CN112956680B - Plant oil body-imitated core-shell liposome and preparation method thereof - Google Patents

Plant oil body-imitated core-shell liposome and preparation method thereof Download PDF

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CN112956680B
CN112956680B CN202110184450.0A CN202110184450A CN112956680B CN 112956680 B CN112956680 B CN 112956680B CN 202110184450 A CN202110184450 A CN 202110184450A CN 112956680 B CN112956680 B CN 112956680B
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oil body
liposome
vegetable oil
body protein
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CN112956680A (en
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唐文婷
蒲传奋
王富丽
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Qingdao Agricultural University
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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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/045—Organic compounds containing nitrogen as heteroatom
    • 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
    • A23L29/03—Organic compounds
    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • 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
    • A23L29/03—Organic compounds
    • A23L29/05—Organic compounds containing phosphorus as heteroatom
    • 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
    • A23L29/238—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seeds, e.g. locust bean gum or guar gum
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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

Abstract

The invention discloses a simulated vegetable oil body core-shell liposome and a preparation method thereof. The plant oil body-imitating core-shell liposome prepared by the invention is based on the unique interfacial activity of plant oil body protein, good water dispersibility and thickening property of nonionic polysaccharide, polar groups on a straight chain and steric hindrance effect of the polar groups, and not only can the stability of the liposome to environmental factors be improved, but also the slow release of a load substance can be realized. The vegetable oil body protein and the nonionic polysaccharide used for stabilizing the liposome are all food sources, the price is low, the liposome can be safely and effectively applied to the field of foods, and the provided method is simple to operate and starts from large-scale popularization and application.

Description

Plant oil body-imitated core-shell liposome and preparation method thereof
Technical Field
The invention relates to the field of health food processing, in particular to a vegetable oil body-imitated core-shell liposome and a preparation method thereof.
Background
Liposomes are ultramicro spherical particles with a bilayer structure formed by self-aggregation of phospholipids dispersed in water. The hydrophilic ends of the phospholipids form the inner and outer surfaces of the vesicles, while the tails of the water-transporting fatty acid chains form the hydrophobic core region of the bilayer. As an excellent transport carrier, the liposome has good biocompatibility, can embed hydrophilic and hydrophobic core materials simultaneously, improves the dispersibility, stability and bioavailability of the core materials, and realizes targeted release and transmission. However, due to the sensitivity of Oswald ripening and environmental factors (such as pH, temperature, oxygen, enzymes, ionic strength), liposomes are susceptible to aggregation, fusion, flocculation, and oxidation during storage and use, resulting in a change in particle size and a loss of structural integrity. The liposome used as an oral preparation is very easily affected by pH and enzyme in the process of digestion and absorption, so that the phospholipid wall is hydrolyzed, the membrane permeability is enhanced, the core material leaks, and the biological functional property of the liposome is greatly influenced. Therefore, the application of liposome as a delivery system in medicine and food is limited to model research on specific functional ingredients or nutritional supplements. Therefore, the targeted controllable modification of the liposome to prepare the stable novel liposome widens the application range of the liposome in the fields of medicine, food and the like, and is a technical problem to be solved in the field.
Disclosure of Invention
The invention aims to provide a plant oil body-like core-shell liposome and a preparation method thereof, and aims to solve the problems.
In order to achieve the purpose, the invention provides the following technical scheme:
one of the technical schemes of the invention is as follows: the natural phospholipid loaded with carotenoid is used as a core, and the vegetable oil body protein and the nonionic polysaccharide coated on the surface are used as shells to form the vegetable oil body imitating core-shell type liposome.
