CN106755243A - A kind of preparation method of konjaku albumen source ACE inhibitor peptides - Google Patents

Abstract

A preparation method of ACE inhibitory peptide derived from konjac protein, which comprises dissolving konjac protein freeze-dried powder in purified water to prepare a protein solution with a mass fraction of 1-3%; heating the protein solution to 35-55°C, and adjusting the pH value to 4.0 ‑9.0, add compound protease and mix evenly, enzymatically hydrolyze to obtain enzymatic hydrolyzate; after inactivating the enzyme in the enzymatically hydrolyzate, adjust the pH value to the isoelectric point of konjac protein; centrifuge, collect the supernatant, and freeze-dry the supernatant into freeze-dried powder to obtain the crude ACE inhibitory peptide derived from konjac protein, and after separation, the ACE inhibitory peptide is obtained. The method provided by the invention has the advantages of simple operation, high product yield and good ACE inhibition effect.

Description

A kind of preparation method of konjac protein source ACE inhibitory peptide

technical field

The invention belongs to the field of preparation of ACE inhibitory peptides, and in particular relates to a preparation method of konjac protein source ACE inhibitory peptides.

Background technique

Konjac is rich in nutrition, rich in glucomannan, starch, protein, vitamins and mineral elements. Among them, the protein can be enzymatically hydrolyzed to obtain biologically active polypeptides with anti-aging, anti-cancer, blood pressure-lowering, immune and other functions, which are good raw materials for the production of functional health food. The protein in konjac is mainly derived from konjac powder. The test results show that the protein content in konjac powder is as high as 23.8%. After degradation, it can produce branched chain amino acid oligopeptides, ACE inhibitory peptides, high F value oligopeptides, mannan peptides, etc. A variety of bioactive peptides.

The natural ACE inhibitory peptide was first discovered by Ferriera in 1965 from the venom of Agkistrodon americana. Subsequently, many studies on ACE inhibitory peptides were carried out rapidly, and short-chain ACE inhibitory peptides were found from a variety of different natural food proteins. Subsequently, ACE inhibitory peptides were discovered from raw materials such as soybean meal, corn, rapeseed meal, pepper seed meal, silver carp, and pig hemoglobin. ACE inhibitory peptide is an angiotensin-converting enzyme inhibitor, which can inhibit the activity of angiotensin-converting enzyme. ACE inhibitory peptides can effectively regulate blood pressure, and at the same time enhance the body's defense against various diseases and maintain its own physiological balance. It has the characteristics of small molecular weight, no digestion, direct absorption, high bioavailability, and no allergy to human body. Therefore, it is necessary to provide a method for preparing ACE inhibitory peptides from konjac.

Contents of the invention

The purpose of the present invention is to provide a method for preparing ACE inhibitory peptide derived from konjac protein, which has simple operation, high product yield and good ACE inhibitory effect.

For realizing above-mentioned purpose of the invention, the technical scheme that the present invention adopts is as follows:

A method for preparing ACE inhibitory peptide derived from konjac protein, which comprises dissolving konjac protein freeze-dried powder in pure water to prepare a protein solution with a mass fraction of 1-3%; heating the protein solution to 35-55°C, and adjusting the pH value to 4.0 -9.0, add compound protease and mix evenly, enzymolyze to obtain enzymolyzate; after inactivating the enzymolyzate, adjust the pH value to the isoelectric point of konjac protein; centrifuge, collect the supernatant, and freeze-dry the supernatant into freeze-dried powder to obtain the crude ACE inhibitory peptide derived from konjac protein, and after separation, the ACE inhibitory peptide is obtained.

The further improvement of the present invention lies in that the amount of compound protease added per gram of protein solution is 3000-6000U.

The further improvement of the present invention is that the temperature for inactivating the enzyme is 90-100° C. and the time is 10-15 minutes.

A further improvement of the present invention is that the centrifugation is performed at a rotational speed of 5000-10000r/min for 10-30min.

A further improvement of the present invention is that the separation is performed using an anion exchange resin 717 .

The further improvement of the present invention lies in that the freeze-drying temperature is -50--110° C., and the freeze-drying time is 10-24 hours.

