48 results about "Pronase" patented technology

A new method of simultaneous and separate extraction of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from a biological sample, indifferently from fresh, frozen, fixed or autoptic tissue with a weight no less than 5 mg. The main steps of the method are: 1/sample lysis in a solution composed of a caotropic agent (urea or guanidine salt), a ionic detergent (SDS or SLS), a proteolytic enzyme (proteinase K, trypsin, chymotrypsin, pepsin or pronase), a reducing agent (β-mercaptoethanol or dithiothreitol); 2/deproteination; 3/precipitation of RNA from aqueous phase; 4/precipitation of DNA from organic phase. The invention includes an extraction kit for nucleic acids, also of viral origin.

The invention provides a new strain FM 603 of bacillus coagulans, and the preservation number of the strain is CGMCC NO.10221. The strain can generate bacteriocin antibacterial substances and achieves antibacterial activity for Gram-positive pathogenic bacteria such as Listeria monocytogenes, staphylococcus aureus, methicillin-resistant staphylococcus aureus, clostridium perfringens and clostridium difficile. The molecular weight of bacteriocin is 4276.45 Da, the partial amino acid sequence is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro, and the bacteriocin is stable under treatment of heat, acid, and pepsase or trypsin and can be degraded easily by pronase to be inactivated. The invention further provides an FM603 fermentation product which can be used as a microbiological feed additive. Animal test results show that the fermentation product can increase the laying rate of laying hens, reduce the feed-egg ratio and improve egg quality; the feed intake and daily gain of piglets are increased, and the feed conversion ratio is reduced; the daily gain and the content of lysozyme in serum of broiler chickens are increased, and the feed conversion ratio and the death rate are reduced.

The invention discloses a method for rapidly separating and culturing human bronchial epithelial cells, and an optimized medium. The optimized medium is prepared from a DMEM culture medium, an F-12 Nutrient mix culture medium, 0.5 mg/ml hydrocortisone, fetal bovine serum, 25mu g/ml epidermal growth factor, 5 mg/ml insulin, 11.7mu M cholera toxin, 10 mM ROCK1 inhibitor, and 10 mM BMP4 antagonist. The method for rapidly culturing the human bronchial epithelial cells by using the optimized medium not only greatly simplifies the steps of culturing epithelial cells which require 3T3 J2 cells as a feeder layer, and only needs to culture the primary human bronchial epithelial cells digested by pronase in the optimized medium, but also can be used for obtaining humane bronchial epithelial cells which are rapidly separated and can be indefinitely subcultured; the subcultured human bronchial epithelial cells are subjected to in vitro expansion and culture; when the human bronchial epithelial cells are subcultured for 20 generations or more, a sufficient amount of seed cells can be obtained so as to be used in a variety of follow-up in vitro and in vivo studies such as in vitro cell interaction model studies.

The invention discloses a building method of a porcine trachea epithelial cell line. The method comprises the following steps of: (1) primary culture: sterilely acquiring newborn piglet tracheas, clearing adhesive tissues, performing digestion and separation by adopting a pronase perfusion cold digestion method to obtain high-purity trachea epithelial cells, suspending the separated primary trachea epithelial cells in a DF12 culture medium, and culturing the cells at the temperature of 37 DEG C under the condition of 5 percent CO2; (2) transfection and screening: extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes, and guiding the eukaryotic expression plasmids pCI-neo-hTERT for coding hTERT and neo genes to the porcine trachea epithelial cell primarily cultured in the step (1) by adopting a liposome transfection method to perform transfection; and (3) screening and amplified culture. The method is easy in culture, the growth speed is high, and the operating method is simple and convenient.

The invention provides a preparation method of low-viscosity and high-purity yeast mannan. The preparation method comprises the following steps: taking the cell wall of saccharomyces cerevisiae as a raw material, adopting water extract and alcohol precipitation method to obtain rough yeast mannan, performing enzymolysis and centrifugation on the rough yeast mannan by neutral protease and pronase, and processing to obtain the product. The preparation method combines secondary digestion, centrifugation, ultrafiltration with spray drying technology, the equipment adopted can meet the requirements of the industrialized production, the yeast mannan obtained has a purity greater than 90 percent, is low in viscosity, belongs to Newtonian fluid, and cannot affect the rheological property of system in actual application.

