CN113975465A - Low-temperature degreasing method based on animal tissue and application thereof - Google Patents
Low-temperature degreasing method based on animal tissue and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The invention provides a low-temperature degreasing method based on animal tissues and application thereof, wherein the low-temperature degreasing method comprises the following steps: (1) taking isolated animal tissues, pretreating, and then treating by using an alkaline reagent; (2) treating the product obtained in the step (1) by using a low-temperature lipase solution; (3) and (3) treating the product obtained in the step (2) by using an acid reagent or a fatty acid dispersant solution, and performing post-treatment to obtain the degreased animal tissue. The low-temperature degreasing method provided by the invention has the advantages that the low-temperature lipase is matched with the acid-base reagent, so that the degreasing efficiency of animal tissues in the preparation process of the biological material is greatly improved, the three-dimensional structure of the biological material is prevented from being damaged, and the low-temperature degreasing method is low in toxicity, safe and pollution-free.
Description
Technical Field
The invention belongs to the technical field of degreasing, and relates to a low-temperature degreasing method based on animal tissues and application thereof.
Background
Lipase, namely triacylglycerol acyl hydrolase, is an important industrial enzyme, can catalyze natural oil and fat to be decomposed into fatty acid, glycerol and monoglyceride or diglyceride, and can catalyze various reactions such as transesterification, esterification and alcoholysis under the conditions of different reaction systems. The lipase is widely existed in animals, plants and microorganisms, the action pH value of the microbial lipase is wider, the action temperature range is wider, the substrate is more specific than that of the animal and plant lipase, the microbial lipase is generally secreted extracellular enzyme, and the microbial lipase which is researched more at present obtains high-purity products, so the microbial lipase is more suitable for industrial production.
The low-temperature microorganisms are microorganisms having growth adaptability in a lower-temperature environment, and are widely distributed in cold regions, such as mountains, glaciers, deep sea, north and south poles, frozen soil and the like. The low-temperature microorganisms can synthesize low-temperature enzymes with high catalytic efficiency at low temperature, and the enzymes degrade biomacromolecules in the environment into small molecules, so that the requirements of self nutrition and normal life activities are ensured. The low-temperature microorganisms producing the low-temperature lipase mostly belong to the genera Aeromonas, Pseudomonas, Penicillium, Candida and the like.
Low-temperature lipase (Cold-temperature lipase) is a lipase which can catalyze the decomposition of grease at a lower temperature, the optimum temperature of the low-temperature lipase is usually about 30 ℃ lower than that of medium-high temperature lipase with the same function, the optimum temperature of the medium-high temperature lipase is usually 50-60 ℃, and the low-temperature lipase can have higher enzyme activity at 0-30 ℃. Basic properties of low-temperature lipases, such as the temperature of action, the optimum pH, the isoelectric point and the molecular weight, vary from enzyme to enzyme, for example, some low-temperature lipases have an optimum pH of 6-9, some low-temperature lipases have an optimum temperature of 30-40 ℃ and some low-temperature lipases have a temperature of 10-15 ℃.
At present, in the field of tissue engineering, the preparation of extracellular matrix materials from animal tissues is a major development. The extracellular matrix is a three-dimensional structure formed by components of a plurality of macromolecular substances such as collagen fibers, glycoprotein, elastin, growth factor and the like, provides a proper place and microenvironment for the survival and the activity of various cells, and regulates the functions of tissues and organs. The extracellular matrix has been widely used clinically as an ideal tissue repair material. In the preparation process of extracellular matrix, degreasing is one of the main technical difficulties, and because fat has certain immunogenicity, the fat in animal tissue materials needs to be removed cleanly to reduce the immunogenicity of the biological materials. The prior animal tissue degreasing methods are various, and the degreasing process is basically carried out by reagents such as a detergent, an organic solvent, an acid-base solution and the like. However, the above-mentioned defatting method is very unstable, and the use of these reagents results in, on one hand, destruction of active ingredients in extracellular matrix materials and incomplete defatting of tissue materials, and on the other hand, the residue of harmful solvents may cause cytotoxicity of biological materials, thereby affecting the repairing effect of biological membrane materials. And most of organic solvents are volatile, have special odor, are inflammable, have high toxicity, and have certain potential safety hazards in use.
