CN114306221A - Compound amino acid oral liquid of sheep placenta for veterinary use and preparation method thereof - Google Patents
Compound amino acid oral liquid of sheep placenta for veterinary use and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biotechnology, and in particular relates to a preparation method and application of a veterinary sheep placenta compound amino acid oral liquid. The method comprises the following steps: placenta pretreatment, degreasing and membrane breaking treatment, tissue homogenate, homogenization, cell breaking and centrifugation for impurity removal. The invention has the advantages that: the process technology is simple and effective, and the yield is high. Can produce full-value amino acid with low cost. No pollutant is generated, and the environment is friendly.
Description
Technical Field
The invention relates to the technical field of biotechnology, and in particular relates to a preparation method and application of a veterinary sheep placenta compound amino acid oral liquid.
Background
Amino acids are important components of biological organisms and play a crucial role in life phenomena. Amino acids are very important substances for the nutrition, survival and development of living organisms, and play an important role in the aspects of substance metabolism regulation and information transmission in living bodies.
Nearly 400 amino acids are found in nature, but only 20 amino acids involved in protein synthesis are called essential amino acids. They exist mainly in two forms, one of which exists in a free state in physiological body fluids (blood plasma, urine), foods (wine, beverage); the other is present in the peptide and protein in a bound state. The invention aims to degrade sheep placenta tissue protein by combining a biochemical technology with a pure physical method to prepare sheep placenta compound (more than or equal to 16) amino acid oral liquid, and clinically takes dogs as target animals to generate remarkable nutrition, immunoregulation and anti-oxidation health care functions.
In China, research and product development aiming at placenta amino acid are few, especially in animal clinic, but research around placenta protein and active peptide is many (Liuyuexin 1985; Huang Tuo Ling 2000; Zhou Jie Jing et al 2015). These studies demonstrate that various placental small molecule substances have diverse biological activities and find clinical applications in medicine and veterinary medicine.
The amino acid industry in China has been on a primary scale, is mainly used for medical clinic, food manufacture and feed additives, and is rarely used for animal medical health care products.
Besides being used as a substrate raw material for protein synthesis, amino acid can also have regulation effects on various life activities of animal organisms through biological activities of the amino acid and metabolites thereof, such as nutrition regulation and substance metabolism, regulation of gene and signal expression of cells, participation in processes of immunity, oxidation resistance, stress resistance and the like of the organisms, and finally influence the growth and development, production performance and health condition of animals. Meanwhile, amino acid as a nutrition additive, a flavoring agent, a feed additive, a medical raw material, a pesticide and the like is widely applied to the aspects of food industry, medicine, agriculture, animal husbandry, human health, health care and the like.
The numerous biological activities of amino acids can be clinically summarized as follows:
(1) the nutrition function is as follows: the most obvious physiological effect of the amino acid is a nutritional effect, sufficient and complete amino acid is a basis for ensuring the synthesis of body protein, and simultaneously participates in the synthesis of important components such as nitric oxide, polyamine, glutathione, nucleic acid, hormone, neurotransmitter and the like in vivo, so that the life activity and metabolism of a body are ensured; participate in the tricarboxylic circulation of the organism to provide energy for the organism, maintain the normal functions of various tissues and organs, and maintain the stability of the internal environment and internal secretion. Amino acids not only supplement nutrition, but also regulate the nutrient balance and nutrient metabolism of the organism and influence the metabolism and life activities of the organism.
(2) And (3) immune regulation: amino acids are involved in immune regulation in the body in a number of ways and mechanisms, mainly in 3 respects: the amino acid is used as basic material for synthesizing protein and nucleic acid, and can promote the synthesis of antibody and cell factor, regulate body fluid and cell immunity and raise the resistance of animal body. Secondly, the nonspecific immunity function of the body is improved and adjusted, the amino acid preparation can obviously improve the phagocytic function of mouse abdominal cavity macrophages and the conversion rate of lymphocytes, and has a synergistic effect on the peripheral blood lymphocyte proliferation reaction induced by PHA, thereby enhancing the nonspecific immunity function of the body. And thirdly, the amino acid can promote the development of immune organs of organisms and obviously improve the index of the immune organs and serology indexes.
(3) Antioxidant function: l amino acid is a natural antioxidant component, is similar to active substances such as low molecular peptide, Q10, VE, coenzyme R and the like, has the function of inducing body cells to secrete endogenous antioxidant substances, has the antioxidant capacity 6 times that of SOD and 40 times that of VE, can eliminate excessive active oxygen free radicals in vivo, prevents the formation of lipid peroxide and malondialdehyde, and has obvious anti-aging effect.
The oral test of the compound amino acid preparation shows that the product can obviously affect the activity change of the blood antioxidant enzymatic system of the test dog, the activity of total superoxide dismutase (T-SOD), the activity of total antioxidant capacity (T-AOC) and the activity of glutathione peroxidase (GSH-Px) are obviously improved, the content of Malondialdehyde (MDA) is obviously reduced, and the antioxidant effect is exact.
(4) Other functions of amino acids
Amino acid and its derivatives have been widely used as food flavoring agent, additive and antioxidant preservative in food industry, calcium supplement food-amino acid hibernating calcium and calcium aspartate are commercialized, and residue-free food-full-value amino acid oral liquid and infusion solution are frequently fresh. Amino acid is also an important medical raw material and a feed additive, and the amino acid is widely used as a medical raw material in medicine or animal clinic, and becomes an ideal component of various complete feeds as a feed additive.