The second technical scheme of the invention is as follows: the preparation method of the plant oil body imitating core-shell type liposome comprises the following steps:
(1) dissolving natural phospholipid, cholesterol, surfactant and carotenoid in solvent, and performing rotary evaporation under reduced pressure to form transparent lipid membrane;
(2) dispersing vegetable oil body protein into a phosphate buffer solution, adding the transparent lipid membrane prepared in the step (1), and performing ultrasonic treatment to prepare a vegetable oil body protein stable liposome;
(3) and (3) adding the plant oil body protein stable liposome prepared in the step (2) into a phosphate buffer solution dissolved with nonionic polysaccharide to prepare the plant oil body imitated core-shell type liposome.
The core-shell liposome imitating the vegetable oil body is actually liposome stabilized by oil body protein and nonionic polysaccharide.
Preferably, the natural phospholipid in the step (1) is egg yolk lecithin; the surfactant is Tween-80; the carotenoid is beta-cryptoxanthin; the solvent is absolute ethyl alcohol; the temperature of the reduced pressure rotary evaporation is 40-50 ℃.
The beta-cryptoxanthin used in the invention can be well encapsulated in the liposome because of the special structure that the beta-cryptoxanthin has 8 isoprene units which are connected end to end.
Preferably, the mass ratio of the egg yolk lecithin to the cholesterol to the tween-80 to the beta-cryptoxanthin is 5:1:1: 0.1.
Preferably, the preparation of the vegetable oil body protein in step (2) comprises: dispersing a vegetable oil body protein raw material in Tris-HCl buffer solution containing sodium chloride and sucrose, stirring, centrifuging, collecting an emulsion layer, removing impurities, dispersing in an organic solvent, performing ultrasonic treatment, centrifuging, precipitating and drying to obtain the vegetable oil body protein.
More preferably, the vegetable oil body protein raw material is full-fat soybean meal, almond meal or corn germ meal; the concentration of Tris-HCl in the Tris-HCl buffer solution is 10mmol/L, the concentration of sodium chloride is 0.5mol/L, the concentration of sucrose is 0.2mol/L, and the pH value is 7.5; the centrifugation temperature is 4 ℃, the rotation speed is 10000r/min, and the time is 30 min.
More preferably, the organic solvent is n-hexane; the ultrasonic wave is ice bath ultrasonic wave; and drying by adopting nitrogen.
More preferably, the mass to volume ratio of the vegetable oil body protein raw material to the Tris-HCl buffer solution containing sodium chloride and sucrose is 5g:100 ml; the volume ratio of the emulsion layer to the organic solvent is 1: 10.
More preferably, the method for removing the impurities comprises the steps of re-dispersing the emulsion layer in Tris-HCl buffer solution, stirring, centrifuging and repeating twice.
Preferably, the ultrasonic treatment in step (2) is carried out at 300W for 1s, and suspended for 1s for 15 min.
Preferably, the phosphate buffer solution in the step (2) and the step (3) has the solubility of 10mmol/L and the pH value of 7.0; in the step (3), the nonionic polysaccharide is guar gum.
The guar gum selected by the invention is used as nonionic polysaccharide, has good water dispersibility and thickening property, has good solubility under the condition of pH value for preparing liposome samples, and the polar group on the straight chain can theoretically form hydrogen bond interaction with polar amino acid residues attached to the surface of liposome phospholipid bilayer by vegetable oil body protein.
More preferably, the volume to mass ratio of the oil body protein to the phosphate buffer is 0.01g to 100 ml; the volume to mass ratio of the guar gum to the phosphate buffer solution is 0.1g to 100 ml; the mass ratio of the vegetable oil body protein to the guar gum is 1: 10.
The beneficial technical effects of the invention are as follows:
the invention is based on the topological structure of vegetable oil body protein, and by using the bio-inspire (bio-inspire) concept in bionics, an oil body protein-single-layer phospholipid composite membrane structure is transplanted to a liposome, and further, nonionic polysaccharide is coated on the surface of the liposome, and the plant oil body protein and nonionic polysaccharide stable simulated core-shell liposome is prepared based on the unique interfacial activity of the vegetable oil body protein and the steric hindrance of the nonionic polysaccharide.
