Background
The RMB spent in China for treating hypertension is nearly 500 million yuan per year, which is only lower than the cost spent in apoplexy. Chemically synthesized antihypertensive drugs such as captopril, aliskiren, losartan and the like have obvious curative effect on treatment of hypertension, but the side effects of the drugs are also obvious, such as rash, proteinuria and the like, and drug dependence can also occur. However, antihypertensive peptides extracted from natural plants, animals and microorganisms have some advantages not possessed by chemically synthesized antihypertensive drugs: firstly, the curative effect is obvious, the blood pressure control effect on patients is obvious, and the effect on normal patients is not influenced; secondly, the safety is high, and the antihypertensive peptide is food-grade enzyme prepared by enzymolysis of food by protease under mild conditions and does not contain chemical synthesis reagents and additives; then, the application is wide, and the natural polypeptide in the food has the effects of reducing blood pressure, preventing tumor, treating blood coagulation and improving immunity; finally, the potential is great, and the ACE inhibitory peptide has better water solubility, so that the ACE inhibitory peptide can be easily added into various existing health care products in the market, and the health care effect of the health care products is improved.
Nereid is a soft invertebrate, and is generally called as scolopendra, Shuibao and the like. The variety of the clamworms in China is rich, more than eighty types exist, and the clamworms are intensively distributed in the east coastal area of China. The Nereid has high nutritive value, rich fat and protein, and multiple minerals and vitamins. Nereid is a traditional marine Chinese medicine, can improve dry mouth and throat, eliminate swelling and pain, remove toxic sores and repair damaged skin, and is known as cordyceps sinensis in the sea. The Chinese invention patent CN201810668300.5 discloses a preparation method of acer truncatum seed active peptide, which is characterized in that acer truncatum seed protein is extracted by ultrasonic assistance, and the acer truncatum seed protein is hydrolyzed by using Alcalase protease to prepare the acer truncatum seed active peptide. Chinese patent CN201811214660.4 discloses an ACE inhibitory active peptide derived from stichopus japonicus, which is prepared by performing enzymolysis on stichopus japonicus by using compound protease, separating by using an ultrafiltration membrane, and separating and purifying by using column chromatography and high performance liquid chromatography to obtain a better ACE inhibitory active peptide. The content of the nereis virens protein is very high, but more applications in baits exist at present, and no report on the separation and purification of the ACE inhibitory active peptide from the perinereis aibuhitensis protein is reported at present.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a method for preparing ACE inhibitory peptide of periceella aibuhitensis protein source, which comprises the steps of extracting the nereis aibuhitensis protein by using an alkali extraction and acid precipitation method, hydrolyzing the nereis aibuhitensis protein by using alkaline protease, optimizing single factors such as solid-to-liquid ratio, enzymolysis pH value and enzymolysis temperature, determining hydrolysis conditions, and performing ultrafiltration on fragments after enzymolysis to obtain the polypeptide with the optimal ACE inhibitory effect.
The invention relates to a method for preparing perinereis aibuhitensis protein source ACE inhibitory peptide, which comprises the following steps:
(1) crushing clamworm and carrying out degreasing treatment;
(2) and (3) extraction of nereis proteins: leaching the pretreated defatted perinereis aibuhitensis powder for 60min by using 1mol/L diluted alkali solution, controlling the temperature at 50-60 ℃, adjusting the pH to 8, centrifuging to remove insoluble substances at the bottom layer, taking supernatant, adjusting the pH to the isoelectric point of perinereis aibuhitensis protein by using hydrochloric acid, standing to ensure that the protein is fully aggregated to preliminarily form precipitate, centrifuging again, filtering to remove the supernatant to obtain precipitated protein, repeatedly adding the precipitate with the pH of 4.5 into distilled water for washing, adjusting the pH of the precipitate to 7.0, fully pre-freezing the obtained precipitated protein, drying the precipitated protein to constant weight by using a freeze-drying method in a freeze-drying machine to obtain perinereis aibuhitensis protein freeze-dried powder, and freezing for later use;
(3) carrying out enzymolysis treatment on nereis protein: weighing nereis protein freeze-dried powder, selecting alkaline protease to prepare an enzymatic hydrolysate, hydrolyzing the nereis protein freeze-dried powder for 4 hours, inactivating enzymes at high temperature after the hydrolysis is finished, centrifuging to obtain a supernatant, and detecting the ACE inhibition rate of each enzymolysis product, wherein the higher the inhibition rate is, the better the enzymolysis effect is;
(4) screening high-activity molecular weight fragments: and (4) passing the centrifuged enzymatic hydrolysate through an ultrafiltration membrane to obtain separation products with different molecular masses.