CN109603195A discloses a degreasing method of animal tissue and its application, comprising the following steps: (1) pretreatment: cleaning animal tissue, sterilizing, cutting into pieces, blocks or mashing into powder; (2) and (3) dehydrating: dehydrating animal tissue with ethanol solution; (3) and (3) extraction: putting the animal tissue into a closed extraction kettle for supercritical carbon dioxide degreasing; (4) separation: and (3) the fat and fat-soluble impurities enter the separation kettle along with the carbon dioxide fluid, the fat and fat-soluble impurities are separated from the carbon dioxide, the carbon dioxide participates in the next extraction cycle, the fat and fat-soluble impurities are removed in the separation kettle, and the carbon dioxide-containing carbon dioxide-based carbon dioxide composite material is obtained through at least two extraction cycles. After the animal tissue degreasing method adopts supercritical carbon dioxide extraction treatment, the degreasing rate of the animal tissue is more than 99 percent, and fat-soluble impurities in the animal tissue can be thoroughly removed; on the other hand, after the treatment of the supercritical carbon dioxide, the potential animal virus removal rate of the animal tissue reaches more than 99 percent. However, the degreasing temperature of the invention is high, and the active ingredients in the extracellular matrix material can be damaged.
Therefore, in the field, it is desired to develop a degreasing method which can avoid the three-dimensional structure of the biomaterial from being damaged, has high degreasing efficiency, low toxicity, safety and no pollution, so as to improve the efficiency of extracellular matrix preparation and further ensure the regeneration and repair activity of the extracellular matrix.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a low-temperature degreasing method based on animal tissues and application thereof, so as to overcome the defects of large pollution, low degreasing efficiency, damage to biological material structures and the like caused by the adoption of a traditional surfactant or organic solvent degreasing method, and realize low-temperature, solvent-free and surfactant-free animal tissue cleaning and efficient degreasing. The low-temperature degreasing method provided by the invention has the advantages that the low-temperature lipase is matched with the acid-base reagent, so that the degreasing efficiency of animal tissues in the preparation process of the biological material is greatly improved, the three-dimensional structure of the biological material is prevented from being damaged, and the low-temperature degreasing method is low in toxicity, safe and pollution-free.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for low temperature degreasing based on animal tissue, said method comprising the steps of:
(1) taking isolated animal tissues, pretreating, and then treating by using an alkaline reagent;
(2) treating the product obtained in the step (1) by using a low-temperature lipase solution;
(3) and (3) treating the product obtained in the step (2) by using an acid reagent or a fatty acid dispersant solution, and performing post-treatment to obtain the degreased animal tissue.
The defatted animal tissue can be stored frozen or subjected to subsequent processing.
At present, most of lipases are medium-high temperature lipases, the activity temperature of the lipase is generally 50-60 ℃, and the enzyme activity is low at low temperature, so the medium-high temperature lipases are difficult to be applied to products with poor thermal stability. The low-temperature lipase has a good application prospect in products with poor thermal stability, compared with medium-high temperature lipase, the low-temperature lipase has the advantages of being beneficial to protecting the characteristics of products, reducing the production energy consumption, having wide pH adaptability, having higher catalytic activity on long-chain glyceride and the like, and the low-temperature lipase can be separated from microorganisms screened in a low-temperature environment and can be applied to a low-temperature degreasing technology.
In the preparation process of the animal-derived biological scaffold material, degreasing is a problem which is difficult to solve, and if a high-temperature degreasing method is used, collagen fibers and a skeleton structure of the biological scaffold material can be damaged, so that the mechanical strength of the biological scaffold material is low, and the induction effect of tissue regeneration is poor. Therefore, the method of using the low-temperature lipase and the acid-base reagent together can effectively degrease at a lower temperature and protect the collagen fiber structure of the biological material.