The plant oil body-imitating core-shell liposome prepared by the invention can improve the stability of the liposome to environmental factors and can realize the slow release of load substances.
The vegetable oil body protein and the nonionic polysaccharide used for stabilizing the liposome are all food sources, the price is low, the liposome can be safely and effectively applied to the field of foods, the provided method is simple to operate, and the method starts from large-scale popularization and application.
Drawings
FIG. 1 is a transmission electron microscope image of the simulated vegetable oil body core-shell liposomes prepared in examples 1-3; wherein FIG. 1(a) is a transmission electron microscope image of the simulated vegetable oil body core-shell liposome prepared in example 1; FIG. 1(b) is a transmission electron microscope image of the core-shell liposome of the vegetable oil-like body prepared in example 2; FIG. 1(c) is a transmission electron microscope image of the core-shell liposome of the vegetable oil body prepared in example 3.
FIG. 2 is a graph showing the change of beta-cryptoxanthin retention rate with time of the simulated plant oil body core-shell liposomes prepared in examples 1 to 3 and the unmodified liposomes prepared in comparative example 1 under the dark storage condition at 4 ℃.
FIG. 3 shows the fatty acid release rate of simulated vegetable oil body core-shell liposomes prepared in examples 1 to 3 and unmodified liposomes prepared in comparative example 1 in an in vitro simulated digestion experiment.
FIG. 4 shows the beta-cryptoxanthin release rates of the simulated vegetable oil body core-shell liposomes prepared in examples 1-3 and the unmodified liposomes prepared in comparative example 1, in an in vitro simulated digestion experiment.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the present invention, examples of which are illustrated in the accompanying drawings, and this detailed description should not be taken to be limiting of the invention, but is rather a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
(1) Dispersing full-fat soybean powder in Tris-HCl (10mmol/L, pH7.5) buffer solution containing 0.5mol/L sodium chloride and 0.2mol/L sucrose at a ratio of 1:10(W/V), stirring, centrifuging (4 deg.C, 10000r/min, 30min), and collecting emulsion layer. And repeating the operations of collecting an emulsion layer, dispersing and centrifuging for 2 times. Dispersing the obtained emulsion layer in n-hexane at a ratio of 1:10(V/V), performing ultrasonic treatment in ice bath, centrifuging (4 ℃, 10000r/min, 10min) to remove the solvent, collecting the precipitated nitrogen, and drying to obtain the oil body protein.
(2) Adding anhydrous ethanol into a mixture of egg yolk lecithin, cholesterol, tween-80 and beta-cryptoxanthin (the mass ratio is 5:1:1:0.1) to uniformly disperse, and carrying out reduced pressure rotary evaporation at 40 ℃ to form a transparent lipid film;
(3) dispersing the oil body protein prepared in the step (1) in a phosphate buffer solution with the concentration of 0.01% (W/V) and the pH value of 10mmol/L and 7.0 to prepare an oil body protein phosphate buffer solution;
(4) adding the transparent lipid membrane prepared in the step (2) into the oleosin phosphate buffer solution prepared in the step (3), carrying out ultrasonic treatment (300W, working for 1s, pausing for 1s, and totally 15min), and then carrying out high-pressure homogenization to obtain a liposome solution with stable oleosin;
(5) dissolving guar gum in phosphate buffer solution of 10mmol/L and pH7.0 at concentration of 0.1% (W/V) to obtain guar gum phosphate buffer solution;
(6) and (3) dropwise adding the liposome solution with stable oleosin prepared in the step (4) into the guar gum phosphate buffer solution prepared in the step (5) in an equal volume under the stirring and conversion state to prepare the vegetable oil body-simulated core-shell liposome.
