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Method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptide through using hairtail

A dipeptidyl peptidase and inhibitory peptide technology is applied in the field of preparation of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides, which can solve the problems of affecting the immune function of the body, large toxic and side effects, and reducing T cell activity. Low production cost, high recovery rate, favorable release effect

Active Publication Date: 2013-10-02
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, DPP-IV inhibitors obtained by chemical synthesis, such as Sitagliptin and Vildagliptin, have been shown in clinical trials to have serious side effects such as thrombocytopenia, diarrhea, decreased T cell activity, and affect the body’s immunity functions, etc.; and obtaining DPP-IV inhibitors from natural products is becoming a new research direction
[0004] At present, there is no report on the preparation of dipeptidyl peptidase IV (DPP-IV) inhibitory peptide by enzymatic hydrolysis of hairtail protein

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Remove the head, tail and viscera of hairtail, wash and weigh 1.0kg hairtail, add 2.0kg of water, and mince it into a uniform fish paste; put the fish paste into an enzymatic hydrolysis tank, adjust the temperature to 45°C, and adjust the pH to 7.2, based on hairtail mass, add 0.3% Neutrase neutral protease, stir and hydrolyze for 6 hours; heat up to 95°C, keep for 15 minutes to inactivate the enzyme; quickly cool the hydrolyzate to 50°C, add 0.5% hairtail mass Flavourzyme flavor protease, Adjust the pH to 7.5, stir and hydrolyze for 6 hours; heat up to 95°C, keep for 15 minutes to inactivate the enzyme; centrifuge the obtained hydrolyzate: 4000rpm, 4°C, 13 minutes, take the supernatant; add the supernatant to the ultrafiltration membrane separator In, the pressure of regulating ultrafiltration membrane is 0.09MPa, and the molecular weight cut-off of ultrafiltration membrane is 3000Da, collects ultrafiltration membrane seepage liquid; Dipeptidyl peptidase IV (commerciall...

Embodiment 2

[0030]Remove the head, tail and viscera of the hairtail, wash and weigh 1.5kg hairtail, add 3.5kg water, mince it into a uniform fish paste; put the fish paste into an enzymatic hydrolysis tank, adjust the temperature to 50°C, and adjust the pH to 6.2, based on hairtail mass, add 0.5% papain, stir and hydrolyze for 5 hours; heat up to 98°C, keep for 14 minutes to inactivate the enzyme; quickly cool the hydrolyzate to 45°C, add 0.8% hairtail mass Flavourzyme flavor protease, adjust pH To 7.2, stir and hydrolyze for 4 hours; heat up to 98°C, keep for 14 minutes to inactivate the enzyme; centrifuge the obtained hydrolyzate: 3800rpm, 5°C, 15 minutes, take the supernatant; add the supernatant to the ultrafiltration membrane separator, The pressure of the ultrafiltration membrane is adjusted to 0.08MPa, and the molecular weight cut-off of the ultrafiltration membrane is 3000Da, and the permeate of the ultrafiltration membrane is collected; the dipeptidyl peptidase IV is immobilized o...

Embodiment 3

[0032] Remove the head, tail and viscera of the hairtail, wash and weigh 2.0kg of hairtail, add 5.0kg of water, and mince it into a uniform fish paste; put the fish paste into an enzymatic hydrolysis tank, adjust the temperature to 55°C, and adjust the pH to 8.0, based on hairtail mass, add 0.8% Alcalase alkaline protease, stir and hydrolyze for 10 hours; raise the temperature to 99°C, keep for 13 minutes to kill the enzyme; quickly cool the hydrolyzate to 55°C, add 0.4% hairtail mass Flavourzyme flavor protease, Adjust the pH to 7.0, stir and hydrolyze for 8 hours; heat up to 99°C, keep for 13 minutes to inactivate the enzyme; centrifuge the obtained hydrolyzate: 3900rpm, 3°C, 14 minutes, take the supernatant; add the supernatant to the ultrafiltration membrane separator In the process, the pressure of the ultrafiltration membrane is adjusted to be 0.07MPa, and the molecular weight cut-off of the ultrafiltration membrane is 3000Da, and the permeate of the ultrafiltration membr...

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Abstract

The invention discloses a method for preparing a DPP-IV inhibitory peptide through using hairtail. The method is characterized in that the method comprises the following steps: adding water to hairtail, and rubbing to form a uniform fish flesh pulp; putting the fish flesh pulp in an enzymatic hydrolysis tank, adding endo protease, and carrying out stirring hydrolysis for 4-12h to obtain a hydrolysate; heating to 95-100DEG C, and maintaining the temperature unchanged for 10-15min for enzyme killing; cooling the hydrolysate to 40-60DEG C, adding exoproteinase, and carrying out stirring hydrolysis for 2-8h; heating to 95-100DEG C, and maintaining the temperature unchanged for 10-15min for enzyme killing; centrifuging the obtained enzyme-killed hydrolysate, and taking the obtained supernatant; adding the supernatant to an ultrafilter membrane separator, adjusting the pressure and the interception molecular weight of the ultrafilter membrane to 0.05-0.1MPa and 3000Da respectively, and collecting an ultrafilter membrane permeate liquid; and separating and purifying the DPP-IV inhibitory peptide through adopting an affinity chromatography process. The method has the advantages of simple technology, low production cost and short production period.

Description

technical field [0001] The present invention relates to the field of biotechnology, and specifically refers to a method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptides by using hairtail as raw material, hydrolyzing hairtail protein with compound enzymes, and using techniques such as ultrafiltration and affinity chromatography . technical background [0002] Hairtail, also known as saury and octopus, is a species of hairtail of the order Perciformes. It is a kind of economic fish with high production in my country's coastal areas. It is delicious and is favored by consumers at home and abroad. Hairtail protein is a complete protein, containing eight essential amino acids, calcium, iron, phosphorus, magnesium, zinc, selenium and other trace elements and multivitamins, rich in nutrition. Traditional Chinese medicine believes that hairtail has the effects of harmonizing the appetite, warming the stomach, nourishing the deficiency, moisturizing the skin and be...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/36C07K1/34C07K1/22
Inventor 靳挺武玉学
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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