CN103880942B - A kind of method utilizing enzymolysis protein to prepare metal chelating peptide - Google Patents

Abstract

The invention provides a method of enzymolyzing whey protein with compound protease and flavor protease to prepare metal (Ca, Fe, Zn) chelating peptides, using whey protein as raw material, enzymolyzing with compound protease and flavor protease, and then separating and purifying The purified specific metal (Ca, Fe, Zn) chelating peptide is obtained, the complete amino acid sequence is: ydt. The metal chelating peptide prepared by the present invention can be used to produce new metal supplements—peptide chelating calcium, peptide chelating iron, and peptide chelating zinc. It has a unique chelating system and transport mechanism, is easily absorbed, is safe and non-toxic , low price, can supplement amino acids and metal ions at the same time, it will become the first choice for calcium, iron and zinc supplements. The invention provides a new idea for the application of whey protein.

Description

A method for preparing metal chelating peptides by enzymatically hydrolyzing proteins

technical field

The invention relates to a metal (Ca, Fe, Zn) chelating peptide, more specifically to a metal chelating peptide prepared by hydrolyzing whey protein with complex protease and flavor protease, and belongs to the field of biotechnology.

Background technique

With the rise of milk-derived active peptide research and product development, it has been found that whey protein contains potential peptides with biological activities such as immune regulation, lowering blood pressure, lowering blood sugar, lowering cholesterol, antioxidant, antibacterial and antiviral. Whey protein-derived bioactive peptides have extremely high nutritional value, food safety and physiological health functions, and play an important role in promoting the growth, development and disease prevention of the body. They can be used as functional foods or food additives or even Drugs are applied to people's daily life.

At present, calcium deficiency is a global nutritional problem. Because our people mainly eat plant-based diets, the phenomenon of calcium deficiency is more serious. Therefore, calcium supplementation has become an important topic in the study of dietary nutrition in our country. Traditional inorganic calcium salts, such as calcium carbonate, calcium phosphate, calcium chloride, etc., have a certain destructive effect on vitamins, and their biological value is low; organic calcium salts, such as calcium citrate, calcium lactate, calcium glucose, etc., although calcium The absorption rate has been improved, but its solubility is large and easy to dissolve, and the price is high. Studies have shown that peptide chelated calcium has become the first choice for calcium supplementation due to its unique chelation system and transport mechanism, easy to be absorbed, safe and non-toxic, low price, and can supplement amino acids and calcium at the same time.

The trace element iron plays an important role in human health, especially in the growth and development of infants and children. Although macromolecular proteins can also bind to iron ions, these macromolecular proteins themselves have the problem of relatively large molecular weights and are difficult to pass through the intestinal mucosa. However, studies have found that amino acid and polypeptide chelated iron can greatly improve the absorption rate of iron ions.

Although zinc sources are very abundant in nature, the absorption and metabolism of zinc in the human body is limited by various factors. Zinc deficiency has become a public health problem in my country and many developing countries. Studies have found that amino acids and small peptides can promote zinc absorption, and the bioavailability of amino acid or peptide metal complexes such as Fe and Zn is higher than that of inorganic salts and has no toxic side effects. Therefore, it is of great significance to research and develop this kind of biological state zinc——proteolyzate chelated zinc.

Therefore, how to obtain peptides with metal chelating activity has become an urgent research direction for the preparation of new metal element supplements.

Contents of the invention

In order to solve the above problems, the present invention provides a metal chelating peptide prepared by hydrolyzing whey protein with complex protease and flavor protease, so that the metal (Ca, Fe, Zn) chelating activity can be realized efficiently.

To achieve the above object, the present invention adopts the following technical solutions:

A metal chelating peptide is a tripeptide composed of 3 amino acids. The amino acid sequence of the polypeptide is: ydt.

The metal is Ca, Fe or Zn.

A method for preparing a metal chelating peptide, using whey protein as a raw material, using complex protease and flavor protease to enzymatically hydrolyze it, separating, purifying, and freeze-drying to obtain the metal chelating peptide.