The structure of soybean oil body protein is that basic protein is embedded in the oil body structure in an anchoring penetration mode, the middle highly hydrophobic region of the basic protein penetrates through a phospholipid monolayer in two rows of antiparallel beta-folds to form hairpin-like (hairpin) configuration, and the top of the hairpin is a proline knot (proline-knob) consisting of 3 prolines and 1 serine. The amphiphilic amino acid residues with positive charges at the nitrogen end and the carbon end of the oil body protein and the phospholipid or fatty acid with negative charges form ionic bonds, the ionic bonds are attached to the surface of the oil body, and the polar amino acid residues are exposed outside, so that the surface of the oil body has hydrophilicity. The oil body protein can be quickly diffused to a fat-water interface and is tightly stacked to form an elastic adsorption layer so as to stabilize an oil body; it can synergistically reduce oil-water interfacial tension with phospholipids.
Example 2
(1) Dispersing almond powder in Tris-HCl (10mmol/L, pH7.5) buffer solution of 0.5mol/L sodium chloride and 0.2mol/L sucrose at a ratio of 1:10(W/V), stirring, centrifuging (4 deg.C, 10000r/min, 30min), and collecting emulsion layer. And repeating the operations of collecting an emulsion layer, dispersing and centrifuging for 2 times. Dispersing the obtained emulsion layer in n-hexane according to a ratio of 1:10(V/V), carrying out ultrasonic treatment in an ice bath, centrifuging (4 ℃, 10000r/min, 10min) to remove the solvent, collecting the precipitated nitrogen, and drying by blowing to obtain the oleosin.
(2) Adding anhydrous ethanol into a mixture of egg yolk lecithin, cholesterol, tween-80 and beta-cryptoxanthin (mass ratio is 5:1:1:0.1) to uniformly disperse the mixture, and carrying out reduced pressure rotary evaporation at 40 ℃ to form a transparent lipid membrane;
(3) dispersing the oil body protein prepared in the step (1) into 10mmol/L phosphate buffer solution with pH of 7.0 at the concentration of 0.01% (W/V) to prepare oil body protein phosphate buffer solution;
(4) adding the transparent lipid membrane prepared in the step (2) into the oleosin phosphate buffer solution prepared in the step (3), carrying out ultrasonic treatment (300W, working for 1s, pausing for 1s, and totally 15min), and then carrying out high-pressure homogenization to obtain a liposome solution with stable oleosin;
(5) dissolving guar gum in phosphate buffer solution of 10mmol/L and pH7.0 at concentration of 0.1% (W/V) to obtain guar gum phosphate buffer solution;
(6) and (5) dropwise adding the liposome solution with stable oleosin prepared in the step (4) into the guar gum phosphate buffer solution prepared in the step (5) in an equal volume manner under a stirring and converting state to prepare the vegetable oil body-simulated core-shell liposome.
Example 3
(1) Dispersing corn germ meal in Tris-HCl (10mmol/L, pH7.5) buffer solution of 0.5mol/L sodium chloride and 0.2mol/L sucrose according to a ratio of 1:10(W/V), stirring, centrifuging (4 ℃, 10000r/min, 30min), and collecting emulsion layer. And repeating the operations of collecting emulsion layer, dispersing and centrifuging for 2 times. Dispersing the obtained emulsion layer in n-hexane at a ratio of 1:10(V/V), performing ultrasonic treatment in ice bath, centrifuging (4 ℃, 10000r/min, 10min) to remove the solvent, collecting the precipitated nitrogen, and drying to obtain the oil body protein.
(2) Adding anhydrous ethanol into a mixture of egg yolk lecithin, cholesterol, tween-80 and beta-cryptoxanthin (the mass ratio is 5:1:1:0.1) to uniformly disperse, and carrying out reduced pressure rotary evaporation at 40 ℃ to form a transparent lipid film;
(3) dispersing the oil body protein prepared in the step (1) in a phosphate buffer solution with the concentration of 0.01% (W/V) and the pH value of 10mmol/L and 7.0 to prepare an oil body protein phosphate buffer solution;
(4) adding the transparent lipid membrane prepared in the step (2) into the oleosin phosphate buffer solution prepared in the step (3), carrying out ultrasonic treatment (300W, working for 1s, pausing for 1s, and totally 15min), and then carrying out high-pressure homogenization to obtain an oleosin stable liposome solution;
(5) dissolving guar gum in phosphate buffer solution with pH of 7.0 and concentration of 0.1% (W/V) in 10mmol/L to obtain guar gum phosphate buffer solution;
(6) and (3) dropwise adding the liposome solution with stable oleosin prepared in the step (4) into the guar gum phosphate buffer solution prepared in the step (5) in an equal volume under the stirring and conversion state to prepare the vegetable oil body-simulated core-shell liposome.
