CN111481675B - Preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate - Google Patents
Preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate Download PDFInfo
- Publication number
- CN111481675B CN111481675B CN202010336860.8A CN202010336860A CN111481675B CN 111481675 B CN111481675 B CN 111481675B CN 202010336860 A CN202010336860 A CN 202010336860A CN 111481675 B CN111481675 B CN 111481675B
- Authority
- CN
- China
- Prior art keywords
- phycocyanin
- sodium alginate
- lysozyme
- solution
- complex coacervate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Urology & Nephrology (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate, which utilizes the electrostatic interaction of proteins with opposite charges and polysaccharide in an aqueous environment to form a soluble complex, thereby obtaining the sodium alginate/lysozyme complex coacervate loaded with phycocyanin. In the product obtained by the method, phycocyanin is preferably complexed with sodium alginate/lysozyme. The phycocyanin-sodium alginate/lysozyme complex coacervate prepared by the invention has the advantages of simple embedding process and good product stability, and has better temperature, illumination and pH stability.
Description
Technical Field
The invention relates to the technical field of protein embedding and slow release, in particular to a preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate.
Background
Spirulina (Spirulina platensis) is a filamentous microalgae rich in proteins, vitamins, essential amino acids, minerals and essential fatty acids, such as gamma-linolenic acid. To date, interest in spirulina has primarily been in its nutritional value. However, recent attention has been focused on the possible pharmacological functions of spirulina, such as cholesterol reduction, improvement of the immune system's defense against cancer, increase of probiotic lactobacilli in the intestinal tract, reduction of toxic kidney damage caused by heavy metals and drugs, and radiation protection.
Phycocyanin (PC) is a protein extracted from Spirulina and rich in nutrition, has remarkable properties of resisting oxidation and inflammation, protecting liver and scavenging free radicals, and can prevent nephrotoxicity induced by cisplatin by inhibiting oxidative stress. Thus, it is a potential therapeutic or protective agent for the treatment of oxidative stress induced diseases. In addition, phycocyanin has shown high therapeutic value in immunomodulation and cancer therapy. However, most natural food pigments are unstable, as is phycocyanin, because the chemical structure contains unsaturated double bonds and other oxidizable groups, and the pigments are oxidized and discolored under the action of oxygen; and the natural pigment is sensitive to factors such as illumination, temperature, metal ions and the like. For example, many natural pigments fade under ultraviolet light, and some fade even in room natural light.
Disclosure of Invention
The invention aims to prepare microcapsule embedding phycocyanin by using a complex coacervate formed by sodium alginate and lysozyme as a wall material so as to improve the stability of the microcapsule embedding phycocyanin.
In order to achieve the purpose, the invention provides a preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate, which uses complex coacervate formed by sodium alginate and lysozyme as a carrier, uses phycocyanin as an embedding object, and obtains the phycocyanin-sodium alginate/lysozyme complex coacervate through electrostatic interaction between macromolecules with opposite charges in an aqueous environment.
A preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate comprises the following steps:
s1, dropwise adding 0.24mg/ml phycocyanin solution into 200000U/ml lysozyme solution, stirring and mixing uniformly, dropwise adding 1-5 mg/ml sodium alginate solution, and stirring for 2-6 h at 4-25 ℃ to obtain an embedding solution; the volume ratio of the phycocyanin solution to the sodium alginate solution to the lysozyme solution is 1; wherein the phycocyanin solution is prepared by dissolving phycocyanin in water; the lysozyme solution is prepared by dissolving lysozyme in water; the sodium alginate solution is obtained by dissolving sodium alginate in water;
s2, taking the embedding solution obtained in the step S1, centrifuging to obtain a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, wherein the precipitate is phycocyanin-sodium alginate/lysozyme complex coacervate. Wherein, the method for detecting the content of phycocyanin in the supernatant refers to the following documents:
Bennett A,Bogorad L.Properties of subunits and aggregates of blue-green algal biliproteins[J].Biochemistry,1971,10(19):3625-3634.
preferably, the centrifugation parameters in step S2 are: the rotating speed is 9000rpm, and the time is 15-60 min.
