CN115191617A - Preparation method of sodium alginate-stabilized hypoglycemic collagen peptide liposome - Google Patents
Preparation method of sodium alginate-stabilized hypoglycemic collagen peptide liposome Download PDFInfo
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- CN115191617A CN115191617A CN202210827054.XA CN202210827054A CN115191617A CN 115191617 A CN115191617 A CN 115191617A CN 202210827054 A CN202210827054 A CN 202210827054A CN 115191617 A CN115191617 A CN 115191617A
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- collagen peptide
- sodium alginate
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- 238000002360 preparation method Methods 0.000 title claims abstract description 69
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method of a sodium alginate-stabilized hypoglycemic collagen peptide liposome, which comprises the following steps: preparing a lipid solution of a hydrophobic compound and glycerol, preparing an aqueous phase comprising collagen peptides, preparing a hydrated lipid solution, extruding, sodium alginate modification. The sodium alginate-stabilized blood sugar-reducing sheep collagen peptide liposome prepared by the invention exerts the blood sugar-reducing function through DPP-IV inhibitory activity, realizes the DPP-IV inhibitory activity of collagen peptide and the guarantee of the blood sugar-reducing activity of collagen peptide, and simultaneously adopts the raw materials to ensure that the sodium alginate-stabilized blood sugar-reducing sheep collagen peptide liposome is more suitable to be added into food.
Description
Technical Field
The invention belongs to the technical field of food, and particularly relates to a preparation method of a sodium alginate-stabilized hypoglycemic collagen peptide liposome.
Background
With the acceleration of the life rhythm of people, poor diet of a plurality of office workers causes a plurality of chronic disease patients. At present, the number of hyperglycemia patients in Chinese residents reaches 1.1 hundred million, and the hyperglycemia patients tend to rise, so that functional food and health-care food are favored by many chronic patients in the day, and the Chinese health-care food and functional food industry reaches the level of 2 trillion in 2021 year, wherein proteins, amino acids and peptides are the varieties with the largest demand.
The hypoglycemic collagen peptide is a functional food with the function of reducing blood sugar. But it, after oral entry into the organism, undergoes a series of barriers which can inactivate them, thus reducing their biological effect. The specific barrier is as follows: (1) the first barrier that collagen peptides experience after oral administration is the low pH environment of the stomach and the inactivation of peptides by pepsin by degradation; (2) the second barrier is the hydrolysis of various digestive enzymes in the intestinal tract, including pepsin, trypsin, chymotrypsin, and the like; (3) the third barrier is a carrier lacking collagen peptide with larger molecular weight on the cell membrane of the small intestine epithelium, so the carrier can pass through the cell membrane after being converted into dipeptide and tripeptide through enzymolysis. Therefore, the bioavailability of the hypoglycemic collagen peptide is very low when the hypoglycemic collagen peptide is directly orally taken, so that a carrier capable of effectively improving the bioavailability of the hypoglycemic collagen peptide is urgently needed.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above problems and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a preparation method of a sodium alginate-stabilized hypoglycemic collagen peptide.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a sodium alginate-stabilized hypoglycemic collagen peptide liposome comprises the following steps:
preparation of lipid solutions of hydrophobic compounds and glycerol: weighing phospholipid, dissolving in glycerol, and stirring to obtain lipid yellow solution;
preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in water; then stirring the collagen peptide solution to completely dissolve the collagen peptide powder, and filtering to remove solid impurities;
preparation of hydrated lipid solution: mixing and stirring a collagen peptide solution and the prepared lipid yellow solution to form a uniform hydrated lipid solution;
extruding: the hydrated lipid solution is subjected to extrusion circulation to obtain collagen peptide liposome preliminarily;
sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, mixing and stirring for 2 hours at normal temperature according to the volume of 1:1, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
As a preferable embodiment of the method for preparing the sodium alginate-stabilized hypoglycemic collagen peptide liposome of the present invention, it comprises preparing an aqueous solution containing collagen peptide, and adjusting the pH of the solution to 7.0.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the step of preparing phospholipid substances from one or more of liquid phospholipid, soybean phospholipid and soybean phosphatidylcholine.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the following steps of preparing an aqueous phase containing collagen peptide, wherein the mass ratio of water addition to phospholipid substances is 5-20.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the following steps of preparing an aqueous phase containing collagen peptide, wherein the mass ratio of water addition to phospholipid substances is 10.
The preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome comprises that the extrusion cycle times in the extrusion are 1-5.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the step of extruding, wherein the number of extrusion cycles is 3.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the step of adding 0.1-0.5% of sodium alginate.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the step of adding 0.3 percent of sodium alginate.
As a preferable scheme of the preparation method of the sodium alginate-stabilized hypoglycemic collagen peptide liposome, the preparation method comprises the step of preparing a lipid solution of a hydrophobic compound and glycerol, wherein the mass ratio of the hydrophobic compound to the glycerol is 3:1.
The invention has the beneficial effects that:
the sodium alginate-stabilized blood sugar reducing sheep collagen peptide liposome prepared by the invention has the blood sugar reducing activity by exerting the blood sugar reducing function through DPP-IV (dipeptidyl peptidase IV) inhibiting activity, and the liposome does not influence the DPP-IV inhibiting activity of the collagen peptide, and enables the collagen peptide to reduce the influence of three digestive barriers of a human body in oral administration so as to keep the blood sugar reducing activity.
The invention adopts glycerin as a membrane fixing agent instead of cholesterol, and is used as a solvent of phospholipid in the liposome, and a plurality of hydrogen bonds can be formed between the molecular layer and the phospholipid and inserted between phospholipid molecular membranes, so that the connection between phospholipid molecules is enhanced, a lipid bilayer membrane is reinforced, and the membrane flow is reduced; macroscopically, due to the characteristic of high viscosity, the liposome can effectively reduce membrane flow and drug permeation when added into the liposome, and improves the viscosity of the common liposome by three to four times. And moreover, as the glycerol is used as a membrane stabilizer, compared with the traditional liposome in which cholesterol is added to adjust the fluidity of a phospholipid molecular membrane, the glycerol has smaller side effect on a human body and is more suitable for being added into food.
The sodium alginate is adopted to modify the liposome, and the sodium alginate is added into the liposome, so that the viscosity of the liposome is improved by about ten times compared with that of the common liposome, membrane flow can be effectively reduced, drug leakage is reduced, and the entrapment rate is improved by about 10%. On the molecular layer, the liposome can react with the surface groups of the liposome through chemical bonds such as hydrogen bonds, ionic bonds and the like, so that the stability of the liposome is improved, and the slow release effect of the embedded activity is improved by about two to three times.
Compared with the traditional homogenizer and ultrasonic homogenization, the extruder extrusion method used by the invention has the advantages of small energy generation, low damage to phospholipid molecular membranes and small damage to collagen peptides.
The invention prepares the sheep collagen peptide for reducing blood sugar into liposome, ensures the bioactivity of the collagen peptide taken by oral administration, expands the application range of the sheep collagen peptide as a health product and the like, realizes the full utilization of resources and has good application prospect.