Comparative example 1
Adding anhydrous ethanol into a mixture of egg yolk lecithin, cholesterol, tween-80 and beta-cryptoxanthin (mass ratio of 5:1:1:0.1) to uniformly disperse, and performing rotary evaporation at 40 ℃ under reduced pressure to form a transparent lipid membrane to obtain the unmodified liposome.
Observing the core-shell liposome of the simulated vegetable oil body prepared in the example 1-3 by a transmission electron microscope; the simulated plant oil core-shell liposomes prepared in examples 1 to 3 and the unmodified liposomes prepared in comparative example 1 were subjected to the measurement of the beta-cryptoxanthin encapsulation efficiency and retention rate and the in vitro simulated digestion experiments.
(1) Observation by a transmission electron microscope: diluting the core-shell liposome imitating the vegetable oil body by 10 times by using phosphate buffer solution (0.05mol/L, pH 7.2), dropwise adding the sample on a copper net, standing at room temperature for 2min, sucking excessive sample from the edge of the copper net by using filter paper, and drying at room temperature. After drying at room temperature, observation was carried out by a transmission electron microscope.
The results are shown in FIG. 1, FIG. 1(a) is a transmission electron microscope image of the simulated plant oil core-shell liposome prepared in example 1; FIG. 1(b) is a transmission electron microscope image of the core-shell liposome of the vegetable oil-like body prepared in example 2; FIG. 1(c) is a transmission electron microscope image of the simulated vegetable oil body core-shell liposomes prepared in example 3. As can be seen from fig. 1, the soybean oil body protein, the almond oil body protein and the beta-cryptoxanthin liposome stabilized by the corn germ oil body protein and the guar gum are irregular spherical particles, wherein the particle size of the beta-cryptoxanthin liposome stabilized by the corn germ oil body protein and the guar gum is the largest.
(2) Determination of beta-cryptoxanthin encapsulation efficiency: the vegetable oil-like core-shell liposomes prepared in examples 1-3 were centrifuged at 4 ℃ for 30min (10000r/min) using an ultrafiltration tube with a molecular weight cut-off of 3000 Da. Determining a beta-cryptoxanthin standard curve by an ultraviolet visible spectrophotometer method, and determining the concentration of free beta-cryptoxanthin in the centrifugal supernatant according to the beta-cryptoxanthin standard curve. The retention rate of beta-cryptoxanthin in the liposome is expressed by the ratio of the amount of beta-cryptoxanthin actually encapsulated to the amount of beta-cryptoxanthin added during the preparation process. The calculation formula is as follows:
beta-cryptoxanthin encapsulation efficiency ═ W1-W2)/W1×100
Wherein W1The amount of beta-cryptoxanthin (mg), W, added to the liposomes2Is the amount of free beta-cryptoxanthin (mg), (W)1-W2) Is beta-cryptoxanthin encapsulation amount.
The beta-cryptoxanthin encapsulation efficiencies of the simulated vegetable oil body core-shell liposomes prepared in examples 1 to 3 and the unmodified liposomes prepared in comparative example 1 were measured to be 95.7%, 90.8%, 93.7% and 87.2%, respectively.