In a preferred mode, the preparation method of the phycocyanin-sodium alginate/lysozyme complex coacervate comprises the following steps:
s1, dropwise adding 1ml of 0.24mg/ml phycocyanin solution into 1ml of 200000U/ml lysozyme solution, uniformly stirring, dropwise adding 1ml of 1mg/ml sodium alginate solution, and stirring for 3 hours at 25 ℃ to obtain an embedding solution;
s2, taking the embedding solution obtained in the step S1, centrifuging at 9000rpm for 30min, taking a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, wherein the precipitate is phycocyanin-sodium alginate/lysozyme complex coacervate. Wherein, the method for detecting the content of phycocyanin in the supernatant refers to the following documents:
Bennett A,Bogorad L.Properties of subunits and aggregates of blue-green algal biliproteins[J].Biochemistry,1971,10(19):3625-3634.
in the product obtained by the method, the phycocyanin-sodium alginate/lysozyme complex coacervate prepared by the electrostatic interaction between macromolecules with opposite charges in an aqueous environment has simple preparation process and good stability.
Compared with the prior art, the invention has the following advantages:
1. the invention prepares the phycocyanin-sodium alginate/lysozyme complex coacervate for the first time, the isoelectric point of the lysozyme adopted by the invention is 11, the optimum pH value of the phycocyanin is 5-6, the lysozyme and the phycocyanin have opposite charges in the range, and in a water environment, the electrostatic interaction of macromolecules with opposite charges can cause the formation of a soluble complex.
2. The sodium alginate serving as a stabilizer is added in the process of preparing the phycocyanin-sodium alginate/lysozyme complex coacervate, so that the stability of the coacervate can be enhanced.
3. The phycocyanin-sodium alginate/lysozyme complex coacervate prepared by the ionic gel method has the advantages of simple embedding process and good product stability, and the phycocyanin-sodium alginate/lysozyme complex coacervate prepared by the method has better temperature, illumination and pH stability.
The method is obtained on the basis of repeated experiments, and the phycocyanin solution, the lysozyme solution and the sodium alginate solution in the step S1 are different in dripping sequence, so that the phycocyanin embedding efficiency and the stability of the complex coacervate can be obviously influenced.
Drawings
FIG. 1 shows the stability of phycocyanin and the complex coacervate of phycocyanin-sodium alginate/lysozyme prepared in example 1 of the present invention after releasing for 30min in disodium hydrogen phosphate-citric acid buffer solution with pH of 7.4 at different temperatures (37-70 deg.C)
FIG. 2 shows the cumulative release of phycocyanin-sodium alginate/lysozyme complex coacervate prepared in example 1 of the present invention in disodium hydrogen phosphate-citric acid buffer solution at pH 3-8.
FIG. 3 shows the cumulative release of phycocyanin and phycocyanin-sodium alginate/lysozyme complex coacervate prepared in example 1 under uvb light and no light in disodium phosphate-citric acid buffer solution at pH 7.4.
Detailed Description
The present invention is further illustrated by the following specific examples.
Phycocyanin-sodium alginate/lysozyme coacervate.
Dropwise adding 1mL of phycocyanin solution into the lysozyme solution by using an injector, stirring under constant magnetic force, and dropwise adding 1mL of sodium alginate solutions with different concentrations by using the injector to ensure that the weight ratio of the sodium alginate to the lysozyme is 1,2,4,5 respectively; followed by magnetic stirring at room temperature for 3h to form an agglomerate; then, the mixture was stirred at 9000r at 4 ℃ for 30min, and the condensate was collected. The coacervate was then washed three times with deionized water to give a phycocyanin-sodium alginate/lysozyme coacervate.
In the product obtained by the method, the phycocyanin-sodium alginate/lysozyme coacervate prepared by the electrostatic interaction between macromolecules with opposite charges in the water-based environment has simple preparation process and good stability.
The preparation method of the phycocyanin-sodium alginate/lysozyme coacervate comprises the following specific steps:
s1, dropwise adding 0.24mg/mL phycocyanin solution, 10mg/mL phycocyanin solution and sodium alginate solutions with different concentrations (1, 2,4,5 mg/mL) according to different dropwise adding sequences.
The specific operations of the different dripping sequences are as follows:
(a) After stirring, the PC solution was added to an excess SA solution to form a phycocyanin sodium alginate intermediate complex, and then the PC-SA mixture was mixed into the LZM solution and stirred for 3 hours to prepare a (PC-SA)/LZM coacervate; (b) After stirring, the PC solution was added to a large excess of the LZM solution to form the PC-LZM intermediate complex, then different concentrations (1,2,4,5mg/mL) of SA solution were mixed into the PC-LZM mixture at weight ratios of SA/LZM of 0.1,0.2,0.4 and 0.5, and stirred for an additional 3 hours to prepare (PC/LZM) -SA coacervate; (c) mixing the SA solution into the LZM solution, wherein the SA: the LZM weight ratios were 0.1,0.2,0.4 and 0.5, and after stirring for 1 hour, SA-LZM coacervate was prepared, followed by addition of PC stock solution to prepare (SA-LZM)/PC coacervate.