The preparation method and the actual operation method used by the invention are simple and easy for large-scale production.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a graph showing the change in zeta potential with the change in storage time at various temperatures measured in example 4;
FIG. 2 is a graph showing the change of the average particle size with the change of the storage time at different storage temperatures;
FIG. 3 is a graph showing the change in encapsulation efficiency with the change in storage time;
FIG. 4 is a graph of the change in PDI as a function of retention time;
FIG. 5 shows the bioactivity of collagen peptide in aqueous collagen peptide solution and in collagen peptide liposome solution over time;
FIG. 6 shows the change of DPP-IV inhibitory activity in the collagen peptide aqueous solution and the collagen peptide liposome solution with time;
FIG. 7 shows the change of the bioavailability of collagen peptide with time at different sodium alginate addition levels;
FIG. 8 shows a group of sodium alginate liposomes prepared according to the present invention under the microscope at a scale of 100 nm;
FIG. 9 shows a single sodium alginate liposome prepared by the present invention under the microscope at 500nm scale;
fig. 10 is a schematic structural diagram of a collagen peptide liposome prepared by the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The liquid phospholipid used in the examples of the present invention is a liquid phospholipid available from engineering technologies, ltd; collagen peptide is disclosed in application No. CN2020110865145Preparing sheepskin collagen peptide powder; the soybean phospholipid is soybean phospholipid powder phospholipid produced by engineering technology Limited liability company; the soybean phospholipid choline is produced by lipid
90G。
Example 1
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing liquid phospholipid, dissolving the liquid phospholipid in glycerol, and stirring at room temperature for 30 min to obtain uniform lipid yellow solution; wherein the feeding mass ratio of the liquid phospholipid to the glycerol is 3:1 (g/g); a yellow solution of lipids was prepared.
Preparation of an aqueous phase containing collagen peptides: weighing collagen peptide powder according to a phospholipid drug ratio of 2:1 (g/g), and dissolving the collagen peptide powder in deionized water, wherein the mass ratio of the water added in the collagen peptide solution to the liquid phospholipid is 10; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution passes through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 100rpm for 30 minutes at 20 c to form a homogeneous hydrated lipid solution.
Extruding: the hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature at a speed of 20 drops per minute, mixing and stirring the two liquids at 50rpm at normal temperature for 2 hours according to the volume ratio of 1:1, and standing at 4 ℃ for 12 hours to obtain the collagen peptide liposome stabilized by sodium alginate.
Example 2
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phospholipid, dissolving the soybean phospholipid in glycerol, and stirring at room temperature for 30 min to obtain uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phospholipids to the glycerol is 3:1 (g/g); a lipid yellow solution was prepared.
Preparation of an aqueous phase containing collagen peptides: weighing collagen peptide powder according to a phospholipid drug ratio of 2:1 (g/g), and dissolving the collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phospholipids is 10; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, mixing and stirring the two liquids at 50rpm at normal temperature for 2 hours according to the volume ratio of 1:1, and standing at 4 ℃ for 12 hours to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 3
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g); a lipid yellow solution was prepared.
Preparation of an aqueous phase containing collagen peptides: dissolving commercially available collagen peptide powder in deionized water, and weighing and dissolving the collagen peptide powder in the deionized water according to a phospholipid drug ratio of 2:1 (g/g), wherein the mass ratio of the added water to soybean phosphatidylcholine is 10 (g/g); the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, mixing and stirring the two liquids at 50rpm at normal temperature for 2 hours according to the volume ratio of 1:1, and standing at 4 ℃ for 12 hours to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 4
The sodium alginate-modified hypoglycemic collagen peptide liposomes prepared in examples 1 to 3 were collected, diluted 100-fold with deionized water, and subjected to particle size, PDI, and zeta potential measurement with a nano laser particle size analyzer. Three replicates were made for each sample, three replicates were machine measured, and the average was taken over three replicates.
The method for measuring the particle size, PDI and zeta potential comprises the following steps: particle size, PDI and zeta potential measurements were carried out using Zetapals, manufactured by Bruker Highen instruments, USA.
Table 1 influence of different wall materials on particle size and PDI of sodium alginate-modified hprp in examples 1 to 3
From table 1, it can be seen that different hydrophobic compounds have significant effects on liposome particle size and PdI, and the soybean phosphatidylcholine purities are in order from small to large: the liquid phospholipid is more than the soybean phospholipid and less than the soybean phosphatidylcholine, and the higher the purity of the soybean phosphatidylcholine is, the smaller the particle size and the PdI value of the soybean phosphatidylcholine are, and the more uniform and stable the liposome is. When the oil content of the soybean phosphatidylcholine is high (liquid phospholipid), phospholipid molecules are easy to agglomerate into larger particles, which is not beneficial to dispersion, so the particle size is larger, and the PdI is also larger. In summary, the hydrophobic compound is preferably soybean phosphatidylcholine in example 3, in combination with the average particle size and the size of PdI.
