CN103965303A - Marine bacillus licheniformis protein and application thereof to cardiovascular field - Google Patents

Abstract

A marine bacillus licheniformis protein. A preparation method comprises the following steps: inoculating a kelp endophytic bacillus licheniformis DNN6 strain to a liquid medium, wherein the liquid medium formula comprises 40-50g / L sucrose and 8-10g / L peptone, and has pH value adjusted to 9; bottling 150-250mL of the liquid medium into each bottle, and conducting shaking culture at temperature of 30-35 DEG C, rotating speed of 150-250 rpm for 4-5 days; centrifuging the fermentation liquid at 8000 r / min to remove thalli, then adding ammonium sulfate, wherein the saturation concentration of ammonium sulfate is 95%, and standing overnight at 4 DEG C; and filtering, dissolving the precipitate with distilled water, then conducting dialysis by using a dialysis bag with cut-off molecular weight of 3600 Dalton for 4 days, and conducting freeze-drying on the products in the dialysis bag, so as to obtain the marine endophytic bacillus licheniformis protein. The invention also provides the application of the protein to the cardiovascular field. The process is simple and low in cost.

Description

A kind of ocean Bacillus licheniformis albumen and application aspect cardiovascular thereof

Technical field

The present invention relates to field of medicaments, particularly raw Bacillus licheniformis DNN6(in a main laminaria bacillus licheniformisdNN6) strain protein and the application aspect cardiovascular.

Background technology

Sea-tangle ( laminaria Japonicaaresch) be a kind of raw brown alga plant in large-scale sea growing in low temperature seawater.It grows in lower marine of water temperature.Sea-tangle is mainly self-sow, also propagates artificially, mainly with dry products, is on sale throughout in city, and quality take that look brown, body is short, the thin and plump person of matter is as good.Sea-tangle has the good reputation of " longevity greens/mustard green ", " vegetables at sea " " containing iodine champion ".

Sea-tangle is as a kind of important oceanic resources, and it eats with pharmaceutical use and is early in the public eye.That the effective constituent of extracting from sea-tangle has is hypoglycemic, reducing blood-fat, antitumor, anti-HIV and strengthen immunologic function, to treatment cardiovascular and cerebrovascular diseases and in, early stage chronic kidney hypofunction etc. all has certain effect.In sea-tangle, also contain some attached microorganisms altogether, but these microorganisms and meta-bolites thereof are not only also fully utilized so far.

Summary of the invention

The object of this invention is to provide raw Bacillus licheniformis DNN6(in a kind of sea-tangle simple to operate, with low cost bacillus licheniformisdNN6) strain protein and the application aspect cardiovascular thereof, to make full use of the common attached microorganism of sea-tangle, overcome the deficiencies in the prior art.

A kind of ocean of the present invention Bacillus licheniformis albumen, as follows preparation:

A. get raw Bacillus licheniformis DNN6(Bacillus licheniformis DNN6 in sea-tangle), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number: CGMCC No:7077 on December 31st, 2012;

B. Bacillus licheniformis DNN6 is inoculated in liquid nutrient medium, the formula of this liquid nutrient medium is: sucrose 40-50 g/L and peptone 8-10 g/L, and pH is deployed into 9;

C. b step finishes rear packing, every bottled 100-200 mL liquid nutrient medium, and temperature 30-35 ℃, rotating speed is 150-200 rpm, shaking table is cultivated 4-5 days;

D. by the fermented liquid of c step centrifugal treating under the condition of 8000 r/min, remove thalline, then add ammonium sulfate, the saturated final concentration of ammonium sulfate is 95%, 4 ℃ of standing over night, filter and will precipitate with after distilled water dissolving, then are 3600 daltonian dialysis tubings dialysis 4 days with molecular weight cut-off, get thing lyophilize in dialysis tubing, obtain ocean Bacillus licheniformis albumen.

The application of a kind of ocean of the present invention Bacillus licheniformis albumen aspect cardiovascular.

Ocean of the present invention Bacillus licheniformis protein preparation method technique is simple, with low cost, the albumen extracting the fermented liquid of raw lichem bacillus strain in sea-tangle has hypotensive activity, the development and the exploitation that can be applicable to Altace Ramipril, have industrial value.

The ocean lichem bacillus strain of applying in the present invention has been preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC No:7077 on December 31st, 2012.

embodiment

The embodiment of the present invention is carried out successively as follows:

A. strain Bacillus licheniformis DNN6 is inoculated in to sucrose 40-50 g/L and peptone 8-10 g/L, in the liquid nutrient medium that pH is 9, every bottled 100-200 mL liquid nutrient medium, temperature 30-35 ℃, rotating speed is 150-200 rpm, shaking table is cultivated 4-5 days.This lichem bacillus strain has been preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC No:7077 on December 31st, 2012.