According to the invention, the low-temperature lipase and the acid-base reagent are matched for use to carry out the degreasing step in the preparation of the extracellular matrix material, wherein the low-temperature lipase can carry out efficient degreasing at a lower temperature, and under the low-temperature condition, the three-dimensional structural integrity of the animal tissue material is ensured, the degreasing efficiency is also improved, so that the degreasing is more thorough, and the immunogenicity of the biological material is reduced. Meanwhile, before the low-temperature lipase solution is used for treatment, an alkaline reagent is used in a matched mode, after the animal tissue material is soaked in an alkaline solution, collagen fiber pores of the animal tissue material are looser, the low-temperature lipase solution can better enter the interior of the tissue material, the degreasing effect after the low-temperature lipase solution is used for treatment, an acidic reagent or a fatty acid dispersing agent solution is used for treatment after the low-temperature lipase solution is used for treatment, and the removed fat can be eluted, so that the purposes of low temperature, high efficiency and thorough degreasing are achieved.
The core of the technology of the invention is that low-temperature lipase is used for catalyzing the decomposition of grease, some low-temperature lipase can reach higher enzyme activity under alkaline conditions, the pores of the biological material treated by the alkaline reagent are loose, and a low-temperature lipase solution is added under a certain alkaline environment, so that the low-temperature lipase solution can rapidly enter the internal structure of the biological material, hydrolyze fat, and enable fatty acid to form soluble soap in situ and enter a water phase. Further dispersing by an acid reagent or a fatty acid dispersing agent, and eluting the animal fat by water, thereby achieving the purpose of low temperature, high efficiency and thorough degreasing.
The low temperature in the low-temperature degreasing method is 4-15 ℃. The low-temperature degreasing method is characterized in that all steps of the method are carried out at a low temperature of 4-15 ℃, degreasing is carried out at the temperature, the three-dimensional structure, collagen fiber and other structures in the biological material are not damaged, the purpose of efficient degreasing can be achieved, and a good method is provided for a degreasing process in the preparation of the biological material.
Preferably, the animal tissue includes any one of an esophagus, stomach, intestine, urethra, or bladder of an animal.
Preferably, the alkaline agent comprises any one of a sodium carbonate solution, a potassium carbonate solution, a sodium hydroxide solution or a potassium hydroxide solution or a combination of at least two thereof.
Preferably, the concentration of the alkaline agent is 0.1-5 mol/L, such as 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L, 1mol/L, 1.5mol/L, 2mol/L, 2.5mol/L, 3mol/L, 3.5mol/L, 4mol/L, 4.5mol/L, or 5 mol/L.
If the concentration of the alkaline agent is less than 0.1mol/L, the treatment degree of the biomaterial is insufficient, and if the concentration of the alkaline agent is more than 5mol/L, the high-concentration alkaline solution causes serious damage to structures such as collagen fibers in the biomaterial, and the like, and the mechanical properties are reduced.
Preferably, the low-temperature Lipase in the low-temperature Lipase solution comprises any one of Lipase ZC1 Lipase, Lipase B5 Lipase, JXJ-7 Lipase, JXJ-54 Lipase, FS119 Lipase or LP28 Lipase. Wherein, Lipase ZC1 Lipase and Lipase B5 Lipase are neutral low temperature Lipase, and the optimum PH value is 7; JXJ-7 lipase, JXJ-54 lipase, FS119 lipase and LP28 lipase are alkaline low temperature lipases with an optimum pH of 8-14, e.g., 8, 9, 10, 11, 12, 13 or 14.
As the preferred technical scheme of the invention, the low-temperature lipase has wide pH adaptability, has higher catalytic activity on long-chain glyceride and can effectively remove fat.
Preferably, the concentration of the low-temperature lipase solution is 10-500U/mL, such as 10U/mL, 20U/mL, 30U/mL, 50U/mL, 80U/mL, 100U/mL, 150U/mL, 200U/mL, 250U/mL, 300U/mL, 350U/mL, 400U/mL, 450U/mL, 480U/mL or 500U/mL, and the like.
If the concentration of the low-temperature lipase solution is lower than 10U/mL, the enzyme activity is low due to too low concentration, lipolysis is not facilitated, and if the concentration of the low-temperature lipase solution is higher than 500U/mL, other components of the biological material, such as growth factors, can be affected, so that the regeneration and repair effects of the biological material are affected.
It is to be noted that U represents an enzyme activity unit, and 10U/mL represents that the enzyme activity per mL of liquid is 10U; enzyme activity units were defined in 1976 as: under specific conditions, 1min of enzyme can convert 1. mu. mol of substrate, i.e.1 IU-1. mu. mol/min. At present, most clinical laboratories at home and abroad often omit the international second word, namely IU is abbreviated as U.