Amino acids as nutritional additives, flavoring agents, feed additives, medicines, pesticides and the like are widely applied in the aspects of food industry, agriculture, animal husbandry, human health, health care and the like, and the most important is the application of the amino acids and the preparations thereof in medical clinic.
(ii) as a nutritional supplement: amino acids are an important component of biological organisms, and the establishment of the los nitrogen balance theory and the discovery by humans that the lack of an amino acid in a normal metabolic tissue protein leads to metabolic disorders throughout the organism, making amino acids a nutritional or medical drug for maintaining human nutrition and treating many diseases: such as amino acid nutrition transfusion, amino acid element meal oral liquid, plasma substitute amino acid, amino acid transfusion agent for hemostasis, etc.
② as a medicine: in the aspect of medicine, the application of the amino acid derivatives as therapeutic agents in clinic is quite active at present, the amino acid derivatives are widely used in the aspects of treating liver diseases, cardiovascular diseases, ulcer diseases, nervous system diseases, inflammation and the like, and the number of the amino acid derivatives used for treatment is hundreds.
③ in animal medicine clinic, the amino acid preparation has therapeutic effect on various epidemic diseases of milk cow, such as low nutrition, postpartum recovery, recovery after illness, stress resistance, etc. However, amino acid preparations for veterinary use are rare, and in particular, amino acid products for dogs are blank.
In the pharmaceutical field, the market promise of amino acid preparations as a product with diverse biological activities is self-evident, especially in the context of products of this type which have not been found in animal medicine in clinical practice to date. The product of each amino acid product has no non-natural extract or synthetic product 2, and has the component characteristics of a preparation, namely, the monomeric amino acid is mainly used as a raw material, and the compound amino acid preparation has slight difference of clinical indications due to different types of the contained amino acid; from the aspect of the characteristics of the preparation, the main forms of the products on the market are solutions, including oral liquid and infusion solution, and the preparation type is emulsion, but is rare.
The production of all these products relies on advances in technology and equipment upgrades. The artificial synthesized products including chemical synthesis, genetic engineering and enzyme engineering are mostly single pure products, and are not used in clinic; while the natural amino acid products extracted by the traditional technology (such as a high-pressure homogenization method, a freeze-thaw method and the like) are mostly composite preparations and are widely applied clinically.
Disclosure of Invention
The invention aims to provide a preparation method and application of the veterinary sheep placenta compound amino acid oral liquid, which has simple and effective process technology, can produce complete amino acid, has low cost and complete nutrition and is environment-friendly.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of a sheep placenta compound amino acid oral liquid for dogs comprises the following steps:
s101 placenta pretreatment:
cleaning fresh and/or thawed sheep placenta, separating, cutting, hemolysis, and sterilizing;
s102 degreasing and membrane breaking treatment:
soaking the hemolyzed placenta with a precooled degreasing membrane breaking agent solution, slowly stirring for 4-8 h, taking out, washing with clear water and deionized water for 1-2 times according to 5-8 times of the weight, and finally washing with distilled water for 1 time;
s103, tissue homogenate:
taking the placenta tissue after the degreasing and membrane breaking treatment, and preparing tissue homogenate by using tissue mashing and colloid mill treatment;
tissue mashing: placenta tissue is prepared according to the following steps of 1: 3(V: V) adding precooling distilled water to dilute and mix evenly, and guiding the mixture into a tissue triturator to triturate and refine into millimeter-sized materials;
homogenizing by a colloid mill: adding 4 times (V: V) of precooled distilled water into the tissue mashing solution according to the placenta material amount to dilute, fully and uniformly mixing, and then homogenizing to obtain a micron-sized material;
mashing the tissue into millimeter level, processing the tissue into micron level by a colloid mill, processing the tissue into nanometer level by a homogenizer, and repeatedly freezing and thawing to crush cells and degrade macromolecules to generate free amino acid;
s104, homogenizing:
homogenizing the placenta tissue homogenate under high pressure to obtain a nanoscale material;
s105, cell disruption:
freezing the homogeneous solution at-20 to-30 ℃ for 16 to 24 hours, then unfreezing the homogeneous solution in a water bath at 20 to 25 ℃, and repeating the unfreezing for 1 to 3 times to prepare a cell freezing and thawing solution;
s106, centrifugal impurity removal:
and centrifuging the frozen and thawed cell liquid after cell disruption at 4 ℃ at 3000-5000 rpm for 15-30 min, sterilizing the supernatant, and blending to obtain the compound amino acid oral liquid.
Further, the step S101 specifically includes:
sterilizing fresh and/or thawed sheep placenta for 15min under the condition that ozone is generated at least 200 g/h;
removing amniotic fluid, dirt, blood clot and other impurities, washing with clear water for 3 times, and washing with 0.25% saline 3 times the weight of the materials for 2 times;
separating chorion, amnion and umbilical cord from the cleaned sheep placenta, shearing the chorion and the umbilical cord into blocks of 0.5-1 multiplied by 0.5-1 cm for later use, and preserving the amnion;
adding the sheared sheep placenta tissues into precooled purified water according to the proportion of 1: 5-9 (W/W) for hemolysis for 24 hours, slowly stirring the mixture, and changing water for 1 time every 4-6 hours; after hemolysis, washing with clean water and purified water for 1 time and 2 times according to the weight of 4-8 times;
soaking the hemolyzed sheep placenta tissue in sheep placenta disinfectant at 20 + -2 deg.C for 4h, taking out, washing with purified water for 2 times, and washing with distilled water for 1 time;
after hemolysis, cleaning the hemolyzed by using clean water for 1 time, cleaning the hemolyzed by using deionized water for 2 times, and draining the hemolyzed for later use;
the preparation method of the sheep placenta disinfectant comprises the following steps: peroxyacetic acid 30ml, 96% ethanol 250ml, distilled water added to 1000 ml.
