CN111568881A - Nucleocapsid slow-release amino acid tonic and preparation method thereof - Google Patents
Nucleocapsid slow-release amino acid tonic and preparation method thereof Download PDFInfo
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- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
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Abstract
The invention discloses a nucleocapsid slow-release amino acid tonic and a preparation method thereof, the nucleocapsid slow-release amino acid comprises an amino acid inner core and one or more layers of shells wrapped outside the amino acid inner core, wherein the one or more layers of shells are made of laminarin, and the preparation method comprises the following steps: and (2) carrying out superfine grinding on the amino acid under the low-temperature condition, carrying out primary coating, drying, carrying out secondary coating, drying, carrying out tertiary coating, and finally obtaining the core-shell structure slow-release amino acid tonic with three shells. The invention utilizes laminarin to wrap amino acid particles to form a core-shell structure, adjusts the half-life period of amino acid in a human body by adjusting the concentration of a wrapping object and the thickness of a wrapping layer, and improves the utilization rate of the amino acid; the laminarin has various pharmacological effects, wraps amino acid, solves the problem of short half-life period of the amino acid after being taken, and conforms to the health preserving concept of medicine and food homology in traditional Chinese medicine.
Description
Technical Field
The invention relates to the technical field of amino acid preparations, in particular to a nucleocapsid slow-release amino acid tonic and a preparation method thereof.
Background
Amino acid is a basic unit for forming protein, is a compound containing compound groups and having amino and carboxyl in molecules, participates in biochemical reaction and physiological function process of human body, is one of the most important nutrient substances of human body, has more than 20 kinds of amino acid for forming protein of human body, but human body can not synthesize all amino acid by self, needs to be supplemented by diet, and if human body can not supplement enough essential amino acid, normal development, growth performance, anti-stress capability, disease resistance and immunity level of human body can be seriously influenced.
Essential amino acids can be supplemented through reasonable diet matching generally, but many people need to take amino acid nutritional agents for supplementing because of insufficient amino acid supplementation caused by unreasonable diet. In the prior art, after the amino acid nutritional agent is taken, the half-life period in a human body is short, so that the utilization rate of the amino acid is insufficient, and therefore, a slow-release amino acid supplement needs to be researched to solve the problem.
Disclosure of Invention
Aiming at the problems, the invention provides a core-shell slow-release amino acid supplement and a preparation method thereof, laminarin is used for coating amino acid particles to form a core-shell structure, the half-life period of amino acid in a human body is adjusted by adjusting the concentration of a coating and the thickness of a coating layer, and the utilization rate of the amino acid is improved.
In order to achieve the above object, the present invention adopts the following technical solutions
A core-shell slow-release amino acid supplement comprises an amino acid inner core and one or more layers of shells wrapping the outer side of the amino acid inner core, wherein the one or more layers of shells are made of laminarin.
Preferably, the core-shell slow-release amino acid supplement is of a layer of core-shell structure and comprises an amino acid inner core and a laminarin shell wrapping the amino acid inner core.
Preferably, the core-shell slow-release amino acid supplement has a three-layer core-shell structure, and comprises an amino acid core, a first shell with a wrapper of laminarin, a second shell with a wrapper of starch, and a third shell with a wrapper of laminarin.
Preferably, the thickness of the first layer of outer shell is smaller than that of the second layer of outer shell, and the thickness of the second layer of outer shell is smaller than that of the third layer of outer shell.
Preferably, the amino acid core is a cyclodextrin inclusion complex of an amino acid.
A method for preparing a nucleocapsid sustained-release amino acid tonic comprises the following steps:
s1, carrying out superfine grinding on the amino acid under the low-temperature condition, and sieving the amino acid with a 600-1000-mesh sieve after grinding to obtain amino acid particles;
s2, adding laminarin into deionized water, homogenizing, and wrapping the amino acid particles to obtain a layer of structure wrapping matter;
s3, drying the one-layer structure wrap generated in the S2, then putting water-soluble starch into deionized water, homogenizing, and carrying out secondary wrapping on the one-layer structure wrap to obtain a two-layer structure wrap;
s4, drying the two-layer structure wrap generated in the S3, then putting laminarin into deionized water, homogenizing, and wrapping the two-layer structure wrap to obtain a three-layer structure wrap;
s5, drying the three-layer wrappage generated in the S4 to obtain the core-shell structure sustained-release amino acid complement with three-layer shells.