In a preferable embodiment of the present invention, when the alkaline protease in the step (3) hydrolyzes the nereis protein, the solid-to-liquid ratio is 1:6 to 1:14 g/mL.
In a preferred embodiment of the present invention, when the alkaline protease in the step (3) hydrolyzes the nereis protein, the pH of the enzymatic hydrolysate is 6 to 14, and more preferably 6 to 12.
As a preferable scheme of the invention, when the alkaline protease in the step (3) hydrolyzes the nereis protein, the enzymolysis temperature is 40-60 ℃.
As a preferable scheme of the invention, when the enzymolysis liquid in the step (4) permeates the ultrafiltration membrane, the size of the ultrafiltration membrane is 1-10 kDa.
Advantageous effects
At present, no report exists for preparing ACE inhibitory active peptide from perinereis aibuhitensis protein. Nereid protein content is very high, but has been used more in bait applications before. The method takes perinereis aibuhitensis protein as a raw material, extracts the nereis aibuhitensis protein by an alkali extraction and acid precipitation method, hydrolyzes the nereis aibuhitensis protein by alkali protease, and obtains the polypeptide with ACE inhibitory activity by an ultrafiltration method, and the method has the advantages of simple extraction method and high application value.
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.
Example 1
(1) Crushing clamworm and carrying out degreasing treatment;
(2) and (3) extraction of nereis proteins: leaching the pretreated defatted perinereis aibuhitensis powder with 1mol/L diluted alkali solution for 60min, controlling the temperature at 50-60 deg.C, adjusting pH to 8, centrifuging for 15min by using a refrigerated centrifuge to remove insoluble substances at the bottom layer, continuously stirring supernatant, simultaneously adding 1mol/L hydrochloric acid into the solution to adjust the pH value to 4.5, standing for 30min to ensure that the protein is fully coagulated to form a precipitate primarily, centrifuging for 15min by using a refrigerated centrifuge again, filtering to remove supernatant to obtain precipitated protein, repeatedly adding the precipitate with the pH value of 4.5 into distilled water for washing, adjusting the pH value of the precipitate to 7.0, fully pre-freezing the obtained precipitated protein, drying the precipitated protein in a freeze dryer by using a freeze-drying method to constant weight to obtain peribalanus bidentate nereis protein freeze-dried powder, and freezing in a freezing chamber at-18 ℃ in a refrigerator for later use;
(3) carrying out enzymolysis treatment on nereis protein: weighing nereis protein freeze-dried powder, selecting alkaline protease, preparing into enzymolysis liquid with a solid-to-liquid ratio of 1:6g/mL and a pH value of 6, carrying out enzymolysis at 60 ℃, hydrolyzing the nereis protein freeze-dried powder for 4h, carrying out high-temperature enzyme deactivation after the hydrolysis is finished, then putting the solution into a refrigerated centrifuge, regulating the rotating speed of the refrigerated centrifuge to 5000r/min, setting the time to 15min, centrifuging, and taking supernatant;
(4) screening high-activity molecular weight fragments: and (3) allowing the centrifuged enzymatic hydrolysate to pass through a 10kDa ultrafiltration membrane to obtain a separated product with the ultrafiltrate of less than 10kDa molecular mass, and detecting the ACE inhibition effect of the separated product, wherein the ACE inhibition rate is 58.3%.
Example 2
The same as example 1, except that the solid-to-liquid ratio in the step (3) is 1:14g/mL, the pH value is 7, and the enzymolysis temperature is 40 ℃; and (4) allowing the centrifuged enzymatic hydrolysate to pass through a 1kDa ultrafiltration membrane to obtain a separated product with the ultrafiltrate of less than 1kDa molecular mass, wherein the ACE inhibition rate of the separated product is 67.4%.