Further, the degreasing film breaking agent in the step S102 is Triton X-100; preferably 0.1% Triton X-100; pre-cooling the degreasing film breaking agent solution to be less than or equal to 6 ℃.
Further, the preparation method of the degreasing film breaking agent comprises the following steps:
preparing 30% stock solution from Triton X-100 for later use;
before use, 30% Triton X-100 stock solution is added with 300 times of PBS to form 0.1% Triton X-100 solution, and the mixture is fully mixed and placed in a water bath at 37-40 ℃ for 2-3 h to be fully dissolved.
Further, in step S103, the material fineness reaches less than 1mm after the placenta tissue is mashed; the fineness of the material after homogenate by a colloid mill is less than or equal to 10 mu m; the homogenate is uniform in emulsification, uniform in dispersion and good in fluidity; storing the placenta tissue homogenate at 15-20 ℃;
tissue trituration main parameters: the rotating speed is more than or equal to 20000rpm, 3 minutes/time and 2-3 times continuously (30 seconds apart), and the temperature is controlled to be less than or equal to 25 ℃;
main parameters of colloid mill homogenate: the rotating speed is more than or equal to 3000rpm, 2 minutes/time and 2-3 times continuously (interval of 30 seconds), and the temperature is controlled to be less than or equal to 30 ℃.
Further, in step S104, the parameters of the high-pressure homogenizer are: working pressure is not less than 23000psi, and continuous homogenization is carried out for 2-3 times at 4 ℃ or under ice bath condition.
Further, in step S105, the cell disruption rate of the frozen and thawed cell solution is not less than 60%.
Further, the sterilization method in step S106 is: adding formaldehyde with the final concentration of 0.1% into the centrifugal supernatant, uniformly mixing, inactivating at 15-25 ℃ for 48 hours, and fully stirring during inactivation;
and S106, adjusting the content of the amino acid to be 2.0-3.0mg/ml, thus obtaining a finished product.
Further, the sheep placenta comprises chorion and umbilical cord.
The invention also provides application of the sheep placenta compound amino acid oral liquid for dogs.
An application of compound amino acid oral liquid prepared from placenta caprae seu ovis for regulating immunity, improving nutrition, resisting aging, and preventing and treating senile diseases.
The invention provides a preparation method of a compound amino acid oral liquid of sheep placenta for dogs, which applies a biotechnology and a physical method, takes healthy sheep placenta tissue protein as a raw material, firstly uses a degreasing film breaking agent to enable tissue cells to generate a degreasing film breaking effect, damages a cell lipid bilayer structure, dissolves cell membrane lipid, destroys protein with weak bonding bonds among molecules, and reduces the bonding force among the molecules, so that the cell membranes are damaged, the toughness of the cell membranes is obviously reduced, the permeability is increased, and a foundation is laid for further breaking of the tissue cells; secondly, homogenizing the tissue by using a high-efficiency colloid mill with a shearing function, and further carrying out high-pressure homogenization treatment on the tissue homogenate to ensure that more than 50 percent of tissue cells are in a crushed or semi-crushed state; and finally, repeatedly freezing and thawing for many times to break more than 60 percent of tissue cells.
The invention provides a preparation method of a compound amino acid oral liquid of sheep placenta for dogs, which takes healthy sheep (sheep, goat) placenta as a raw material and degrades tissue protein into free protein amino acid by combining biotechnology with a physical method. The technology of the present invention can be summarized into 2 stages, namely, amino acid and preparation. The specific process for preparing the amino acid comprises the following steps: the placental tissue is first pre-treated to purify, chop it, to greatly increase the treated surface area of the material, and to cause tissue damage by cutting action. Then, biological degreasing and membrane breaking techniques are used to remove fat and cause damage to cell membranes and cell structures. The third step is tissue homogenate treatment, and the high-shear colloid mill can generate strong physical actions such as shearing force, friction force, high-frequency vibration, high-speed vortex and the like, can generate effective crushing, refining, emulsifying and dispersing effects on materials, not only enables the materials to achieve ultrafine crushing and emulsifying, but also can prevent the aggregation of free cells and generate good dispersing effect. And fourthly, further degrading the tissue homogenate by high-pressure homogenization. The tissue homogenate is non-uniform homogenate in which tissue fragments, free cells, macromolecular substances and polypeptides are mixed, after the treatment by a homogenizer with stronger degradation effect, the produced homogenate is still composed of the tissue fragments, the free cells, the macromolecular substances and the polypeptides, but small molecular components appear, and the components of the free cells, the macromolecular substances and the polypeptides are more than those of the tissue fragments, so that the cell disruption and the protein degradation are more facilitated. Finally, the cells are broken by repeated freeze thawing, and macromolecules are degraded to generate free amino acids. The cell disruption solution after freeze thawing still has tissue fragments, cell fragments and macromolecules, so the cell disruption solution is a tissue emulsion and can form final oral liquid only by preparation.