(3) Determination of beta-cryptoxanthin retention: the vegetable oil-like core-shell liposomes prepared in examples 1 to 3 and the unmodified liposomes prepared in comparative example 1 were stored at 4 ℃ for 90 days under dark storage conditions. The beta-cryptoxanthin entrapment amount is measured every 15 days according to the method for measuring the beta-cryptoxanthin entrapment rate, and the beta-cryptoxanthin retention rate (%) is the ratio of the beta-cryptoxanthin entrapment amount to the initial beta-cryptoxanthin entrapment amount in the liposome after different storage time.
The results are shown in FIG. 2, and it can be seen from FIG. 2 that the retention rates of the four β -cryptoxanthin liposomes decrease with the increase of the storage time. After 90 days of storage, about 50% of beta-cryptoxanthin in the unmodified liposome is reserved, and the beta-cryptoxanthin reservation rate of the liposome stabilized by the vegetable oil body protein and the nonionic polysaccharide can be maintained above 75%, which shows that the leakage of the beta-cryptoxanthin is effectively inhibited by introducing the vegetable oil body protein and the nonionic polysaccharide.
(4) In vitro simulated digestion experiments: simulated gastric digest, consisting of 2g/LNaCl, 7mL/L HCl and 3.2g/L porcine pepsin, was adjusted to pH 1.2. Simulated gastric digestive juice was mixed with the vegetable oil-mimetic core-shell liposomes prepared in examples 1 to 3 and the unmodified liposomes prepared in comparative example 1 at a ratio of 1:1 mass ratio, adjusting the pH to 6.8, and continuously shaking in a water bath (100rpm) at 37 ℃ to simulate gastric juice digestion for 2 h.
The simulated intestinal digestive juice contained 2.5mL of pancreatic lipase (4.8mg/mL), 4mL of porcine bile extract (5mg/mL) and 1mL of calcium chloride solution (750mM), and was adjusted to pH 7.0. The simulated gastric digest was mixed with simulated small intestine digest (1: 1w/w ratio) and incubated at 37 ℃ for 2h, maintaining the pH of the solution at 7.0 with NaOH solution throughout the digestion. The volume of NaOH solution consumed during the experiment was recorded to calculate the fatty acid (FAA) release rate, according to the following formula:
Figure BDA0002942449060000101
in the formula VNaOHIs the volume of NaOH (mL), C, required to neutralize the fatty acids liberatedNaOHIn terms of the molar concentration of NaOH, M is the average molecular weight (g/mol) of the liposome, and M is the mass (g) of the liposome.
The measurement results are shown in fig. 3, and it can be seen from fig. 3 that the FFA release rate of the unmodified β -cryptoxanthin liposome after the simulated intestinal digestion is finished is 53.5%, and the FFA release rates of the β -cryptoxanthin liposome stabilized by three vegetable oil body proteins of soybean, almond and maize germ and guar gum are 44.02%, 42.08% and 43.22% respectively, which are all less than the FFA release rate (53.5%) of the unmodified β -cryptoxanthin liposome, which indicates that the vegetable oil body proteins and guar gum can enhance the stability of the liposome in the simulated intestinal digestion environment. This is probably due to the fact that oleosin and guar gum form a dense structure on the surface of the phospholipid bilayer, reducing lipase penetration; the partially hydrolyzed vegetable oil body protein fragments and free phospholipid molecules adsorb to the membrane surface, inhibiting lipase activity.