The enzymolysis conditions are: substrate concentration 5%, enzymolysis pH 7.0, temperature 49°C, enzymolysis time 7 hours, enzyme-substrate weight ratio 1:25.

The enzyme is a composite protease and a flavor protease, and the weight compounding ratio of the two enzymes is composite protease:flavor protease=2:1.

The specific steps of separation and purification are as follows: the enzymatic hydrolysis product is first separated by TOYOPEARLDEAE-650M anion exchange chromatography, and the eluent is a 0.02mol/L pH9.0 phosphate buffer with a concentration gradient of 0-0.5M NaCl, and the flow rate is 0.5 mL/min, the elution peak was measured at 214nm; the peak with the highest metal chelating activity was collected, and then separated by SephadexG-25 gel filtration chromatography, the eluent was deionized water, and the flow rate was 0.3mL/min, The elution peak was measured at 214nm; the peak with the highest metal chelating activity was collected and further separated by semi-preparative RP-HPLC-C18 reverse-phase high-performance liquid chromatography. The separation condition was 0-30% acetonitrile by volume The solution was gradient eluted as an eluent with a flow rate of 4mL/min, and the elution peak with the highest metal chelating activity was collected, and then further separated by analytical RP-HPLC-C18 reversed-phase high-performance liquid chromatography. The volume ratio is 0-10% acetonitrile solution, the flow rate is 1mL/min, the peaks are collected for metal chelating activity determination, the eluted peak has the highest metal chelating activity, and the metal chelating peptide is obtained.

The present invention is based on the fact that the polypeptide has an action site for chelation with metal ions, and can form a stable compound with it, and the polypeptide-metal chelate has a unique chelation system and transport mechanism, is easily absorbed, and can supplement amino acids and metals at the same time Theoretical basis, using whey protein from whey as the raw material, through the control of the cleavage conditions of complex protease and flavor protease, the peptide with high metal (Ca, Fe, Zn) chelating activity is cleaved to make the metal chelate activity is achieved efficiently. The invention provides a new idea for the application of whey protein.

Description of drawings

Figure 1 RP-HPLC-C18 chromatogram of purified whey protein-derived metal chelating peptides; WPH-2 has peaks with the highest metal (Ca, Fe, Zn) chelating activity.

Detailed ways

Example 1

The preparation method is as follows:

The whey protein (WPC80) used in this technique was purchased from HILMAR (Texas, USA), and the enzyme was purchased from Novozymes Biotechnology Co., Ltd. (Tianjin, China). Single factor experiments were used to investigate four enzymatic hydrolysis factors, namely substrate concentration (1%, 3%, 5% and 7%), enzymatic hydrolysis temperature (40, 50, 55 and 60°C), enzyme Compound ratio (composite protease: flavor protease = 1:1, 1:2, 1:3 and 2:1w/w), enzyme-substrate ratio (1:100, 1:50, 1:25 and 1: 20w/w) and enzymatic hydrolysis time (1,3,5,7,9 hours). Weigh a certain amount of whey protein and dissolve it in distilled water, then adjust its pH to 7.0 with 2mol/L NaOH. First heat the solution in a water bath to the required temperature, then add the corresponding amount of enzyme according to different enzyme-substrate ratios, and start the reaction according to the predetermined reaction time. Then inactivate the enzyme in a boiling water bath for 10 minutes, and then centrifuge at 10,000 rpm for 10 minutes after cooling. After the supernatant was collected, the metal (Ca, Fe, Zn) chelating activity was measured to determine the optimal enzymatic hydrolysis conditions. The enzymolysis conditions to obtain the enzymolysis liquid with maximum metal chelating activity are: substrate concentration 5%, enzymolysis pH 7.0, temperature 49°C, enzymolysis time 7 hours, enzyme-substrate ratio 1:25 (w/w); the enzyme is a compound protease and a flavor protease, and the compounding ratio of the two enzymes is compound protease: flavor protease=2:1 (w/w).

Weigh 5.0 g of whey protein and dissolve it in 100 ml of distilled water, then adjust its pH to 7.0 with 2 mol/L NaOH. First heat the solution to 49°C in a water bath, then add the corresponding amount of enzyme according to the enzyme-substrate ratio of 1:25, the mass ratio of compound protease to flavor protease is 2:1, and the enzymatic hydrolysis time is 7 hours . Then inactivate the enzyme in a boiling water bath for 10 minutes, then centrifuge at 10,000 rpm for 10 minutes after cooling, and collect the supernatant for later use.

The supernatant was separated by TOYOPEARLDEAE-650M anion exchange chromatography (length 50cm, outer diameter 1.6cm). The eluent was a 0.02mol/L pH9.0 phosphate buffer containing 0-0.5M NaCl in a concentration gradient. The flow rate was 0.5mL/min, collect samples of each peak and determine the metal (Ca, Fe, Zn) chelation activity.

The elution peak with the highest metal (Ca, Fe, Zn) chelating activity separated by TOYOPEARLDEAE-650M anion exchange chromatography is separated in the next step, and SepadexG-25 gel filtration chromatography (length 100cm, outer diameter 2.0cm) The peaks with the highest metal (Ca, Fe, Zn) chelating activity were collected and further separated by semi-preparative RP-HPLC-C18 reversed-phase high-performance liquid chromatography. The eluent contained 100% (v/v) water From the beginning, to the end of the mixture of 30% acetonitrile and 70% water (v/v), gradient elution was carried out, the elution time was 50min, the flow rate was 4mL/min, and the samples with the highest metal (Ca, Fe, Zn) chelation The active peak is the elution peak with a retention time of 20.681min, and then further separated by analytical RP-HPLC-C18 reverse-phase high-performance liquid chromatography, the eluent is 0-10% (v/v) acetonitrile solution, The flow rate was 1mL/min, and the peaks were collected for metal (Ca, Fe, Zn) chelation activity determination. The elution peak with a retention time of 25.727min had the highest metal (Ca, Fe, Zn) chelation activity, and the described metal chelating peptides.

The chelating effect of metal chelating peptides on calcium ions was determined by the o-cresolphthalein colorimetric method. Add 1 mL of 5 mmol/L CaCl ₂ and 2 mL of 0.2 mol/L phosphate buffer (pH 8.0) into a stoppered test tube, then add 1 mL of albumin peptide solution, and incubate at 37 ° C for 2 h in a constant temperature heating water bath shaker , after taking it out, centrifuge at 10000r/min at room temperature for 10min. Take 1 mL of the supernatant, add 5 mL of o-cresolphthalein chromogenic solution, and shake well. After standing for 10 minutes, measure the absorbance value at 570nm with a spectrophotometer, and substitute the value into the standard curve to calculate the amount of soluble calcium binding.

Preparation of standard curve: Take standard Ca working solution (10ug/mL) 0, 0.2, 0.4, 0.6, 0.8, 1.0mL respectively in 10mL test tubes, add deionized water 1.0, 0.8, 0.6, 0.4, 0.2, 0mL, Add 5mL o-cresolphthalein chromogenic solution, shake well, let it stand for 10min, and measure the absorbance at 570nm with a spectrophotometer. Taking the soluble calcium content (ug/mL) as the abscissa and the absorbance value as the ordinate, the formula of the standard curve is: y=0.0974x-0.0402, R ² =0.9996.

The chelating effect of metal chelating peptides on iron ions was determined by o-phenanthroline colorimetric method. Put 0.05g of the sample in a 100mL beaker, add 2mL of concentrated hydrochloric acid, and after the sample is completely dissolved, dilute it to a 100mL volumetric flask with distilled water. Accurately draw 5mL of sample solution into a 50mL volumetric flask, add 1mL of 1mol/L HCl solution, 1mL of 10% hydroxylamine hydrochloride, 1mL of 0.12% phenanthroline, then add 5mL of 10% sodium acetate, dilute to the mark with water, and shake uniform. Use the reagent blank solution without adding iron as the reference solution, measure the absorbance at a wavelength of 510nm, and substitute the value into the standard curve to calculate the iron binding amount.

