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A method of ultrasonic-assisted enzymatic hydrolysis to prepare visceral antioxidant peptides

A technology of single-ring thorns and antioxidant peptides, which is applied to the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of shortening proteolysis reaction time, long reaction time, and low activity of products, and achieves improved induction Effects of oxidative damage, increased utilization, and high antioxidant peptide activity

Active Publication Date: 2021-12-10
CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the disadvantages of low proteolysis degree, low product activity, and long reaction time of the traditional preparation method of the monocylic spinypin The problem of wasting waste resources in the viscera of Echinacea; improve the product activity of the antioxidant peptide of the viscera of Echinacea monocyclic, and shorten the reaction time of proteolysis

Method used

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  • A method of ultrasonic-assisted enzymatic hydrolysis to prepare visceral antioxidant peptides
  • A method of ultrasonic-assisted enzymatic hydrolysis to prepare visceral antioxidant peptides
  • A method of ultrasonic-assisted enzymatic hydrolysis to prepare visceral antioxidant peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Effect of ultrasonic-assisted enzymatic hydrolysis method on hydrolysis degree of viscera polypeptide

[0049] The reaction rate of the entire reaction is measured by the degree of hydrolysis (DH). The determination of the degree of hydrolysis refers to the formaldehyde titration method in GB 5009.235-2016. First, the amino acid nitrogen is calculated, and then the degree of protein hydrolysis is calculated. The calculation formulas are:

[0050] The formula for amino acid nitrogen calculation is as follows:

[0051]

[0052] The formula for calculating the degree of hydrolysis is as follows:

[0053]

[0054] In formula (1):

[0055] V 1 : NaOH titration volume (mL)

[0056] V 2 : NaOH titration volume in blank experiment (mL)

[0057] C: NaOH solution concentration (mol / L)

[0058] In formula (2):

[0059] m: mass of amino acid nitrogen in protein hydrolyzate (g)

[0060] M: total nitrogen mass of the sample (g), M = 0.05 x sample g (g)

[006...

Embodiment 2

[0075] Example 2 Separation and purification of the hydrolyzate of the monocyclic echinacea protein and sequence identification of the polypeptide

[0076] The protein hydrolyzate of Echinacea monocyclon was separated and purified by ultrafiltration and Sephadex G-25 gel chromatography. First, the hydrolyzate was separated into fractions of different molecular weights by ultrafiltration, then the fraction with the highest antioxidant activity was purified by Sephadex G-25 gel chromatography, and finally the fraction with the highest activity was purified by LC-MS / MS The filtrate was used for peptide identification.

[0077] Antioxidant activity assay method:

[0078] (1) Determination of DPPH clearance rate

[0079] First weigh 0.004 g of DPPH and dissolve it with 100 mL of absolute ethanol to make a 0.1 mmol / L DPPH solution. Take 2 mL of sample solution and 2 mL of DPPH solution, and fully react in the dark for 30 min, measure its absorbance at a wavelength of 517 nm, and ...

Embodiment 3

[0098] Example 3 The visceral polypeptide of the monocyclic spinyweed improves H 2 o 2 RAW264.7 cells induced injury

[0099] The activity of RAW264.7 cells was used to measure the effect of the visceral polypeptide of C. monocylicus, and the determination of cell activity refers to the CCK-8 method. First explore whether the polypeptide has cytotoxicity to RAW264.7 cells, and then explore whether the polypeptide has cytotoxicity on H 2 o 2 Whether the induced injury of RAW264.7 cells has improvement effect.

[0100] Cytotoxicity determination of RAW264.7 cells by the visceral polypeptide of monocyclic spinypinus:

[0101] Seed RAW264.7 cells on a 96-well cell culture plate at a density of 1.0×10 4 After culturing in a carbon dioxide incubator for 24 h, the growth medium was removed, and then DMEM growth medium with antioxidant peptide concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, and 2 mg / mL was added, respectively. Six replicate wells were set up in each group, and the...

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Abstract

The invention discloses a preparation method of visceral antioxidative peptide of monocyclic echinacea. The preparation process is as follows: first, degrease, dry and pulverize the viscera of C. monoringensis; then add protease combined with ultrasonic-assisted enzymolysis; after enzymolysis, high temperature inactivates protease, and centrifuge to collect the supernatant; then add granular activated carbon for decolorization and deodorization; The filtrate was collected by ultrafiltration and Sephadex G-25 gel chromatography; finally, the P1, P2, and P3 polypeptides were identified by LC-MS / MS. The ultrasound-assisted enzymatic hydrolysis method of the present invention prepares the antioxidative peptide of the viscera of Echinococcus monoringensis, shortens the reaction time, improves the effect of enzymatic hydrolysis, effectively solves the problem of waste resources of the viscera of Echinococcus monocyclicum, and obtains High-efficiency antioxidant peptide products. And the results of sequence analysis showed that the antioxidant peptide P1 in this study has higher antioxidant activity than the published antioxidant peptides of the monocyclic spinyweed.

Description

technical field [0001] The invention belongs to the field of deep processing of Echinococcus monocylicus, and in particular relates to a method for preparing visceral antioxidant peptides of Echinococcus monocylicus by ultrasonic-assisted enzymolysis. Background technique [0002] Urechisunicinctus, also known as "sea intestine", belongs to the phylum Echiurioidea, class Echiurida, order Xenopneusta, and family Urechidae. It is a cylindrical or oval marine invertebrate that mainly lives on sandy or muddy bottoms in shallow waters and obtains nutrients through filter feeding. Due to its unique morphology, its burrow is "U" shaped, which helps to maintain aerobic conditions, and it can survive in a wide range of water temperature, water salinity, pH and dissolved oxygen conditions. It is mainly distributed in Japan, South Korea and China's Bohai Sea and Yellow Sea region. It has a unique taste and a smooth taste, and is deeply loved by people in coastal areas. Echinacea mono...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06C07K7/06C07K7/08C07K1/36C07K1/34C07K1/16C07K1/14A61K38/08A61K38/10A61P39/06
CPCC12P21/06C07K7/06C07K7/08A61P39/06A61K38/00
Inventor 贺晓云陆佳俊黄昆仑刘清亮许文涛孙艳丽周秀芝张瑞英
Owner CHINA AGRI UNIV