Preferably, the acidic reagent includes any one of a phosphoric acid solution, an acetic acid solution, or a hydrochloric acid solution.
Preferably, the solute content of the acidic reagent is 0.1% to 5% by mass, for example, 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%.
If the mass percentage of the solute in the acidic reagent is lower than 0.1%, the decomposed fat is not thoroughly eluted, and if the mass percentage of the solute in the acidic reagent is higher than 5%, the acidic reagent with higher concentration can damage structures such as collagen fibers and elastic fibers, and the mechanical properties of the biological material are influenced.
Preferably, the fatty acid dispersant solution includes any one of a sodium tripolyphosphate solution, a potassium tripolyphosphate solution, a sodium hexametaphosphate solution, a potassium hexametaphosphate solution, a sodium phosphate solution, a potassium phosphate solution, a sodium pyrophosphate solution, a potassium pyrophosphate solution, a sodium silicate solution, a potassium silicate solution, a sodium metasilicate solution, or a potassium metasilicate solution, or a combination of at least two thereof.
Preferably, the mass percentage of solute in the fatty acid dispersant solution is 0.1% to 5%, for example, 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%.
Preferably, the pretreatment of step (1) comprises removing surface impurities and fat lumps and cleaning.
Preferably, the washing comprises washing with purified water.
Preferably, the treatment of step (1) comprises soaking.
Preferably, the temperature of the treatment in step (1) is 4 to 15 ℃, such as 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃ or 15 ℃.
Preferably, the treatment time in step (1) is 0.5-5 h, such as 0.5h, 0.8h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5 h.
Preferably, the step (1) further comprises the following steps after the treatment with the alkaline reagent: animal tissue was washed to neutrality.
Preferably, the treatment of step (2) comprises soaking.
Preferably, the pH value of the treatment in step (2) is 7-14, for example, 7, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5 or 14, and the pH value of the soaking solution is adjusted to 7-14 by using an acid-base reagent during soaking.
Preferably, the temperature of the treatment in step (2) is 4 to 15 ℃, such as 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃ or 15 ℃.
Preferably, the treatment time in the step (2) is 4-12 h, such as 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h or 12 h.
Preferably, the processing of step (2) is followed by the following steps: animal tissue was washed to neutrality.
Preferably, the treatment of step (3) comprises soaking.
Preferably, the temperature of the treatment in step (3) is 4 to 15 ℃, such as 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃ or 15 ℃.
Preferably, the treatment time in the step (3) is 1-8 h, such as 1h, 2h, 3h, 4h, 5h, 6h, 7h or 8 h.
And (3) cleaning the post-treatment by using purified water.
As a preferred embodiment of the present invention, the low temperature degreasing method comprises the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning, treating with an alkaline reagent, and cleaning the animal tissue to be neutral;
(2) adjusting the pH value of the low-temperature lipase solution to 7-14, then treating the product obtained in the step (1) by using the solution, and then cleaning animal tissues to be neutral;
(3) and (3) treating the product obtained in the step (2) by using an acid reagent or a fatty acid dispersant solution, and then cleaning the product by using purified water to obtain the degreased animal tissue.
In a second aspect, the present invention provides an application of the low temperature degreasing method of the first aspect in tissue engineering fields such as biomaterial preparation, acellular matrix preparation, or animal-derived material degreasing.
Compared with the prior art, the invention has at least the following beneficial effects:
the low-temperature degreasing process is carried out in stages, firstly, alkaline reagent is used for acting to loosen the animal tissue material, then, low-temperature lipase is used for acting on the animal tissue material, the low-temperature lipase can rapidly enter pores inside the tissue under the condition of the optimal pH value of the low-temperature lipase to completely hydrolyze fat, and further, the decomposed fat is eluted by acid reagent or fatty acid dispersant solution, so that the high-efficiency degreasing is realized (the fat content of the degreased biological material is 0.096-0.295%). In addition, the low-temperature degreasing method can avoid the damage of the three-dimensional structure of the biological material, and has the advantages of low toxicity, safety and no pollution.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In this embodiment, a method for low temperature degreasing based on animal tissue is provided, the method comprising the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning with purified water, soaking with alkaline reagent at 10 deg.C for 3h, and cleaning the animal tissue to neutrality;
(2) adjusting the pH value of the low-temperature lipase solution to 8, soaking the product obtained in the step (1) in the low-temperature lipase solution at 10 ℃ for 8h, and then cleaning animal tissues to be neutral;
(3) and (3) soaking the product obtained in the step (2) by using an acid reagent at 10 ℃ for 5h, and then cleaning by using purified water to obtain the degreased animal tissue.