Example 3
The same as example 1, except that the solid-to-liquid ratio in the step (3) is 1:8g/mL, the pH value is 9, and the enzymolysis temperature is 45 ℃; and (4) allowing the centrifuged enzymolysis liquid to penetrate through a 3kDa ultrafiltration membrane to obtain a separated product with the ultrafiltrate of which the molecular mass is less than 3kDa, wherein the ACE inhibition rate of the separated product is 60.3%.
Example 4
The same as example 1, except that the solid-to-liquid ratio in the step (3) is 1:10g/mL, the pH value is 11, and the enzymolysis temperature is 55 ℃; and (4) allowing the centrifuged enzymatic hydrolysate to pass through a 3kDa ultrafiltration membrane to obtain a separated product with the ultrafiltrate of less than 3kDa molecular mass, wherein the ACE inhibition rate of the separated product is 63.7%.
Example 5
The same as example 1, different from example 1, in the step (3), the solid-to-liquid ratio is 1:12g/mL, the pH value is 12, the enzymolysis temperature is 50 ℃, and the ACE inhibition rate of the finally obtained separation product is 55.2%.
Evaluation of Performance
A. The single-factor test optimizes the preparation process of the ACE inhibitory peptide.
(1) Effect of solid-liquid ratio on ACE inhibition ratio
Taking 5 clean test tubes, respectively labeling, adding 1g of peribalanus bidentate nereis protein freeze-dried powder, respectively performing enzymolysis for 45min by using alkaline protease under the conditions that the pH =10 and the temperature is 50 ℃, wherein the solid-to-liquid ratio is 1:6g/mL, 1:8g/mL, 1:10g/mL, 1:12g/mL and 1:14g/mL, centrifuging and filtering for 15min, collecting supernatant, and respectively determining the ACE inhibition rate.
(2) Influence of pH value of enzymolysis liquid on ACE inhibition rate
Taking 5 clean test tubes, respectively labeling, adding 1g of Perinereis aibuhitensis protein freeze-dried powder, performing enzymolysis for 45min by using alkaline protease at the temperature of 50 ℃ and under the condition that the solid-to-liquid ratio is 1:10g/mL, respectively, centrifuging and filtering for 15min, collecting supernatant, and respectively determining the ACE inhibition rate.
(3) Effect of enzymolysis temperature on ACE inhibition ratio
Taking 5 clean test tubes, respectively labeling, adding 1g of Perinereis aibuhitensis protein freeze-dried powder, respectively performing enzymolysis at 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ for 45min under the conditions that the pH =10 and the solid-to-liquid ratio is 1:10g/mL, centrifuging and filtering for 15min, collecting supernatant, and respectively determining the ACE inhibition rate.
FIG. 1 shows that when the solid-liquid ratio is 1: 6-1: 14g/mL, the ACE inhibition rate firstly rises and then falls along with the rise of the solid-liquid ratio, and reaches the highest value when the solid-liquid ratio is 1:10 g/mL.
FIG. 2 shows that pH can affect the spatial structure of the enzyme, even denature the enzyme in severe cases, and the change of the enzyme activity can affect the enzymolysis effect and further affect the ACE inhibition rate. When the pH value of the enzymolysis liquid is 6-14, the ACE inhibition rate firstly rises and then falls along with the rise of the pH, and the ACE inhibition rate is highest when the pH is = 10.
FIG. 3 shows that the enzyme is a protein with a catalytic effect, the enzyme structure is damaged by overhigh temperature, the enzyme activity is reduced, the enzymolysis effect is reduced by the reduction of the enzyme activity, and the ACE inhibition rate is finally influenced. When the enzymolysis temperature is between 40 ℃ and 60 ℃, the ACE inhibition rate firstly rises and then falls along with the rise of the temperature, and reaches the highest value when the temperature is 50 ℃.
The foregoing is illustrative of the preferred embodiments of the present invention only and is not intended to limit the process of the present invention in any way. According to the preparation method of the perinereis aibuhitensis protein source ACE inhibitory peptide, the perinereis aibuhitensis protein source ACE inhibitory peptide can be prepared under the condition that the pH value of the enzymolysis liquid in the step (3) is 14. The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.