The formation processes of the coacervate are different, and the amount and the stability of the phycocyanin embedded in the coacervate obtained by the three methods are slightly different. The phycocyanin in the scheme b has higher embedding rate and stability and better embedding effect.
The phycocyanin-sodium alginate/lysozyme coacervate is prepared by the method of the preferable scheme b of the method of the invention.
S2, centrifuging the phycocyanin-sodium alginate/lysozyme coacervate prepared in the step S1 for 30min at 4 ℃ under 9000r, collecting lower coacervate, washing with deionized water for three times, and removing the phycocyanin and the like which are not adsorbed and combined to obtain the phycocyanin-sodium alginate/lysozyme coacervate.
The method removes the phycocyanin, the sodium alginate and the lysozyme which are not adsorbed and combined by washing with deionized water for three times; the protein content in the eluent is zero, which proves that the phycocyanin-sodium alginate/lysozyme coacervate is washed clean.
In a preferred mode, the preparation method of the phycocyanin-sodium alginate/lysozyme coacervate in the step S1 comprises the following steps:
after stirring for 30min, adding the PC solution into a large excess of LZM solution to form a PC-LZM intermediate complex, then dropwise adding an SA solution with the concentration of 1mg/mL into the PC-LZM mixture, wherein the weight ratio of SA/LZM is 0.1, then moving to a magnetic therapy stirrer, and continuously stirring for 3 hours to obtain phycocyanin-sodium alginate/lysozyme coacervate;
in a preferable mode, the phycocyanin-sodium alginate/lysozyme coacervate in the step S2 is prepared by washing with deionized water three times.
Different metal ions have different influences on the stability of the natural pigment, some of the metal ions can fade the pigment, but some of the metal ions can play a role in stabilizing the pigment.
The microcapsule technology is a technology of coating a solid or liquid into fine particles using a film-forming material. Is a micro-packaging technology for storing solid, liquid and gas, and can control the release rate under the influence of certain conditions. Specifically, it means that a target (core or internal phase) is completely covered with various natural or synthetic polymer continuous films (walls or external phases) without damaging the original chemical properties of the target, and then the function of the target is externally exhibited again gradually by some external stimulation or slow-release action. The particle size of the microcapsule prepared usually can be 0.2-1000 μm, and the wall material thickness is 0.2-10 μm. With the progress of the technology, nanocapsules with a size of 1-1000 nm can be prepared. The microcapsule can improve the stability of the product and prevent the mutual interference among various components.
Since 1989, the ionic gel method is proposed for the first time, and is widely applied due to the function of the ionic gel method in the aspect of forming microcapsules, and the specific operations are as follows: under the stirring at room temperature, sodium Tripolyphosphate (TPP) without toxic and side effects is dripped into a Chitosan (CS) acetic acid solution, and phosphate radical ions with negative charges and protonated amino groups with positive charges on chitosan molecular chains induce ionic gelation through electrostatic attraction. The method has simple operation and mild reaction condition, and does not need to use organic solvent. Therefore, the method for preparing the microcapsule by adopting the ionic gel method to improve the stability of the phycocyanin is significant and urgently needed.
A method for embedding phycocyanin comprises the steps of utilizing a coacervate formed by sodium alginate and lysozyme as a carrier, utilizing phycocyanin as an embedding object, and obtaining phycocyanin-sodium alginate/lysozyme coacervate through electrostatic interaction between positive charges and negative charges.
The preparation method of the phycocyanin-sodium alginate/lysozyme polymer comprises the following specific steps:
s1, preparing phycocyanin-sodium alginate/lysozyme coacervate from a phycocyanin solution in an ionic gel mode;
the concrete operation of forming the agglomerate is as follows:
mixing and dripping 0.24mg/mL phycocyanin solution and sodium alginate and lysozyme solution in different mixing sequences, and stirring for 3h to obtain phycocyanin-sodium alginate/lysozyme coacervate;
the mass ratio of the sodium alginate solution to the lysozyme solution is 0.1,0.2,0.4 and 0.5;
s2, centrifuging the condensate obtained in the step S1 at 4 ℃ for 30min-1h under the condition of 9000r, and respectively collecting supernatant and lower-layer condensate;
and (3) dissolving the lower-layer condensed substance prepared in the step (S1) by using proper deionized water, and centrifugally cleaning for 3 times at 4 ℃ and 9000r to obtain a relatively pure phycocyanin-sodium alginate/lysozyme condensate.