The invention discloses a method for evaluating the antioxidant activity of fruit and vegetable food and functional health product. The method comprises the following steps: preprocessing a sample to be detected: making a raw material to be detected into water homogenate or directly taking the oral liquid type health food as the sample; co-processing and extracting the sample by multiple enzymes in sequence: processing the sample by pepsin, processing the sample by trypsin, processing the sample by pronase E, and processing the sample by plant composite hydrolase Viscozyme L; removing the reducing sugar in the extract through a cold ethyl acetate extraction technology; and finally analyzing the antioxidant substance content and activity by a conventional method. During the extraction process of substances with antioxidant activity, multiple enzymes are used to carry out enzymatic hydrolysis so as to simulate the human digestion process, at the same time, a cell level antioxidant activity analysis method is used, and thus an antioxidant activity evaluation method, which has stronger biological relevance and is more close to animal experiment level, is established.

The invention discloses a hepatic stellate cell separation and preparation method. The hepatic stellate cell is prepared through the steps of perfusion, stripping, and density gradient separation. The perfusion is carried out successively with a perfusion solution I, a perfusion solution II and a perfusion solution III, wherein the perfusion solution I comprises a buffer solution I; the perfusion solution II is prepared by adding 0.22-0.38 mg of pronase E to 1 ml of a buffer solution II; and the perfusion solution II is prepared by adding 0.52-0.68 mg of collagenase D to 1 ml of the buffer solution II. The invention belongs to the technical field of cell separation, wherein cell surface substrates are completely enzymolyzed successively by means of single enzyme and then compound enzymes matched with the buffer solution II, so that yield of the hepatic stellate cells are increased.

The invention discloses a preparing method of zebrafish embryo single-cell suspension. The preparing method sequentially comprises the following steps of 1, taking a zebrafish embryo, and adding an aqueous solution of Pronase E; 2, conducting incubation at 37 DEG C for 4-5 min; 3, utilizing a 2-mL disposable dropper for gently blow-sucking the embryo so that the chorion can be peeled off, moving the embryo into a 1.5-mL non-enzyme centrifugal tube, and quickly sucking away Pronase E; 4, adding an E3 culture medium for washing, and then removing the culture medium, wherein the step is repeated3-5 times; 5, adding dispase which is pre-cooled on ice; 6, conducting incubation at 37 DEG C for 2-5 min, wherein intermittent vibration is conducted in the incubation period; 7, adding FBS for terminating digestion; 8, making suspension obtained in step 7 pass through a 70-micrometer screen, and then making a filtrate pass through a 40-micrometer screen; 9, conducting 200-500*g centrifuging on the screened suspension for 3-5 min; 10, taking HBSS re-suspension cells which are pre-cooled on ice and contain 1% BSA; 11, conducting 200-500*g centrifuging on the suspension obtained in step 10 for3-5 min; 12, repeating steps 10 and 11 and conducting washing once; 13, taking IESC re-suspension cells pre-cooled on ice to obtain the zebrafish embryo single-cell suspension.

The invention provides a cattle trophoderm stem cell system establishment method which comprises the following steps: inoculating hemiblastula of which zona pellucida is removed through pronase treatment into a feed layer in mouse embryonic fibroblast subjected to mitomycin treatment and Wnt-3A mouse subcutaneous connective tissue cells, adding 2i small-molecule inhibitor culture solution to perform continuous cell culture of cells, and establishing a cattle trophoderm stem cell system. The method can be used for obtaining and culturing cattle trophoderm stem cells under in-vitro conditions for a long time. The cattle trophoderm stem cell system establishment method can be applied to large animal transgenosis and somatic cell cloning and can be further used for cattle embryo implantation and placenta differentiation.

