CN106565839B

CN106565839B - Method for extracting trypsin inhibitor from pitaya seeds

Info

Publication number: CN106565839B
Application number: CN201610893580.0A
Authority: CN
Inventors: 汪财生; 陈煜�; 钱国英; 郑云浩; 李思; 俞超
Original assignee: Zhejiang Wanli College
Current assignee: Zhejiang Wanli College
Priority date: 2016-10-13
Filing date: 2016-10-13
Publication date: 2020-01-03
Anticipated expiration: 2036-10-13
Also published as: CN106565839A

Abstract

The invention relates to a method for extracting trypsin inhibitor from dragon fruit seeds, which is characterized by comprising the following steps: freezing fresh pitaya at-30 to-10 ℃ for 45 to 50 hours, and taking out the epidermis after thawing; filtering to remove liquid, washing residual pulp on the surface of the seed with clear water to obtain solid, drying, and pulverizing into powder; adding the powder into a citric acid-sodium citrate buffer solution, heating to 60-70 ℃, and keeping the temperature for 1.5-2.5 h; centrifuging at 9000-11000 rpm, taking supernatant, and performing suction filtration on the supernatant by using qualitative filter paper to obtain filtrate; adsorbing and eluting in an anion exchange fiber column by using NaCl-Tris-HCl buffer solution; eluting the anion exchange fiber column by using NaCl-Tris-HCl buffer solution containing 0-1M NaCl in an equal gradient manner; detecting under the wavelength of 230nm and 280nm, collecting the eluent under 0-1M NaCl, membrane concentrating and desalting under a 3.5KD tangential flow ultrafiltration membrane, and then carrying out vacuum freeze drying to obtain the trypsin inhibitor.

Description

Method for extracting trypsin inhibitor from pitaya seeds

Technical Field

The invention relates to a method for extracting a trypsin inhibitor, in particular to a method for extracting the trypsin inhibitor from pitaya seeds.

Background

Pitaya, also known as red dragon fruit, Xianmei fruit, and Yulong fruit, is a plant of the family Cactaceae, genus Petasites. The fruit is oval, 10-12 cm in diameter, red or yellow in peel, and has a green rounded triangle-shaped frond, white, red or yellow in pulp, and black seeds. The pitaya fruit is rich in nutrition and unique in function, and contains plant albumin and betanin which are few in common plants, abundant vitamins, water-soluble dietary fibers and the like. In the natural state, the fruit ripens in summer and autumn, and is light and juicy. A dragon fruit black seed is rich in content, and contains a large amount of unsaturated fatty acid and protein. But the protein in the dragon fruit seeds is not fully utilized at present.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for extracting trypsin inhibitor from pitaya seeds, which has good inhibition effect, low cost and less side reaction aiming at the current situation of the prior art.

The technical scheme adopted by the invention for solving the technical problems is as follows: the method for extracting the trypsin inhibitor from the dragon fruit seeds is characterized by comprising the following steps:

freezing fresh pitaya at-30 to-10 ℃ for 45 to 50 hours, and taking out the epidermis after thawing; filtering to remove liquid, washing the residual pulp on the surface of the seeds with clear water to obtain solid, drying at 45-55 ℃ for 24-48h, and crushing into dragon fruit seed powder;

adding the obtained pitaya seed powder into a citric acid-sodium citrate buffer solution, heating to 60-70 ℃, and keeping the temperature for 1.5-2.5 h; centrifuging at 9000-11000 rpm, taking supernatant, and performing suction filtration on the supernatant by using qualitative filter paper to obtain filtrate;

the concentrations of citric acid and sodium citrate in the citric acid-sodium citrate buffer solution are both 90-110 mmol/ml, and the pH value is 5-8; the ratio of the pitaya seed powder to the citric acid-sodium citrate buffer solution is 1:10-20g/ml,

adsorbing and eluting the obtained filtrate in a DEAE-52 anion exchange fiber column, wherein a buffer system used for elution is NaCl-Tris-HCl buffer solution, and the pH value is 7.0-7.2;

eluting a DEAE-52 anion exchange fiber column by using NaCl-Tris-HCl buffer solution containing 0-1M NaCl in an equal gradient at the flow rate of 1 mL/min; detecting under the wavelength of 230nm and 280nm, collecting eluent under 0-1M NaCl, membrane-concentrating and desalting under a 3.5KD tangential flow ultrafiltration membrane to one fifth of the original volume, and then carrying out vacuum freeze drying for 46-50h at the temperature of-90-70 ℃ under the pressure of 120-140 MPa to obtain the trypsin inhibitor.

