafiltration, separation and purification in this invention have clear sequences, small molecular weights, and are easy to utilize and absorb.

[0037] II. The polypeptide powder prepared by this invention can effectively reduce the levels of total triglycerides and total cholesterol in the serum of hyperlipidemic mice. Attached Figure Description

[0038] Figure 1 The structure of an oyster polypeptide;

[0039] Figure 2 The gel chromatography pattern of oyster polypeptides;

[0040] Figure 3 This refers to the total TC content in mouse blood.

[0041] Figure 4 This represents the total TG content in mouse blood. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below.

[0043] Unless otherwise specified, all raw materials used in this invention are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0044] Example 1

[0045] Oyster peptides with fat-reducing effects are produced through the following process:

[0046] (1) After removing the shell from the oyster, take the adductor muscle tissue and heat it at 100℃ for 5 minutes.

[0047] (2) Pepsin was added to the oyster homogenate and hydrolyzed under acidic conditions, followed by hydrolysis under alkaline conditions by trypsin and alkaline protease.

[0048] (3) Add the enzymatic hydrolysate after step (2) to an 80% ethanol solution, precipitate at 4°C for 15 hours, and then centrifuge to obtain the supernatant.

[0049] (4) Filter the supernatant obtained in step (3) and retain polypeptides with a molecular weight of 1000 Da;

[0050] (5) Separation and purification by gel column chromatography: The above-mentioned truncated enzymatic hydrolysate was dissolved in a polypeptide solution with a concentration of 200 mg / mL (polypeptide concentration 200 mg / mL), centrifuged at 4℃ and 10000 r / min for 20 min, and the supernatant was added to a Sephadex LH-20 dextran gel chromatography column at a volume ratio of 1 mL: 25 mL, eluted with ultrapure water, and the absorbance was detected at a wavelength of 214 nm and an absorbance curve was plotted (e.g., Figure 2 The components were collected, eluted, and then freeze-dried into powder.

[0051] like Figure 2 As shown, after gel chromatography separation, the oyster adductor muscle polypeptide can be separated into three components at a wavelength of 214 nm, with peak times of 2 min, 6.5 min and 9 min, respectively.

[0052] (6) Based on the oral lipid-lowering effect on the hyperlipidemic mouse model, component 2 in step (5) was determined to be the polypeptide component with the best lipid-lowering effect.

[0053] (7) The oyster peptides in component 2, which showed the best effect, were identified by UPLC-MS as follows: AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM, KHSVITG, EVDDAGG, LDVSWASD, FCENVCPY, TAGSTTCLD, DNMSIMV. The specific structural formulas are as follows: Figure 1 As shown.

[0054] In step (2), when the pepsin is added for hydrolysis, the pH of the homogenate is maintained at 1-3, the hydrolysis time is 2-4 hours, the hydrolysis temperature is 30℃, the amount of pepsin added is 2.5%, and the activity of pepsin is 300U / mg. In step (2), when the trypsin is added for hydrolysis, the pH of the homogenate is maintained at 7.8, the hydrolysis time is 1-2 hours, the hydrolysis temperature is 37℃, the amount of trypsin added is 2.5%, the activity of trypsin is 10000U / mg, and the activity of alkaline protease is 40000U / g.

[0055] Polypeptide sequence identification methods

[0056] (1) Liquid phase conditions

[0057] The instrument was an ultra-high performance liquid chromatograph (UHPLC), with a C18 column. Mobile phase A was double-distilled water containing 0.1% formic acid, and mobile phase B was acetonitrile solution containing 0.1% formic acid. The flow rate was 0.5 mL / min, and the temperature was 40℃. The gradient conditions were as follows: 0–2.5 min, maintaining 99% A and 1% B; 2.5–5 min, increasing B from 1% to 5% and decreasing A from 99% to 95%; 5–10 min, increasing B from 5% to 10% and decreasing A from 95% to 90%; 10–30 min, increasing B from 10% to 25% and decreasing A from 90% to 75%; 31–35 min, increasing B from 25% to 40% and decreasing A from 75% to 60%; 36–40 min…

[0058] (2) Mass spectrometry conditions

[0059] The mass spectrometer was a Thermo QE Orbitrap. Ion mode was ESI+, mass range 50–2000 m / z; capillary voltage was 3.0 kV; sampling cone voltage was 35.0 V; ion source temperature was 105 °C; desolventizing temperature was 350 °C; cone gas flow rate was 50.0 L / h; desolventizing gas flow rate was 600.0 L / Hr; collision energy was 6.0 e V; collision gas flow rate was 0.6 mL / min; scan time was 0.26 sec; internal scan time was 0.02 sec.

