158 results about "Collagenase" patented technology

The invention discloses starch-collagen composite hydrogel as well as a preparation method and application thereof. Collagen is catalyzed by microbial transglutaminase to form a covalent cross-linked network, and meanwhile, hydroxypropyl starch is introduced to construct a physical interpenetrating network through molecular chain penetration and hydrogen-bond interaction; and chondroitin sulfate is further integrated to strengthen the network structure and biological activity through electrostatic interaction. The method is mild in condition and does not need a toxic chemical cross-linking agent, the obtained composite hydrogel has an interpenetrating double-network structure and has excellent mechanical properties, enzymatic degradation resistance and cell affinity, the compression modulus of the composite hydrogel is remarkably improved, the composite hydrogel can keep structural stability for a long time in a collagenase environment, cell adhesion and proliferation can be effectively promoted, and the composite hydrogel has a good application prospect. The hydrogel can be widely applied to tissue engineering scaffolds, wound dressings, drug sustained-release carriers and cartilage repair materials.

The invention relates to the technical field of medical cosmetology, in particular to a porous hollow glass bead capable of slowly releasing collagen and application of the porous hollow glass bead. The porous hollow glass bead capable of slowly releasing the collagen comprises a porous hollow glass bead body, the outer wall of the porous hollow glass bead body is provided with a pore channel structure penetrating through the exterior and an inner cavity of the hollow glass bead body, and the pore channel and the inner cavity of the porous hollow glass bead body are filled with collagen gel. The compressive strength of the hollow glass beads is utilized to improve the mechanical supporting effect, the collagen gel is subjected to catalytic decomposition under the action of collagenase in a body so as to achieve the slow-release effect, the outer walls of the hollow glass beads slow down the reaction time through physical isolation, and then the slow-release time is prolonged; meanwhile, ions such as Si and Ca released by the hollow glass beads can promote cells to secrete collagen, the biological activity is improved, and the three beneficial effects of physical support, collagen slow release and biological safety are achieved.

The invention relates to a method for extracting high-purity extracellular vesicles from liver tissues of pelteobagrus vachelli, which comprises the following steps of: firstly, mixing collagenase II and collagenase IV, performing enzymolysis on the tissues, adding a CaCl2 solution, incubating, and digesting the liver tissues into a tissue suspension; then removing cells and fragments through low-speed centrifugation, and filtering to remove impurities by using needle filters of 0.8 [mu] m and 0.22 [mu] m; and finally, further purifying by combining ultracentrifugation with a high-precision iodixanol density gradient centrifugation method, and merging density layers with high purity to obtain high-purity EVs. The method has the characteristics of high extraction purity, high efficiency and simplicity and convenience in operation, and has a wide application prospect.

The invention discloses an eggshell membrane peptide for enhancing bone mineral density as well as a preparation method and application thereof, belongs to the technical field of egg by-product processing, and particularly relates to a preparation method of the eggshell membrane peptide for enhancing bone mineral density, which comprises the following steps: carrying out vacuum freeze drying and crushing on an eggshell membrane to obtain eggshell membrane powder; mixing the eggshell membrane powder with deionized water to obtain an eggshell membrane solution; adding keratinase, performing enzymolysis, and taking supernate; adding collagenase, carrying out enzymolysis, and centrifuging to take supernate, so as to obtain primary enzymatic hydrolysate; adding alkaline phosphatase, performing enzymolysis and centrifugation, and taking supernate to obtain enzymatic hydrolysate; and performing ultrafiltration and drying to obtain the eggshell membrane peptide. According to the eggshell membrane peptide for enhancing the bone mineral density, keratin cross-linking is destroyed through enzymolysis of keratinase, active peptide capable of stimulating osteoblast proliferation is released through enzymolysis of collagenase, phosphopeptide calcium binding sites are further activated through enzymolysis of alkaline phosphatase, calcium salt deposition is promoted, and the eggshell membrane peptide for enhancing the bone mineral density is provided.

