CN116284231A - Oyster adductor muscle polypeptide with fat reducing effect and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of polypeptides, and discloses an oyster adductor muscle polypeptide with a fat-reducing effect and its preparation method and application. In order to develop oyster protein resources, the preparation method and application of an oyster adductor muscle polypeptide with a fat-reducing effect are disclosed. The oyster polypeptide is a short peptide or a mixture of at least two short peptides; the short peptide is a mixture of DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP, QMFPYMPN; Hydrolyzed by trypsin and alkaline protease, then centrifuged and filtered to retain molecules of 500-1500Da; and then separated and purified to freeze-dry the obtained polypeptide powder, which has a fat-reducing effect.

Description

Oyster adductor muscle polypeptide with fat-reducing effect, preparation method and application thereof

technical field

The invention relates to the technical field of polypeptides, in particular to an oyster adductor muscle polypeptide with the effect of reducing fat, a preparation method and application thereof.

Background technique

Hyperlipidemia is a chronic disease with abnormal lipid content in the blood. It is closely related to the occurrence of fatty liver, diabetes and cardiovascular diseases, and poses a great threat to human health. Studies in recent years have shown that peptides have the functions of lowering blood fat, lowering blood sugar, anti-tumor, anti-oxidation, anti-inflammation, immune regulation, and protecting liver and kidney functions. Oysters are a shellfish belonging to the Oyster family of the mollusk Bivalvia (Bivalvia). Oysters are rich in protein, and protein is a high-quality aquatic protein. Compared with oyster protein, oyster peptide has excellent physical and chemical properties: higher activity, can play its unique physiological role in the case of trace and low concentration; Oyster peptides do not cause diarrhea. At present, less attention has been paid to the application of oyster peptide adductor muscle protein hydrolyzate peptide to the lipid-lowering function. Hydrolyzed polypeptide, has inhibitory activity on pancreatic lipase, and has lipid-lowering effect on mice fed a high-fat diet.

Contents of the invention

In order to develop oyster protein resources, the object of the present invention is to provide a fat-reducing oyster adductor muscle polypeptide and its preparation method and application, that is, to provide oyster polypeptide with fat-reducing effect and polypeptide powder containing oyster polypeptide ; Purification and preparation method of oyster polypeptide and polypeptide powder;

The present invention provides following technical scheme:

Oyster polypeptide with fat-reducing effect, the oyster polypeptide is a short peptide or a mixture of at least two short peptides;

The polypeptide is a mixture of DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP, 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.

The preparation method of the above-mentioned oyster polypeptide comprises the following steps:

(1) Remove the shell from the oyster and take the adductor muscle tissue, then heat it at 100°C for 5 minutes;

(2) adding pepsin to the homogenate of oysters for enzymolysis under acidic conditions, followed by enzymolysis under alkaline conditions with trypsin and alkaline protease;

(3) adding the enzymolysis liquid after step (2) to a total volume of 75% ethanol solution, alcohol precipitation at 4°C for 24 hours, and then centrifuging to obtain the supernatant;

(4) filtering the supernatant described in step (3), and retaining molecules with a molecular weight cut-off of 500 to 1500 Da;

(5) Separation and purification gel column chromatography: dissolve the above-mentioned intercepted enzymatic hydrolyzate in a polypeptide solution with a concentration of 200mg/mL, centrifuge at 4°C and 10000r/min for 20min, and add the supernatant to dextran according to the volume ratio of 1mL: 25mL Gel Sephadex LH-20 chromatography column, eluted with ultrapure water, detected with 214nm wavelength and drawn absorbance curve, collected and eluted components and lyophilized into powder;

(6) According to the oral fat-reducing effect on the hyperlipidemic mouse animal model, determine that component 2 in step (5) is the polypeptide component with the best lipid-lowering effect.

As a preferred method of the present invention, when pepsin is enzymolyzed in step (2), the pH value of the homogenate is maintained at 1 to 3, the enzymolysis time is 2 to 4 hours, the enzymolysis temperature is 35 to 38°C, and the pepsin addition is 2 ~4%, the activity of pepsin is 300~500U/mg.

As a preference of the method of the present invention, when trypsin and alkaline protease are added in step (2) for double enzymatic hydrolysis, the pH value of the homogenate should be kept at 7.0 to 8.6, the enzymolysis time is 1 to 2 hours, and the enzymolysis temperature is 35 to 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.

A polypeptide powder comprising the above-mentioned oyster polypeptide.

