CN102093473A - Preparation method of bacteriostatic active protein of Sinonovacula constricta Lamarck epipsymbiosis strain - Google Patents

Abstract

The invention discloses a low-cost preparation method of bacteriostatic active protein of a Sinonovacula constricta Lamarck epipsymbiosis strain, which is simple to operate. The method comprises the following sequential steps: grinding clean and fresh Sinonovacula constricta flesh, evenly spreading 200mL of grinding fluid on a beef extract peptone plate culture medium, and culturing at a constant temperature of 37 DEG C for 2 days; selecting a single colony, repeatedly carrying out streak culture to obtain pure strains, and detecting the bacteriostatic activity of the pure strains by using a filter paper method; taking the strains of which the diameter of the inhibition zone is greater than or equal to 10mm, and culturing the strains in a pH 5 Sabouraud's liquid culture medium, which uses glucose as the carbon source, at 35 DEG C for 120 hours; centrifugating at 4000 r/min to remove thallus, and adding ammonium sulfate into the supernatant, wherein the saturation final concentration of the ammonium sulfate is 90%; and standing over night at 4 DEG C, centrifugating at 10000 r/min for 30 minutes, dissolving the precipitate in a PBS (phosphate buffer solution), dialyzing with a dialysis bag of which the molecular retention is 3500 daltons, and carrying out freeze-drying on the substances in the bag filter.

Description

The Yi razor clam proteic preparation method of bacterial strain bacteriostatic activity that grows nonparasitically upon another plant altogether

Technical field

The present invention relates to the proteic preparation method of a kind of bacterial activity, particularly a kind of Yi razor clam simple to operate, with low cost proteic preparation method of bacterial strain bacteriostatic activity that grows nonparasitically upon another plant altogether.

Background technology

The Yi razor clam ( Sinonovacula constrictaIamarck) popular name razor clam, blue or green son, bamboo blood clam, extra large razor clam, be under the jurisdiction of Mollusca (Mollusca), lamellibranchiata (LameUibranchia), Eulamellibranchia (Eulamellibranchia), razor clam section (Pharellidae), being China and japonic kind, also is one of important shellfish of the coastal breed of China over more than 100 year.Yi razor clam meat flavour deliciousness, nutritious, protein content 5.5%, fat 0.8%, sugar 1.8% and inorganic salt 1.1%.The soft body of Yi razor clam also has the effect of qi-restoratives, to the deficiency of Yin, deficiency of blood best results, postpartum, after being ill uses it more.In recent years, the research of seashells has caused that people greatly pay attention to, the multiple biologically active peptides that is had in the seashells, polysaccharide, unsaturated fatty acids etc. have become the important raw and processed materials of marine health food and functional food, with Pacific oyster (Crassostrea gigas) pepsin hydrolysis, obtain having the hydrolysate that suppresses the ACE ability as Bordenave etc.; Clam (Meretrix meretrix (Linnaeus)) polysaccharide has two-ways regulation function etc. to the delayed type hypersensitivity (DTH) that the endoxan (CTX) of various dose causes.But, the research about the Yi razor clam at present focuses mostly on aspect dry products processing and the Analysis of Nutritional, and is very few to its bioactive research, and grow nonparasitically upon another plant altogether bacterial strain and the meta-bolites thereof of Yi razor clam are not fully used, the range of product dullness has limited the development of Yi razor clam aquaculture to a certain extent.

Summary of the invention

The present invention is in order to solve the above-mentioned technical problem that prior art exists, and a kind of Yi razor clam simple to operate, with low cost proteic preparation method of bacterial strain bacteriostatic activity that grows nonparasitically upon another plant altogether is provided.

Technical solution of the present invention is: a kind of Yi razor clam proteic preparation method of bacterial strain bacteriostatic activity that grows nonparasitically upon another plant altogether is characterized in that carrying out as follows successively:

A. after getting clean, fresh Yi razor clam meat and grinding, 200 mL lapping liquids are evenly coated on the beef extract-peptone plate culture medium 37 ℃ of constant temperature culture 2 days;

B. picking list bacterium colony is streak culture repeatedly to pure bacterial strain, detects the bacteriostatic activity of pure bacterial strain with the filter paper method;

C. get the bacterial strain of antibacterial circle diameter, in the Sha Shi liquid nutrient medium of being carbon source pH5 of glucose, cultivate 120 h for 35 ℃ more than or equal to 10 mm;

D.4000r/min centrifugal removal thalline, in supernatant liquor, add ammonium sulfate, the saturated final concentration of ammonium sulfate is 90%, 4 ℃ of standing over night, centrifugal 30 min of 10000 r/min, to precipitate with after the dissolving of PBS damping fluid, with molecular retention amount 3500 daltonian dialysis tubing dialysis 4 days, get thing lyophilize in the dialysis tubing again.