Step S1, phycocyanin-sodium alginate/lysozyme coacervate is prepared in an ionic gel mode.
The preparation method of the phycocyanin-sodium alginate/lysozyme in the step S1 comprises the following steps:
1mL of prepared 10mg/mL lysozyme solution is taken respectively, sodium alginate solution and phycocyanin solution with the weight ratio of sodium alginate to lysozyme being 0.1,0.2,0.4 and 0.5 are prepared respectively, and the three solutions are dripped and mixed according to different mixing sequences. Stirring for 3-5 h at room temperature to form a condensate; then centrifuging the obtained sample at 4 ℃ under 9000r for 30min-1h; collecting supernatant and lower layer aggregate, respectively, dissolving the aggregate with deionized water, centrifuging at 4 deg.C and 9000r, and cleaning for 3 times to obtain phycocyanin-sodium alginate/lysozyme aggregate.
The phycocyanin-sodium alginate/lysozyme coacervate in the step S2 can be cleaned for 3 times by centrifugation to remove redundant salt and protein.
Phycocyanin used in the following examples was purchased from Zhejiang Binmei Bioltd, CAS No.: 11016-15-2; lysozyme was purchased under the product number of Shanghai Biotechnology engineering, inc.: a610308-0005; CAS number: 12650-88-3; sodium alginate was purchased in alatin cat #: s100126-500g; CAS number: 9005-38-3.
Example 1
A preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate comprises the following steps:
s1, dropwise adding 1ml of 0.24mg/ml phycocyanin solution into 1ml of 200000U/ml lysozyme solution, uniformly stirring, dropwise adding 1ml of 1mg/ml sodium alginate solution, and stirring for 3 hours at 25 ℃ to obtain an embedding solution;
s2, centrifuging the embedding solution obtained in the step S1 at 9000rpm for 30min, taking a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, wherein the precipitate is phycocyanin-sodium alginate/lysozyme complex coacervate. Wherein, the detection method of phycocyanin in the supernatant refers to the following documents:
Bennett A,Bogorad L.Properties of subunits and aggregates of blue-green algal biliproteins[J].Biochemistry,1971,10(19):3625-3634.
this embodiment may include pretreatment steps such as solution preparation, deionized water preparation, etc.
Example 2
A preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate comprises the following steps:
s1, dropwise adding 1ml of 0.24mg/ml phycocyanin solution into 1ml of 200000U/ml lysozyme solution, stirring and uniformly mixing, dropwise adding 1ml of 2mg/ml sodium alginate solution, and stirring for 3 hours at 25 ℃ to obtain an embedding solution;
s2, taking the embedding solution obtained in the step S1, centrifuging at 9000rpm for 30min, taking a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, wherein the precipitate is phycocyanin-sodium alginate/lysozyme complex coacervate. Wherein, the detection method of phycocyanin in the supernatant refers to the following documents:
Bennett A,Bogorad L.Properties of subunits and aggregates of blue-green algal biliproteins[J].Biochemistry,1971,10(19):3625-3634.
example 3
A preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate comprises the following steps:
s1, dropwise adding 1ml of 0.24mg/ml phycocyanin solution into 1ml of 200000U/ml lysozyme solution, uniformly stirring, dropwise adding 1ml of 4mg/ml sodium alginate solution, and stirring for 3 hours at 25 ℃ to obtain an embedding solution;
s2, centrifuging the embedding solution obtained in the step S1 at 9000rpm for 30min, taking a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, wherein the precipitate is a phycocyanin-sodium alginate/lysozyme complex coacervate; wherein, the detection method of phycocyanin in the supernatant refers to the following documents:
Bennett A,Bogorad L.Properties of subunits and aggregates of blue-green algal biliproteins[J].Biochemistry,1971,10(19):3625-3634.