Preferably, in S2, S3 and S4, the coating is performed by an atomized spraying technique, and the spraying process is as follows: the air inlet temperature is 60-75 ℃, and the air quantity is 40-60m3H, the atomization pressure is 0.3-0.5MPa, and the flow rate is 10-20 ml/min.
Preferably, in S2, S3 and S4, ultrasonic wave assistance is adopted during wrapping, and the ultrasonic wave frequency is 50k-100 kHz.
Preferably, in S3, S4 and S5, the drying temperature is 75-90 ℃ and the time is 20-40 min.
Preferably, in S2, the weight ratio of laminarin to deionized water is (2-5): 8; in S4, the weight ratio of laminarin to deionized water is (4-6): 8.
preferably, in S2 and S4, laminarin is homogenized with deionized water at a temperature of 40-60 deg.C.
Laminarin (Laminarin), also called Laminarin, brown algae starch, mainly contains brown algae (such as sodium alginate, also called algin, the content in kelp is about 19.7%), brown algae starch (also called Laminarin, algal sulphated polysaccharide, brown algae polysaccharide, the content in kelp is about 1%), algin (also called fuco, the main component is fucoidan, also called fucoidan), etc. the Laminarin is formed by connecting beta (1-3) -glucan and some beta (1-6) -glycosidic bonds, is a neutral glucan of chemical book, widely exists in brown algae, also is the main chemical component in laminaria of pteridophyceae or laminaria of laminariaceae, has the pharmacological actions of regulating immunity, resisting tumor, resisting blood coagulation, regulating blood fat, reducing blood sugar, resisting radiation, resisting oxidation, resisting bacteria, resisting virus, etc., and also has the pharmacological actions of reducing blood pressure, resisting fatigue, etc Resisting pulmonary interstitial fibrosis, regulating tyrosine kinase activity, resisting mutation, etc. Laminarin can repair glomerulus to keep the activity of the glomerulus so as to reduce the hardening speed of the glomerulus, can slow but actually repair the glomerulus, can prevent the fibrosis trend of capillary vessels, can metabolize salt and sugar absorbed by a human body out of the human body directly in small intestine, reduces the burden of the kidney and plays a role in protecting the kidney.
The invention has the beneficial effects that:
1. the invention utilizes laminarin to wrap amino acid particles to form a core-shell structure, adjusts the half-life period of amino acid in a human body by adjusting the concentration of a wrapping object and the thickness of a wrapping layer, and improves the utilization rate of the amino acid.
2. And (3) wrapping by adopting a spraying technology, controlling the thickness of the coating by controlling the spraying time, and adjusting the compactness of the coating by adjusting the frequency of ultrasonic waves.
3. The proportion of the amino acid particles, the amino acid with the one-layer core-shell structure and the amino acid with the three-layer core-shell structure is adjusted, so that the amino acid can be supplemented in time and in a longer slow release period, and the amino acid sustained-release tablet is suitable for all people needing to supplement the amino acid.
4. The laminarin has various pharmacological effects, wraps amino acid, solves the problem of short half-life period of the amino acid after being taken, and conforms to the health preserving concept of medicine and food homology in traditional Chinese medicine.
Drawings
FIG. 1 is a schematic diagram of the structure of one layer of core-shell amino acids in the present invention;
FIG. 2 is a schematic diagram of the structure of amino acids in a three-layer core-shell structure according to the present invention;
FIG. 3 is a graph of the release rate of various embodiments of the extended release amino acid supplement of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
A core-shell slow-release amino acid supplement has a core-shell structure containing amino acid core and laminarin shell wrapping the amino acid core.