The invention relates to the field of relic protection, and discloses a method for simulating enzyme ageing of printing and drawing type silk relics in tombs. The method comprises steps as follows: (1) antique printing and drawing silk fabric is treated with a solution A, washed with deionized water, subjected to ultraviolet sterilization treatment for 30-50 min after being air-dried and sheared into small pieces B; (2) 800-1,000 parts by mass of a buffer solution are prepared, 1/10000-2/10000 parts by mass of pronase from streptomyces gris are added, then, an enzyme promoter is added, and a solution C is prepared; (3) B is completely soaked in the solution C and continuously aged in a sterile incubator at 38+/-1 DEG C, and an enzyme ageing sample simulating the printing and drawing type silk relics in tombs can be obtained after 40-60 days. Through integration of multiple factors of enzyme for ageing of the relics, the enzyme ageing mode of the printing and drawing type silk relics in tombs can be effectively simulated, and a very important role is played on research of the enzyme ageing mechanism of the printing and drawing type silk relics and protection of relics.

The invention discloses a flame-retardant wool fabric finishing method. The method includes the steps of adding a monopotassium phosphate aqueous solution to preprocess a wool fabric, dispersing globular proteins slightly cross-linked between fiber cells of the wool fabric, and conducting later-period enzyme processing; conducting enzymolysis preprocessing on the wool fabric through mixed protease obtained by mixing ficus proteinase, pronase and bromelain, making mixed protease adsorbed to surfaces of wool fibers and penetrate through the whole fibers, dispersing mixed protease along the fibers, and conducting hydrolysis, so hydrolysis is continuously conducted in sebum layers of wool fibers, and it is ensured that inner layers of wool fibers are prevented from damage; modifying the wool fabric through alkaline protease. The content of disulfide bonds in the wool fibers is decreased, the content of sulfydryl groups is increased, and the flame retardance of the wool fabric is improved; meanwhile, the washable degree of the wool fabric can be improved, the influences on the whiteness and hand feeling of wool are small, and high economic benefits are achieved.

The invention provides a microbiological feed additive. The feed additive contains bacillus coagulans FM603 or a fermentative substance of the bacillus coagulans FM603; the fermentative substance of the bacillus coagulans FM603 contains bacteriocin and has antibacterial activity on gram-positive pathogenic bacteria such as Listeria monocytogenes, staphylococcus aureus, methicillin resistant staphylococcus aureus, clostridium perfringens, clostridium firmus and the like. The molecular weight of the bacteriocin is 4276.45 Da, a partial amino acid sequence is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro, and the bacteriocin is more stable under the processing of heat, acid, pepsin or trypsin, is likely to be degraded by pronase and loses activity. With the adoption of the feed additive, the egg laying rate of laying hens can be increased, the feed-egg ratio is reduced, and the egg quality is improved; the feed intake and the daily gain of piglets are increased, and the feed-meat ratio is decreased; the daily gain and the serum muramidase content of broilers are increased, and the feed-meat ratio and the death rate are decreased.

The invention provides a wheat germ protein hydrolysate and a preparation method and application thereof, and relates to the technical field of wheat germ protein derivatives. The preparation method of the wheat germ protein hydrolysate adopts wheat germ powder as a raw material to carry out alkali extraction and acid precipitation so as to obtain wheat germ protein, and then the obtained wheat germ protein is enzymatically decomposed by pronase or protease N so as to obtain the wheat germ protein hydrolysate. Tests show that the wheat germ protein hydrolysate has the remarkable activity of resisting adhesion of helicobacter pylori to gastric mucosal cells, and can be used for preventing helicobacter pylori infection. Compared with existing medicine, the medicine has the advantages of being natural, safe, low in price, high in efficiency, mild, not prone to production of generate drug-resistant bacteria and the like. A new utilization way for wheat processing by-products, and the additional value of wheat processing is increased.