The present invention relates to a cartilage tissue single cell extraction kit and an extraction method thereof, the kit comprises: (1) a first enzyme for enzymatic hydrolysis, the first enzyme for enzymatic hydrolysis comprising one or more of trypsin, chondroitinase ABC and fucosidase; (2) a second enzyme for enzymolysis, wherein the second enzyme for enzymolysis comprises collagenase II, neutral protease II, hyaluronidase and Dnase I; and (3) a basic solution, wherein the basic solution comprises a basic culture medium and / or a buffer solution. The invention also provides a method for extracting single cells from cartilage tissues by using the kit. The cartilage tissue single-cell suspension prepared by the method has the advantages of high cell yield, high activity and the like, and has a wide application prospect.

The invention provides a preparation method of a single-cell sequencing suspension fixedly preserved in situ. The invention provides digestive juice for preparing a single-cell suspension. The digestive juice is a solution composed of collagenase II and neutral protease. The invention also provides a kit containing the digestive juice. The invention provides application of the digestive juice to preparation of a kit and application of the kit to in-situ fixed preservation of cells and preparation of a single-cell suspension. The invention also provides a preparation method of the in-situ immobilized preserved cell and the single-cell suspension, and the single-cell suspension prepared by the preparation method.

The present invention provides a composition comprising collagenase as an enzyme; calcium, histidine, and glycine as coenzymes, and a method for stabilizing enzymes, the method comprising the preparation of the composition by adding histidine and glycine to collagenase. According to the present invention, the enzyme composition and method for stabilizing enzymes can minimize or inhibit the agglutination of enzymes, maintain the concentration of freeze-dried enzymes even during long-term storage, and provide a formulation that has excellent rehydration time and hygroscopicity when restored to a liquid state, as well as superior enzyme activity.

The invention relates to a collagenase-loaded LDH (layered double hydroxide) / EDTA (ethylene diamine tetraacetic acid) layered double hydroxide nano material as well as a preparation method and application thereof. The collagenase-loaded LDH / EDTA layered double hydroxide nano material is prepared from a zinc-aluminum layered double hydroxide LDH carrier, ethylenediamine tetraacetic acid EDTA inserted between layers of the LDH carrier through anion exchange, and collagenase loaded on the surface of the LDH carrier through an electrostatic adsorption effect. The invention further discloses a preparation method of the collagenase-loaded LDH / EDTA layered double hydroxide nano material for preparing the collagenase-loaded LDH / EDTA layered double hydroxide nano material for preparing the collagenase-loaded LDH / EDTA layered double hydroxide nano material.

The present application relates to a kind of anti-aging composition and its application of brightening and shrinking pores.The composition includes the following components: halocyprid extract, purple magnolia extract, desert rose leaf cell extract, rain lily labiatae flower extract and white lily extract, and the mass ratio of the halocyprid extract, purple magnolia extract, desert rose leaf cell extract, rain lily labiatae flower extract and white lily extract is (0.2-2):(0.001-0.1):(0.001-0.1):(0.001-0.1):(0.001-0.1).The composition of the present application can effectively improve the clearance rate of collagenase, maintain the elasticity and tightness of skin, have good anti-aging and pore-shrinking effect, and can also effectively remove hydroxyl radicals, have good whitening and brightening effect.

The invention relates to a low-temperature gradient enzymolysis technology for collagen peptide in the body wall of a sea cucumber, and discloses mild decomposition and efficient extraction of collagen in the body wall of the sea cucumber by virtue of a multi-section temperature zone reaction, enzyme seed compounding control and on-line regulation system. And the collagen peptide with concentrated molecular weight, strong biological activity and high terminal product quality is prepared by the low-temperature gradient enzymolysis technology. The method is characterized by comprising the following steps: cutting the body wall of the sea cucumber to obtain a particle size suitable for enzymolysis, adding the cut body wall of the sea cucumber into a neutral buffer solution, stirring at normal temperature, and adjusting the pH value to 6.5-7.5, so that the tissue of the body wall of the sea cucumber is fully expanded and uniformly dispersed to be more suitable for the subsequent enzymolysis process; the method comprises the following steps: carrying out staged reaction on the body wall of the sea cucumber and an enzyme at different temperatures, carrying out fine separation and terminal treatment on an enzymatic hydrolysate, and immediately cooling a system to 4 DEG C after the reaction of the enzymatic hydrolysate is finished.