The making of standard curve: draw standard solution 0, 2.0, 4.0, 6.0, 8.0, 10.0mL of 10ug/mL iron, place in 50mL volumetric flask respectively, add 1mL of HCl solution of 1mol/L, 1mL of 10% hydroxylamine hydrochloride, Add 1 mL of 0.12% o-phenanthroline, then add 5 mL of 10% sodium acetate, dilute to the mark with water, and shake well. Using the reagent blank solution without adding iron as the reference solution, measure the absorbance at a wavelength of 510nm, and draw a standard curve, the formula of the standard curve is: y=0.1717x+0.003, R ² =0.9994.

The chelating effect of metal chelating peptides on zinc ions was determined by EDTA titration. Weigh 100 mg of the chelate compound in a 100 mL small beaker, add 50 mL of water, and add a few drops of 6 mol/L hydrochloric acid. Shake well, heat it on a water bath to dissolve it completely, cool it down to 100mL, draw 10mL from it into the Erlenmeyer flask, make 3 parts, add 10mL of NH ₃ -NH ₄ CI buffer solution (pH10), appropriate amount of chrome black T indicator , and then use 0.0lmol/LNa ₂ EDTA to drop to blue; record the number of milliliters of EDTA consumed, and calculate the zinc content of the chelate.

Using TOYOPEARLDEAE-650M anion exchange chromatography, SephadexG-25 molecular sieve, RP-HPLC reversed-phase high-performance liquid chromatography and other separation and purification methods, the efficient separation and purification of whey protein metal chelating peptide with significant activity is realized.

The purified specific metal chelating peptide has very high metal (Ca, Fe, Zn) chelating activity. It can be seen from Table 1 that compared with the enzymatic hydrolysis solution, the metal chelating ability of WPH-2 has been greatly improved. improve.

The metal chelating ability of the specific metal chelating peptide of table 1 purification

The amino acid sequence of the purified specific metal chelating peptide was determined by ESI mass spectrometer ((WATERSMALDISYNAPTQ-TOFMS, WatersCo., U.S.A). The amino acid sequence of the metal chelating peptide is: ydt.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCELISTING

<110> Fuzhou University

<120> A method for preparing metal chelating peptides by enzymolysis of proteins

<130>1

<160>1

<170>PatentInversion3.3

<210>1

<211>3

<212>PRT

<213> metal chelating peptide

<400>1

TyrAspThr

1

Claims (3)

1. a metal chelating peptide, is characterized in that: the aminoacid sequence of described peptide is: ydt.

2. a kind of metal chelating peptide according to claim 1, is characterized in that: described metal is Ca, Fe or Zn.

3. a preparation method for a kind of metal chelating peptide as claimed in claim 1, is characterized in that: take whey-protein as raw material, adopts compound protease and flavor protease is composite carries out enzymolysis to it, and separation and purification, lyophilize obtain metal chelating peptide, described enzymatic hydrolysis condition is: concentration of substrate 5%, and enzymolysis pH is 7.0, temperature 49 DEG C, enzymolysis time are 7 hours, enzyme-substrate weight proportion is 1:25, described enzyme is compound protease and flavor protease, and the composite ratio of weight of two kinds of enzymes is compound protease: flavor protease=2:1, the concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARLDEAE-650M anion-exchange chromatography to be separated, the phosphoric acid buffer of elutriant to be concentration gradient the be 0.02mol/LpH9.0 containing 0-0.5MNaCl, flow velocity is 0.5mL/min, and elution peak is measured under 214nm, collect the peak with the highest metal chelating activity, then be separated by SephadexG-25 gel filtration chromatography, elutriant is deionized water, and flow velocity is 0.3mL/min, and elution peak measures under 214nm, collect the peak with the highest metal chelating activity, half preparation RP-HPLC-C18 RPLC is utilized to be separated further again, separation condition is that 0-30% acetonitrile solution is as elutriant gradient elution by volume ratio, flow velocity is 4mL/min, collect the elution peak with the highest metal chelating activity, recycling analysis mode RP-HPLC-C18 RPLC is separated further again, elutriant is volume ratio 0-10% acetonitrile solution, flow velocity is 1mL/min, collect each peak and carry out metal-chelating vitality test, obtain elution peak and there is the highest metal chelating activity, obtain described metal chelating peptide.