Wherein the animal tissue is sheep esophageal tissue; the alkaline reagent is sodium hydroxide solution with the concentration of 3 mol/L; the low-temperature lipase in the low-temperature lipase solution is JXJ-7 lipase, and the concentration of the low-temperature lipase solution is 300U/mL; the acid reagent is hydrochloric acid solution, and the mass percentage of solute in the acid reagent is 3%.
Example 2
In this embodiment, a method for low temperature degreasing based on animal tissue is provided, the method comprising the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning with purified water, soaking with alkaline reagent at 4 deg.C for 5h, and cleaning the animal tissue to neutrality;
(2) adjusting the pH value of the low-temperature lipase solution to 7, soaking the product obtained in the step (1) in the low-temperature lipase solution at 4 ℃ for 12 hours, and then cleaning animal tissues to be neutral;
(3) and (3) soaking the product obtained in the step (2) by using an acid reagent at the temperature of 4 ℃ for 8h, and then cleaning by using purified water to obtain the degreased animal tissue.
Wherein the animal tissue is porcine stomach; the alkaline reagent is a sodium carbonate solution with the concentration of 1 mol/L; the low-temperature Lipase in the low-temperature Lipase solution is Lipase ZC1 Lipase, and the concentration of the low-temperature Lipase solution is 10U/mL; the acid reagent is acetic acid solution, and the mass percentage of solute in the acid reagent is 0.1%.
Example 3
In this embodiment, a method for low temperature degreasing based on animal tissue is provided, the method comprising the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning with purified water, soaking with alkaline reagent at 15 deg.C for 0.5h, and cleaning the animal tissue to neutrality;
(2) adjusting the pH value of the low-temperature lipase solution to 10, soaking the product obtained in the step (1) in the low-temperature lipase solution at 15 ℃ for 4h, and then cleaning animal tissues to be neutral;
(3) soaking the product obtained in the step (2) in a fatty acid dispersant solution at 15 ℃ for 1h, and then cleaning the product with purified water to obtain the degreased animal tissue.
Wherein the animal tissue is intestinal tract of cattle; the alkaline reagent is a sodium carbonate solution with the concentration of 5 mol/L; the low-temperature lipase in the low-temperature lipase solution is JXJ-54 lipase, and the concentration of the low-temperature lipase solution is 500U/mL; the fatty acid dispersant solution is sodium tripolyphosphate solution, and the mass percentage of solute in the fatty acid dispersant solution is 3%.
Example 4
In this embodiment, a method for low temperature degreasing based on animal tissue is provided, the method comprising the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning with purified water, soaking with alkaline reagent at 8 deg.C for 4h, and cleaning the animal tissue to neutrality;
(2) adjusting the pH value of the low-temperature lipase solution to 12, soaking the product obtained in the step (1) in the low-temperature lipase solution at 12 ℃ for 6 hours, and then cleaning animal tissues to be neutral;
(3) and (3) soaking the product obtained in the step (2) by using a fatty acid dispersant solution at the temperature of 6 ℃ for 7 hours, and then cleaning by using purified water to obtain the degreased animal tissue.
Wherein the animal tissue is pig esophagus; the alkaline reagent is potassium hydroxide solution with the concentration of 1 mol/L; the low-temperature lipase in the low-temperature lipase solution is FS119 lipase, and the concentration of the low-temperature lipase solution is 100U/mL; the fatty acid dispersant solution is sodium hexametaphosphate solution, and the mass percentage of solute in the fatty acid dispersant solution is 0.1 percent.