The invention provides a preparation method of a compound amino acid oral liquid of sheep placenta for dogs. The technical process not only can efficiently and industrially prepare the compound placenta amino acid preparation, but also overcomes the technical defects of various hydrolysis methods from the technical characteristics and ensures the full-value characteristics and the activity of the product amino acid.
The preparation method of the sheep placenta compound amino acid oral liquid for dogs provided by the invention has the remarkable technical characteristics that: the biological membrane breaking technology is combined with three-level (mechanical crushing, homogenate, homogenization and cell freeze thawing) physical degradation technology to form a complete and effective technical process. The tissue particles homogenized by the colloid mill reach micron-sized (mum), and the tissue particles treated by the homogenizer become nano-sized (nm), so that the process not only is greatly favorable for cell pulverization (membrane breaking), but also can directly break partial cells, thereby improving the pulverization efficiency of the tissue cells. The invention is also characterized in that:
(1) the degreasing and film breaking technology is the first application of the technology process. Due to the application of the degreasing film breaking agent, cell membrane lipid is dissolved, the cell lipid bilayer structure is damaged, partial intermolecular binding protein is damaged, and the probability of cell breakage is greatly increased; meanwhile, the further degradation of macromolecules is facilitated due to the dissolving effect of cytoplasm. This is one of the technical features of the present invention.
The degreasing film breaking agent is 0.1 percent TritonX-100PBS solution, TritonX-100 (polyethylene glycol octyl phenyl ether) is a nonionic surfactant, is an excellent cleaning agent, a dispersing agent and an oil-in-water system emulsifier, is used in the field of biomedicine, and has the functions of dissolving lipid and cracking cells.
According to the technical safety specification of chemicals (GB/T16483-2008), it is shown that: TritonX-100 is a stable chemical preparation, is a non-hazardous article, and has no toxic effect on human and animal health and environment.
(2) Good tissue homogenization and cell dispersion. Thanks to the design of tissue homogenate and high-pressure homogenization in the technical process, in the tissue homogenate link, due to the powerful shearing force, friction force, high-frequency vibration and high-speed vortex action of equipment, shearing, emulsification and dispersion effects are generated on materials, and tissue homogenate liquid consisting of tissue fragments, free cells, macromolecular substances and polypeptide is formed. The high-pressure homogenizing device has stronger shearing and emulsifying functions, so that the tissue homogenate is further refined, small molecular substances appear in the formed homogenate, and the components of free cells, macromolecular substances and polypeptide are more than tissue fragments. By the effective and progressive crushing and cell dispersion, the tissue protein is effectively degraded, and a foundation is laid for further cell disruption and protein degradation.
(3) Simple process, high yield and environment protection. As a complete technical process, the method has the characteristics of simplicity, effectiveness, no complex process design and relatively simple equipment requirement. The yield of finished products reaches more than 60 percent and is obviously higher than the similar technical level. The whole process is mainly a physical method, no pollutant is generated, and no adverse effect is caused to the environment.
Compared with the prior art, the preparation method of the compound sheep placenta amino acid oral liquid for dogs has the advantages that:
(1) the process technology is simple and effective, and the yield is high.
(2) Can produce full-value amino acid with low cost.
(3) No pollutant is generated, and the environment is friendly.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not for limiting the scope of the present invention.
A preparation method of a sheep placenta compound amino acid oral liquid for dogs comprises the following steps:
s101 placenta pretreatment:
cleaning fresh and/or thawed sheep placenta, separating, cutting, hemolysis, and sterilizing;
s102 degreasing and membrane breaking treatment:
soaking the hemolyzed placenta with a precooled degreasing membrane breaking agent solution, slowly stirring for 4-8 h, taking out, washing with clear water and deionized water for 1-2 times according to 5-8 times of the weight, and finally washing with distilled water for 1 time;
s103, tissue homogenate:
taking the placenta tissue after the degreasing and membrane breaking treatment, and preparing tissue homogenate by using tissue mashing and colloid mill treatment;
tissue mashing: placenta tissue is prepared according to the following steps of 1: 3(V: V) adding precooling distilled water to dilute and mix evenly, and guiding the mixture into a tissue triturator to triturate and refine into millimeter-sized materials;
homogenizing by a colloid mill: adding 4 times (V: V) of precooled distilled water into the tissue mashing solution according to the placenta material amount to dilute, fully and uniformly mixing, and then homogenizing to obtain a micron-sized material;
mashing the tissue into millimeter level, processing the tissue into micron level by a colloid mill, processing the tissue into nanometer level by a homogenizer, and repeatedly freezing and thawing to crush cells and degrade macromolecules to generate free amino acid;
s104, homogenizing:
homogenizing the placenta tissue homogenate under high pressure to obtain a nanoscale material;
s105, cell disruption:
freezing the homogeneous solution at-20 to-30 ℃ for 16 to 24 hours, then unfreezing the homogeneous solution in a water bath at 20 to 25 ℃, and repeating the unfreezing for 1 to 3 times to prepare a cell freezing and thawing solution;
s106, centrifugal impurity removal:
and centrifuging the frozen and thawed cell liquid after cell disruption at 4 ℃ at 3000-5000 rpm for 15-30 min, sterilizing the supernatant, and blending to obtain the compound amino acid oral liquid.