The dragon fruit is preferably red-pulp dragon fruit.

Compared with the prior art, the extraction method of the trypsin inhibitor provided by the invention overcomes the technical prejudice, adopts the dragon fruit seeds as the raw material, is matched with a specific extraction process, and adopts the citric acid-sodium citrate buffer solution heating method to extract the trypsin inhibitor from the dragon fruit seeds, so that the method is simple and convenient to operate, and the obtained trypsin inhibitor has high activity and good inhibition effect on trypsin. In the prior art, the trypsin inhibitor is usually extracted from crops such as soybean, potato, pigeonpea, mung bean, tartary buckwheat and the like, the method is complex, and the activity of the obtained trypsin inhibitor is low.

Drawings

FIG. 1 shows the relationship between the inhibition of trypsin activity by the addition of

extracts

1 and 2 in the present invention.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

The method for extracting the trypsin inhibitor from the dragon fruit seeds comprises the following steps:

picking red meat dragon fruits which are picked in 10 months, mature and fresh, 300-. Freezing in a refrigerator at-20 deg.C for 48 hr, thawing, peeling, filtering with gauze, washing residual pulp on seed surface with clear water, drying in oven at 50 deg.C for 36 hr, pulverizing with Chinese herbal medicine pulverizer to obtain dragon fruit seed powder, packaging in sealed bag, and placing in a refrigerator at-20 deg.C for use.

Adding a citric acid-sodium citrate buffer solution with the pH value of 6.25 into the dragon fruit seed powder, wherein the ratio of the dragon fruit seed powder to the citric acid-sodium citrate buffer solution is 1:10 g/ml; heating to 60 deg.C, holding the temperature for 2h, centrifuging at 10000r/min for 10min, vacuum filtering with qualitative filter paper, and removing oil component in the solution to obtain filtrate.

Adsorbing and eluting the obtained filtrate in a DEAE-52 anion exchange fiber column, wherein the elution conditions are as follows: the buffer system is NaCl-Tris-HCl buffer solution with the pH value of 7.0, the DEAE-52 anion exchange fiber column is eluted isocratically by NaCl-Tris-HCl buffer solution containing 0-1M NaCl at the flow rate of 1mL/min, the content of the extract in the eluent is monitored under the wavelength of 230nm and 280nm, and the peak collection is carried out.

Respectively collecting the eluates under 0M NaCl to obtain extract 1; extract 2 was obtained as an eluate under 1M NaCl. Respectively concentrating and desalting to one fifth of the original volume by a membrane under a 3.5KD tangential flow ultrafiltration membrane; vacuum freeze drying at-80 deg.C under vacuum degree of 130Pa for 48 hr; thus obtaining the trypsin inhibitor.

The inhibition effect of the prepared trypsin inhibitor is verified:

reagent: porcine trypsin, purchased from Yuye Biopsis; ethyl N-benzoyl-L-arginine (BAEE), available from alatin bio; sodium dihydrogen phosphate and disodium hydrogen phosphate, available from national pharmaceutical group chemical agents, ltd; distilled water was provided by the laboratory.

The instrument comprises the following steps: shimadzu, Japan, UV-2600 visible spectrophotometer; TLE104E electronic balance, mettler-toledo instruments (shanghai) ltd; haier BCD-215DC refrigerator, haier island gmbh; RC-200 medium and low pressure chromatography system, Guangzhou Rui Bo apparatus science and technology, Inc.