B. by the centrifugal removal thalline of fermented liquid 8000 r/min, then add ammonium sulfate, the saturated final concentration of ammonium sulfate is 95%, 4 ℃ of standing over night, filter and will precipitate with after distilled water dissolving, then be 3600 daltonian dialysis tubings dialysis 4 days with molecular weight cut-off, get thing lyophilize in dialysis tubing, obtain ocean Bacillus licheniformis albumen, SDS-PAGE electrophoresis result shows that this throw out is protein.

Get freeze-drying sample and with PBS solution, be dissolved into the solution of 1 mg/mL, use respectively the dosage of 180 mg/kg and 18 mg/kg, jugular vein administration SD rat (The 2nd Army Medical College Experimental Animal Center provides), two dosage all can make systolic pressure decline 3.17 ± 0.1 KPa; Isolated heart pours into the shrinkage amplitude that 18 mg/kg can reduce SD isolated rat heart.

C. the impact on rat blood pressure

Laboratory animal: male SD rat is purchased from The 2nd Army Medical College Experimental Animal Center.Experimental session, keeps Animal House room temperature 22 ℃ of left and right, relative humidity 70% left and right, early 8 extremely late 8 automatic illuminatings.Animal ad lib, freely drinks water.

The preparation of ductus arteriosus: PE10(internal diameter 0.3 cm, external diameter 0.5 cm) and PE50(internal diameter 0.58 mm, external diameter 0.96 mm) two kinds of specification conduits are prepared ductus arteriosus, PE10 pipe is docked by thin wire with PE50 pipe, after heating, press the protuberance that joint forms, cooling rear extraction iron wire, can apply after inspection joint is firm, unobstructed.

20% urethanum (urethane), 5 mL/kg intraperitoneal anesthesia rats, lain on the back and be fixed on animal constant temperature autopsy table, a separated side femoral artery, distal end ligation, proximal part inserts the arterial cannulation of filling with 1% heparin-saline, the opposite side connecting tee pipe of intubate is by by PowerLab MLT1199 invasive blood pressure measuring system, dynamically recording arteriotony.After blood pressure stabilization, by jugular vein intubate, to DNN6 strain protein (180 mg/kg and 18 mg/kg), and recording blood pressure changes.Experimental result shows that two dosage all can make systolic pressure decline 3.17 ± 0.1 KPa, have the potentiality of exploitation depressor.

D. the foundation of Isolated Rat heart model: by SD for rat after urethane (1.2 gkg-1) and heparin (400 IUkg-1) mixed solution intraperitoneal anesthesia, opening rapidly chest cores, by classical Langendorff method, through aorta ascendens, drive in the wrong direction and insert intrusion pipe, make it be placed in aortic valve top, in order to coronary perfusion.Adopt KH liquid to carry out balance perfusion, when front 30 min of perfusion and perfusion, fill with mixed gas (95% O2+5% CO2), make the KH liquid of balance perfusion fully reach oxygen saturation, pH value maintains 7.35 ~ 7.45, keeping filling temperature is 37.0 ℃ ~ 37.5 ℃, and injection pressure is 60 mmHg.Two suspension electrocardiographic recording electrodes are positioned over to pulmonary conus respectively and apex is guided out electrocardiogram(ECG, and the cardiac function index such as recorded heart rate.Experimental result shows 4 min after the K-H liquid perfusion heart of the albumen (18 mg/kg) with containing bacterial strain DNN6, and heart contraction amplitude reduces by 23%.With K-H liquid, recover after perfusion, heart contraction amplitude and rest tension recover gradually again.The hypotensive activity of bacterial strain DNN6 albumen may be relevant with reduction heart function.

Claims (2)

1. an ocean Bacillus licheniformis albumen, is characterized in that: preparation as follows:

A. get raw Bacillus licheniformis DNN6(in sea-tangle bacillus licheniformisdNN6), this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number: CGMCC No:7077 on January 10th, 2013;

B. Bacillus licheniformis DNN6 is inoculated in liquid nutrient medium, the formula of this liquid nutrient medium is: sucrose 40-50 g/L and peptone 8-10 g/L, and pH is deployed into 9;

C. b step finishes rear packing, every bottled 100-200 mL liquid nutrient medium, and temperature 30-35 ℃, rotating speed is 150-200 rpm, shaking table is cultivated 4-5 days;

D. by the fermented liquid of c step centrifugal treating under the condition of 8000 r/min, remove thalline, then add ammonium sulfate, the saturated final concentration of ammonium sulfate is 95%, 4 ℃ of standing over night, filter and will precipitate with after distilled water dissolving, then are 3600 daltonian dialysis tubings dialysis 4 days with molecular weight cut-off, get thing lyophilize in dialysis tubing, obtain ocean Bacillus licheniformis albumen.

2. the application of ocean according to claim 1 Bacillus licheniformis albumen aspect cardiovascular.