Further preferably, the preparation method of the above-mentioned polypeptide powder comprises the following steps:

(1) Take the adductor muscle tissue after shelling the oyster and heat it at 100°C for 5 minutes;

(2) adding pepsin to the homogenate of oysters for enzymolysis under acidic conditions, followed by enzymolysis under alkaline conditions with trypsin and alkaline protease;

(3) adding the enzymolysis solution after step (2) to an ethanol solution with a total volume of 80%, alcohol precipitation at 4° C. for 24 hours, and then centrifuging to obtain the supernatant;

(4) filtering the supernatant described in step (3), and retaining molecules with a molecular weight cut-off of 500 to 1500 Da;

(5) Separation and purification gel column chromatography: dissolve the above-mentioned intercepted enzymatic hydrolyzate in a polypeptide solution with a concentration of 200mg/mL, centrifuge at 4°C and 10000r/min for 20min, and add the supernatant to dextran according to the volume ratio of 1mL: 25mL Gel Sephadex LH-20 chromatography column, eluted with ultrapure water, detected with 214nm wavelength and drawn absorbance curve, collected and eluted components and lyophilized into powder;

(6) According to the oral fat-reducing effect on the hyperlipidemic mouse animal model, determine that component 2 in step (5) is the polypeptide component with the best lipid-lowering effect.

As a preferred method of the present invention, when pepsin is enzymatically hydrolyzed in step (2), the pH value of the homogenate is maintained at 1 to 3, the enzymolysis time is 2 to 4 hours, the enzymolysis temperature is 35 to 38°C, and the pepsin addition is 2-4%, the activity of pepsin is 300-500U/mg.

As a preference of the method of the present invention, when trypsin and alkaline protease are added in step (2) for double enzymatic hydrolysis, the pH value of the homogenate should be kept at 7.0 to 8.6, the enzymolysis time is 1 to 2 hours, and the enzymolysis temperature is 35 to 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.

Application of the above-mentioned oyster polypeptide or polypeptide powder in the preparation of oral liquid with fat-reducing effect.

The beneficial effects of the present invention are as follows:

1. The sequence of the polypeptide components obtained after hydrolysis, ultrafiltration, separation and purification of the present invention is clear, the molecular weight is small, and it is easy to use and absorb;

2. The polypeptide powder prepared by the present invention can effectively reduce the content of total triglyceride and total cholesterol in the serum of hyperlipidemia mice.

Description of drawings

Fig. 1 is oyster polypeptide structural formula;

Fig. 2 is the gel chromatographic separation spectrum of oyster polypeptide;

Fig. 3 is the total TC content of mouse blood;

Figure 4 is the total TG content in mouse blood.

Detailed ways

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

Unless otherwise specified, the raw materials used in the present invention can be purchased from the market or commonly used in the field. If not specified, the methods in the following examples are all conventional methods in the field.

Example 1

Oyster peptides with fat-reducing effects, made through the following process:

(1) Remove the shell from the oyster and take the adductor muscle tissue, then heat it at 100°C for 5 minutes;

(2) adding pepsin to the homogenate of oysters for enzymolysis under acidic conditions, followed by enzymolysis under alkaline conditions with trypsin and alkaline protease;

(3) adding the enzymolysis liquid after step (2) to an ethanol solution with a total volume of 80%, alcohol precipitation at 4° C. for 15 h, and then centrifuging to obtain the supernatant;

(4) filter the supernatant described in step (3), and cut off the polypeptide of molecular weight 1000Da;

(5) Separation and purification gel column chromatography: dissolve the above-mentioned intercepted enzymatic hydrolyzate in a polypeptide solution with a concentration of 200mg/mL (peptide concentration is 200mg/mL), centrifuge at 4°C and 10000r/min for 20min, and the supernatant is mixed according to the volume ratio 1mL: 25mL was added to Sephadex LH-20 chromatographic column of Sephadex, eluted with ultrapure water, detected with 214nm wavelength and drawn absorbance curve (as shown in Figure 2), collected and eluted components and frozen dry into powder;

As shown in Figure 2, it shows that after separation by gel chromatography, oyster adductor muscle polypeptide can be separated into three components at a wavelength of 214nm, and the peak times are 2min, 6.5min and 9min respectively.

(6) According to the oral fat-reducing effect on the hyperlipidemic mouse animal model, determine that component 2 in step (5) is the polypeptide component with the best lipid-lowering effect.

(7) The oyster polypeptides in component 2 with the best effect were identified by UPLC-MS and their peptide sequences were divided into: AEELGIQ, GANSIEL, VTHCNVK, TSGFSDSGY, WTGFSFM, KHSVITG, EVDDAGG, LDVSWASD, FCENVCPY, TAGSTTCLD, DNMSIMV. The specific structural formula is shown in Figure 1.

During pepsin enzymolysis in step (2), keep the pH of the homogenate at 1-3, enzymolysis time 2-4 hours, enzymolysis temperature 30°C, pepsin addition 2.5%, pepsin activity 300U/mg When adding trypsin enzymolysis in step (2), keep the pH value of homogenate to be 7.8, enzymolysis time 1～2 hour, enzymolysis temperature 37 ℃, trypsin addition 2.5%, the activity of trypsin 10000U/mg , Alkaline protease added activity 40000U/g.

Peptide sequence identification method

(1) Liquid phase conditions

The instrument is an ultra-high performance liquid chromatograph, the chromatographic column is a C18 chromatographic column, the mobile phase A is double distilled water containing 0.1% formic acid, the mobile phase B is acetonitrile solution containing 0.1% formic acid, and the flow rate is 0.5mL/min. The temperature is 40°C, and the gradient conditions are 0-2.5 minutes to maintain 99% solution A and 1% solution B; 2.5-5 minutes, increase solution B from 1% to 5%, and adjust solution A from 99% to 95%; 5-10 minutes , liquid B increased from 5% to 10%, liquid A decreased from 95% to 90%; 10-30min, liquid B increased from 10% to 25%, liquid A decreased from 90% to 75%; 31-35min, B Solution increased from 25% to 40%, A solution decreased from 75% to 60%; 36 ~ 40min.