Example 5
In this embodiment, a method for low temperature degreasing based on animal tissue is provided, the method comprising the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning with purified water, soaking with alkaline reagent at 12 deg.C for 2h, and cleaning the animal tissue to neutrality;
(2) adjusting the pH value of the low-temperature lipase solution to 7, soaking the product obtained in the step (1) in the low-temperature lipase solution at the temperature of 8 ℃ for 10 hours, and then cleaning animal tissues to be neutral;
(3) and (3) soaking the product obtained in the step (2) by using a fatty acid dispersant solution at the temperature of 8 ℃ for 6 hours, and then cleaning by using purified water to obtain the degreased animal tissue.
Wherein the animal tissue is stomach of sheep; the alkaline reagent is potassium hydroxide solution with the concentration of 2 mol/L; the low-temperature Lipase in the low-temperature Lipase solution is Lipase B5, and the concentration of the low-temperature Lipase solution is 400U/mL; the fatty acid dispersant solution is potassium silicate solution, and the mass percentage of solute in the fatty acid dispersant solution is 5%.
Example 6
This example is different from example 1 only in that the concentration of the alkali agent used in step (1) is 0.05mol/L, and the other conditions are the same as example 1.
Example 7
This example is different from example 1 only in that the concentration of the alkali agent used in step (1) was 6mol/L, and the other conditions were the same as example 1.
Example 8
This example is different from example 1 only in that the concentration of the low-temperature lipase solution used in step (2) was 5U/mL, and the other conditions were the same as example 1.
Example 9
This example is different from example 1 only in that the solute content of the acidic reagent used in step (3) is 5% by mass, and the other conditions are the same as example 1.
Example 10
This example is different from example 4 only in that the solute content of the fatty acid dispersant solution used in step (3) is 0.05% by mass, and the other conditions are the same as example 4.
Comparative example 1
This comparative example is different from example 1 only in that the step of soaking treatment with an alkaline agent is not included in step (1), and the other conditions are the same as example 1.
Comparative example 2
This comparative example is different from example 1 only in that step (2) is not included, and the other conditions are the same as example 1.
Comparative example 3
This comparative example is different from example 1 only in that the step of soaking treatment with an acidic agent is not included in step (3), and the other conditions are the same as example 1.
The animal tissues after degreasing obtained in examples 1 to 10 and comparative examples 1 to 3 were subjected to a performance test by the following method:
(1) and (3) degreasing effect detection: detecting the fat content by adopting an acid hydrolysis method in GB 5009.6-2006 national standard food fat determination;
(2) tensile strength: the tensile strength was measured according to the method specified in GB/T1040.3-2006 model 2;
(3) standard collagen content: detection was carried out according to the method for determining hydroxyproline in appendix B in YY/T1511-2017.
The results of the performance tests are shown in table 1.
TABLE 1
As can be seen from the test data of table 1, the fat content of the defatted biomaterial prepared by the methods provided in examples 1 to 5 of the present invention was below 0.300% (0.096% to 0.295%), the tensile strength was above 5MPa (5.98MPa to 8.51MPa), and the standard collagen content was above 97 wt% (97.8 wt% to 99.5 wt%).
The fat content of the defatted biomaterial produced by the method provided in example 6 was increased compared to example 1; the tensile strength of the defatted biomaterial prepared by the method provided in example 7 was reduced.
Compared with example 1, the fat content of the defatted biological material prepared by the method provided by example 8 is obviously increased;
compared with example 1, the fat content of the degreased biological material prepared by the method provided by example 9 is not greatly different, and the tensile strength is reduced;
compared with example 4, the fat content of the defatted biological material prepared by the method provided in example 10 is increased;
the fat content of the defatted biomaterial prepared by the methods provided in comparative examples 1-3 was significantly increased compared to example 1.
The data in the table above show that the degreasing process in the preparation of the biomaterial by the method provided by the invention can effectively reduce the fat content in the biological tissue on one hand, thereby reducing the immunogenicity of the biomaterial, and on the other hand, the collagen content of the material is not affected and the tensile strength is high when the degreasing process is performed by using the treatment steps and the treatment method provided by the invention, which indicates that the collagen in the biomaterial is not damaged by the preparation method, so that the performance of the biomaterial is not affected. Therefore, the degreasing method provided by the invention can be used in the preparation process of the biological material without using different tissues of animals, can ensure the integrity of the three-dimensional structure of the biological material while effectively and thoroughly removing fat, and can be effectively used for guiding the growth of tissue cells.