The application provides a preparation method of non-denatured type II collagen for increasing bone density and improving bone elasticity, and belongs to the technical field of biotechnology. The application adopts EDTA-citric acid buffer containing beta-mercaptoethanol to decalcify cartilage, adopts supercritical CO2 extraction technology to defat, adopts alkaline protease and collagenase II to perform matrix directional enzymolysis, and performs gradient digestion; then three-phase centrifugal separation is adopted to separate non-denatured type II collagen, and biomimetic H column chromatography purification is adopted, so that non-denatured type II collagen with high extraction rate, high purity and high structural integrity can be obtained.

The invention discloses an in-vitro biochemical test method for evaluating the anti-wrinkle effect of cosmetics and raw materials. The in-vitro biochemical test method comprises the following steps: step 1, preparing a working solution; 2, preparing a sample solution; step 3, adding an enzyme working solution; step 4, loading samples in groups; 5, adding a substrate and reacting; step 6, developing treatment; step 7, determining absorbance; 8, calculating, analyzing and processing data; and step 9, result judgment: analyzing and evaluating the anti-wrinkle effect of the sample through a statistical means. According to the method, based on a collagenase inhibition test, when peptide bonds between GLY and GLY in Z-GLY-PRO-GLY-GLY-PRO-ALA-OH are specifically hydrolyzed by collagenase, GLY-PRO-ALA is released, and the activity of the collagenase is indirectly evaluated by measuring the change of a light absorption value at 565 nm after the GLY-PRO-ALA reacts with a color developing agent, so that whether a sample to be detected has an anti-wrinkle effect or not is judged. The method is economical and fast, the test process is controllable, the test system is standardized, and result deviation caused by individual difference can be avoided.

The invention relates to a preparation method of a placenta trophoblast-derived transporter and application of the placenta trophoblast-derived transporter in a medicine for treating spontaneous abortion, which comprises the following steps of: taking out a placenta of a suckling mouse, cutting the placenta into fine tissue blocks, resuspending by using digestive juice, digesting in a water bath kettle at 37 DEG C for 20 minutes, and then stopping digestion by using 1640 culture containing high-temperature heat-inactivated fetal calf serum; diluting and neutralizing collagenase digestive juice according to 5 times of volume; and centrifuging the collagenase digestive juice for multiple times to obtain a precipitate, namely the exosome. And carrying out density gradient centrifugation on the coarse migration body by using Optiprep as a density medium, washing, precipitating and centrifuging by using PBS on an ultra-high-speed centrifuge by using a horizontal rotor to obtain the placental trophoblast source migration body. The invention finds that the generation of the placental trophoblast cell source migration body is related to the physiological process of pregnancy for the first time, and the placental trophoblast cell source migration body can enter the circulatory system and reach the placenta through in-vitro supplementation.

The invention relates to the technical field of neutrophile granulocyte separation, in particular to a mouse neutrophile granulocyte separation method which comprises the following steps: (1) anesthetizing and disinfecting a mouse; (2) tissue sampling; (3) tissue digestion; (4) preparing a cell suspension; (5) purifying cells; (6) cell culture; according to the method disclosed by the invention, isoflurane is adopted in the anesthesia disinfection step, so that central nervous activity of mice can be rapidly inhibited, tissue damage caused by stress reaction is avoided, and chemical stimulation can be reduced while body surface microorganisms are cleared through combination of ethanol disinfection and sterile normal saline flushing, and aortic tissues with intact activity are provided for subsequent material taking. In the compound enzyme digestive juice, collagenase I can specifically degrade aortic wall collagenous fibers, trypsin dissociates adhesion connection between cells, neutral protease assists in decomposing elastic fibers, and DNA enzyme I is supplemented to inhibit cell aggregation, so that the contradiction between incomplete digestion and large cell damage of the traditional enzyme juice is effectively solved.

The present application relates to cell preparation process in the field of bioengineering, disclose a kind of high-efficiency preparation mouse primary hepatocyte method, comprising: perfusion needle is placed in reverse direction to liver direction through inferior vena cava, utilize the blood vessel clamp with elastic micro-tooth damping surface to lock the perfusion needle and inferior vena cava wall;The overflow damping of hepatic portal vein outflow end is dynamically regulated by blood vessel clamp, to construct controlled positive pressure permeation environment in liver vascular bed;Collagenase solution is radially permeated to liver parenchyma using the environment, the present application makes enzyme solution radial deflection and cross blood sinus barrier into di's cavity by constructing micro-pressure environment, eliminate the digestion dead zone originally due to flow path preference, ensure that enzyme molecule and extracellular matrix are uniformly contacted in global dimension, realize the collaborative promotion of tissue cutting efficiency and total amount of cell release, ensure that the output cell population has highly consistent physiological state.

The invention provides cross-linked recombinant collagen gel. The cross-linked recombinant collagen gel consists of the following components in percentage by mass: 0.4-1.6% of recombinant III-type humanized collagen; 0.2%-2% of a D-core; 0.1% to 0.5% of carbomer; 0.1% to 0.6% of triethanolamine; 1.0%-10% of glycerol; the collagen gel disclosed by the invention has the advantages of low immunogenicity, high safety, no toxicity, no stimulation, good biocompatibility, structural stability, collagen enzymolysis resistance and the like, and meanwhile, the invention also provides a preparation method and application of the cross-linked recombinant collagen gel.

A method of isolating mature adipocytes from a tissue is disclosed. The disclosed method comprises incubating the tissue in a solution comprising an amphiphilic agent and an enzyme. The amphiphilic agent may be Pluronic F-68 and the enzyme may be collagenase. Methods of culturing mature adipocytes comprising culturing mature adipocytes isolated by one of the methods disclosed herein and methods of differentiating fat cells comprising culturing mature adipocytes according to any one of the methods disclosed herein are also disclosed. A kit comprising an amphiphilic agent and an enzyme is disclosed.

The invention relates to an anti-aging, brightening and pore-shrinking composition and application thereof. The composition comprises the following components: an artemia extract, a magnolia liliiflora extract, a desert rose leaf cell extract, a lilium lanceolarium extract and a lilium fortunei extract. The mass ratio of the artemia extract to the magnolia liliiflora extract to the rosa deserticola leaf cell extract to the lilachium lanceolatum extract to the lilium fortunei extract is (0.2 to 2) to (0.001 to 0.1) to (0.001 to 0.1) to (0.001 to 0.1) to (0.001 to 0.1) to (0.001 to 0.1). The composition provided by the invention can effectively improve the collagenase clearance rate, maintain the elasticity and compactness of the skin, achieve good anti-aging and pore-shrinking effects, also can effectively remove hydroxyl radicals, and has good whitening and brightening effects.

Compositions and methods are provided of a therapeutic nanoparticle composed of collagenase IV linked via a linker (e.g. cathepsin B cleavable linker) to ferumoxytol (iron oxide). The collagenase IV is key in the composition intended for the breakdown of the tumor wall as the collagenase. Such compositions and methods are aimed at solving at the same time two of the main challenges of current approaches in the treatment of glioblastoma multiforme (GBM) which are low specificity and poor uptake.

The invention provides application of inflammatory mediator combined collagenase in construction of an animal corneal thinning model, and belongs to the technical field of construction of animal models. According to the method, an inflammatory mediator and collagenase are combined, a novel mouse keratoconus model with the thinned cornea is constructed and obtained, and the deficiency of a keratoconus animal model is made up. Researches show that after the inflammatory mediator is combined with collagenase to construct a mouse cornea thinning model, the cornea thickness of a mouse can be obviously thinned, and the occurrence and development of keratoconus are promoted.

The invention belongs to the technical field of nano preparations, and particularly relates to a preparation method and application of extracellular vesicles of tissue interstitial fluid. The extracellular vesicles of the tissue interstitial fluid are prepared by combining collagenase II and amylase, the prepared extracellular vesicles of the tissue interstitial fluid are high in yield and good in quality, and the extracellular vesicles can be used for tissue repair and regeneration, tissue microenvironment research, liquid biopsy or preparation of engineered extracellular vesicles for preventing or treating inflammatory diseases.