Further, the step S101 specifically includes:
sterilizing fresh and/or thawed sheep placenta for 15min under the condition that ozone is generated at least 200 g/h;
removing amniotic fluid, dirt, blood clot and other impurities, washing with clear water for 3 times, and washing with 0.25% saline 3 times the weight of the materials for 2 times;
separating chorion, amnion and umbilical cord from the cleaned sheep placenta, shearing the chorion and the umbilical cord into blocks of 0.5-1 multiplied by 0.5-1 cm for later use, and preserving the amnion;
adding the sheared sheep placenta tissues into precooled purified water according to the proportion of 1: 5-9 (W/W) for hemolysis for 24 hours, slowly stirring the mixture, and changing water for 1 time every 4-6 hours; after hemolysis, washing with clean water and purified water for 1 time and 2 times according to the weight of 4-8 times;
soaking the hemolyzed sheep placenta tissue in sheep placenta disinfectant at 20 + -2 deg.C for 4h, taking out, washing with purified water for 2 times, and washing with distilled water for 1 time;
after hemolysis, cleaning the hemolyzed by using clean water for 1 time, cleaning the hemolyzed by using deionized water for 2 times, and draining the hemolyzed for later use;
the preparation method of the sheep placenta disinfectant comprises the following steps: peroxyacetic acid 30ml, 96% ethanol 250ml, distilled water added to 1000 ml.
Further, the degreasing film breaking agent in the step S102 is Triton X-100; preferably 0.1% Triton X-100; pre-cooling the degreasing film breaking agent solution to be less than or equal to 6 ℃.
Further, the preparation method of the degreasing film breaking agent comprises the following steps:
preparing 30% stock solution from Triton X-100 for later use;
before use, 30% Triton X-100 stock solution is added with 300 times of PBS to form 0.1% Triton X-100 solution, and the mixture is fully mixed and placed in a water bath at 37-40 ℃ for 2-3 h to be fully dissolved.
Further, in step S103, the material fineness reaches less than 1mm after the placenta tissue is mashed; the fineness of the material after homogenate by a colloid mill is less than or equal to 10 mu m; the homogenate is uniform in emulsification, uniform in dispersion and good in fluidity; storing the placenta tissue homogenate at 15-20 ℃;
tissue trituration main parameters: the rotating speed is more than or equal to 20000rpm, 3 minutes/time and 2-3 times continuously (30 seconds apart), and the temperature is controlled to be less than or equal to 25 ℃;
main parameters of colloid mill homogenate: the rotating speed is more than or equal to 3000rpm, 2 minutes/time and 2-3 times continuously (interval of 30 seconds), and the temperature is controlled to be less than or equal to 30 ℃.
Further, in step S104, the parameters of the high-pressure homogenizer are: working pressure is not less than 23000psi, and continuous homogenization is carried out for 2-3 times at 4 ℃ or under ice bath condition.
Further, in step S105, the cell disruption rate of the frozen and thawed cell solution is not less than 60%.
Further, the sterilization method in step S106 is: adding formaldehyde with the final concentration of 0.1% into the centrifugal supernatant, uniformly mixing, inactivating at 15-25 ℃ for 48 hours, and fully stirring during inactivation;
and S106, adjusting the content of the amino acid to be 2.0-3.0mg/ml, thus obtaining a finished product.
Further, the sheep placenta comprises chorion and umbilical cord.
The invention also provides application of the sheep placenta compound amino acid oral liquid for dogs.
An application of compound amino acid oral liquid prepared from placenta caprae seu ovis for regulating immunity, improving nutrition, resisting aging, and preventing and treating senile diseases.
Example 1
A preparation method of a sheep placenta compound amino acid oral liquid for dogs comprises the following steps:
s101 placenta pretreatment:
sterilizing fresh placenta caprae seu ovis for 15min under the condition of ozone generation of 200 g/h;
removing amniotic fluid, dirt, blood clot and other impurities, washing with clear water for 3 times, and washing with 0.25% saline 3 times the weight of the materials for 2 times;
separating chorion, amnion and umbilical cord from cleaned placenta caprae seu ovis, cutting chorion and umbilical cord into pieces of 0.5 × 0.5cm, and storing amnion;
adding the minced sheep placenta tissue into precooled purified water according to the proportion of 1:9(W/W) for hemolysis for 24 hours, slowly stirring the mixture, and changing water for 1 time every 6 hours; after hemolysis, washing the hemolyzed by 4 times of the hemolyzed by a single step for 1 time, and then washing the hemolyzed by a single step for 2 times;
soaking the hemolyzed sheep placenta tissue in sheep placenta disinfectant at 20 deg.C for 4h, taking out, washing with purified water for 2 times, and washing with distilled water for 1 time;
after hemolysis, cleaning the hemolyzed by 4 times of the hemolyzed by a patient for 1 time, cleaning the hemolyzed by the hemolyzed blood for 2 times for standby use;
the preparation method of the sheep placenta disinfectant comprises the following steps: peroxyacetic acid 30ml, 96% ethanol 250ml, distilled water added to 1000 ml.