Considering the storage stability of the liposomes in example 3, the liposomes prepared in example 3 were wrapped with tinfoil and stored in a constant temperature and humidity chamber at three temperatures of 4 ℃. + -. 0.5 ℃, 25 ℃. + -. 0.5 ℃ and 37 ℃. + -. 0.5 ℃ in the dark, and the samples were photographed and the average particle size, pdI, zeta potential and encapsulation rate of the samples were measured every 5 days in a simulated refrigerator, room temperature and storage acceleration experiment. Particle size, pdi and zeta potential were measured as described in example 4, and encapsulation efficiency was measured as follows:
taking 0.5ml of each of the sodium alginate-modified collagen peptide liposomes prepared in the embodiment 3, placing the liposomes into a top sleeve of a Millipore ultrafiltration centrifuge tube (with the molecular weight cutoff of 100000 Da), placing the liposomes into a centrifuge 7000r/min for centrifugation for 15min, taking down the top sleeve, sucking liquid in the tube, measuring the content of the collagen peptide in the liquid, obtaining the concentration of free collagen peptide, and then utilizing the formula: e% = (x-x) 0 ) The encapsulation efficiency of the liposomes in the example was determined by/x × 100%, where E% is the encapsulation efficiency of the collagen peptide liposomes, x is the total amount of collagen peptide in the prepared liposomes, and x is 0 The total amount of free collagen peptide not encapsulated by the liposomes.
The data obtained for the zeta potential, average particle diameter, encapsulation efficiency and PdI as a function of the storage time are recorded in FIGS. 1 to 4.
As can be seen from FIGS. 1 to 4, the increase of particle size of the liposomes in 40 days at 4 ℃ is no more than 30nm, pdI is always below 0.3, the change of encapsulation efficiency is no more than 8%, and the zeta potential is less than-30 mV, which indicates that the liposomes have better storage stability, stable morphology and less leakage of the embedded collagen peptide under refrigeration.
The encapsulation efficiency of the product prepared in example 3 can reach 60% or more after the preparation is completed, and as the storage time increases, although the time increases, the encapsulation efficiency can be maintained to be high at a low storage temperature (4 ℃), and the encapsulation efficiency can be maintained to be good at normal temperature or a high storage temperature (37 ℃).
The liposomes of example 3 were examined for their gastrointestinal digestive stability, and the in vitro stability of the liposomes was evaluated by the tolerance of the encapsulated collagen peptide to conditions mimicking the pH of the gastrointestinal tract and the ability to resist degradation by digestive tract enzymes. The method for determining the in vitro simulated gastrointestinal digestion stability of the liposome comprises the following steps:
simulated artificial gastric fluid (SGF, containing 0.32% pepsin, pH 1.2) and simulated artificial intestinal fluid (SIF, containing 1% trypsin, pH 6.8, bile salt, 0.5M CaCl) were prepared 2 And 7.5M NaCl). 5mL of collagen peptide-loaded liposomes or collagen peptide solution diluted twice to the same peptide concentration was added to 5mL of artificial gastric juice and incubated at 37 ℃ with 100rpm shaking. Sampling 500. Mu.l at specified time intervals, immediately terminating the degradation reaction with 0.1M NaOH; after 1 hour, the artificial intestinal juice was added at 1:1 (v/v) for 2 hours and 500. Mu.l samples were taken at specified time intervals. All samples were subsequently treated with triton x-100 to release encapsulated collagen peptides, analyzed for residual collagen peptide concentration by BCA, and the bioavailability of residual collagen peptides was plotted against incubation time, and DPP-IV inhibitory activity against incubation time. Bioavailability (%) = c/c0 × 100%, c: collagen peptide concentration at a time point; c0: an initial collagen peptide concentration; bioavailability refers to the change in collagen peptide activity of a product during gastrointestinal digestion, i.e., the proportion of collagen peptides that can be utilized in vivo. Wherein, the DPP-IV inhibitory activity is measured by adopting a kit.
