A nano-liposome loaded with fish collagen peptide, its preparation method and application

Abstract

The present invention discloses a nano-liposome loaded with fish collagen peptides, its preparation method and application, belonging to the fields of polypeptide and bioproduction processing. In the present invention, fish collagen peptide solid powder is dissolved in pure water, and further, soybean lecithin and phytosterol are added to the collagen polypeptide solution. The above substances are fully mixed by high-speed homogenization and whipping, and then the emulsion is introduced into a microfluidic homogenizer for high-pressure homogenization. Subsequently, the emulsion is diluted and added to tangential flow ultrafiltration, and diluted and ultrafiltered repeatedly for multiple times to separate the liposome from the free peptide, obtain high-purity liposomes, and at the same time realize the recovery and reuse of the free peptide. Compared with the current mainstream preparation methods, its preparation method has the advantages of not introducing organic solvents, simplifying the process, recycling the unentrapped raw materials, and reducing raw material waste.

Description

Technical Field

[0001] The present invention relates to a nano-liposome loaded with fish collagen peptide, its preparation method and application, and belongs to the fields of polypeptide and bioproduction processing. Background Art

[0002] Fish collagen peptide is mainly a product prepared by protease hydrolysis of collagen extracted from fish processing by-products such as fish scales, skin, and bones. Collagen peptide has functional activities such as antioxidant, prevention of cardiovascular and other related diseases, and promotion of the synthesis of skin collagen and hyaluronic acid. Collagen peptide is mainly absorbed into the blood by intestinal epithelial cells in the form of oligopeptides. However, oral collagen peptide is easily affected by the acid-base environment and digestive enzymes in the gastrointestinal tract and is degraded by blood peptidases after being absorbed into the blood, resulting in a gradual decrease in the retention of collagen peptide in the blood, thereby reducing its oral bioavailability. According to research, it is necessary to orally intake 15 - 30 g of collagen peptide daily to exert its biological activity.

[0003] Nano-liposome uses amphiphilic lipids as wall materials to encapsulate polypeptides through forms such as thin-film hydration and reverse evaporation, and then prepares nano-liposome emulsion by microfiltration and high-pressure homogenization methods. Due to its high similarity to the human cell membrane structure, it can achieve efficient delivery of polypeptides through endocytosis, macropinocytosis and other ways. Phospholipid is a by-product of vegetable oil refining and processing. Because it can be obtained in large quantities, has a low cost, and has excellent emulsifying properties, it has become an ideal wall material for nano-carriers and is widely used in the fields of drug and nutrition delivery. However, due to the limitation of the oil-water interfacial tension performance of phospholipids, the particle size of the liposomes formed by itself is difficult to reduce to below 200 nm, and the stability is also poor. Usually, cholesterol is needed to form nano-liposome emulsion. However, cholesterol increases the burden of lipid metabolism in the body and has a very adverse effect on human health.

[0004] Phytosterol is a non-saponifiable by-product generated during the extraction and processing of legume and cereal oils. It has a high melting point, poor fluidity, and a low price, and has not been fully utilized at present. Although the structure of phytosterol is similar to that of cholesterol, it will not be absorbed by the human body and can combine with cholesterol in the human intestine to reduce the absorption of cholesterol in the intestine and lower the cholesterol intake level. Phytosterol is a good choice to replace cholesterol as the wall material of nano-liposome. Adding phytosterol to nano-liposome can reduce membrane fluidity, increase the stability of nano-liposome, and facilitate the application of nano-liposome in complex food systems.

[0005] Currently, the mainstream methods for preparing liposomes include the thin-film dispersion method, ethanol injection method, reverse evaporation method, etc. However, due to the involvement of rotary evaporation and a large amount of organic reagents, these methods have certain safety risks. Microfluidic homogenization technology is an efficient homogenization method for the preparation of nanoparticles, cells, and other biological materials. It realizes the refinement and homogenization of materials through high-speed jetting. This technology is widely used in fields such as drug delivery, bioengineering, synthesis of nanomaterials, and biological experimental research, and can produce nanoliposomes with uniform particles and stable properties. Summary of the Invention

[0006] To solve the above technical problems, the object of the present invention is to provide a nano-liposome loaded with fish collagen peptide and its preparation method.