The applicant states that the present invention is illustrated by the above examples of the method for low-temperature degreasing based on animal tissues and the application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A low-temperature degreasing method based on animal tissues is characterized by comprising the following steps:
(1) taking isolated animal tissues, pretreating, and then treating by using an alkaline reagent;
(2) treating the product obtained in the step (1) by using a low-temperature lipase solution;
(3) and (3) treating the product obtained in the step (2) by using an acid reagent or a fatty acid dispersant solution, and performing post-treatment to obtain the degreased animal tissue.
2. The method of claim 1, wherein the animal tissue comprises any one of esophagus, stomach, intestine, urethra, or bladder.
3. The low-temperature degreasing method as claimed in claim 1 or 2, wherein the alkaline reagent comprises any one of sodium carbonate solution, potassium carbonate solution, sodium hydroxide solution or potassium hydroxide solution or a combination of at least two of the above solutions;
preferably, the concentration of the alkaline reagent is 0.1-5 mol/L.
4. The low-temperature degreasing method as claimed in any one of claims 1 to 3, wherein the low-temperature Lipase in the low-temperature Lipase solution comprises any one of Lipase ZC1 Lipase, Lipase B5 Lipase, JXJ-7 Lipase, JXJ-54 Lipase, FS119 Lipase or LP28 Lipase;
preferably, the concentration of the low-temperature lipase solution is 10-500U/mL.
5. The low-temperature degreasing method as claimed in any one of claims 1 to 4, wherein the acidic reagent comprises any one of a phosphoric acid solution, an acetic acid solution, or a hydrochloric acid solution;
preferably, the mass percentage of the solute in the acidic reagent is 0.1-5%;
preferably, the fatty acid dispersant solution includes any one or a combination of at least two of a sodium tripolyphosphate solution, a potassium tripolyphosphate solution, a sodium hexametaphosphate solution, a potassium hexametaphosphate solution, a sodium phosphate solution, a potassium phosphate solution, a sodium pyrophosphate solution, a potassium pyrophosphate solution, a sodium silicate solution, a potassium silicate solution, a sodium metasilicate solution, or a potassium metasilicate solution;
preferably, the mass percentage of the solute in the fatty acid dispersant solution is 0.1-5%.
6. The low-temperature degreasing method as claimed in any one of claims 1 to 5, wherein the pretreatment of step (1) comprises removing surface impurities and fat lumps and washing;
preferably, the washing comprises washing with purified water;
preferably, the treatment of step (1) comprises soaking;
preferably, the temperature of the treatment in the step (1) is 4-15 ℃;
preferably, the treatment time in the step (1) is 0.5-5 h;
preferably, the step (1) further comprises the following steps after the treatment with the alkaline reagent: animal tissue was washed to neutrality.
7. The low-temperature degreasing method as claimed in any one of claims 1 to 6, wherein the treatment of step (2) comprises soaking;
preferably, the pH value of the treatment in the step (2) is 7-14;
preferably, the temperature of the treatment in the step (2) is 4-15 ℃;
preferably, the treatment time in the step (2) is 4-12 h;
preferably, the processing of step (2) is followed by the following steps: animal tissue was washed to neutrality.
8. The low-temperature degreasing method as claimed in any one of claims 1 to 7, wherein the treatment of step (3) comprises soaking;
preferably, the temperature of the treatment in the step (3) is 4-15 ℃;
preferably, the treatment time in the step (3) is 1-8 h;
and (3) cleaning the post-treatment by using purified water.
9. Low-temperature degreasing method according to any of claims 1-8, wherein the low-temperature degreasing method comprises the steps of:
(1) taking isolated animal tissue, removing surface impurities and fat blocks, cleaning, treating with an alkaline reagent, and cleaning the animal tissue to be neutral;
(2) adjusting the pH value of the low-temperature lipase solution to 7-14, then processing the product obtained in the step (1), and then cleaning animal tissues to be neutral;
(3) and (3) treating the product obtained in the step (2) by using an acid reagent or a fatty acid dispersant solution, and then cleaning the product by using purified water to obtain the degreased animal tissue.
10. Use of the method of low temperature degreasing according to any of claims 1-9 in the preparation of biological material, in the preparation of acellular matrix or in the degreasing of animal derived material.
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