[0060] Peptide fat reduction efficacy test

[0061] The fat-reducing effect of peptides on mice

[0062] Model mouse experimental methods

[0063] Sixty male SPF-grade C57BL6 / J mice were used in the following trials: a control group fed a standard diet and treated with pure water via gavage; a model group fed a commercially available high-fat diet and treated with pure water via gavage; a positive control group fed a commercially available high-fat diet and treated with simvastatin (20 mg / kg / d) via gavage; and an example group fed a commercially available high-fat diet and treated with a polypeptide prepared according to the method of this invention (100 mg / kg / d) via gavage. After two weeks of acclimatization, the mice were randomly divided into four groups of eight mice each, and fed continuously for 16 weeks. During this period, mice were administered a volume equal to their body weight via gavage daily, and their food intake was recorded weekly.

[0064] After the last feeding in week 16, the animal was fasted for 12 hours, weighed, and euthanized by cardiac blood collection and neck dislocation. Liver tissue and blood samples were collected at 1500g and centrifuged for 15 minutes. Serum was collected and stored at -80℃.

[0065] Testing indicators: ① Measure the heart index and liver index (the ratio of heart and liver weight to body weight).

[0066] ②The serum triglyceride (TC) and total cholesterol (TG) levels were determined according to the instructions of the Nanjing Jiancheng reagent kit.

[0067] Table 1 Mouse cardiac and liver indices

[0068]

[0069]

[0070] like Figure 3 As shown, component 2 obtained after gel chromatography separation can effectively reduce the total cholesterol content in the blood of obese mice.

[0071] like Figure 4 As shown, component 2 obtained after gel chromatography separation can effectively reduce the total triglyceride content in the blood of obese mice. In summary, the separated and purified components of oyster adductor muscle polypeptide powder prepared by this method, namely the polypeptide mixture of DIFMAWP, GSSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVALCFMYLMY, GPMMRGP, and QMFPYMPN, can effectively reduce the blood lipid level of obese mice.

Claims

1. A polypeptide from adductor muscle of oyster having fat-reducing efficacy, characterized in that, The polypeptide with fat-reducing efficacy in the adductor muscle of oyster is a mixture of DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP and QMFPYMPN. The amino acid sequence of each short peptide is: DIFMAWP: Asp-Ile-Phe-Met-Ala-Trp-Pro; GSWSCDGCL: Gly-Ser-Trp-Ser-Cys-Asp-Gly-Cys-Leu; GPMGPRM: Gly-Pro-Met-Gly-Pro-Arg-Met; FSMPPQMFPY: Phe-Ser-Met-Pro-Pro-Gln-Met-Phe-Pro-Tyr; AVLCFMYLMY: Ala-Val-Leu-Cys-Phe-Met-Tyr-Leu-Met-Tyr; GPMMRGP: Gly-Pro-Met-Met-Arg-Gly-Pro; QMFPYMPN: Gln-Met-Phe-Pro-Tyr-Met-Pro-Asn.

2. The method for preparing the oyster adductor muscle polypeptide with fat-reducing effect as described in claim 1, characterized in that, The method comprises the following steps: (1) After the oyster is shelled, the adductor muscle tissue is taken and mixed uniformly after heating to obtain an oyster homogenate; (2) The oyster homogenate is added with pepsin under acidic conditions for enzymolysis, and then added with trypsin and alkaline protease under alkaline conditions for double-enzyme enzymolysis to obtain an enzymolysis liquid; (3) The enzymolysis liquid after the enzymolysis in step (2) is added with ethanol for alcohol precipitation, and then centrifuged to take the supernatant; (4) The supernatant in step (3) is filtered, and molecules with a molecular weight of 500-1500 Da are intercepted; (5) Gel column chromatography for separation and purification: the above-mentioned intercepted enzymolysis material is dissolved in a polypeptide solution, centrifuged, and the supernatant is added to a dextran gel Sephadex LH-20 chromatography column for elution, and each component after the collection of elution is a short peptide, namely DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP and QMFPYMPN.

3. The production method as claimed in claim 2, characterized in that In step (2), when the pepsin is used for enzymolysis, the pH value of the homogenate is maintained at 2.5-3.5, the enzymolysis time is 3-5 hours, and the enzymolysis temperature is 30-37 ℃.

4. The production method as claimed in claim 2, characterized in that In step (2), when the double-enzyme enzymolysis of trypsin and alkaline protease is performed, the pH value of the homogenate is maintained at 7.0-8.6, the enzymolysis time is 1-2 hours, and the enzymolysis temperature is 35-38 ℃.

5. The production method as claimed in claim 2, characterized in that, In step (3), the alcohol precipitation condition is: alcohol precipitation at 2-6 ℃ for 12-18 h.

6. The production method as claimed in claim 2, characterized in that, In step (5), the centrifugation is performed at 2-6 ℃ and 8000-12000 r / min for 20-30 min.

7. The polypeptide with fat-reducing efficacy in the adductor muscle of oyster according to claim 1 is used for preparing a fat-reducing efficacy oral liquid.

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