CN106565839B - Method for extracting trypsin inhibitor from pitaya seeds - Google Patents

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 dragon fruit at -30--10°C for 45-50 hours, and taking out the epidermis after thawing; filtering After removing the liquid, the obtained solid is washed with clean water and the remaining pulp on the surface of the seeds is dried, and then pulverized into powder; the powder is added to the citric acid-sodium citrate buffer solution, heated to 60-70 ° C, and the constant temperature is 1.5-2.5 h Then centrifuge at 9000～11000rpm, take the supernatant, and the supernatant adopts qualitative filter paper to carry out suction filtration to obtain the filtrate; carry out adsorption and elution with NaCl-Tris-HCl buffer in the anion exchange fiber column; ～1M NaCl-Tris-HCl buffer isogradient elution anion exchange fiber column; detection at wavelengths of 230nm and 280nm, collecting the eluent under 0～1M NaCl, under 3.5KD tangential flow ultrafiltration membrane Concentrate and desalt, and then freeze-dry in vacuo to obtain trypsin inhibitor.

Description

A kind of method for extracting trypsin inhibitor from dragon fruit seeds

technical field

The present invention relates to a method for extracting trypsin inhibitor, in particular to a method for extracting trypsin inhibitor from dragon fruit seeds.

Background technique

Dragon fruit, also known as red dragon fruit, dragon bead fruit, fairy honey fruit, jade dragon fruit, belongs to the cactus family and is a plant of the genus Tianchi. The fruit is oval in shape, 10-12 cm in diameter, with red or yellow skin, green rounded triangular fronds, white, red or yellow pulp, and black seeds. Dragon fruit is rich in nutrients and unique in function. It contains vegetable albumin, betaside, rich in vitamins and water-soluble dietary fiber, which are rare in general plants. In its natural state, the fruit ripens in summer and autumn, with a light and juicy taste. A dragon fruit is rich in black seeds, and dragon fruit seeds contain a lot of unsaturated fatty acids and protein. But at present, the protein in dragon fruit seeds has not been fully utilized.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for extracting trypsin inhibitor from dragon fruit seeds with good inhibitory effect, low cost and few side reactions according to the current situation of the prior art.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is: the method for extracting trypsin inhibitor from dragon fruit seeds is characterized in that comprising the following steps:

Freeze the fresh dragon fruit at -30～-10℃ for 45～50h, take out the epidermis after thawing; filter out the liquid, wash the remaining pulp on the surface of the seeds with clean water, and dry at 45～55℃ for 24-48h , crushed into dragon fruit seed powder;

The obtained dragon fruit seed powder is added to the citric acid-sodium citrate buffer solution, heated to 60-70 ° C, and the constant temperature is 1.5 to 2.5 hours; then centrifuged at 9000 to 11000 rpm, and the supernatant is taken, and the supernatant is used qualitative filter paper Carry out suction filtration to obtain a 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 is 5-8; the ratio of the dragon fruit seed powder to the citric acid-sodium citrate buffer solution 1:10-20g/ml,

The obtained filtrate was adsorbed and eluted in a DEAE-52 anion exchange fiber column, and the buffer system used for the elution was NaCl-Tris-HCl buffer, and the pH was 7.0-7.2;

The DEAE-52 anion exchange fiber column was eluted with isogradient NaCl-Tris-HCl buffer containing 0-1M NaCl at a flow rate of 1mL/min; detected at wavelengths of 230nm and 280nm, and collected the washes under 0-1M NaCl. Deliquoring, concentrating and desalting to one-fifth of the original volume under a 3.5KD tangential flow ultrafiltration membrane, and then vacuum freeze-drying at 120-140MPa and -90--70°C for 46-50h to obtain trypsin inhibitor.

The dragon fruit is preferably a red-fleshed dragon fruit.

Compared with the prior art, the extraction method of trypsin inhibitor provided by the present invention overcomes the technical prejudice, adopts dragon fruit seeds as raw materials, cooperates with a specific extraction process, and adopts the heating method of citric acid-sodium citrate buffer solution from dragon fruit. The trypsin inhibitor is extracted from fruit seeds, the method is simple and easy to operate, the obtained trypsin inhibitor has high activity, and has a good inhibitory effect on trypsin. The prior art usually extracts from soybean, potato, more bean, mung bean, tartary buckwheat and other crops, the method is complicated, and the obtained trypsin inhibitor has low activity.

Description of drawings

Figure 1 shows the relationship between the addition of extract 1 and extract 2 to the inhibition of trypsin activity in the embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

The method for extracting trypsin inhibitor from dragon fruit seeds is as follows:

Pick a batch of red-fleshed dragon fruit picked in October, ripe and fresh, 300-500g/piece. Put it in a -20℃ refrigerator for 48 hours, after thawing and peeling, filter the liquid with gauze, wash the remaining pulp on the surface of the seeds with water, then put it in an oven to dry at 50℃ for 36 hours, and pulverize it with a Chinese herbal medicine grinder to get the dragon. The fruit seed powder is placed in a sealed bag and placed in a -20°C refrigerator for later use.