The present invention is simple to operate, with low cost, the bacteriostatic activity albumen bacteriostatic activity that is extracted from the Yi razor clam is grown nonparasitically upon another plant the fermented liquid of bacterial strain altogether is strong, have and remove the superoxide anion activity preferably, and extra large shrimp nauplius larva had certain toxicity, can be applicable to the aspects such as additive of antitumor medicine, biological pesticide and food fresh keeping, sanitas and makeup, have industrial value.

Embodiment:

The embodiment of the invention is carried out as follows:

A. get fresh Yi razor clam,, Yi razor clam meat fully ground make Yi razor clam meat lapping liquid, 200 mL lapping liquids are evenly coated on the beef extract-peptone plate culture medium, 37 ℃ of constant temperature culture 2 days with after the sterile water wash 5 times.

B. picking list bacterium colony is streak culture repeatedly to pure strain, detects the bacteriostatic activity of pure bacterial strain with the filter paper method:

With intestinal bacteria Escherichia coli(EC), Bacillus proteus Proteus vulgaris (PV), streptococcus aureus Staphylococcus aureus (SA), gas bacillus Enterobacter aerogenes (EA) and subtilis Bacillus subtilis (BS) be inoculated in respectively in the beef extract-peptone liquid nutrient medium, shaking culture 24 h do indicator, and above-mentioned bacterial classification all can be available from the female willing biochemical industry in Shanghai company limited.

Get indicator liquid 100 μ L in the beef extract-peptone solid medium with liquid-transfering gun respectively, being coated with rod with aseptic triangle smears evenly, then beef extract-peptone solid medium flat board is divided into six districts, each district puts into a filter paper respectively, again respectively with liquid-transfering gun pipette the Yi razor clam altogether the tunning sample 10 μ L of attached pure bacterial strain sabouraud culture medium splash into filter paper central authorities, place 37 ℃ of incubators to cultivate.3 repetitions are done in each processing, and positive control is 75% alcohol, and negative control is a sterilized water.Observe the growing state of indicator after three days, measure the diameter of inhibition zone.The result shows: all strain fermentation products substantially all possess bacteriostatic action to indicator, especially to subtilis, streptococcus aureus and Bacillus proteus, colibacillary fungistatic effect highly significant.

C. get the bacterial strain of antibacterial circle diameter, in the Sha Shi liquid nutrient medium of being carbon source pH5 of glucose, cultivate 120 h for 35 ℃ more than or equal to 10 mm;

D.4000r/min centrifugal removal thalline, in supernatant liquor, add ammonium sulfate, the saturated final concentration of ammonium sulfate is 90%, 4 ℃ of standing over night, centrifugal 30 min of 10000 r/min, to precipitate with after the dissolving of PBS damping fluid, with molecular retention amount 3500 daltonian dialysis tubing dialysis 4 days, get thing lyophilize in the dialysis tubing again.

The obtained freeze-drying product are carried out the polyacrylate hydrogel electrophoresis, and the result shows: thing is a bacteriostatic activity protein in the cryodesiccated dialysis tubing.

Experiment and result:

1. suppress the active detection of phytopathogen: under aseptic condition, pipette Yi razor clam pure bacterial strain fermentation liquor 1.5 mL that grow nonparasitically upon another plant altogether with liquid-transfering gun, join in the sabouraud culture medium that is cooled to about 40 ℃ and shake up, making its final volume is 15 ml, be that the punch tool of 0.8cm is got the eugonic phytopathogen bacterium of colony edge cake and is inoculated on the sabouraud culture medium flat board that contains fermented liquid with the aperture respectively after solidifying, 1 bacterium cake of each culture dish inoculation, 3 repetitions of every kind of plant pathogenic bacteria.Join substratum in contrast with blank sabouraud culture medium, place 27 ℃ interior cultivation of constant incubator.After cultivating 7 d, take pictures, measure diameter, method is to measure 2 times according to right-angled intersection, represents the size of colony diameter with its mean value.Calculation formula according to inhibiting rate is as follows: pure increment=bacterium colony mean diameter-bacterium cake diameter, inhibiting rate=(contrast pure increment-handle pure increment)/pure increment of contrast is calculated.The result shows among the present invention that the fermented liquid of implementing from middle obtained strains is withered for cotton, the ancient sickle-like bacteria of standing grain, fusarium moniliforme, mucormycosis, capsicum green grass or young crops are withered, tomato gray mould, corn are early used as a servant inhibiting rate near 100%; Reach 87% for pink, the melon withered inhibiting rate of apple; Reach 31% to avenging the withered inhibiting rate of mould leaf, above-mentioned bacterial classification all can be available from the female willing biochemical industry in Shanghai company limited.This shows that the Yi razor clam pure bacterial strain of growing nonparasitically upon another plant altogether has very strong inhibition phytopathogen activity, its activated protein can be used as the preparation that pulvis or liquid preparation are used for biological pesticide.