[0007] The technical solutions adopted to achieve the object of the present invention are as follows:

[0008] Dissolve the solid powder of fish collagen peptide (FCP) in pure water, and further add different proportions of soy lecithin and phytosterol to the collagen polypeptide solution. Homogenize the above substances by high-speed homogenization to make them fully mixed, and then introduce the emulsion into a microfluidic homogenizer for high-pressure homogenization. Subsequently, dilute the emulsion and add it to tangential flow ultrafiltration, and dilute and ultrafilter it repeatedly for multiple times to separate the liposomes from the free peptides, obtain high-purity liposomes, and at the same time realize the recovery and reuse of the free peptides.

[0009] The preparation method of NL-FCP specifically includes the following steps:

[0010] (1) Dissolve the solid powder of FCP in water to prepare an FCP solution with a mass concentration of 10 - 40%. Add phytosterol and soy lecithin to the FCP solution according to a mass ratio of 1:5 - 1:50, and homogenize for 5 - 10 minutes under the condition that the rotational speed of the high-speed shear homogenizer is 10000 - 17000 rpm to make the emulsion fully mixed and uniform.

[0011] (2) Introduce the emulsion after high-speed homogenization treatment into a high-pressure microfluidic device and circulate and homogenize it 1 - 5 times under the condition of a homogenization pressure of 40 - 200 Mpa.

[0012] (3) Dilute the NL-FCP emulsion treated by the microfluidic homogenizer 5 - 10 times with pure water, and then add it to tangential flow ultrafiltration to separate NL-FCP from free FCP, and obtain an NL-FCP emulsion with an encapsulation efficiency of over 90% through multiple cycles.

[0013] (4) Spray-dry the NL-FCP emulsion to obtain an NL-FCP powder product.

[0014] Preferably, in step (1) of the present invention, the optimization of the material addition ratio is carried out. The mass concentration of FCP is 40%, and the mass ratio of FCP:soybean lecithin:phytosterol = 60:30:1. Under this material selection condition, the encapsulation efficiency of NL-FCP is relatively high and the stability is good.

[0015] Preferably, in step (2) of the present invention, a high-pressure microfluidization homogenization device is used, the homogenization pressure is 80 Mpa, and the number of homogenization times is 3 times. Under this process condition, the particle size of NL-FCP can be reduced and the stability of the nanoliposome can be increased.

[0016] Preferably, in step (3) of the present invention, tangential flow ultrafiltration is used, and the tangential flow ultrafiltration device used is numbered MasterRex ® 07514-10, and the specification of the tangential filter membrane used is 10 kDa. The unentrapped components can pass through this specification of tangential filter membrane, while the nanoliposomes will be retained, thus realizing the separate collection of NL-FCP and unentrapped FCP.

[0017] The technical advantages of the present invention are mainly reflected in:

[0018] (1) Compared with the mainstream liposome preparation methods such as the thin film dispersion method and the ethanol injection method, the present invention reduces the cumbersome processes such as rotary evaporation and does not introduce organic solvents, which conforms to the characteristics of being green and low-cost and is suitable for large-scale production.

[0019] (2) The present invention separates liposomes and free polypeptides through a tangential flow ultrafiltration device. The separation of liposomes and free polypeptides and the recovery of free collagen polypeptides are realized, and the raw materials can be supplemented for re-embedding, reducing the waste of raw materials.

[0020] (3) This invention is more suitable for industrial production by direct hydration, without adding organic solvents such as ethanol and simplifying the process. It also recycles the unentrapped collagen peptides, avoids waste, and reduces the industrial production cost. Description of the Drawings

[0021] The following further details the specific embodiments of the present invention in conjunction with the drawings. Hereinafter, the high-pressure microfluidization conditions are defaulted to a pressure of 200 MPa and a cycle of 3 times.

[0022] Figure 1 Effect of adding different contents of soybean lecithin (compared with the mass of FCP) on the encapsulation efficiency of NL-FCP (A) 10%; (B) 20%; (C) 30%; (D) 40%.

[0023] Figure 2Effect of adding different contents of phytosterol (compared with the mass of FCP) at a ratio of soy lecithin to FCP of 2:1 on the encapsulation efficiency, particle size, ζ-potential and stability of NL-FCP (A) Encapsulation efficiency; (B) Particle size of NL-FCP at room temperature (25°C) and after 7 days of storage; (C) ζ-potential of NL-FCP at room temperature (25°C) and after 7 days of storage.

[0024] Figure 3 Effect of different pressures on the encapsulation efficiency, particle size, ζ-potential of NL-FCP under the condition of FCP:soy lecithin:phytosterol mass ratio of 60:30:1 (A) Encapsulation efficiency; (B) Particle size; (C) ζ-potential.