The further improvement of the present invention lies in that the enzymolysis temperature is 35-55° C. and the time is 3-6 hours.

Compared with the prior art, the present invention has the beneficial effects:

In the present invention, konjac protein freeze-dried powder is used as raw material, and compound protease is used for enzymolysis, and the enzymolysis conditions are as follows: the substrate concentration is 2-10%, the amount of enzyme added is 1000-5000U/g, and the enzymolysis temperature is 40-60°C. The pH value is 5-7, and the ACE inhibitory peptide is obtained through inactivation, centrifugation and separation, and the inhibitory activity of the konjac ACE inhibitory peptide is 15%-50% as detected by ultraviolet spectrophotometry. The method provided by the invention is simple in operation, high in product yield and good in ACE inhibition effect, and provides a certain reference for fully and rationally developing and utilizing the high-quality resource of konjac protein freeze-dried powder.

detailed description

The present invention will be described in further detail below in conjunction with the examples.

The present invention comprises the following steps:

Enzymolysis process:

Dissolve konjac protein freeze-dried powder in pure water to make a protein solution; heat the protein solution on a constant temperature magnetic stirrer; adjust the pH, add compound protease and mix evenly, the enzymatic hydrolysis reaction conditions are: pH value 4.0-9.0, bottom The mass fraction of the substance is 1-3%, the amount of enzyme added is 3000-6000U/g, the enzymolysis time is 3-6h, and the enzymolysis temperature is 35-55°C; the enzymolysis solution is placed in a 90-100°C constant temperature water bath Inactivate the enzyme for 10-15min, wait until the temperature of the enzymolysis solution drops to room temperature, adjust its pH to the isoelectric point of konjac protein; centrifuge at 5000-10000r/min for 10-30min, collect the supernatant, and take a certain amount of the supernatant to measure its ACE inhibition Rate;

Determination of ACE inhibition rate:

Detection by Cushman assay: take 75uL, HHL (hippuryl-histidyl-leucine) and 25uL, ACE inhibitor (polypeptide solution) and mix well. Incubate in a 37°C water bath for 5min, add 25uL, 25mu/mL ACE borate solution, and react in a 37°C water bath for 40min. After the end, add 200uL, 1.0moL/L hydrochloric acid solution to destroy the reaction environment and terminate the reaction process. Then, 1.7 mL of ethyl acetate was added to each test tube, and mixed evenly; 1.2 mL of ethyl acetate layer was pipetted into another test tube, placed in an oven at 100°C, and the solvent was evaporated for 40 min. Take it out and cool it, add 3mL of deionized water, mix well and measure its absorbance at a wavelength of 228nm.

Table 1 ACE inhibition rate detection method

The calculation formula is:

In the formula: A ₁ —the absorbance value of the solution where both ACE inhibitory peptide and ACE exist

A ₂ —the absorbance value of the solution without ACE inhibitory peptide

A ₃ —Absorbance value of the blank solution in the presence of ACE and HHL

Detection of activity by UV spectrophotometry:

The reaction solution and the blank solution were prepared, and the other conditions of the two were the same, and the konjac ACE inhibitory peptide was added to the reaction solution. Draw the hippuric acid standard curve equation with the absorbance as the ordinate and the concentration as the abscissa. The content of hippuric acid in the blank solution and the reaction solution was measured, and the inhibitory activity of the ACE inhibitory peptide was calculated.

Example 1

Dissolve konjac protein freeze-dried powder in pure water to prepare a protein solution with a mass concentration of 1%; heat the protein solution to 35°C on a constant temperature magnetic stirrer, adjust the pH value to 4.0, then add compound protease and mix evenly, The enzymolysis reaction was carried out with a constant temperature magnetic stirrer, the enzymolysis time was 3 hours, and the enzymolysis temperature was 35°C to obtain an enzymolysis solution; wherein, the amount of compound protease added to each gram of protein solution was 3000U. Place the enzymolysis solution in a constant temperature water bath at 90°C to inactivate the enzyme for 15 minutes. After the temperature of the enzymolysis solution drops to room temperature, adjust its pH value to the isoelectric point of konjac protein; centrifuge at 8000r/min for 20 minutes, collect the supernatant, and take a certain amount The ACE inhibition rate of the supernatant was measured to be >30%, and the supernatant was freeze-dried at -50°C for 24 hours to form a freeze-dried powder to obtain the crude ACE inhibitory peptide derived from konjac protein, which was separated by anion exchange resin 717 to obtain the ACE inhibitory peptide. The inhibitory activity of the konjac protein-derived ACE inhibitory peptide was measured to be 18.6%.

Example 2

Dissolve konjac protein freeze-dried powder in pure water to prepare a protein solution with a mass concentration of 1.5%; place the protein solution on a constant temperature magnetic stirrer and heat it to 45°C; adjust the pH value to 5.0, then add compound protease to mix evenly, and The enzymolysis reaction was carried out with a constant temperature magnetic stirrer, the enzymolysis time was 4 hours, and the enzymolysis temperature was 40°C to obtain an enzymolysis solution; wherein, the amount of compound protease added to each gram of protein solution was 4000U. Place the enzymolysis solution in a constant temperature water bath at 95°C to inactivate the enzyme for 15 minutes. After the temperature of the enzymolysis solution drops to room temperature, adjust its pH value to the isoelectric point of konjac protein; centrifuge at 9500r/min for 15 minutes, collect the supernatant, and take a certain amount The ACE inhibition rate of the supernatant was measured to be >50%, and the supernatant was freeze-dried at -110°C for 10 hours to form a freeze-dried powder to obtain the crude ACE inhibitory peptide derived from konjac protein, which was separated by anion exchange resin 717 to obtain the ACE inhibitory peptide. The inhibitory activity of the konjac protein-derived ACE inhibitory peptide was measured to be 31.2%.

Example 3

Dissolve konjac protein freeze-dried powder in purified water to prepare a protein solution with a mass concentration of 2.0%; heat the protein solution to 55°C on a constant temperature magnetic stirrer, adjust the pH value to 6.0, add compound protease and mix evenly, The enzymolysis reaction was carried out with a magnetic stirrer, the enzymolysis time was 3 hours, and the enzymolysis temperature was 45°C to obtain an enzymolysis liquid; wherein, the amount of compound protease added to each gram of protein solution was 5000U. Place the enzymolysis solution in a constant temperature water bath at 97°C to kill the enzyme for 10 minutes. After the temperature of the enzymolysis solution drops to room temperature, adjust its pH value to the isoelectric point of konjac protein; centrifuge at 10000r/min for 10 minutes, collect the supernatant, and take a certain amount The ACE inhibition rate of the supernatant was measured to be >60%, and the supernatant was freeze-dried at -70°C for 15 hours to form a freeze-dried powder to obtain the crude product of konjac protein source ACE inhibitory peptide, which was separated by anion exchange resin 717 to obtain the ACE inhibitory peptide. The inhibitory activity of the konjac protein-derived ACE inhibitory peptide was measured to be 37.0%.

Example 4

Dissolve konjac protein freeze-dried powder in pure water to prepare a protein solution with a mass concentration of 2.5%; put the protein solution on a constant temperature magnetic stirrer and heat it to 40°C, adjust the pH value to 7.0, then add compound protease to mix evenly, and The enzymolysis reaction was carried out with a constant temperature magnetic stirrer, the enzymolysis time was 5 hours, and the enzymolysis temperature was 50°C to obtain an enzymolysis solution; wherein, the amount of compound protease added to each gram of protein solution was 6000U. Put the enzymolysis solution in a constant temperature water bath at 90°C to kill the enzyme for 15 minutes. After the temperature of the enzymolysis solution drops to room temperature, adjust its pH value to the isoelectric point of konjac protein; centrifuge at 5000r/min for 30 minutes, collect the supernatant, and take a certain amount The ACE inhibition rate of the supernatant was measured to be >50%. The supernatant was freeze-dried at -80°C for 20 hours to form a freeze-dried powder to obtain the crude ACE inhibitory peptide derived from konjac protein. After separation by anion exchange resin 717, the ACE inhibitory peptide was obtained. The inhibitory activity of the konjac protein-derived ACE inhibitory peptide was measured to be 33.0%.