Trypsin activity assay:

trypsin can catalyze the hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) into H-benzoyl-L-arginine (BA). BA has a maximum absorption peak at a wavelength of 253 nm. Therefore, a straight line is obtained by plotting the relation between the light absorption value of the measurement system under the enzyme catalysis reaction system A253 and the time, and the head and tail values of the straight line are substituted into the formula (1) to calculate the enzyme activity.

P＝(A₂－A₁)/0.003TW (1)

Wherein P is the amount of trypsin per 1mg of test sample (U/mg);

A₁is the light absorption value at the beginning of the straight line;

A₂the absorbance value at the end of the straight line;

t is A₁To A₂Time of reading, min;

w is the amount of test product trypsin contained in the determination solution, mg;

0.003 is the change in absorbance per minute, i.e.equivalent to 1 trypsin unit.

The specific determination method comprises the following steps: weighing substrate BAEE0.086g, and dissolving in 1000mL of 0.067mol/L phosphate buffer solution with pH of 6.8 to obtain BAEE solution; and (3) regulating the substrate BAEE solution by using a phosphate buffer solution to ensure that A253nm is between 0.575 and 0.585 by taking water as a blank. And (3.0 mL) of the substrate solution is taken, 100uL of distilled water is added, 200uL of trypsin aqueous solution with the concentration of 0.020-0.030 mg/mL is added after the substrate solution is uniformly mixed, the mixture is rapidly and uniformly mixed, the timing is immediately carried out, and the light absorption value of A253nm is measured at room temperature. A mixture of 3mL of BAEE solution and 300uL of distilled water was used as a blank control, and the A253nm value was read every 1min for 6min, and the absorbance value was plotted as ordinate and the time as abscissa.

The change of the light absorption value in every 1min is controlled to be between 0.015 and 0.018, and the time in a linear relation is not less than 3 min. Substituting the head and tail values on the straight line into the formula (1) to calculate the trypsin activity. The smaller the angle between the graph and the x-axis, the higher the inhibition.

Determination of trypsin inhibition by

extracts

1 and 2 prepared in this example:

the prepared extract 1 and extract 2 were dissolved in water, respectively, to prepare an extract solution having a concentration of 1 mg/mL.

A mixture of 3mL of BAEE, 200uL of distilled water and 100uL of the extract solution was used as a blank. And uniformly mixing 3mL of BAEE solution with 100uL of each component protein extracting solution, adding 200uL of trypsin aqueous solution with the concentration of 0.020-0.030 mg/mL, uniformly mixing within 1s, immediately timing in a spectrophotometer, and measuring the light absorption value of A253nm at room temperature to obtain the trypsin activity added with the inhibitor.

Trypsin activity after each extract was added was calculated according to the formula (1), and trypsin inhibition ratio of each extract was calculated according to the measurement results.

Inhibition rate-trypsin activity/trypsin activity after addition of each extract

Trypsin activity and trypsin activity added to each extract are shown in fig. 1.

As can be seen from FIG. 1, the trypsin inhibition rate of extract 1 was 90%, and the trypsin inhibition rate of extract 2 was 99%. The inhibitor prepared by the invention has very good inhibiting effect on trypsin and can be used as a novel trypsin inhibitor.

Claims

1. A method for extracting trypsin inhibitor from dragon fruit seeds is characterized by comprising the following steps:

the concentrations of citric acid and sodium citrate in the citric acid-sodium citrate buffer solution are both 90-110 mmol/ml, and the pH value is 5-8; the ratio of the pitaya seed powder to the citric acid-sodium citrate buffer solution is 1:10-20 g/ml;

eluting a DEAE-52 anion exchange fiber column by using NaCl-Tris-HCl buffer solution containing 0-1M NaCl in an equal gradient at the flow rate of 1 mL/min; detecting under the wavelength of 230nm and 280nm, collecting eluent under 0-1M NaCl, membrane-concentrating and desalting under a 3.5KD tangential flow ultrafiltration membrane to one fifth of the original volume, and then carrying out vacuum freeze drying for 46-50h at the temperature of 120-140 MPa, -90-70 ℃ to obtain the trypsin inhibitor;

the dragon fruit is red-pulp dragon fruit.