The invention belongs to the technical field of collagen peptide preparation, and particularly relates to a proline aminopeptidase mutant and application thereof in collagen peptide preparation. The amino acid sequence of the proline aminopeptidase mutant is as shown in SEQ ID NO. 2. The proline aminopeptidase mutant provided by the invention has the advantages of high enzyme activity, wide temperature and pH adaptation range, capability of adapting to a long-time high-temperature environment of collagen enzymolysis and the like, and is suitable for deep hydrolysis of collagen. Through cooperation with neutral protease, papain and collagenase, collagen raw materials from fish skin can be effectively degraded, the yield of low-molecular-weight peptide is increased, and collagen peptide with the molecular weight smaller than 1000 Da and accounting for 90% or above, collagen tripeptide accounting for 39% or above, collagen dipeptide accounting for 2% or above and the content of characteristic dipeptide Hyp-Gly being remarkably increased is generated. The method has important application value in the field of oriented preparation of collagen dipeptide.

The invention belongs to the technical field of biology, and particularly relates to a nucleus pulposus cell separation and culture method. According to the method, a step-by-step enzyme digestion method is adopted, NB6 collagenase is firstly used for primary digestion, and then compound enzyme liquid composed of trypsin and hyaluronidase is adopted for subsequent digestion; and culturing in combination with a culture vessel coated with a specific concentration of extracellular matrix component and a special culture medium. According to the method, on the premise of not depending on an immortalization technology, the primary nucleus pulposus cells with good activity can be efficiently obtained, stable amplification and long-term passage of the primary nucleus pulposus cells in vitro are realized, and specific marker expression and biological functions of the nucleus pulposus cells can be effectively maintained. The invention provides a stable cell source and technical platform for the development of cell therapy products and the research of spinal degenerative diseases.

The invention discloses brown cattle bone marrow collagen polypeptide with an immunoregulation effect as well as a preparation method and application of the brown cattle bone marrow collagen polypeptide, and belongs to the technical field of animal-derived food processing. The preparation method comprises the following steps: pretreating brown cattle bone marrow; the method comprises the following steps: dissolving pretreated brown cattle bone marrow in water, firstly adding alkaline protease to carry out enzymolysis, then simultaneously adding collagenase U and basophilic protease into a system, carrying out coupled enzymolysis under the protection of nitrogen, then cooling the system to 0-4 DEG C, regulating the pH value to 3-4, then carrying out autolysis for 10-30 minutes, and finally carrying out enzyme deactivation; centrifuging an enzyme deactivation product, taking supernatant, adjusting the pH value to be neutral, concentrating, dialyzing and freeze-drying to obtain crude collagen peptide; and carrying out primary purification and secondary refining on the crude collagen peptide to obtain the collagen peptide. The preparation process is simple, the molecular weight of the prepared polypeptide is 366-1514 Da, and the polypeptide has immunoregulation and antioxidant activity, can be used for preparing food, health care products or medicines for improving immunity and resisting oxidation, and realizes high-value utilization of livestock and poultry byproducts.

The invention provides a preparation process of collagen peptide, and relates to the technical field of collagen peptide. The preparation process of the collagen peptide comprises the following steps: performing primary enzymolysis and secondary enzymolysis on degreased and impure protein-removed pigskin pieces, performing enzymolysis by adopting a compound enzyme in the primary enzymolysis, and performing enzymolysis by adopting collagenase in the secondary enzymolysis; the compound enzyme comprises trypsin, papain and alkaline protease; after secondary enzymolysis, filtering to obtain a first filtrate, adsorbing the first filtrate by using macroporous resin, and then filtering to obtain a second filtrate; performing deodorization treatment on the second filtrate by using a food additive, and then filtering to obtain a third filtrate; the food additives comprise beta-cyclodextrin and fructo-oligosaccharide. The preparation process not only can effectively remove bitter taste and fishy smell of the collagen peptide, but also has higher yield.