(2) Mass Spectrometry Conditions

The mass spectrometer was a Thermo QE Orbitrap. The ion mode is ESI+, the mass range is 50-2000m/z; the capillary voltage (Capillary) is 3.0kV; the sampling cone is 35.0V; the ion source temperature is 105°C; the solvent removal temperature is 350°C; the cone gas flow rate is 50.0L /h; Desolvent gas flow: 600.0L/Hr; Collision energy: 6.0e V; Collision gas flow: 0.6m L/min; Scan time: 0.26sec; Internal scan time: 0.02sec.

Fat Loss Efficacy Test of Peptides

Fat-reducing effect of peptides on mice

Experimental method for model mice

60 male SPF grade C57BL6/J mice. The blank group was fed with ordinary feed and intervened by intragastric administration of pure water; the model group was fed with commercial high-fat feed and intervened by intragastric administration of pure water; Feed feeding, and oral administration of simvastatin-positive drug (20mg/kg/d), the example group was fed with commercial high-fat feed, and intervened by intragastric administration of polypeptide (100mg/kg/d) prepared by the method of the present invention. After two weeks of adaptive feeding, the mice were randomly divided into 4 groups according to body weight and weight, with 8 mice in each group, and fed continuously for 16 weeks. During this period, the same volume of body weight was orally administered every day, and the food intake of the mice was recorded every week.

After the last feeding in the 16th week, the rats were fasted for 12 hours, their body weight was weighed, blood was collected from the heart, and they were killed by neck dislocation. Liver tissue and blood samples were collected and centrifuged at 1500 g for 15 min, and the serum was collected and stored at -80°C.

Detection indicators: ① Determination of heart index and liver index (the ratio of heart and liver weight to body weight)

② Determination of triglyceride TC and total cholesterol TG content in serum according to the instructions of Nanjing Jiancheng kit

Table 1 Mouse heart index and liver index

As shown in FIG. 3 , it indicates that fraction 2 obtained after separation by gel chromatography can effectively reduce the total cholesterol content in the blood of obese mice.

As shown in Figure 4, it is shown that the component 2 obtained after separation by gel chromatography can effectively reduce the total triglyceride content in the blood of obese mice. In summary, the oyster adductor muscle polypeptide powder prepared by this method has The isolated and purified components, namely DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP, and QMFPYMPN polypeptide mixtures, can effectively reduce blood lipid levels in obese mice.

Claims (7)

1. The oyster shell-closing muscle polypeptide with the fat reducing effect is characterized in that the oyster shell-closing muscle polypeptide with the fat reducing effect is a short peptide or a mixture of at least two short peptides;

the short peptide is DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP, 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 oyster adductor polypeptide with fat reducing effect according to claim 1, comprising the steps of:

(1) Removing shell from Concha Ostreae, taking the muscle tissue of the closed shell, and heating and mixing to obtain homogenate of Concha Ostreae;

(2) Sequentially adding pepsin into the homogenate of oyster for enzymolysis under an acidic condition, and then carrying out enzymolysis by trypsin and alkaline protease under an alkaline condition to obtain an enzymolysis solution;

(3) Adding ethanol into the enzymolysis liquid obtained in the step (2), precipitating the ethanol, and centrifuging to obtain supernatant;

(4) Filtering the supernatant fluid obtained in the step (3) and intercepting molecules with the molecular weight of 500-1500 Da;

(5) Separating and purifying gel column chromatography: dissolving the trapped zymolyte in a polypeptide solution, centrifuging, adding the supernatant into a Sephadex LH-20 chromatographic column of Sephadex, eluting, and collecting and eluting components to obtain the short peptide DIFMAWP, GSWSCDGCL, GPMGPRM, FSMPPQMFPY, AVLCFMYLMY, GPMMRGP, QMFPYMPN.

3. The process according to claim 2, wherein in the step (2), the pH of the homogenate is maintained at 2.5 to 3.5 during the enzymolysis of pepsin for 3 to 5 hours at 30 to 37 ℃.

4. The preparation method of claim 2, wherein in the step (2), trypsin and alkaline protease are added for enzymolysis, the pH value of the homogenate is kept at 7.0-8.6, the enzymolysis time is 1-2 hours, and the enzymolysis temperature is 35-38 ℃.

5. The method of claim 2, wherein in step (3), the conditions for alcohol precipitation are: alcohol depositing at 2-6 deg.c for 12-18 hr.

6. The process according to claim 2, wherein in step (5), the mixture is centrifuged at 8000 to 12000r/min at 2 to 6℃for 20 to 30 minutes.

7. The use of oyster adductor polypeptide with fat reducing effect according to claim 1 in the preparation of fat reducing oral liquid.

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