During the simulated gastrointestinal fluid incubation period, a volume of liposome sample was periodically taken and the amount of free β -cryptoxanthin in the digestion product was determined as in (2). The amount of free beta-cryptoxanthin in the digested product minus the amount of free beta-cryptoxanthin in the undigested system is reported as the amount of beta-cryptoxanthin released. The beta-cryptoxanthin release rate calculation formula is as follows:
beta-cryptoxanthin release rate (%) ═ beta-cryptoxanthin release amount/beta-cryptoxanthin encapsulated amount × 100
The results of the assay are shown in fig. 4, and it can be seen from fig. 4 that β -cryptoxanthin is slowly released from the four liposomes during the simulated gastric fluid digestion stage. At the end of simulated gastric fluid digestion, the beta-cryptoxanthin release rate of the unmodified liposome is 8.3 percent, which is obviously higher than that of the other three (1.9-3.5 percent). After entering simulated small intestine digestion, the four samples of the beta-cryptoxanthin are all rapidly released. The beta-cryptoxanthin release rate of the unmodified liposome is higher than that of the three surface-modified liposomes in the whole simulated in-vitro digestion stage. After 4h of digestion, the release rates of beta-cryptoxanthin in the liposome stabilized by three vegetable oil body proteins of soybean, almond and maize germ and guar gum are 40.5%, 43.8% and 56.8%, respectively, while the release rate of beta-cryptoxanthin in the unmodified liposome is 89.3%. The steric hindrance of the guar gum and the structural solidification effect of the oleosin on the phospholipid bilayer are shown, the damage effect of digestive enzyme on the constructed transmission carrier is reduced, and the release of the beta-cryptoxanthin is delayed.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (1)

1. A kind of kernel-shell liposome of artificial vegetable oil body, characterized by, load the natural phosphatide of carotenoid as the core, vegetable oil body protein and nonionic polysaccharide that the surface coats are the outer cover, form the kernel-shell liposome of artificial vegetable oil body;
the preparation method of the vegetable oil body-imitated core-shell type liposome comprises the following steps:
(1) dissolving natural phospholipid, cholesterol, surfactant and carotenoid in solvent, and performing rotary evaporation under reduced pressure to form transparent lipid membrane;
(2) dispersing vegetable oil body protein into a phosphate buffer solution, adding the transparent lipid membrane prepared in the step (1), and performing ultrasonic treatment to prepare a vegetable oil body protein stable liposome;
(3) adding the plant oil body protein stable liposome prepared in the step (2) into a phosphate buffer solution dissolved with nonionic polysaccharide to prepare a vegetable oil body-simulated core-shell type liposome;
the natural phospholipid in the step (1) is egg yolk lecithin; the surfactant is Tween-80; the carotenoid is beta-cryptoxanthin, wherein the mass ratio of egg yolk lecithin, cholesterol, tween-80 and the beta-cryptoxanthin is 5:1:1: 0.1; the solvent is absolute ethyl alcohol; the temperature of the reduced pressure rotary evaporation is 40-50 ℃;
the preparation method of the vegetable oil body protein in the step (2) comprises the following steps: dispersing a vegetable oil body protein raw material in a Tris-HCl buffer solution containing sodium chloride and sucrose, stirring, centrifuging, collecting an emulsion layer, removing impurities, dispersing in an organic solvent, performing ultrasonic treatment, centrifuging, precipitating and drying to obtain the vegetable oil body protein;
the vegetable oil body protein raw material is full-fat soybean meal, almond meal or corn germ meal; the concentration of Tris-HCl in the Tris-HCl buffer solution is 10mmol/L, the concentration of sodium chloride is 0.5mol/L, the concentration of sucrose is 0.2mol/L, and the pH value is 7.5; the centrifugation temperature is 4 ℃, the rotation speed is 10000r/min, and the time is 30 min;
the mass to volume ratio of the vegetable oil body protein raw material to Tris-HCl buffer solution containing sodium chloride and sucrose is 5g:100 ml; the volume ratio of the emulsion layer to the organic solvent is 1: 10;
the ultrasonic treatment method in the step (2) comprises the steps of working for 1s under the power of 300W, suspending for 1s and lasting for 15 min;
the phosphate buffer solution in the step (2) and the step (3) has the solubility of 10mmol/L and the pH value of 7.0; the nonionic polysaccharide in the step (3) is guar gum; wherein the volume-to-mass ratio of the oleosin to the phosphate buffer is 0.01g:100 ml; the volume to mass ratio of the guar gum to the phosphate buffer is 0.1g to 100 ml; the mass ratio of the oil body protein to the guar gum is 1: 10.
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