[0025] Figure 4 Effect of different cycle numbers on the encapsulation efficiency, particle size, ζ-potential of NL-FCP under the condition of FCP:soy lecithin:phytosterol mass ratio of 60:30:1 (A) Encapsulation efficiency; (B) Particle size; (C) ζ-potential.

[0026] Figure 5 Performance characterization of NL-FCP after tangential flow ultrafiltration treatment

[0027] Figure 6 Particle size and ζ-potential distribution diagrams of NL-FCP after tangential flow ultrafiltration treatment. (A) Particle size; (B) ζ-potential.

[0028] Figure 7 Images of NL-FCP at different magnifications of transmission electron microscope (TEM). (A) 2 μm; (B) 500 nm; (C) 100 nm; (D) 50 nm.

[0029] Figure 8 NL-FCP emulsion and spray-dried powder. (A) Emulsion; (B) Powder product.

[0030] Figure 9 Pharmacokinetic curves of total Hyp (A) and free Hyp (B) of oral fish collagen peptide and NL-FCP

[0031] Figure 10 Area under the concentration-time curve of oral fish collagen peptide and NL-FCP Detailed implementation manners

[0032] Example 1

[0033] This example provides the preparation of fish collagen peptide nanoliposomes, and the specific technical scheme steps are as follows:

[0034] Weigh 30 g of FCP and dissolve it in 45 mL of pure water, and fully dissolve it under a magnetic stirrer. Weigh 15 g of soy lecithin and 0.5 g of phytosterol and add them to the FCP solution, and then homogenize at a high speed of 13,000 rpm for 5 min to fully mix the materials and form an emulsion. Introduce the emulsion into a high-pressure microfluidic device and circulate it 3 times under the condition of a pressure of 80 Mpa.

[0035] The Lowry method was used to determine the protein concentration, so as to determine the encapsulation rate of collagen peptide nanoliposomes. After diluting the collagen peptide nanoliposome emulsion by n times, add an equal volume of 5% Triton X-100 to demulsify it, and use the Lowry protein concentration determination method to measure the total protein concentration as G1. Then take 5 mL of the diluted sample and add it to an ultrafiltration tube with a specification of 10 kDa, centrifuge at 2000×g for 10 min, and use the Lowry method to measure the protein concentration of the lower layer as G2. The encapsulation rate of NP-FCP is calculated according to the following formula (1):

[0036] Encapsulation rate (%) = ……………………………… (1)

[0037] In the formula, G1 is the total protein concentration;

[0038] G2 is the protein concentration of the filtrate after ultrafiltration;

[0039] n is the dilution factor of the sample before ultrafiltration.

[0040] Dilute the NL-FCP emulsion treated by microfluidic homogenization 5 times with pure water, add it to a tangential flow ultrafiltration device for ultrafiltration, and circulate it 3 times. Separate the free collagen peptides of NL-FCP to obtain liposomes with a higher encapsulation rate ( Figure 5 ), recover the tangential filter filtrate, and it can be reused after concentration. The NL-FCP powder obtained by spray drying is as Figure 8 shown.

[0041] Example 2

[0042] This example provides a method for characterizing the stability and morphology of the NP-FCP prepared in Example 1.

[0043] Dissolve the NP-FCP powder obtained in Example 1 at a concentration of 5 mg / mL, put it into the container pool specified by the system, and at 25°C, use dynamic light scattering (DLS) to measure the average particle size and measure the surface charge of the liposomes using the same system ( Figure 6 )

[0044] Take 10 μL of the nanoliposomes and dilute them in 990 μL of pure water. Drop the diluted liposomes onto a copper mesh coated with a carbon film, let it stand for 3 min, then stain with 2% phosphotungstic acid for 5 min, and dry it. Finally, observe the micro-morphology under a transmission electron microscope at 200 kV ( Figure 7 ).

[0045] Example 3

[0046] This example provides a method for evaluating the oral bioavailability of the NP-FCP prepared in Example 1.

[0047] After male SD rats at 7-8 weeks of age were fed with AIN-93M standard feed and sufficient water for 1 week to adapt, they were divided into a blank group (normal saline) and a test group (fish collagen peptide, collagen peptide nanoliposomes), with 6 rats in each group. The gavage dose was calculated based on the hydroxyproline content of 82 mg / kg. Blood was collected from the anticoagulant tube at the tail of the rats at 0, 0.5, 1, 2, 4, and 8 h after gavage. After the blood samples were allowed to stand at 4°C for 30 min, they were centrifuged at 3000 rpm / min for 15 min, and the supernatant was stored at -80°C. The plasma samples were used for the determination of the total hydroxyproline and free hydroxyproline contents.