The resulting data for bioavailability and DPP-IV inhibitory activity over time during gastrointestinal digestion are reported in tables 5 and 6.
According to fig. 5-6, the bioavailability of the collagen peptide is increased and the DPP-IV inhibitory activity of the liposome embedded in the liposome is increased compared with that of the free collagen peptide aqueous solution, because the phospholipid bilayer of the liposome protects the collagen peptide from gastric acid in the gastrointestinal tract and enzyme damage and inactivation, which shows that the prepared collagen peptide liposome has the effects of protecting the collagen peptide from gastrointestinal tract digestion and protecting the DPP-IV inhibitory activity from damage.
Example 5
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 5:1 (g/g); the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: and (3) extruding and circulating the hydrated lipid solution at 25 ℃ for 3 times (manually extruding and passing through a 100nm polycarbonate membrane), wherein the particle size of the liposome is about 80-120 nm, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 6
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 10 (g/g); the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 7
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving commercially available collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 20; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 8
The sodium alginate-modified collagen peptide liposomes prepared in examples 5 to 7 were collected, diluted 100-fold with deionized water, and the particle size, PDI, and zeta potential were measured with a nano laser particle size analyzer. Each sample is subjected to three repetitions, three groups of parallel samples are measured by a machine, the average value is obtained by the three repetitions, the encapsulation efficiency and the encapsulation capacity are determined, and the calculation method of the encapsulation capacity is as follows:
the encapsulating amount of the liposome is calculated based on the encapsulating rate of the liposome, the characteristic is the mass of the collagen peptide which can be embedded in the unit mass of the liposome, and the calculating formula of the encapsulating amount is as follows: z theory (mg/g) = x/m; zitual (mg/g) = ztheoretical × E; in the formula, x is the total amount of collagen peptide in the prepared liposome, and m is the liposome mass, and the obtained data are recorded in table 2.
TABLE 2 Mass ratio of Water addition to Soybean Phosphatidylcholine in collagen peptide solutions in examples 5-7 influence of entrapment efficiency, entrapment amount, particle size, pdI and zeta potential in sodium alginate-modified collagen peptide liposomes
As can be seen from table 2, the mass ratio of the water addition amount in the collagen peptide solution to the soybean phosphatidylcholine in examples 5 to 7 has a significant effect on the entrapment rate, the particle size, and the PdI of the prepared sodium alginate-modified collagen peptide liposome, and as the mass ratio increases, the entrapment rate and the entrapment amount both show a tendency of increasing first and then decreasing, and when the mass ratio is 10; similarly, the average particle size and PdI both showed a tendency to decrease. The mass ratio is not greatly influenced by the zeta potential, and the solutions are in a stable state (zeta < -30). Therefore, the mass ratio of the added water amount in the collagen peptide solution in the hydrated lipid solution to the soybean phosphatidylcholine is preferably 10.
Example 9
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to soybean phosphatidylcholine is 10 (g/g); the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then maintaining 50rpm at normal temperature, and removing solid impurities by passing the obtained clear solution through a water film with the aperture of 0.22 mu m; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the hydrated lipid solution was passed through 1 extrusion cycle (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, mixing and stirring for 2 hours at normal temperature according to the volume of 1:1, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 10
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 10; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then maintaining 50rpm at normal temperature, and removing solid impurities by passing the obtained clear solution through a water film with the aperture of 0.22 mu m; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, mixing and stirring for 2 hours at normal temperature according to the volume of 1:1, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 11
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 10; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 5 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 12
The sodium alginate-modified collagen peptide liposomes prepared in examples 9 to 11 were collected, diluted 100-fold with deionized water, and the encapsulation efficiency, particle size, PDI, and zeta potential were measured with a nano laser particle size analyzer. Three replicates were made for each sample, three replicates were machine measured, the average was taken over the three replicates and the resulting data is recorded in table 3.