The invention discloses a preserved meat product preservation method. The method comprises the following steps: (1) screening a culture for fermenting a preserved meat product, and establishing preserved meat fermentation conditions; (2) determining the types and the amount of enzymes and enzymolysis conditions, adding papain, pancrelipase, calpains, pronase and other enzyme preparations into the salting process by taking the texture, flavor, color and the like as indexes, and determining the enzyme addition amount, the enzymolysis time, the enzymolysis temperature and other enzymolysis method conditions; (3) analyzing the acid value and change of peroxide value in the preserved meat production process, and selecting an antioxidant; and (4) preparing an edible film, establishing a coating technology, wherein microbes, the acid value, the peroxide value and the TBA value are used as indexes, different composite edible film materials such as chitosan, modified starch and soy isolate protein are used, and nisin or nisin are added, and performing antiseptic validity treatment on the product by adopting dipping, spraying and other different coating technologies. The method is easy to operate and obvious in effect.

The invention belongs to the technical field of organic selenium content testing, and discloses a method for determining the form of selenium in selenium-rich tea trees and application. The method is characterized by comprising the following steps: preparing a KBH4 solution, a KI solution, an HCl solution and a mixed standard working solution; pretreating a sample and preparing a sample solution; analyzing the reference conditions by using a liquid chromatography-atomic fluorescence spectrometer; performing qualitative analysis on the forms of selenium in the mixed standard working solution and the sample solution; and quantitatively determining the corresponding peak area in the chromatogram of the sample solution according to the standard curve. According to the method, the forms of two kinds of inorganic selenium and three kinds of organic selenium in a selenium-rich tea tree tissue sample can be simply and quickly determined, the stability of the selenium forms is ensured by adopting a freeze drying technology and a diammonium hydrogen phosphate-formic acid mobile phase system, and meanwhile, a Tris-HCl buffer solution and a pronase E ultrasonic extraction system are adopted, so that the sample extraction rate is improved to the greatest extent on the premise of ensuring the stability of the selenium form. The composition and content of the selenium form in a selenium-rich tea tree can be truly reflected.

The invention discloses a collagen peptide treatment production line and a process thereof. The collagen peptide treatment production line comprises a production mechanism, wherein the production mechanism comprises a supporting base supported and fixed to the ground; a production storage tank is transversely arranged at the top end of the supporting base; a feeding open groove is formed in the middle part of the top end of the production storage tank; the top end of the feeding open groove communicates with a protective cover in a sealed mode; a quantitative feeder is arranged at the top end of the protective cover; the bottom end of the quantitative feeder communicates with a guide pipe in a sealed mode; and a mixing and stirring groove is formed in the middle part of the side wall of the production storage tank. The collagen peptide treatment production line and the process provided by the invention have the beneficial effects that through the arrangement of the production storage tank, the quantitative feeder and a hydrolyzer, collagen peptide production raw materials can be conveniently put from the upper side; the hydrolysis conversion of collagen pronase is realized through the preparation of the hydrolyzer; and the collagen pronase is converted into triple-helix polypeptide, a semi-finished product which is converted together with the triple-helix polypeptide is put into the production storage tank, and stirring blades are driven to rotate through a step motor arranged at the outside.

The invention discloses a double staining method for a bovine in-vitro fertilization blastocyst. The method comprises the steps that the bovine in-vitro fertilization blastocyst which is cultured for 7-9 days is processed with 0.5% pronase for 55 seconds, washed in a PBS for 2-3 times, stained in PI (1% TritonX-100/PBS) with the concentration of 100 mug/ml for 20 seconds, washed in the PBS for 2-3 times, transferred into Hoechst33342 with the concentration of 45 mug/ml to be stained for 50 seconds, washed in the PBS for 2-3 times and then processed through preforming; the bovine in-vitro fertilization blastocyst is placed under an inverted fluorescence microscope after preforming is finished, observing and photographing are performed under ultraviolet light, cell nucleuses of an inner cell mass (ICM) are blue, and cell nucleuses of trophoderm cells are red. According to the double staining method, staining can be completed in 3 minutes, but at least 47 minutes are needed in previous reports; accordingly, the time needed by double staining of the blastocyst is greatly shortened, the efficiency is improved, and the bovine blastocyst quality can be quickly evaluated.