Triple-enzyme hydrolysis preparation method of anti-tumor polypeptides of spirulina

A spirulina, anti-tumor technology, applied in the biological field, can solve the problem that the anti-tumor peptide is in the primary stage

Active Publication Date: 2015-04-29
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it has been reported that antioxidant peptides, ACE inhibitory peptides, and bacteriostatic peptides have been successfully extracted from spirulina protein hydrolyzate, but the research on anti-tumor peptides is still in its infancy.

Method used

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  • Triple-enzyme hydrolysis preparation method of anti-tumor polypeptides of spirulina
  • Triple-enzyme hydrolysis preparation method of anti-tumor polypeptides of spirulina
  • Triple-enzyme hydrolysis preparation method of anti-tumor polypeptides of spirulina

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] (1) Take 10 g of spirulina powder, prepare a solution with a concentration of 5% (w / v) with ultrapure water, freeze it in a -20 °C refrigerator for 4 hours, and then thaw it in a 37 °C water bath for 2 hours, and freeze it repeatedly Melt 5 times. The sample was homogenized in an ice bath for 2 minutes, and the rotation speed was 5000rpm for 30s→10000rpm for 1min→5000rpm for 30s. Then in an ice bath with a power of 460W ultrasonic 20min (each ultrasonic 6 s, interval 9 s). 4°C, 8000r / min, centrifuge for 30 min.

[0029] (2) Prepare the spirulina protein obtained in step (1) into a 2% (w / w) protein solution, add pepsin so that the concentration ratio of the enzyme to the substrate is 4% (w / w), and adjust the temperature to 37 ℃, pH 1.0, enzymatic hydrolysis for 1 h. Then adjust the pH to 7.0, add trypsin according to the ratio of 3% (w / w) of the enzyme to the substrate for 2 hours. Then add chymotrypsin according to the ratio of 4% (w / w) enzyme to substrate, and enzy...

Embodiment 2

[0034] (1) Take 20 g of spirulina powder, prepare a solution with a concentration of 5% (w / v) with ultrapure water, freeze it in a -20 ℃ refrigerator for 5 hours, and then thaw it in a 37 ℃ water bath for 2.5 hours, and repeat this process Freeze and thaw 4 times. The sample was homogenized in an ice bath for 70s, and the rotation speed was 4000rpm for 20s→12000rpm for 30s→4000rpm for 20s. Then in an ice bath, sonicate at a power of 500 W for 20 min (every sonication is 5 s, with an interval of 7 s). 4°C, 8000r / min, centrifuge for 40 min.

[0035] (2) Prepare the spirulina protein obtained in step (1) into a 3% (w / w) protein solution, add pepsin so that the concentration ratio of the enzyme to the substrate is 5% (w / w), and adjust the temperature to 37 ℃, pH 1.5, enzymatic hydrolysis for 1.5 h. Then the pH was adjusted to 7.5, and trypsin was added for 3 hours according to the enzyme-to-substrate ratio of 4% (w / w). Then add chymotrypsin according to the enzyme-to-substrate...

Embodiment 3

[0039] (1) Take 25 g of spirulina powder, prepare a solution with a concentration of 5% (w / v) with ultrapure water, freeze it in a -20 °C refrigerator for 6 hours, and then thaw it in a 37 °C water bath for 3 hours, and freeze it repeatedly Melt 3 times. The sample was homogenized in an ice bath for 150s, and the rotation speed was 3000rpm 50s→8000rpm 50s→3000rpm 50s. Then ultrasound was performed at 600 W for 15 minutes in an ice bath (4 seconds for each ultrasound, with an interval of 6 seconds). 4°C, 8000r / min, centrifuge for 45 min.

[0040] (2) Prepare the spirulina protein obtained in step (1) into a 4% (w / w) protein solution, add pepsin so that the concentration ratio of the enzyme to the substrate is 5.5% (w / w), and adjust the temperature to 37 ℃, pH 2.0, enzymatic hydrolysis for 2 h. Then adjust the pH to 8.0, and add trypsin to hydrolyze for 4 hours according to the ratio of enzyme to substrate of 3% (w / w). Then add chymotrypsin according to the ratio of 4% (w / w)...

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Abstract

The invention provides a triple-enzyme hydrolysis preparation method of anti-tumor polypeptides of spirulina. The method comprises the following steps: preparing a solution having a concentration of 5 percent from spirulina powder with ultrapure water; repeatedly freezing and thawing, homogenizing and performing ultrasonic treatment, centrifuging to obtain supernate, freezing and drying for later use; adding the prepared protein fluid into pepsin for enzyme hydrolysis, controlling the pH value, adding trypsin for enzyme hydrolysis, adding chymotrypsin for enzyme hydrolysis, controlling the pH value, deactivating enzyme, cooling, and centrifuging to obtain supernate; sequentially filtering with ultrafiltration membranes having molecular weight cut-off of 10KD, 5KD and 3KD respectively to obtain three kinds of spirulina protein enzymatic hydrolysates; carrying out sephadex G15 column chromatography on 0-3K of enzymatic hydrolysate, eluting with water, and collecting four polypeptide ingredients, namely anti-tumor polypeptides of spirulina. The obtained spirulina polypeptide and monopeptide ingredients can be used for establishing a theoretical basis for development and utilization of anti-tumor medicines and health foods.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a three-enzyme hydrolysis preparation method of spirulina anti-tumor polypeptide. Background technique [0002] Bioactive peptides are special protein fragments that have a positive effect on the function or state of the body and ultimately affect the health of the body. Compared with proteins, the advantages of small-molecule peptide fragments are mainly reflected in: easier to be absorbed and utilized by the human body; high activity, which can exert its unique physiological effects at a small concentration; small molecular weight, easy to modify and transform, can By artificial chemical synthesis etc. Compared with single amino acids, small molecule peptides not only have special physiological activities, but also have incomparable advantages of amino acids in absorption channels and absorption speed. Studies have confirmed that there are special oligopeptide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K7/06C07K1/36C07K1/34C07K1/16A61P35/00
Inventor 张学武王竹君
Owner SOUTH CHINA UNIV OF TECH
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