TABLE 3 influence of number of extrusion cycles on encapsulation efficiency, particle size, pdI and zeta potential in sodium alginate-modified collagen peptide liposomes in examples 9 to 11
As can be seen from table 3, the improvement effect of the encapsulation efficiency shows a tendency of increasing first and then decreasing, the average particle diameter, and the improvement effect of Pdi shows a tendency of decreasing, considering the combination of the encapsulation efficiency, the amount of encapsulation, the average particle diameter, pdi, zeta potential, and combining the time and cost of the increase of the cycle number, the preference for the cycle number is 3.
Example 13
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 10; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 14
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to the soybean phosphatidylcholine is 10; the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.3% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 15
Preparation of lipid solutions of hydrophobic compounds and glycerol: weighing soybean phosphatidylcholine, dissolving the soybean phosphatidylcholine in glycerol, and stirring at room temperature for 30 minutes to obtain a uniform lipid yellow solution; wherein the feeding mass ratio of the soybean phosphatidylcholine to the glycerol is 3:1 (g/g).
Preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in deionized water, wherein the mass ratio of the added water to soybean phosphatidylcholine is 10 (g/g); the collagen peptide solution was then stirred at 20 ℃ for 10 minutes until the collagen peptide powder was completely dissolved, forming a clear solution. Then, the obtained clear solution is passed through a water film with the aperture of 0.22 mu m at normal temperature to remove solid impurities; the pH of the aqueous phase was then adjusted to 7.0.
Preparation of hydrated lipid solution: the collagen peptide solution was mixed with the lipid yellow solution prepared above and stirred at 20 ℃ for 30 minutes to form a homogeneous hydrated lipid solution.
Extruding: the above hydrated lipid solution was passed through 3 extrusion cycles (manual extrusion through a 100nm polycarbonate membrane) at 25 ℃. The particle size of the liposome is about 80-120 nm after extrusion circulation, and the collagen peptide liposome is obtained primarily.
Sodium alginate modification: dripping the collagen peptide liposome suspension into 0.5% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, keeping 50rpm at normal temperature according to the volume of 1:1, mixing and stirring for 2 hours, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
Example 16
The sodium alginate-modified hypoglycemic collagen peptide liposomes prepared in examples 13 to 15 were collected, diluted 100-fold with deionized water, and the particle size, PDI, zeta potential, and DPP-IV inhibitory activity were measured using a nano laser particle size analyzer. Three replicates were made for each sample, three replicates were machine measured, and the average was taken over three replicates.
The resulting data are recorded in table 4.
TABLE 4 influence of sodium alginate addition amount on encapsulation efficiency, encapsulation amount, particle size, pdI, zeta potential and DPP-IV inhibitory Activity in sodium alginate-modified collagen peptide liposomes in examples 13 to 15
As can be seen from table 4, the addition amount of sodium alginate in examples 13 to 15 has a significant effect on the particle size and PdI of the prepared sodium alginate-modified collagen peptide liposome, and as the addition amount of sodium alginate increases, the encapsulation efficiency, the encapsulation amount, the average particle size, pdI, and the absolute value of zeta potential all show a tendency to increase. The reason is that the thickness of the sodium alginate wrapped on the liposome is increased along with the increase of the addition amount of the sodium alginate, so that the particle size and PdI are increased, the encapsulation efficiency is increased because the addition amount of the sodium alginate has little influence on the DPP-IV inhibitory activity, and the influence degree of the modification of the sodium alginate on the DPP-IV inhibitory activity of the collagen peptide per se is small.
