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Preparation method of whitmania pigra anticoagulation active peptide

A technology of wide-body golden leeches and anticoagulant activity, which is applied in the field of active peptides, can solve the problems of complex operation, large loss of active peptides, and low proteolysis rate, and achieve high yield, simple operation, and anticoagulant activity Good results

Inactive Publication Date: 2016-11-09
袁春华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention: Aiming at the problems that the current preparation method of the active peptide of the wide-bodied leech leech has low proteolysis rate, large loss of active peptide, complicated operation, and is not suitable for large-scale production and application in industrialization, the present invention provides A method for preparing the anticoagulant active peptide of leech chinensis. The invention extracts the crude extract of alkaline protease by cultivating Bacillus subtilis, and separates it into three phases after standing by salting out, isoelectric point precipitation and co-solvent precipitation. , take mesophase enzyme protein precipitation, separate and purify to obtain highly active alkaline protease, and then use it to enzymatically hydrolyze the isolated protein solution of the wide-bodied leech to obtain the anti-coagulant active peptide of the wide-bodied leech

Method used

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Examples

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Effect test

example 1

[0016] Weigh 4g of sodium chloride, 0.1g of potassium dihydrogen phosphate, 0.01g of magnesium sulfate heptahydrate, 1g of peptone, and 5g of glucose, add 95mL of deionized water, stir at 200r / min for 20min, heat to boiling, and keep warm for 20min to obtain culture Bacillus subtilis was inoculated into the culture solution according to the inoculum size of 8%, and cultured at 30°C for 22 hours. Measure 2mL of the culture solution, mix it with 1.5L of deionized water, add 40g of sodium chloride and 1.5g of dihydrogen phosphate respectively Potassium, 0.15g magnesium sulfate heptahydrate, 11g beef extract, 11g yeast extract powder, 22g sucrose, adjust the pH to 10.2 with a mass concentration of 1% sodium hydroxide solution, and ferment at 30°C for 110h with shaking at 120r / min. Place the fermentation broth in a centrifuge, centrifuge at 12000r / min for 30min, take the supernatant to obtain a crude enzyme solution; weigh 18g of ammonium sulfate, place it in a 50°C drying oven, dry...

example 2

[0019]Weigh 4.5g sodium chloride, 0.15g potassium dihydrogen phosphate, 0.015g magnesium sulfate heptahydrate, 1.5g peptone, 5.5g glucose, add 98mL deionized water, stir at 250r / min for 25min, heat to boiling, keep warm The culture solution was obtained in 25 minutes. According to the inoculum size of 9%, Bacillus subtilis was inoculated into the culture solution, and cultured at 31°C for 23 hours. Measure 2.5mL of the culture solution, mix it with 1.55L of deionized water, add 41g of sodium chloride, 1.55 g potassium dihydrogen phosphate, 0.15g magnesium sulfate heptahydrate, 11.5g beef extract, 11.5g yeast extract powder, 23g sucrose, adjust the pH to 10.3 with a mass concentration of 1% sodium hydroxide solution, at 31°C, at 125r / Min shaking and fermenting for 115 hours, put the fermentation liquid in a centrifuge, centrifuge at 12000r / min for 35 minutes, take the supernatant to obtain a crude enzyme liquid; weigh 20g of ammonium sulfate and place it in a 52°C drying oven t...

example 3

[0022] Weigh 5g of sodium chloride, 0.2g of potassium dihydrogen phosphate, 0.02g of magnesium sulfate heptahydrate, 2g of peptone, and 6g of glucose, add them into 100mL of deionized water, stir at 300r / min for 30min, heat to boiling, and keep warm for 30min to obtain the culture Bacillus subtilis was inoculated into the culture solution according to the inoculum size of 10%, and cultured at 32°C for 24 hours. Measure 3mL culture solution, mix it with 1.6L deionized water, add 42g sodium chloride and 1.6g dihydrogen phosphate respectively Potassium, 0.16g magnesium sulfate heptahydrate, 12g beef extract, 12g yeast extract powder, 24g sucrose, adjust the pH to 10.5 with a mass concentration of 1% sodium hydroxide solution, and shake and ferment at 130r / min for 120h at 32°C. Place the fermentation broth in a centrifuge, centrifuge at 12000r / min for 40min, take the supernatant to obtain a crude enzyme solution; weigh 25g of ammonium sulfate, place it in a 55°C drying oven, dry it...

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Abstract

The invention discloses a preparation method of a whitmania pigra anticoagulation active peptide and belongs to the technical field of active peptides. Aiming at the problems of an existing preparation method of the whitmania pigra anticoagulation active peptide that the protein hydrolysis rate is relatively low, the loss of the active peptide is relatively large, the operation is complicated and the method is not suitable for industrial large-scale production, the invention provides the preparation method of the whitmania pigra anticoagulation active peptide. The preparation method comprises the following steps: culturing bacillus subtilis; extracting an alkaline protease rough extracting solution; carrying out salting-out, isoelectric precipitation and co-solvent precipitation, and standing and dividing into three phases; taking middle-phase zymoprotein and precipitating; separating and purifying to obtain high-activity alkaline protease; and carrying out enzymolysis on whitmania pigra protein isolate liquid to obtain the whitmania pigra anticoagulation active peptide. The whitmania pigra anticoagulation active peptide prepared by the preparation method is high in yield, good in anticoagulation activity and simple in operation.

Description

technical field [0001] The invention relates to a preparation method of an anticoagulant active peptide of the broad-bodied leeches, belonging to the technical field of active peptides. Background technique [0002] In traditional Chinese medicine, leeches are widely used because of their anticoagulant, antithrombotic, and blood lipid-lowering effects. [0003] For the treatment of hypertension, blood stasis, swelling and pain, etc., its specific active ingredients are still unclear for many years. In recent years, the research on its effective active ingredients has become a hot spot. Lv Dan et al. found that the moisture content of living leeches broad-bodied is 85.61%, the crude protein content is 11.24%, the sugar content is 1.43%, the total fat content is 0.95%, and the ash content is 0.77%. Besides water, proteins make up most of the composition of this mollusk. Jiang Peifen performed electrophoresis analysis on several protein leeches and found that the protein ban...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/14C12N9/56
CPCC12P21/06C07K1/14C12N9/54C12Y304/21062
Inventor 袁春华王龙
Owner 袁春华
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