Example 5

Dissolve konjac protein freeze-dried powder in purified water to prepare a protein solution with a mass concentration of 3.0%; heat the protein solution to 50°C on a constant temperature magnetic stirrer, adjust the pH value to 8.0, and then add 6000U/g compound protease to mix Evenly, the enzymolysis reaction was carried out in a constant temperature magnetic stirrer, the enzymolysis time was 6 hours, and the enzymolysis temperature was 55°C to obtain an enzymolysis solution; wherein, the amount of compound protease added to each gram of protein solution was 3000U. Place the enzymolysis solution in a constant temperature water bath at 95°C to kill the enzyme for 10 minutes. After the temperature of the enzymolysis solution drops to room temperature, adjust its pH value to the isoelectric point of konjac protein; centrifuge at 7000r/min for 20 minutes, collect the supernatant, and take a certain amount The ACE inhibition rate of the supernatant was measured to be >40%, and the supernatant was freeze-dried at -90°C for 20 hours to form a freeze-dried powder to obtain the crude product of konjac protein source ACE inhibitory peptide, which was separated by anion exchange resin 717 to obtain the ACE inhibitory peptide. The inhibitory activity of the konjac protein-derived ACE inhibitory peptide was measured to be 23.3%.

Example 6

Dissolve konjac protein freeze-dried powder in pure water to prepare a protein solution with a mass concentration of 3.0%; heat the protein solution to 50°C on a constant temperature magnetic stirrer, adjust the pH value to 9.0, and then add 6000U/g compound protease to mix Evenly, the enzymolysis reaction was carried out in a constant temperature magnetic stirrer, the enzymolysis time was 6 hours, and the enzymolysis temperature was 55°C to obtain an enzymolysis solution; wherein, the amount of compound protease added to each gram of protein solution was 3000U. Place the enzymolysis solution in a constant temperature water bath at 100°C to kill the enzyme for 12 minutes. After the temperature of the enzymolysis solution drops to room temperature, adjust its pH value to the isoelectric point of konjac protein; centrifuge at 10,000r/min for 10 minutes, collect the supernatant, and take a certain amount The ACE inhibition rate of the supernatant was measured to be >40%, and the supernatant was freeze-dried at -90°C for 20 hours to form a freeze-dried powder to obtain the crude ACE inhibitory peptide derived from konjac protein, which was separated by anion exchange resin 717 to obtain the ACE inhibitory peptide.

Claims (7)

1. A preparation method of konjac protein source ACE inhibitory peptide, characterized in that, konjac protein freeze-dried powder is dissolved in purified water to form a protein solution with a mass fraction of 1-3%; the protein solution is heated to 35-55°C , adjust the pH value to 4.0-9.0, add compound protease and mix evenly, enzymolyze to obtain the enzymolyzate; after the enzymolyzate is inactivated, adjust the pH value to the isoelectric point of konjac protein; centrifuge, collect the supernatant, and The supernatant is lyophilized into a lyophilized powder to obtain a crude product of the konjac protein source ACE inhibitory peptide, and after separation, the ACE inhibitory peptide is obtained. 2. the preparation method of a kind of konjac protein source ACE inhibitory peptide according to claim 1, is characterized in that, the amount of adding compound protease in every gram of protein solution is 3000-6000U. 3. The preparation method of a kind of konjac protein source ACE inhibitory peptide according to claim 1, characterized in that, the temperature for inactivating the enzyme is 90-100°C, and the time is 10-15min. 4. the preparation method of a kind of konjac protein source ACE inhibitory peptide according to claim 1, is characterized in that, centrifugation is to carry out 10-30min under the rotating speed of 5000-10000r/min. 5. The preparation method of a kind of konjac protein source ACE inhibitory peptide according to claim 1, characterized in that, the separation is carried out by anion exchange resin 717. 6 . The method for preparing ACE inhibitory peptide derived from konjac protein according to claim 1 , wherein the freeze-drying temperature is -50--110° C., and the freeze-drying time is 10-24 hours. 7. The method for preparing ACE inhibitory peptide derived from konjac protein according to claim 1, characterized in that the enzymolysis temperature is 35-55°C and the time is 3-6h.