Based on the increase of the added amount of sodium alginate, more sodium alginate needs to be used, and relatively speaking, more processing time is needed for higher added amount of sodium alginate, in the case that the average particle size is 249.5nm in example 14, the average particle size is larger due to the continuous increase of the content of sodium alginate, and in the case that the added amount of sodium alginate is further increased, pdI is more than 0.3, and in combination with the above consideration, the preferable concentration of 0.3% used in example 14 is the concentration of sodium alginate. A means of
Examples 13-15 the gastrointestinal digestive stability of liposomes, the in vitro stability of liposomes was evaluated by the tolerance of the encapsulated collagen peptide to the environment mimicking the gastrointestinal pH and the resistance to degradation by digestive enzymes, and figure 7 shows the effect of different sodium alginate addition levels on the bioavailability of liposomes in the gastrointestinal tract.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A preparation method of a sodium alginate-stabilized hypoglycemic collagen peptide liposome is characterized by comprising the following steps: the method comprises the following steps:
preparation of lipid solutions of hydrophobic compounds and glycerol: weighing phospholipid substances, dissolving in glycerol, and stirring to obtain lipid yellow solution;
preparation of an aqueous phase containing collagen peptides: dissolving collagen peptide powder in water; then stirring the collagen peptide solution to completely dissolve the collagen peptide powder, and filtering to remove solid impurities;
preparation of hydrated lipid solution: mixing and stirring a collagen peptide solution and the prepared lipid yellow solution to form a uniform hydrated lipid solution;
extruding: the hydrated lipid solution is subjected to extrusion circulation to obtain collagen peptide liposome preliminarily;
sodium alginate modification: dripping the collagen peptide liposome suspension into 0.1% sodium alginate aqueous solution at normal temperature according to the speed of 20 drops per minute, mixing and stirring for 2 hours at normal temperature according to the volume of 1:1, and standing for 12 hours at 4 ℃ to obtain the collagen peptide liposome stabilized by the sodium alginate.
2. The method for preparing the sodium alginate-stabilized hypoglycemic collagen peptide liposome according to claim 1, wherein the method comprises the following steps: the preparing an aqueous solution comprising collagen peptides further comprises adjusting the pH of the solution to 7.0.
3. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome of claim 1, wherein the method comprises the following steps: the phospholipid material comprises one or more of liquid phospholipid, soybean phospholipid, and soybean phosphatidylcholine.
4. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome of claim 1, wherein the method comprises the following steps: the preparation method comprises the following steps of preparing an aqueous phase containing collagen peptide, wherein the mass ratio of the water addition amount to the phospholipid substances is 5-20.
5. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome as claimed in claim 1 or 4, wherein: the preparation method comprises the following steps of preparing an aqueous phase containing collagen peptide, wherein the mass ratio of water addition to phospholipid substances is 10.
6. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome of claim 1, wherein the method comprises the following steps: in the extrusion, the number of extrusion cycles is 1 to 5.
7. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome according to claim 1 or 6, wherein the method comprises the following steps: in the extrusion, the number of extrusion cycles was 3.
8. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome of claim 1, wherein the method comprises the following steps: the addition amount of the sodium alginate is 0.1-0.5%.
9. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposomes according to claim 1 or 8, wherein the method comprises the following steps: the addition amount of the sodium alginate is 0.3%.
10. The method for preparing sodium alginate-stabilized glycemic collagen peptide liposome of claim 1, wherein the method comprises the following steps: in the preparation of the lipid solution of the hydrophobic compound and the glycerol, the mass ratio of the hydrophobic compound to the glycerol is 3:1.
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| CN114668152A (en) * | 2022-03-14 | 2022-06-28 | 北京林业大学 | ACE inhibitory peptide liposome with high stability and directional release in small intestine and preparation method thereof |
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