S102 degreasing and membrane breaking treatment:
soaking the hemolyzed placenta with precooled defatted membrane breaking agent solution, slowly stirring for 4h, taking out, washing with 5 times of water for 2 times, washing with deionized water for 1 time, and finally washing with distilled water for 1 time;
the degreasing film breaking agent is Triton X-100; preferably 0.1% Triton X-100; and pre-cooling the degreasing and membrane breaking agent solution to 6 ℃.
The preparation method of the degreasing film breaking agent comprises the following steps: preparing 30% stock solution from Triton X-100 for later use; before use, 30% Triton X-100 stock solution is added with 300 times of PBS to form 0.1% Triton X-100 solution, and the mixture is fully mixed and placed in a 37 ℃ water bath for 3 hours to be fully dissolved.
S103, tissue homogenate:
taking the placenta tissue after the degreasing and membrane breaking treatment, and preparing tissue homogenate by using tissue mashing and colloid mill treatment;
tissue mashing: placenta tissue is prepared according to the following steps of 1: 3(V: V) adding precooling distilled water to dilute and mix evenly, and guiding the mixture into a tissue triturator to triturate and refine into millimeter-sized materials;
homogenizing by a colloid mill: adding 4 times (V: V) of precooled distilled water into the tissue mashing solution according to the placenta material amount to dilute, fully and uniformly mixing, and then homogenizing to obtain a micron-sized material;
mashing the tissue into millimeter level, processing the tissue into micron level by a colloid mill, processing the tissue into nanometer level by a homogenizer, and repeatedly freezing and thawing to crush cells and degrade macromolecules to generate free amino acid;
after the placenta tissue is mashed, the fineness of the material reaches less than 1 mm; the fineness of the material after homogenate by a colloid mill is less than or equal to 10 mu m; the homogenate is uniform in emulsification, uniform in dispersion and good in fluidity; storing the placenta tissue homogenate at 15 ℃;
tissue trituration main parameters: rotating at 20000rpm for 3 min/time and continuously for 2 times (at intervals of 30 seconds), and controlling the temperature to 25 ℃;
main parameters of colloid mill homogenate: rotating speed of 3000rpm, 2 min/time, continuous 3 times (interval of 30 s), controlling temperature less than or equal to 30 ℃.
S104, homogenizing: homogenizing the placenta tissue homogenate under high pressure to obtain a nanoscale material; parameters of the high-pressure homogenizer: working pressure 23000psi, 4 ℃ continuous homogenizing for 3 times;
s105, cell disruption: freezing the homogeneous solution at-20 deg.C for 24 hr, thawing in 25 deg.C water bath, repeating for 2 times, and making into cell freezing and thawing solution; the cell breakage rate of the cell freezing and thawing solution is 65.6 percent.
S106, centrifugal impurity removal: centrifuging the frozen and thawed cell solution after cell disruption at 4 deg.C and 4000rpm for 30min, adding 0.1% final concentration formaldehyde into the supernatant, mixing, inactivating at 25 deg.C for 48 hr, and stirring thoroughly; sterilizing the supernatant, and adjusting the amino acid content to 3.0mg/ml to obtain the compound amino acid oral liquid.
The sheep placenta comprises chorion and umbilical cord.
Example 2
A preparation method of a sheep placenta compound amino acid oral liquid for dogs comprises the following steps:
s101 placenta pretreatment:
sterilizing fresh and/or thawed sheep placenta for 15min under the condition of 200g/h ozone generation;
removing amniotic fluid, dirt, blood clot and other impurities, washing with clear water for 3 times, and washing with 0.25% saline 3 times the weight of the materials for 2 times;
separating chorion, amnion and umbilical cord from cleaned placenta caprae seu ovis, cutting chorion and umbilical cord into pieces of 1 × 1cm, and storing amnion;
adding the minced sheep placenta tissue into precooled purified water according to the proportion of 1:9(W/W) for hemolysis for 24 hours, slowly stirring the mixture, and changing water for 1 time every 4 hours; after hemolysis, washing the hemolyzed by 8 times of the hemolyzed by a single cartridge for 2 times;
soaking the hemolyzed sheep placenta tissue in sheep placenta disinfectant at 18 deg.C for 4h, taking out, washing with purified water for 2 times, and washing with distilled water for 1 time;
after hemolysis, cleaning the hemolyzed by 8 times by weight for 1 time, cleaning the hemolyzed by the same method for 2 times, and draining the hemolyzed blood;
the preparation method of the sheep placenta disinfectant comprises the following steps: peroxyacetic acid 30ml, 96% ethanol 250ml, distilled water added to 1000 ml.
S102 degreasing and membrane breaking treatment:
soaking the hemolyzed placenta with precooled defatted membrane breaking agent solution, slowly stirring for 8h, taking out, washing with clear water for 2 times, then washing with deionized water for 1 time, and finally washing with distilled water for 1 time according to 8 times of the weight;
the degreasing film breaking agent is Triton X-100; preferably 0.1% Triton X-100; and pre-cooling the degreasing film breaking agent solution to 4 ℃.
The preparation method of the degreasing film breaking agent comprises the following steps: preparing 30% stock solution from Triton X-100 for later use; before use, 30% Triton X-100 stock solution is added with 300 times of PBS to form 0.1% Triton X-100 solution, and the mixture is fully mixed and placed in a water bath at 40 ℃ for 2 hours to be fully dissolved.