The preparation method comprises the following steps:
s1, carrying out superfine grinding on the amino acid under the low-temperature condition, and sieving the amino acid with a 600-1000-mesh sieve after grinding to obtain amino acid particles;
s2, adding laminarin into deionized water, wherein the weight ratio of laminarin to deionized water is 3: homogenizing at 45 deg.C, and coating amino acid particles by atomizing spray to obtain a layer of structure coating;
and S3, drying the layer of structure wrap generated in the step S2 at the drying temperature of 80 ℃ for 30min to obtain the core-shell structure slow-release amino acid tonic with a layer of shell.
As shown in FIG. 1, a layer of laminarin is used to wrap the amino acid particles, so as to slow down the release rate of the amino acid and prolong the half-life period of the amino acid particles; simultaneously makes full use of the pharmacological action of laminarin.
Example two
A core-shell slow-release amino acid supplement has a three-layer core-shell structure, including an amino acid core, a first shell containing laminarin as a wrap, a second shell containing starch as a wrap, and a third shell containing laminarin as a wrap.
The thickness of the first layer of shell is smaller than that of the second layer of shell, and the thickness of the second layer of shell is smaller than that of the third layer of shell.
The preparation method comprises the following steps:
s1, carrying out superfine grinding on the amino acid under the low-temperature condition, and sieving the ground amino acid with a 1000-mesh sieve to obtain amino acid particles;
s2, adding laminarin into deionized water, wherein the weight ratio of laminarin to deionized water is 3: homogenizing at 45 deg.C, wrapping amino acid particles by atomization spray method with ultrasonic wave of 60kHz to obtain a layer of structure wrap;
s3, drying the one-layer structure wrap generated in the S2 at 75 ℃ for 20min, adding water-soluble starch into deionized water, homogenizing, wrapping the one-layer structure wrap in an atomizing spraying mode for the second time, and wrapping by adopting ultrasonic assistance at the ultrasonic frequency of 80kHz to obtain a two-layer structure wrap;
s4, drying the two-layer structure wrap generated in the S3 at 80 ℃ for 30min, and then putting laminarin into deionized water, wherein the weight ratio of laminarin to deionized water is 5: 8, homogenizing at the temperature of 45 ℃, wrapping the two-layer structure wrapping object by adopting an atomization spraying mode, and obtaining a three-layer structure wrapping object by adopting ultrasonic assistance and the ultrasonic frequency of 100kHz during wrapping;
and S5, drying the three-layer wrap generated in the S4 at the drying temperature of 85 ℃ for 40min to obtain the core-shell structure slow-release amino acid tonic with the three-layer shell.
As shown in FIG. 2, the amino acid microparticles are coated with three layers of shells, wherein two layers are laminarin, which further slows down the release rate of amino acid and prolongs the half-life period of the amino acid microparticles than the first embodiment; simultaneously makes full use of the pharmacological action of laminarin.
EXAMPLE III
A core-shell slow-release amino acid supplement has a three-layer core-shell structure, including an amino acid core, a first shell containing laminarin as a wrap, a second shell containing starch as a wrap, and a third shell containing laminarin as a wrap.
Wherein the amino acid inner core is cyclodextrin inclusion compound of amino acid.
The preparation method comprises the following steps:
s1, carrying out superfine grinding on amino acid at low temperature, sieving with a 800-mesh sieve after grinding to obtain amino acid particles, and carrying out cyclodextrin inclusion treatment on the amino acid particles to obtain a cyclodextrin inclusion compound of the amino acid;
s2, adding laminarin into deionized water, wherein the weight ratio of laminarin to deionized water is 1: 2, homogenizing at 50 ℃, wrapping the cyclodextrin inclusion compound of the amino acid by adopting an atomization spraying mode, and obtaining a layer of structural wrapping matter by adopting ultrasonic wave assistance and the ultrasonic frequency of 80kHz during wrapping;
s3, drying the one-layer structure wrap generated in the S2 at the drying temperature of 80 ℃ for 20min, then putting water-soluble starch into deionized water, homogenizing, secondarily wrapping the one-layer structure wrap in an atomizing spraying mode, and obtaining a two-layer structure wrap by adopting ultrasonic assistance and ultrasonic frequency of 60 kHz;
s4, drying the two-layer structure wrap generated in the S3 at 85 ℃ for 30min, and then putting laminarin into deionized water, wherein the weight ratio of laminarin to deionized water is 3: 4, homogenizing at 50 ℃, and then wrapping the two-layer structure wrapping object by adopting an atomization spraying mode, wherein ultrasonic assistance is adopted during wrapping, and the ultrasonic frequency is 100kHz, so that a three-layer structure wrapping object is obtained;
and S5, drying the three-layer wrap generated in the S4 at the drying temperature of 90 ℃ for 40min to obtain the core-shell structure slow-release amino acid tonic with the three-layer shell.
