CN108047307B

CN108047307B - Edible and medicinal fungus source selenium chelating peptide and preparation method thereof

Info

Publication number: CN108047307B
Application number: CN201810148454.1A
Authority: CN
Inventors: 赵立娜; 陈紫红; 刘斌; 于志颖; 林占熺; 吕旭聪; 李鑫
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2018-02-13
Filing date: 2018-02-13
Publication date: 2020-01-10
Anticipated expiration: 2038-02-13
Also published as: CN108047307A

Abstract

The invention belongs to the technical field of biology, and particularly relates to edible and medicinal fungus source selenium chelating peptide. The edible and medicinal fungus source selenium chelating peptide is Grifola frondosa selenium chelating peptide, and the amino acid sequence of the selenium chelating peptide is as follows: EAMY. The preparation method comprises the following steps: taking grifola frondosa protein as a raw material, carrying out enzymolysis on the grifola frondosa protein by adopting alkaline protease, and separating, purifying, freezing and drying enzymolysis liquid to obtain the selenium chelating peptide. The invention prepares the peptide with high selenium chelating activity by controlling the cutting condition of alkaline protease and the separation and purification condition of enzymolysis liquid, so that the selenium chelating activity can be efficiently realized, and a new idea is provided for the resource utilization of edible and medicinal fungi grifola frondosa.

Description

Edible and medicinal fungus source selenium chelating peptide and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to edible and medicinal fungus source selenium chelating peptide.

Background

Selenium (Se) is one of the essential trace elements for human body, and has important nutrition physiological effect. However, the availability of selenium from food to the human body is often insufficient, and this deficiency can lead to the development of a number of diseases. Therefore, it is essential to add an appropriate selenium source to the diet. Selenium has various supplementary forms, mainly including organic selenium and inorganic selenium. Sodium selenite and sodium selenate are the main inorganic selenium, and have high toxicity at a lower level. Research shows that inorganic selenium is absorbed in human body in a small part and is mostly discharged out of the body along with urine, and the bioavailability is low. In contrast, organic selenium has a higher bioavailability than inorganic selenium and is less toxic.

Grifola frondosa (A) and (B)Grifola frondosa) Also called Bayesian fungus, Yuanqingyuan, Hebei, Shixingchi, chestnut mushroom, Qianfo fungus, etc., and Maitake mushroom in Japan. Since the last 80 s, scientists mainly in Japan and China have conducted extensive research on the biology, chemistry and pharmacology of Grifola frondosa, and the Grifola frondosa is proved to be edible and medicinal mushrooms with special value. The active component grifola frondosa D-fraction extracted from grifola frondosa has strong anticancer effect and is known as 'anticancer wonderful flower'. Grifola frondosa is also a popular edible fungus, and the meat quality of the Grifola frondosa is crisp and tender, is similar to that of magnolia, and is delicious and tasty. Grifola frondosa is rich in high-quality protein and rich in vitamins and dietary fibers, and the nutritional values of the grifola frondosa are known by more and more people.

The grifola frondosa is rich in nutrition, the content of the nutrients of the grifola frondosa is detected by the institute of nutrition and food sanitation and the center of quality control of agricultural departments, and each 100 g of the grifola frondosa contains 25.2 g of protein, so that the grifola frondosa is subjected to deep processing of protein resources by using a biological technology, and the bioactive peptides are subjected to screening, extraction and separation by jointly applying ultrafiltration, gel filtration and high performance liquid chromatography to obtain pure polypeptides, and the amino acid sequence of the polypeptides is determined by flight time mass spectrometry. The polypeptide can not only convert inorganic selenium into organic selenium by combining selenium element through reaction, but also can be actively absorbed in the integral form of a complex by virtue of the absorption and transportation characteristics of the polypeptide in intestinal tract digestion. The polypeptide still has the biological activities of resisting tumors, regulating cholesterol, reducing blood sugar and the like, has important research significance and value, can improve the added value of the grifola frondosa, and enriches the types of grifola frondosa functional foods.

Disclosure of Invention

The invention aims to provide a selenium chelating peptide as an edible and medicinal fungus source aiming at the defects of the prior art. The method utilizes alkaline protease to carry out enzymolysis on the grifola frondosa protein, the prepared selenium chelating peptide has high activity, and inorganic selenium can be converted into organic selenium; provides a new idea for the resource utilization of the grifola frondosa.