[0048] Accurately weigh 0.1 g of the powdered test substance into an ampoule, add 2 mL of 6 mol / L hydrochloric acid solution, seal the bottle with nitrogen, hydrolyze at 110°C for 16 h, then take it out and cool. Adjust the pH value of the hydrolysis solution to the range of 6-8 with 2 mL of 6 mol / L sodium hydroxide solution. Take 50 μL of the hydrolysis solution into a 1.5 mL centrifuge tube, add 50 μL of chloramine T reagent, mix and let it stand at room temperature for 20 min; then add 50 μL of the color reagent and 100 μL of water, mix well and seal; place the test tube in a 60°C water bath and heat for 20 min; finally, transfer the liquid to a 96-well microplate and measure its absorbance at 560 nm. Prepare a hydroxyproline standard solution (0.469, 0.938, 1.875, 3.75, 7.5, 15 μg / mL), and make a standard curve according to the above method to obtain the standard curve y = 0.0215x + 0.0925 (R 2 = 0.9989). The plasma samples were alkali-hydrolyzed with 4 mol / L NaOH at 100°C for 30 min; the chloroamine T method was used to determine the total hydroxyproline and free hydroxyproline contents in the plasma, and the absorbance value was detected at a wavelength of 560 nm and the hydroxyproline content was calculated

[0049] Taking the blood collection time as the abscissa and the plasma peptide-bound hydroxyproline content as the ordinate, plot the pharmacokinetic curve. Calculate the area under the blood concentration-time curve (AUC) and the incremental area under the curve (iAUC) of each group from 0 to 8 h according to the following formula (3) by mathematical integration. The calculation results are as followsFigure 9 and Figure 10 As shown in Figure 10 , the bioavailability of fish collagen peptide after liposome encapsulation increased significantly, while the content of free hydroxyproline released by peptidase hydrolysis was lower than that of directly orally administered fish collagen peptide.

[0050] iAUC 受试制剂 = AUC 受试制剂 - AUC 空白对照 …………………………(3)

[0051] The above embodiments illustrate and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention, rather than limiting the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the claimed invention.

Claims

1. A preparation method of nano-liposomes loaded with fish collagen peptide (FCP), characterized in that, The nano-liposomes contain fish collagen peptides, soy lecithin and phytosterols, and are prepared by combining high-speed shear homogenization, high-pressure microfluidization technology and tangential flow ultrafiltration. The preparation method of the nano-liposomes loaded with fish collagen peptides specifically includes the following steps: (1) Dissolve the solid powder of fish collagen peptides in water to prepare an FCP solution with a mass concentration of 10-40%. Add phytosterols and soy lecithin to the FCP solution at a mass ratio of 1:5-1:50, and homogenize for 5-10 minutes under the condition that the rotation speed of the high-speed shear homogenizer is 10,000-17,000 rpm to make the emulsion fully mixed and uniform. (2) Introduce the emulsion treated by high-speed shear homogenization into a high-pressure microfluidization device, and circulate and homogenize 1-5 times under the condition of a homogenization pressure of 40-200 Mpa to obtain an NL-FCP emulsion. (3) After diluting the NL-FCP emulsion treated by the high-pressure microfluidization device 5-10 times with pure water, add it to tangential flow ultrafiltration to separate NL-FCP from free FCP, and obtain an NL-FCP emulsion with an encapsulation efficiency of over 90% through multiple cycles. (4) Spray-dry the NL-FCP emulsion to obtain an NL-FCP powder product. Among them, in step (3), tangential flow ultrafiltration is used, and the tangential flow ultrafiltration device used is numbered MasterRex®07514-10, and the specification of the tangential filter membrane used is 10 kDa.

2. The preparation method of the nano-liposome loaded with fish collagen peptide according to claim 1, wherein, In step (1), the mass concentration of FCP is 40%, and the mass ratio of FCP:soy lecithin:phytosterol is 60:30:

1.

3. The preparation method of the nano-liposome loaded with fish collagen peptide according to claim 1, characterized in that, In step (2), a high-pressure microfluidization device is used, the homogenization pressure is 80 Mpa, and the number of homogenization times is 3 times.

Images