The invention discloses a method for extracting and separating cord blood and placenta stem cells of sika deer, and belongs to the technical field of biomedicine. The problems that an existing stem cell extraction and separation method is low in efficiency, poor in cell activity, low in purity and large in pollution risk are solved. The method comprises the following steps: collecting cord blood and placenta after delivery of sika deer; diluting the cord blood, performing density gradient centrifugation, collecting the albuginea layer enriched with the stem cells, adding an erythrocyte lysate to remove residual erythrocytes, and centrifuging and collecting the stem cells; the method comprises the following steps: cutting placenta tissues into pieces, putting the placenta tissues into trypsin-EDTA digestive juice containing collagenase, carrying out ultrasonic-assisted digestion, and centrifuging to obtain stem cells enriched at the bottom of a centrifugal tube; and finally, resuspending the collected stem cells in a culture medium containing fetal calf serum and growth factors, adjusting the cell concentration, and inoculating into a cell culture bottle for culture. According to the method, the extraction efficiency and quality of the sika deer cord blood and placenta stem cells can be remarkably improved, and the activity and safety of the cells are ensured.

The present invention relates to a multifunctional hemostatic sponge, its preparation method, and application. The sponge comprises a matrix formed by cross-linking methacryloylated collagen and epigallocatechin gallate-modified nanohydroxyapatite, with the mass ratio of methacryloylated collagen to epigallocatechin gallate-modified nanohydroxyapatite being 2 to 30:1. By combining ColMA with HAP@EGCG and optimizing the ratio, the resulting hemostatic sponge exhibits excellent resistance to collagenase degradation, as well as hemostatic, anti-inflammatory, and antibacterial effects. It can promote tissue regeneration and repair, provide greater patient comfort, and enhance therapeutic efficacy.

The invention relates to collagenase as well as a coding gene and application thereof. The collagenase provided by the invention is a protein (a) or (b), wherein (a) is a protein composed of an amino acid sequence as shown in SEQ ID No: 1; and (b) a protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a), has collagen hydrolysis or fat dissolving activity and is derived from (a). The collagenase provided by the invention can be applied to cell separation and tissue digestion, fat dissolution or collagen dissolution, and can also be applied to preparation of medicines for treating Doltt's contracture, Petroleum disease and lumbar disc herniation and degreasing in the field of beauty.

The application provides king salmon bone collagen peptide for promoting longitudinal growth of bone, a preparation method and application, and relates to the technical field of collagen peptides. The preparation method comprises the following steps: (1) treating king salmon bone raw materials by steam explosion to obtain pretreated king salmon bone; then mixing the pretreated king salmon bone with pepsin and performing first enzymolysis under pressure to obtain a first enzymolysis mixture; mixing the first enzymolysis mixture with collagenase and ficin, performing second enzymolysis under pressure, collecting an enzymolysis liquid, and performing ultrafiltration on the enzymolysis liquid to obtain collagen peptide. In the application, pepsin is used for first enzymolysis under pressure, then collagenase and ficin are used for compounding, the synergistic effect of the three kinds of proteases makes the collagen be fully enzymolyzed, the diversity of the cleavage site is improved, the molecular weight of the polypeptide is reduced, the activity of the polypeptide is improved, the longitudinal growth of the bone is promoted, and the bone density is improved.

The invention discloses a preparation method and application of a prostate cancer tissue single-cell suspension. The preparation method of the prostate cancer tissue single-cell suspension comprises the following steps: S1, tissue pretreatment: putting a prostate cancer tissue sample into a low-temperature pretreatment buffer solution, and cutting the prostate cancer tissue sample into tissue fragments; s2, circular enzymolysis: mixing the tissue fragments with a composite enzymatic hydrolysate, and performing digestion for multiple rounds under an oscillation condition; after each round of digestion, collecting supernate into a stop solution containing serum, repeatedly digesting the remaining tissues for multiple times, and combining the supernate to obtain a cell crude suspension; wherein the composite enzymatic hydrolysate contains collagenase, protease, nuclease, an androgen receptor stimulant and / or a ROCK inhibitor; and S3, sequentially carrying out separation and purification on the cell crude suspension through filtration and density gradient centrifugation to obtain the prostate cancer tissue single-cell suspension.