Add a citric acid-sodium citrate buffer solution with a pH of 6.25 to the dragon fruit seed powder, and the ratio of the dragon fruit seed powder and the citric acid-sodium citrate buffer solution is 1:10g/ml; heat to 60°C and keep the constant temperature for 2h , centrifuge at 10000r/min for 10min, use qualitative filter paper for suction filtration, remove the oil and fat components in the solution, and obtain a filtrate.

The obtained filtrate was adsorbed and eluted on a DEAE-52 anion exchange fiber column, and the elution conditions were: the buffer system was NaCl-Tris-HCl buffer with pH 7.0, and the NaCl-Tris-HCl buffer containing 0-1M NaCl was used The DEAE-52 anion exchange fiber column was eluted isocratically with a flow rate of 1 mL/min. The content of the extract in the eluate was monitored at wavelengths of 230 nm and 280 nm, see peak collection.

The eluate under 0M NaCl, namely extract 1; the eluate under 1M NaCl, namely extract 2, were collected. The membranes were concentrated and desalted under a 3.5KD tangential flow ultrafiltration membrane to one-fifth of the original volume; vacuum freeze-dried at a vacuum degree of 130Pa and a temperature of -80°C for 48h; that is, trypsin inhibitor was obtained.

Validation of the inhibitory effect of the prepared trypsin inhibitor:

Reagents: porcine trypsin, purchased from Yuanye Biological Company; N-benzoyl-L-arginine ethyl ester (BAEE), purchased from Aladdin Biological Company; sodium dihydrogen phosphate, disodium hydrogen phosphate, purchased from Sinopharm Group Chemical Reagent Co., Ltd.; distilled water provided by the laboratory.

Instruments: Japan Shimadzu UV-2600 visible light spectrophotometer; TLE104E electronic balance, METTLER TOLEDO Instruments (Shanghai) Co., Ltd.; Haier BCD-215DC refrigerator, Qingdao Haier Co., Ltd.; RC-200 medium and low pressure chromatography system , Guangzhou Ruibai Instrument Technology Co., Ltd.

Trypsin activity assay:

Trypsin can catalyze the hydrolysis of p-N-benzoyl-L-arginine ethyl ester (BAEE) to H-benzoyl-L-arginine (BA). BA has a maximum absorption peak at 253 nm wavelength. Therefore, by measuring the relationship between the absorbance value and time of the enzyme catalyzed reaction system A253, a straight line is obtained by plotting, and the first and last values of the straight line are taken and substituted into formula (1) to calculate the enzyme activity.

P=(A ₂ -A ₁ )/0.003TW (1)

In the formula, P is the amount of trypsin per 1 mg of the test product (U/mg);

A ₁ is the absorbance value at the beginning of the line;

A ₂ is the absorbance value at the end of the straight line;

T is the time from A ₁ to A ₂ reading, min;

W is the amount of trypsin contained in the test solution, mg;

0.003 is the change in absorbance value per minute, which is equivalent to 1 trypsin unit.

The specific determination method is as follows: Weigh 0.086 g of the substrate BAEE, dissolve it in 1000 mL of 0.067 mol/L phosphate buffer with a pH of 6.8 to obtain a BAEE solution; take water as a blank, adjust the substrate BAEE solution with a phosphate buffer to make it A253nm is between 0.575 and 0.585. Take 3.0 mL of the substrate solution, add 100 uL of distilled water, and after mixing, add 200 uL of trypsin aqueous solution with a concentration of 0.020-0.030 mg/mL, mix quickly and time it immediately, and measure the A253nm absorbance at room temperature. In addition, 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 a total of 6min. The absorbance value was used as the ordinate and the time as the abscissa.

Control the change of absorbance value within 1min between 0.015-0.018, and the time of linear relationship shall not be less than 3min. Substitute the first and last values on the straight line into formula (1) to calculate the trypsin activity. The smaller the angle between the graph line and the x-axis, the higher the inhibition rate.

Determination of the inhibitory effect of extract 1 and extract 2 prepared in this example on trypsin:

The prepared extracts 1 and 2 were respectively dissolved in water to prepare extract solutions with a concentration of 1 mg/mL.

A mixture of 3mL of BAEE, 200uL of distilled water and 100uL of extract solution was used as a blank. Take another 3 mL of BAEE solution and mix it with 100 uL of each component protein extract, then add 200 uL of trypsin enzyme solution with a concentration of 0.020-0.030 mg/mL, mix within 1 s and put it in a spectrophotometer for immediate timing, at room temperature The absorbance at A253 nm was measured to obtain the trypsin activity after adding the inhibitor.

The trypsin activity after adding each extract was calculated according to formula (1), and the trypsin inhibition rate of each extract was calculated according to the measurement results.

Inhibition rate = trypsin activity - trypsin activity/trypsin activity after adding each extract

Trypsin activity and trypsin activity of each extract added are shown in Figure 1.

It can be seen from Figure 1 that the inhibitory rate of extract 1 to trypsin reached 90%, and the inhibitory rate of extract 2 to trypsin reached 99%. It shows that the inhibitor prepared by the present invention has a very good inhibitory effect on trypsin, and can be used as a new trypsin inhibitor.

Claims (1)

1. A method for extracting trypsin inhibitor from 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 1:10-20 g/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 120-140 MPa, -90-70 ℃ to obtain the trypsin inhibitor;

the dragon fruit is red-pulp dragon fruit.

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