2. the mensuration of temperature sensitivity: the Yi razor clam pure bacterial strain fermentation liquor of growing nonparasitically upon another plant is altogether carried out following processing respectively: 20 ℃, 40 ℃, 60 ℃, 80 ℃, boiling water (100 ℃) water-bath 1h, 121 ℃ of autoclaving 30min.Detect the bacteriostatic activity of pure bacterial strain after handling according to the method described above with the filter paper method, the result shows that the Yi razor clam pure bacterial strain fermentation liquor of growing nonparasitically upon another plant altogether has patience preferably to temperature, from 20 ℃ to 121 ℃ bacteriostatic activity is arranged all, and activity does not weaken.Illustrate that prepared activated protein has heat impedance, enlarged its range of application, can be applicable to must be in the product that high heat condition is handled.

3. proteic pH sensitivity testing: the prepared albumen of the embodiment of the invention is dissolved in respectively (the pH value is 3,4,5,6,7,8,9,10,11) is made into the fermented liquid that concentration is 3 mg/ml in the distilled water that has mixed up the pH value, its fermented liquid is carried out bacteriostatic experiment (method is the same), measure bacteriostatic diameter at last.The result shows: during different pH value, albumen is little to withered grass subtilis and Bacillus proteus influence, has enlarged its range of application.

4. extra large shrimp toxicity test: get the prepared protein sample of the embodiment of the invention (8 mg/mL) 0.05mL and be added in 3 band scale test tube, in every test tube, add the extra large shrimp nauplius larva of about 4 mL seawater and 10 health again, add to 5 Ml with seawater at last; Other gets 3 test tubes, also adds the extra large shrimp nauplius larva of 10 health, adds to 5 mL with seawater again, as blank.Put under the fluorescent lamp the extra large shrimp of counting survival behind 24 h for 24 ℃.Each tests triplicate, and experimental result shows that the shrimp survival rate is 26.67%, and none death of blank, so this albumen has certain extra large shrimp toxicity activity.Sea shrimp larva discovers that once by many active testing system applies extra large shrimp toxicity method and 9KB (human nasopharyngeal carcinoma) cytotoxicity experiment present good dependency (P=01036, kappa=0156).Cytotoxic ED50 generally is 1/10 of extra large shrimp poison LD50, and available extra large shrimp toxicity method replaces expensive and numerous and diverse vivo and vitro anti-tumor experiment, and guidance is to the separation of tool cell toxicant and 3PS (mouse leukemia in the P388, body) activity extract.Sea shrimp toxicity method tool is (24h) fast, cheap and simple advantages such as (as not needing aseptic technique), and this experimental result shows that the active protein that the present invention prepares has anti-tumor activity, can be applicable to preparing anti-tumor medicine.

Claims (1)

1. a Yi razor clam proteic preparation method of bacterial strain bacteriostatic activity that grows nonparasitically upon another plant altogether is characterized in that carrying out as follows successively:

A. after getting clean, fresh Yi razor clam meat and grinding, 200 mL lapping liquids are evenly coated on the beef extract-peptone plate culture medium 37 ℃ of constant temperature culture 2 days;

B. picking list bacterium colony is streak culture repeatedly to pure bacterial strain, detects the bacteriostatic activity of pure bacterial strain with the filter paper method;

C. get the bacterial strain of antibacterial circle diameter, in the Sha Shi liquid nutrient medium of being carbon source pH5 of glucose, cultivate 120 h for 35 ℃ more than or equal to 10 mm;

D.4000r/min centrifugal removal thalline, in supernatant liquor, add ammonium sulfate, the saturated final concentration of ammonium sulfate is 90%, 4 ℃ of standing over night, centrifugal 30 min of 10000 r/min, to precipitate with after the dissolving of PBS damping fluid, with molecular retention amount 3500 daltonian dialysis tubing dialysis 4 days, get thing lyophilize in the dialysis tubing again.