Performing cyclodextrin inclusion treatment on the amino acid particles, and then wrapping the amino acid particles by adopting three layers of shells, wherein two layers are laminarin, which further slows down the release speed of the amino acid and prolongs the half-life period of the amino acid compared with the second embodiment; simultaneously makes full use of the pharmacological action of laminarin.
Experimental example 1
Detecting the slow release performance and half-life period of the core-shell slow-release amino acid:
taking lysine in essential amino acids as an example, the sustained-release amino acids prepared in the first example, the second example and the third example are respectively taken as a sample A, a sample B and a sample C, 0.400g of the sample A, the sample B and the sample C are respectively taken, 0.400g of lysine particles are taken as a comparative sample D, the samples are respectively put into a phosphate buffer solution simulated intestinal juice (pH 7.45), and the samples are stirred at the speed of 120r/min at the temperature of (36.8 +/-0.8) DEG C and released for 360 min; at intervals, 1mL of the solution was aspirated, and the lysine content was determined while supplementing the same volume of the mock solution. The lysine content was determined by ninhydrin color development at 570nm using a UV-visible spectrophotometer and the percent release was calculated from the measured lysine content of the solution over time.
Experimental results, as shown in fig. 3, four curves respectively show the release rates of sample a, sample B, sample C and comparative sample D over six hours, from which it can be concluded that the release rates of sample a, sample B and sample C are significantly lower than comparative sample D, whereas the release rate of sample C is the lowest among sample a, sample B and sample C.
Experimental example two
Detecting the half-life period of the nucleocapsid slow-release amino acid:
the detection method comprises the following steps:
taking lysine among essential amino acids as an example, sustained-release amino acids prepared by example one, example two and example three were respectively designated as sample a, sample B and sample C, and lysine microparticles without any treatment were taken and designated as comparative sample D.
1. Taking four rabbits with the same weight, respectively making the sample A, the sample B, the sample C and the comparison sample D into drinking water and then correspondingly feeding, and recording the feeding time.
2. After feeding for 2 hours, 5ml of rabbit ear vein blood is extracted and placed in a beaker, blood is extracted once every 2 hours for 12 times, and before blood extraction, a heparin wetting needle tube is used for anticoagulation.
3. The blood is extracted by a syringe, dropped into a test tube containing 5ml of 5 percent trichloroacetic acid, fully shaken up, then centrifuged for 5min by a centrifuge with the centrifugation speed of 1500rpm/min, and then the supernatant is taken for marking for standby.
4. Preparing standard solution, preparing 0.03% of each 0.5ml of the solution of the sample A, the sample B, the sample C and the comparative sample D, adding the solution into a test tube containing 5ml of 5% trichloroacetic acid, and shaking uniformly for later use.
5. And preparing colorimetric tubes corresponding to the sample A, the sample B, the sample C and the comparative sample D respectively.
6. The half-lives of each of sample A, sample B, sample C and comparative sample D were calculated from the measured blood lysine content using a UV-visible spectrophotometer measurement at 570 nm.
The experimental results are as follows:
the half-life of comparative sample D was calculated to be 21.3 hours, the half-life of sample a was calculated to be 27.6 hours, the half-life of sample B was calculated to be 34.5 hours, and the half-life of sample C was calculated to be 39.1 hours, from which it was found that the half-life of the encapsulated sustained-release amino acid in the animal body was significantly increased.