S103, tissue homogenate:
taking the placenta tissue after the degreasing and membrane breaking treatment, and preparing tissue homogenate by using tissue mashing and colloid mill treatment;
tissue mashing: placenta tissue is prepared according to the following steps of 1: 3(V: V) adding precooling distilled water to dilute and mix evenly, and guiding the mixture into a tissue triturator to triturate and refine into millimeter-sized materials;
homogenizing by a colloid mill: adding 4 times (V: V) of precooled distilled water into the tissue mashing solution according to the placenta material amount to dilute, fully and uniformly mixing, and then homogenizing to obtain a micron-sized material;
mashing the tissue into millimeter level, processing the tissue into micron level by a colloid mill, processing the tissue into nanometer level by a homogenizer, and repeatedly freezing and thawing to crush cells and degrade macromolecules to generate free amino acid;
after the placenta tissue is mashed, the fineness of the material reaches less than 1 mm; the fineness of the material after homogenate by a colloid mill is less than or equal to 10 mu m; the homogenate is uniform in emulsification, uniform in dispersion and good in fluidity; storing the placenta tissue homogenate at 15 ℃;
tissue trituration main parameters: rotating at 25000rpm for 3 min/time, continuously for 2 times (at intervals of 30 seconds), and controlling the temperature to be 20 ℃;
main parameters of colloid mill homogenate: rotate at 5000rpm for 2 minutes/time and continue for 2 times (30 second intervals), and control the temperature at 28 ℃.
S104, homogenizing: homogenizing the placenta tissue homogenate under high pressure to obtain a nanoscale material; parameters of the high-pressure homogenizer: working pressure 25000psi, and continuously homogenizing for 2 times under ice bath condition;
s105, cell disruption: freezing the homogeneous solution at-30 deg.C for 16 hr, thawing in 20 deg.C water bath, repeating for 3 times to obtain cell freezing and thawing solution; the cell breakage rate of the cell freezing and thawing solution is 68.4 percent.
S106, centrifugal impurity removal: centrifuging the frozen and thawed cell solution after cell disruption at 4 deg.C and 5000rpm for 15min, adding formaldehyde with final concentration of 0.1% into the supernatant, mixing, inactivating at 15 deg.C for 48 hr, and stirring thoroughly; sterilizing the supernatant, and adjusting the content of amino acid to 2.0mg/ml to obtain the compound amino acid oral liquid.
The sheep placenta comprises chorion and umbilical cord.
Example 3
Influence of defatted membrane breaking agent on degradation degree of sheep placenta tissue
And (3) determining the effect of the degreasing film breaking agent on the degradation of the placenta material tissue by measuring the absorbance (OD value) of the homogenate of the placenta material treated by the degreasing film breaking agent and comparing the absorbance with the absorbance of the untreated material.
Test materials and methods
1. The pretreated placental material was taken in 3 batches (for test 005, for test 008, for test 009), 500 g each, for testing. Each group of placental material was divided equally into 2 groups of 250 grams each. The group 1 was a test group, treated with a degreasing rupture agent for 4 hours, and the group 2 was a control group without treatment.
2. The test and control materials were diluted with 2 times (500ml) of purified water (1: 2), treated with high speed tissue triturator (20000 rpm or more) for 3min, and then repeated 2 times.
3. Diluting the mashed material with 7 times (1725ml) of purified water (1: 7) based on placenta weight, and homogenizing in colloid mill at 3000rpm for 10min to obtain homogenate.
4. Taking 3 tubes of homogenate of each test group and the control group respectively, and taking 18 tubes of materials for detection in each batch (3 tubes of the test group and 3 tubes of the control group). And (4) adjusting the distilled water to zero at 560nm, measuring the absorbance of each tube, and visually comparing the change of the degradation degree of the tissues.
The results are summarized in table 1.
TABLE 1 Effect of degreasing film-breaking agent on degree of degradation of Material tissue
As can be seen from table 1, the absorbance of the test material homogenate was significantly reduced and the difference was significant compared to the control (P < 0.05). This is because the tissue degradation degree of the test material is increased after the treatment of the degreasing film-breaking agent, the homogenate particles become smaller, the color of the homogenate becomes lighter, and the OD value is reduced.
Example 4
The compound amino acid oral liquid prepared in the embodiment 1 is detected to obtain 16L amino acids in total. The method comprises the following steps: aspartic Acid (ASP), Threonine (THR), Serine (SER), glutamic acid (GLU), Proline (PRO), Glycine (GLY), alanine (ALA), Valine (VAL), Methionine (MET), Isoleucine (ILE), Leucine (LEU), Tyrosine (TYR), Phenylalanine (PHE), Histidine (HIS), Lysine (LYS), Arginine (ARG).
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various changes may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are included in the protective scope of the present invention.
It should be noted that, in the foregoing embodiments, various specific technical features and steps described in the above embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features and steps are not described separately.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (11)
1. A preparation method of a compound placenta amino acid oral liquid for animals is characterized by comprising the following steps:
s101, placenta pretreatment;
s102 degreasing and membrane breaking treatment:
soaking the hemolyzed placenta with precooled defatted membrane breaking agent solution, slowly stirring, and washing with water for 3-5 times;
s103, tissue homogenate:
taking the placenta tissue after the degreasing and membrane breaking treatment, and preparing tissue homogenate by using tissue mashing and colloid mill treatment;
s104, homogenizing:
homogenizing the placenta tissue homogenate under high pressure;
s105, cell disruption:
freezing the homogeneous solution at-20 to-30 ℃ for 16 to 24 hours, then unfreezing the homogeneous solution in a water bath at 20 to 25 ℃, and repeating the unfreezing for 1 to 3 times to prepare a cell freezing and thawing solution;
s106, centrifugal impurity removal:
centrifuging the frozen cell melt solution at low temperature after cell disruption, taking supernatant, sterilizing and blending to obtain the compound amino acid oral liquid.
2. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein the step S101 comprises the following steps: cleaning fresh and/or thawed placenta, cutting, and hemolysis,
sterilizing fresh and/or thawed sheep placenta for 15min under the condition that ozone is generated at least 200 g/h;
removing amniotic fluid, dirt, blood clot and other impurities, washing with clear water for 3 times, and washing with 0.25% saline 3 times the weight of the materials for 2 times;
shearing the washed chorion, amnion and umbilical cord of the placenta into blocks of 0.5-1 × 0.5-1 cm for later use;
adding the sheared sheep placenta tissue into deionized water precooled to 4-8 ℃ according to the proportion of 1: 5-9 (W/W) for hemolysis for 24 hours, slowly stirring the mixture, and changing water for 1 time every 4-6 hours;
after hemolysis, cleaning the hemolyzed by using clean water for 1 time, cleaning the hemolyzed by using deionized water for 2 times, and draining for later use.
3. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein the preparation method comprises the following steps: the degreasing film breaking agent in the step S102 is Triton X-100;
preferably 0.1% Triton X-100,
and adding a release agent, stirring for 4-8 h, taking out, washing with clear water and deionized water for 1-2 times respectively according to 5-8 times of the weight, and finally washing with distilled water for 1 time.
4. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 3, wherein the preparation method of the defatted membrane breaking agent comprises the following steps:
preparing 30% stock solution from Triton X-100 for later use;
before use, 30% Triton X-100 stock solution is added with 300 times of PBS to form 0.1% Triton X-100 solution, and the mixture is fully mixed and placed in a water bath at 37-40 ℃ for 2-3 h to be fully dissolved.
5. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein the preparation method comprises the following steps: step S103, mashing tissues, namely, mashing placenta tissues according to the weight ratio of 1: 3(V: V) adding precooling distilled water to dilute and mix evenly, and guiding the mixture into a tissue triturator to triturate and refine into millimeter-sized materials;
the colloid mill homogenate is specifically that the tissue trituration liquid is diluted by adding 4 times (V: V) of precooled distilled water according to the placenta material amount, and after being fully and uniformly mixed, the tissue trituration liquid is homogenized into a micron-sized material;
the tissue mashing main parameters are as follows: the rotating speed is more than or equal to 20000rpm, 3 minutes/time, 2-3 times continuously, the interval is 30 seconds, and the temperature is controlled to be less than or equal to 25 ℃;
the main parameters of the colloid mill homogenate are as follows: the rotating speed is more than or equal to 3000rpm, 2 minutes/time, 2-3 times continuously, the interval is 30 seconds, and the temperature is controlled to be less than or equal to 30 ℃.
After the placenta tissue is mashed, the fineness of the material reaches less than 1 mm; the fineness of the material after homogenate by a colloid mill is less than or equal to 10 mu m; the homogenate is uniform in emulsification, uniform in dispersion and good in fluidity; storing the placenta tissue homogenate at 15-20 ℃.
6. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein the preparation method comprises the following steps: step S104, homogenizing the placenta tissue homogenate under high pressure, wherein the parameters of a high-pressure homogenizer are as follows: working pressure is not less than 23000psi, and continuous homogenization is carried out for 2-3 times at 4 ℃ or under ice bath condition.
7. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein in step S105, the cell breakage rate of the cell frozen and thawed liquid is not less than 60%.
8. The method for preparing the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein the centrifugation conditions in step S106 are as follows: centrifuging at 3000-5000 rpm for 15-30 min at 4 ℃; the sterilization method comprises the following steps: and (3) adding formaldehyde with the final concentration of 0.1% into the centrifugal supernatant, uniformly mixing, inactivating for 48 hours at 15-25 ℃, fully stirring during inactivation, and adjusting the content of amino acid to be 2.0-3.0mg/ml to obtain a finished product.
9. The preparation method of the placenta compound amino acid oral liquid for veterinary use according to claim 1, wherein the preparation method comprises the following steps: the placenta is sheep placenta, and the sheep placenta comprises chorion, amnion and umbilical cord.
10. A compound placenta amino acid oral liquid for animals prepared by the method of claims 1-9.
11. The use of the placenta compound amino acid oral liquid for veterinary use according to claim 10 for preparing a medicine or health product or food for immune regulation, nutrition improvement, anti-aging and senile disease prevention and treatment of dogs.
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| TNSN97094A1 (en) * | 1996-06-14 | 2005-03-15 | Bayer Corp | HUMAN BIKUNIN |
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| TNSN97094A1 (en) * | 1996-06-14 | 2005-03-15 | Bayer Corp | HUMAN BIKUNIN |
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| CN112516058A (en) * | 2020-11-25 | 2021-03-19 | 郑楠 | Amino acid-loaded modified amniotic membrane mask and preparation method thereof |
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