In order to achieve the purpose, the invention adopts the following technical scheme:

the edible and medicinal fungus source selenium chelating peptide is Grifola frondosa selenium chelating peptide, and the amino acid sequence of the edible and medicinal fungus source selenium chelating peptide is as follows: EAMY.

The preparation method of the edible and medicinal fungus source selenium chelating peptide comprises the following steps: taking grifola frondosa protein as a raw material, carrying out enzymolysis on the grifola frondosa protein by adopting alkaline protease, and separating, purifying, freezing and drying enzymolysis liquid to obtain the selenium chelating peptide.

The enzymolysis conditions are as follows: the concentration of the substrate is 3wt%, the enzymolysis pH is 10.0, the temperature is 50 ℃, the enzymolysis time is 1h, and the enzyme-substrate mass ratio is 1: 20.

The specific steps of the separation and purification are as follows: passing the enzymolysis solution through a 0.22 mu m filter membrane, and dividing the enzymolysis solution into solutions with different molecular weights through ultrafiltration membranes of 3 kDa and 10kDa for freeze-drying; measuring and collecting peak with highest selenium chelating activity, separating with Sephadex G-25 gel filtration chromatography, eluting with deionized water at flow rate of 0.3ml/min, and measuring the elution peak at 214 nm; collecting a peak with the highest selenium chelating activity, performing further separation by using semi-preparative RP-HPLC-C18 reversed-phase high performance liquid chromatography, wherein the separation condition is to use acetonitrile solution with volume fraction of 0-50% as eluent for gradient elution, the flow rate is 4 ml/min, the eluent begins until the water content is 100% and the mixed solution of acetonitrile with volume ratio of 50% and water with volume ratio of 50% is ended, performing gradient elution, collecting elution peaks with volume ratio of 25% and 75% of water, and analyzing by adopting an LC/MS liquid mass spectrometer to obtain the peak with retention time of 12.38min as the selenium chelating characteristic peptide.

The invention has the beneficial effects that:

based on the fact that the polypeptide has action sites chelated with mineral element ions and can form a stable compound with the polypeptide, and the polypeptide-mineral element chelate has a unique chelate system and a transport mechanism, is easy to absorb and can supplement amino acid and mineral elements simultaneously, grifola frondosa protein from grifola frondosa is used as a raw material, and the peptide with high selenium chelating activity is prepared by controlling the cutting condition of alkaline protease and the separation and purification condition of enzymolysis liquid, so that the selenium chelating activity is realized efficiently; provides a new idea for the resource utilization of the edible and medicinal fungi grifola frondosa.

Drawings

FIG. 1 is a LC/MS microarray of purified grifolan selenium-chelating peptide.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

Example 1

A preparation method of edible and medicinal fungus source selenium chelating peptide comprises the following specific steps:

the grifola frondosa protein adopted by the invention is prepared by laboratories, and the enzyme is purchased from Beijing Soilebao science and technology Limited (China. Beijing).

3.0g of grifolan was weighed out and dissolved in 100ml of distilled water, and then the pH was adjusted to 10.0 with 2mol/L NaOH. Firstly heating the solution to 50 ℃ in a water bath, then adding a corresponding amount of enzyme according to the enzyme-substrate mass ratio of 1:20, and carrying out enzymolysis for 1 h. Then, the enzyme was inactivated in a boiling water bath for 10 minutes, cooled and centrifuged at 10000rpm for 10 minutes. Collecting the supernatant for later use, wherein the enzyme is alkaline protease.

Separating the supernatant by using a membrane ultrafiltration technology, firstly, passing the supernatant solution through a 0.22 mu m filter membrane, then separating the supernatant solution into solutions with different molecular weights by using ultrafiltration membranes of 3 kDa and 10kDa, freeze-drying, measuring and collecting samples in various molecular weight ranges, and measuring the selenium chelation activity;

separating the sample with highest selenium chelating activity by membrane ultrafiltration with Sephadex G-25 gel filtration chromatography (length 100cm, outer diameter 2.0 cm) at flow rate of 0.3mL/min and deionized water as eluent, and measuring the peak at 214 nm; collecting a peak with the highest selenium chelating activity, performing further separation by using semi-preparative RP-HPLC-C18 reversed-phase high performance liquid chromatography, wherein the separation condition is to use 0-50% (v/v) acetonitrile solution as eluent for gradient elution, the flow rate is 4 mL/min, the eluent starts until the volume of water is 100%, the eluent finishes the mixed solution of acetonitrile with the volume ratio of 50% and water with the volume ratio of 50%, performing gradient elution, collecting the elution peaks with the volume ratio of 25% acetonitrile and 75% water, analyzing by using an LC/MS liquid mass spectrometer to obtain the peak with the retention time of 12.38min as the selenium chelating characteristic peptide, and performing freeze drying to obtain the selenium chelating peptide.

