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

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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 t

Method used

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Examples

Experimental program
Comparison scheme
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Example Embodiment

[0015] Example 1

[0016] Weigh 4 g of sodium chloride, 0.1 g of potassium dihydrogen phosphate, 0.01 g of magnesium sulfate heptahydrate, 1 g of peptone, and 5 g of glucose respectively, add them to 95 mL of deionized water, stir at 200 r / min for 20 min, heat to boiling, and keep the culture for 20 min. Bacillus subtilis was inoculated into the culture solution according to the inoculum volume of 8%, incubated at 30°C for 22 hours, 2mL of the culture solution was weighed, mixed with 1.5L of deionized water, 40g of sodium chloride and 1.5g of dihydrogen phosphate were added respectively. Potassium, 0.15g magnesium sulfate heptahydrate, 11g beef extract, 11g yeast extract powder, 22g sucrose, adjust pH to 10.2 with 1% sodium hydroxide solution, ferment at 30°C with shaking at 120r / min for 110h, The fermentation broth was placed in a centrifuge, centrifuged at 12,000 r / min for 30 min, and the supernatant was taken to obtain a crude enzyme liquid; 18 g of ammonium sulfate was wei...

Example Embodiment

[0018] Example 2

[0019]Weigh 4.5g of sodium chloride, 0.15g of potassium dihydrogen phosphate, 0.015g of magnesium sulfate heptahydrate, 1.5g of peptone, and 5.5g of glucose, respectively, add them into 98mL of deionized water, stir at 250r / min for 25min, heat to boiling, and keep warm. 25min to get the culture solution, according to the inoculum amount of 9%, inoculate Bacillus subtilis into the culture solution, incubate at 31 ℃ for 23h, measure 2.5mL culture solution, mix with 1.55L deionized water, add 41g sodium chloride, 1.55mL respectively g potassium dihydrogen phosphate, 0.15g magnesium sulfate heptahydrate, 11.5g beef extract, 11.5g yeast extract powder, 23g sucrose, adjust pH to 10.3 with 1% sodium hydroxide solution, at 31 °C, at 125r / Min shake fermentation for 115h, put the fermentation liquid in a centrifuge, centrifuge at 12000r / min for 35min, take the supernatant to obtain crude enzyme liquid; weigh 20g of ammonium sulfate and place it in a drying oven at 52...

Example Embodiment

[0021] 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, respectively, add them to 100mL of deionized water, stir at 300r / min for 30min, heat to boiling, and keep the culture for 30min. Bacillus subtilis was inoculated into the culture solution according to the inoculum volume of 10%, incubated at 32°C for 24 hours, 3mL of the culture solution was weighed, mixed with 1.6L of deionized water, and 42g of sodium chloride and 1.6g of dihydrogen phosphate were added respectively. Potassium, 0.16g magnesium sulfate heptahydrate, 12g beef extract, 12g yeast extract powder, 24g sucrose, adjust pH to 10.5 with 1% sodium hydroxide solution, ferment at 32°C with shaking at 130r / min for 120h. The fermentation broth was placed in a centrifuge, centrifuged at 12,000 r / min for 40 min, and the supernatant was taken to obtain a crude enzyme liquid; 25 g of ammonium sulfate was weigh...

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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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