example 4
A preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate comprises the following steps:
s1, dropwise adding 1ml of 0.24mg/ml phycocyanin solution into 1ml of 200000U/ml lysozyme solution, uniformly stirring, dropwise adding 1ml of 5mg/ml sodium alginate solution, and stirring for 3 hours at 25 ℃ to obtain an embedding solution;
s2, taking the embedding solution obtained in the step S1, centrifuging at 9000rpm for 30min, taking a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, wherein the precipitate is phycocyanin-sodium alginate/lysozyme complex coacervate. Wherein, the detection method of phycocyanin in the supernatant refers to the following documents:
Bennett A,Bogorad L.Properties of subunits and aggregates of blue-green algal biliproteins[J].Biochemistry,1971,10(19):3625-3634.
the thermal stability of the phycocyanin-sodium alginate/lysozyme complex coacervate prepared in the embodiment 1 of the invention is measured, and the result is shown in fig. 1, and the result shows that the stability of the phycocyanin is obviously reduced at the temperature of more than 53 ℃, and the phycocyanin-sodium alginate/lysozyme complex coacervate prepared by the ion crosslinking method is more stable than the phycocyanin. This is probably because the stability of phycocyanin is protected after the complex coacervate is formed.
The phycocyanin-sodium alginate/lysozyme complex coacervate prepared in example 1 of the present invention was taken and measured for pH stability, and the results are shown in fig. 2, and evaluated by monitoring the change in phycocyanin concentration with time (0 to 12 hours) under continuous shaking conditions. All formulations exhibited a two-step release profile, including a burst release within 2 hours followed by a sustained slower release for up to 12 hours. The reason for this is probably due to phycocyanin physically adsorbed to phycocyanin-sodium alginate/lysozyme complex coacervate, and further, phycocyanin was not substantially released when pH =3, the release rate at pH =8, which indicates that the closer the pH value was to alkalinity, the faster the release rate of complex coacervate was unstable.
The phycocyanin-sodium alginate/lysozyme complex coacervate prepared in the invention example 1 was taken, and the light stability thereof was measured, and the result is shown in fig. 3; in FIG. 3, "light + phycocyanin" is the stability of phycocyanin in disodium hydrogenphosphate-citric acid buffer solution at pH7.4 under uvb illumination, and "light + example 1" is the stability of phycocyanin-sodium alginate/lysozyme complex coacervate prepared in example 1 in disodium hydrogenphosphate-citric acid buffer solution at pH7.4 under uvb illumination; "dark + phycocyanin" is the stability of phycocyanin in disodium hydrogen phosphate-citric acid buffer solution with pH of 7.4 under the condition of no light; "dark + example 1" is the stability of the phycocyanin-sodium alginate/lysozyme complex coacervate prepared in example 1 in sodium phosphate dibasic-citric acid buffer solution at ph7.4 in the absence of light; the result shows that the phycocyanin shows a loss tendency under the uvb illumination condition, the complex coacervate is released more rapidly under the illumination condition than the complex coacervate without illumination, when the complex coacervate is released for 4 hours continuously, the release end point is reached, and the complex coacervate with illumination at the final release end point is higher than that of the sample without illumination, which shows that the illumination can accelerate the release of the phycocyanin-sodium alginate/lysozyme complex coacervate.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (3)
1. A preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate is characterized in that the embedding step comprises the following steps:
s1, dropwise adding 0.24mg/ml phycocyanin solution into 200000U/ml lysozyme solution, stirring and mixing uniformly, dropwise adding 1-5 mg/ml sodium alginate solution, and stirring for 2-6 h at 4-25 ℃ to obtain an embedding solution; the volume ratio of the phycocyanin solution to the sodium alginate solution to the lysozyme solution is 1;
and S2, taking the embedding solution obtained in the step S1, centrifuging to obtain a precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, and obtaining the phycocyanin-sodium alginate/lysozyme complex coacervate.
2. The method for preparing phycocyanin-sodium alginate/lysozyme complex coacervate according to claim 1, wherein the centrifugation parameters of step S2 are: the rotating speed is 9000rpm, and the time is 15-60 min.