1. Sequencing

The amino acid sequence of the selenium chelate peptide prepared in example 1 was determined by ESI mass spectrometry (WATERS MALDI SYNAPT Q-TOF MS, Waters co., u.s.a), and the amino acid sequence was: EAMY.

2. Selenium chelating ability test of selenium chelating peptide:

the chelation of the selenium chelating peptide to selenium ions is determined by a colorimetric method, and the selenium content is determined by a 3,3' -diaminobenzidine colorimetric method.

1) And (3) preparing a standard curve:

selenium standard solution: 2.1940g of dried sodium selenite is accurately weighed, dissolved in distilled water, the volume is constant to 1L, selenium stock solution containing 1g/L of selenium is prepared, and the selenium stock solution is diluted into 5mg/mL of selenium standard solution when in use.

Taking 5 conical flasks, putting 2.0, 4.0, 6.0, 8.0 and 10.0 mL of selenium standard solutions respectively, adding distilled water to 40mL of distilled water, adjusting the pH to 2 ~ by using 1mol/L HCl, adding 2mL of 0.2mol/L EDTA-2Na solution, then adding 2mL of 0.5wt% 3,3' -Diaminobenzidine (DBA), placing in a water bath at 60 ℃ for 50min (avoiding light) to shake, taking out, adjusting the pH to 7.0 ~.5 by using 1mol/L NaOH solution, accurately adding 10mL of toluene, shaking for 2min, standing for 3 ~ min to layer, and then measuring the absorbance of the solution at 420nm (taking a blank of toluene), wherein y represents the absorbance, and x represents the standard mass concentration/(mug/mL) of selenium.

2) Weighing 6mL of polypeptide solution, adding sodium selenite (0.5 mol/L) according to the volume ratio of sodium selenite solution (0.5M) to the polypeptide solution being 4:6, stirring uniformly, adjusting pH to 9.0, continuously reacting for 90min at 50 ℃, cooling 4500r/min, centrifuging for 10min to remove solids, adding 5-fold volume fraction of 95% ethanol solution, precipitating and standing for 12h, centrifuging at 4500r/min for 10min to remove supernatant, washing with equal volume of absolute ethyl alcohol for several times, drying to obtain chelate powder, weighing 0.2g of grifolan peptide-selenium chelate, placing into a 100mL small beaker, adding 5mL of digestive juice, digesting on an electric furnace until colorless and transparent, adjusting pH to 7.0 by using 40wt% of solution and 5wt% of solution to adjust pH to constant volume, fixing to 50mL, placing the solution to be tested into a 10mL centrifuge tube (storage), taking 0.5mL of sample solution, adding 40mL of distilled water, adjusting pH to 24.0.0 by using 1mol/L of NaOH solution, adding to adjust pH to 863 min, adding standard solution of toluene to obtain standard solution, shaking, placing the sample into a standard toluene solution, shaking for measuring absorbance at 35 mL, adding 2min, shaking for measuring, adding standard pH 3-2 mL of toluene, adding standard solution, shaking for measuring, adding standard pH 3, shaking for measuring, 2min, adding standard pH, shaking, adding standard solution, shaking for measuring, adding 2mL of toluene, adding standard solution, dissolving, adding standard solution, standing for measuring, dissolving, standing for measuring, adding standard toluene, dissolving:

in the formula: p is the standard mass concentration/(mug/mL) which is equivalent to selenium and is found from the standard curve; v is the volume/mL of the sample obtained by toluene extraction; m is the mass/g of the sample; n is the volume fraction/% of the sample volume for determination in the total volume; the results were: the selenium chelating power of EAMY is 49.34 mg/g.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Sequence listing

<110> Fujian agriculture and forestry university

<120> edible and medicinal fungus source selenium chelating peptide and preparation method thereof

<130>1

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>4

<212>PRT

<213> amino acid sequence (Grifola frondosa sequence)

<400>1

Glu Ala Met Tyr

1

Claims

1. The edible and medicinal fungus source selenium chelating peptide is characterized in that: the edible and medicinal fungus source selenium chelating peptide is Grifola frondosa selenium chelating peptide, and the amino acid sequence of the selenium chelating peptide is as follows: EAMY.