Pharmacological action of laminarin
1. The laminarin for regulating the immune function has the obvious function of regulating the immune function of the organism. Fucoidan isolated from brown algae regulates the complement system by interfering with the activation of C1 or inhibiting the cleavage of C4 into two fragments C4b and C4a and inhibiting the interaction of C4b and C2 to block the formation of C3 transferase in the classical pathway; secondly, the binding of factor B to C3B is inhibited by influencing the stability of the lyase (properdin), thereby preventing the formation of C3 transferase in the alternative pathway. Besides affecting complement activity, brown algae polysaccharide also has obvious enhancement effect on specific immune function of organisms. As an important immune function regulator, laminarin can also obviously influence the nonspecific immune function of the body. The glycolipid substance in thallus laminariae can be combined with immunostimulant complex, and used as liposome for presenting microorganism and tumor antigen.
2. The antitumor effect of laminarin sulfate can inhibit the growth of tumor cell directly and inhibit the growth and diffusion of tumor cell through strengthening body's immunity. Laminarin can reduce the expression of Bcl-2 protein of breast cancer cells and liver cancer cells, and can reduce the effective therapeutic dose of 5-Fu, MTX, MMC, ADM and CTX; inducing in vitro HL-60 apoptosis, wherein the regulation of apoptosis may be negative; the expression of NF-kB is regulated and has time difference. In addition, the laminarin sulfate can interfere the stimulation of Bgfgr angiogenesis signals in tumor tissues, so that the angiogenesis of the tumor tissues is blocked, and the tumor growth is inhibited; can be used for resisting leukopenia caused by chemotherapy drug cyclophosphamide, and has leukocyte increasing effect. The antitumor action mechanism of laminarin is related to directly killing tumor cells, inducing apoptosis, inhibiting angiogenesis and regulating the immune function of the organism.
3. The blood lipid regulation function of plasma Triglyceride (TG), cholesterol (TC) and Low Density Lipoprotein (LDL) are promotion factors of atherosclerosis, LDL elevation is a risk factor of myocardial infarction, High Density Lipoprotein (HDL) can bring redundant cholesterol in peripheral blood back to liver to be metabolized to generate bile acid, and the HDL elevation is a safety factor for resisting myocardial infarction due to chemical book. The laminarin can reduce the cholesterol content in blood plasma by 13-17%, the low density lipoprotein content by 20-25%, the high density lipoprotein content by 16%, reduce the atherosclerosis index of arteries, and reduce the concentration of lipid peroxide in blood plasma. The extract of thallus laminariae with different solvents has blood lipid reducing effect. In the research aspect of reducing blood fat, laminarin can effectively prevent and treat atherosclerosis and other cardiovascular and cerebrovascular diseases by improving blood fat metabolism.
4. The fucoidan sulfate with anticoagulant effect can increase animal bleeding amount, prolong bleeding time, and inhibit thrombosis of artery and vein of rat. Has inhibitory effect on platelet aggregation caused by thrombin, CaCl2, arachidonic acid, adenosine diphosphate, etc.
5. The fucoidan with anti-oxidation effect can obviously inhibit cell oxidative hemolysis and has good protection effect on lipid peroxidation.
6. The laminarin with the function of reducing blood sugar can not only reduce the blood sugar of a diabetic mouse and participate in sugar metabolism, but also regulate the protein metabolism of the diabetic mouse, and promote islet cells to secrete insulin while reducing the blood sugar.
7. The sodium alginate with the radiation-resistant effect has the effects of eliminating and preventing absorption of radioactive elements 54Mn, 90Sr, 109Cd, 133Sn and 133 Ra. It can also reduce the incidence of malignant tumor and leukemia of rats irradiated by 131I and 137 Cs.
8. Antibacterial and antiviral thallus water decoction has certain inhibiting effect on Microsporum canis, Trichophyton rubrum, Sporothrix, Trichophyton gypseum, Epidermophyton floccosum, etc.; laminarin can resist RNA and DNA virus, inhibit poliovirus type III, Coxsackie type B3 and A16 virus, echovirus type IV, etc., and also has HIV virus resisting effect.
9. The protective function of laminarin has a certain protective function on inhalation benzene poisoning, laminarin has a certain antagonistic protective function on mouse bone marrow suppression caused by benzene poisoning, and the action mechanism of laminarin is related to the functions of immunoregulation, mutation resistance, oxidation resistance and the like of organisms.
10. Other effects Laminarin also has various biological activities of lowering blood pressure, regulating tyrosine kinase activity, resisting pulmonary interstitial fibrosis, resisting mutation, resisting fatigue, etc.
The invention utilizes laminarin to wrap amino acid particles to form a core-shell structure, adjusts the half-life period of amino acid in a human body by adjusting the concentration of a wrapping object and the thickness of a wrapping layer, and improves the utilization rate of the amino acid; in actual use, the proportion of the amino acid particles, the amino acid with the one-layer core-shell structure and the amino acid with the three-layer core-shell structure is adjusted, so that the amino acid can be supplemented in time and in a longer slow release period, and the amino acid slow-release capsule is suitable for all people needing to supplement the amino acid. The laminarin has various pharmacological effects, wraps amino acid, solves the problem of short half-life period of the amino acid after being taken, and conforms to the health preserving concept of medicine and food homology in traditional Chinese medicine.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Finally, it should be noted that the above-mentioned preferred embodiments of the present invention are provided merely to help illustrate the present invention. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (10)
1. A core-shell slow-release amino acid supplement comprises an amino acid inner core and one or more layers of shells wrapping the outer side of the amino acid inner core, wherein the one or more layers of shells are made of laminarin.
2. The core-shell slow-release amino acid supplement of claim 1, wherein the core-shell slow-release amino acid supplement has a core-shell structure comprising an amino acid core and a laminarin shell covering the amino acid core.
3. The core-shell slow-release amino acid supplement of claim 1, wherein the core-shell slow-release amino acid supplement has a three-layer core-shell structure comprising an amino acid core, a first outer shell with a coating of laminarin, a second outer shell with a coating of starch, and a third outer shell with a coating of laminarin.
4. The supplement of nucleocapsid sustained-release amino acid of claim 3, wherein the first shell has a smaller thickness than the second shell and the second shell has a smaller thickness than the third shell.
5. The nucleocapsid sustained-release amino acid supplement of claim 1, wherein the amino acid core is a cyclodextrin inclusion of an amino acid.
6. The method for preparing a nucleocapsid sustained-release amino acid supplement of claim 3, comprising the steps of:
s1, carrying out superfine grinding on the amino acid under the low-temperature condition, and sieving the amino acid with a 600-1000-mesh sieve after grinding to obtain amino acid particles;
s2, adding laminarin into deionized water, homogenizing, and wrapping the amino acid particles to obtain a layer of structure wrapping matter;
s3, drying the one-layer structure wrap generated in the S2, then putting water-soluble starch into deionized water, homogenizing, and carrying out secondary wrapping on the one-layer structure wrap to obtain a two-layer structure wrap;
s4, drying the two-layer structure wrap generated in the S3, then putting laminarin into deionized water, homogenizing, and wrapping the two-layer structure wrap to obtain a three-layer structure wrap;
s5, drying the three-layer wrappage generated in the S4 to obtain the core-shell structure sustained-release amino acid complement with three-layer shells.
7. The method for preparing the nucleocapsid sustained-release amino acid supplement of claim 6, wherein in S2, S3 and S4, ultrasonic wave is used for assisting in packaging, and the ultrasonic wave frequency is 50k-100 kHz.
8. The method for preparing the nucleocapsid sustained-release amino acid supplement of claim 6, wherein the drying temperature in S3, S4 and S5 is 75-90 ℃ and the time is 20-40 min.
9. The method for preparing the nucleocapsid sustained-release amino acid supplement of claim 6, wherein in S2, the weight ratio of laminarin to deionized water is (2-5): 8; in S4, the weight ratio of laminarin to deionized water is (4-6): 8.
10. the method of preparing the nucleocapsid sustained-release amino acid supplement of claim 6, wherein the temperature of the homogenization of laminarin with deionized water in S2 and S4 is 40-60 ℃.
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