3. The method for preparing phycocyanin-sodium alginate/lysozyme complex coacervate according to claim 1, comprising the steps of:
s1, dropwise adding 1ml of 0.24mg/ml phycocyanin solution into 1ml of 200000U/ml lysozyme solution, uniformly stirring, dropwise adding 1ml of 1mg/ml sodium alginate solution, and stirring for 3 hours at 25 ℃ to obtain an embedding solution;
and S2, centrifuging the embedding solution obtained in the step S1 at 9000rpm for 30min, taking the precipitate, washing the precipitate with deionized water until no phycocyanin exists in the supernatant, and obtaining the phycocyanin-sodium alginate/lysozyme complex coacervate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010336860.8A CN111481675B (en) | 2020-04-26 | 2020-04-26 | Preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010336860.8A CN111481675B (en) | 2020-04-26 | 2020-04-26 | Preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111481675A CN111481675A (en) | 2020-08-04 |
| CN111481675B true CN111481675B (en) | 2022-10-21 |
Family
ID=71810499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010336860.8A Active CN111481675B (en) | 2020-04-26 | 2020-04-26 | Preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111481675B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115176960B (en) * | 2022-07-04 | 2024-02-20 | 中国海洋大学 | Phycocyanin-rich thermostable type blue pigment and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102640933A (en) * | 2012-04-17 | 2012-08-22 | 中国科学院烟台海岸带研究所 | Phycocyanin microcapsule and preparation method of phycocyanin microcapsule |
| CN103341161B (en) * | 2013-06-20 | 2015-12-23 | 湖北省潜江市华山水产食品有限公司 | A kind of preparation method of sodium alginate-lysozyme compound microcapsule |
-
2020
- 2020-04-26 CN CN202010336860.8A patent/CN111481675B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| "Preparation, Characterization, and Antioxidant Properties of Phycocyanin Complexes Based on Sodium Alginate and Lysozyme";Bian-Wen Qiao et al.;《Frontiers in Nutrition》;20220524;第9卷;文献编号890942第1-11页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111481675A (en) | 2020-08-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111481675B (en) | Preparation method of phycocyanin-sodium alginate/lysozyme complex coacervate | |
| WO2007109244A2 (en) | Novel nanoparticles for delivery of active agents | |
| JPH0411254B2 (en) | ||
| Suzery et al. | Improvement of stability and antioxidant activities by using phycocyanin-chitosan encapsulation technique | |
| Jain et al. | Lectin conjugated gastro-retentive microspheres of amoxicillin for effective treatment of Helicobacter pylori | |
| Walke et al. | Physicochemical and functional characterization of chitosan prepared from shrimp shells and investigation of its antibacterial, antioxidant and tetanus toxoid entrapment efficiency | |
| CN108541866A (en) | A kind of cinnamic acid-sodium alginate-chitosan nanoparticle and preparation method thereof | |
| Volodkin et al. | Model system for controlled protein release: pH-sensitive polyelectrolyte microparticles | |
| Yanagawa et al. | Characterization of marigranules and marisomes, organized particles with elastin-like structures | |
| CN109276560B (en) | Lactoferrin-containing pH response type microcapsule and preparation method and application thereof | |
| Zhao et al. | Synthesis and applications of pectin-based nanomaterials | |
| CN114569582A (en) | Enzyme preparation, preparation method and application thereof | |
| Dong et al. | Encapsulation of vitamin C by a double-layer zein/chitosan structure with improved stability and controlled release | |
| Zhou et al. | A non-thermal modification method to promote the interaction of zein-alginate oligosaccharides composites for better encapsulation and stability—Cold plasma | |
| CN108324954B (en) | Phenylboronic acid hyperbranched modified magnetic nanosphere and preparation method and application thereof | |
| Zhu et al. | Fabrication of a ferritin–casein phosphopeptide–calcium shell–core composite as a novel calcium delivery strategy | |
| CN115530317B (en) | Method for improving stability and steady embedding of carotenoid in mango peel | |
| McManus et al. | Co-encapsulation by Flash NanoPrecipitation of Insulin, Trypsin Inhibitor and Caprate Permeabilization Enhancer for Oral Administration | |
| CN116831287A (en) | Pea protein-curcumin nanoparticle and preparation method thereof | |
| Jiang et al. | Polyanion/gelatin complexes as pH‐sensitive gels for controlled protein release | |
| CA2551564C (en) | A simplified method to retrieve chitosan from acidic solutions thereof | |
| CN110934304B (en) | Loaded vitamin D 3 Preparation method of modified chitosan nanoparticles | |
| JP4665131B2 (en) | POLYMER ELECTROLYTE COMPOSITE AND METHOD FOR PRODUCING THE SAME | |
| CN109096118B (en) | Aminofullerene derivative and preparation method and application thereof | |
| Zahrani et al. | Release profile of Andrographis paniculata leaf extract nanocapsule as α-glucosidase inhibitors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |