CN112790384A

CN112790384A - Preparation method of gel-coated beta-carotene nanoparticles

Info

Publication number: CN112790384A
Application number: CN202110051168.5A
Authority: CN
Inventors: 钟芳; 陈羚; 梁蓉; 刘飞
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2021-05-14
Anticipated expiration: 2041-01-14
Also published as: CN112790384B

Abstract

The invention discloses a preparation method of gel-coated beta-carotene nanoparticles, which is characterized by comprising the following steps: the method comprises the following steps: preparing an organic phase, preparing a water phase, mixing, and removing the organic phase to obtain beta-carotene nanoparticles; the invention can control the small intestine fixed-point release of the beta-carotene nanoparticles, promote the beta-carotene nanoparticles to be transported into the systemic circulation, exert the biological activity of diminishing inflammation and losing weight, and have good application prospect in the fields of food and health care products.

Description

Preparation method of gel-coated beta-carotene nanoparticles

Technical Field

The invention relates to the technical field of food additives, in particular to a preparation method of gel-coated beta-carotene nanoparticles.

Background

Recent research reports show that the beta-carotene not only has the functions of promoting immune response and relieving chronic diseases, but also can improve obesity. When β -carotene is ingested, it is usually present as its metabolite retinol/retinoic acid and stored in the liver as retinol palmitate. The functional characteristics of the beta-carotene, such as enhancing immunity and relieving chronic diseases, are mainly characterized in that the form 1) of the retinoic acid interacts with transcription factors of nuclear receptor superfamily, such as Retinoic Acid Receptors (RAR), retinoic acid X receptors (RXRs), peroxisome proliferator-activated receptors (PPAR gamma) and the like; 2) interfere with the activity of other transcription factors, such as activin-1, nuclear factor kappa B (NF-kappa B) or CCAAT enhancer binding proteins (C/EBPs); 3) regulating NF-kB or Nrf2 signal path related to inflammation and oxidative stress, and inhibiting the expression of proinflammatory factors; 4) by eliminating active components and the like. If the beta-carotene molecules can be carried to the systemic circulation through the particles, are not metabolized and are stored in the adipose tissues in the form of original molecules, the activity of key transcription factors for controlling the growth of the adipocytes can be influenced, and the size of the adipocytes can be regulated, so that the purpose of losing weight is achieved. However, in the natural uptake of β -carotene or in the conventional vector design, β -carotene molecules are difficult to maintain their original state and are absorbed and utilized by the body.

Recent studies show that the vectorization of nutrients is related to the transport and absorption mode of the nutrients, and the metabolic behaviors of the nutrients in the body can be changed and the existing forms of the nutrients in the body can be further changed by regulating the release and transport mode of the nutrients in the gastrointestinal tract and the absorption mode of the nutrients in small intestinal epithelial cells. The design of the protein nanoparticle carrier is proved to be capable of effectively improving the transport efficiency of beta-carotene molecules in small intestinal epithelial cells, reducing the metabolic behavior of the beta-carotene in the small intestinal cells and transporting more beta-carotene into systemic circulation. However, the carrier has poor gastrointestinal stability and is difficult to maintain in the oral route of ingestion.

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 in view of the above and/or problems occurring in the existing beta-carotene nanoparticles.

Therefore, one of the objects of the present invention is to overcome the disadvantages of the existing β -carotene nanoparticle products and to provide a method for preparing gel-coated β -carotene nanoparticles.

To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a method for preparing gel-encapsulated beta-carotene nanoparticles, comprising the steps of:

preparing an organic phase: dissolving carotene in an organic solvent to serve as an organic phase;

preparing an aqueous phase: selecting a water phase wall material to be dissolved in ultrapure water to prepare a water phase;

removing the organic phase after mixing to prepare the beta-carotene nanoparticles: mixing the organic phase and the water phase, performing high-speed dispersion under high-speed centrifugation, and then homogenizing; removing an organic phase from the homogenized mixed suspension by rotary evaporation through a rotary evaporator to obtain beta-carotene nanoparticles;

preparing protein gel: taking protein for hydration, heating at high temperature for a period of time after hydration, cooling, mixing the cooled protein solution with beta-carotene nanoparticles, cooling and forming protein gel; homogenizing to obtain gel-coated beta-carotene nanoparticles: and dispersing the protein gel at a high speed, diluting the protein gel with deionized water, and homogenizing the protein gel to obtain the beta-carotene nano-particles wrapped by the gel.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: preparing organic solvent in organic phase comprises one or more of ethyl acetate, dichloromethane, chloroform, methanol, acetonitrile, and ethanol.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: the water phase wall material adopted in the preparation of the water phase comprises one or more of protein, modified starch, phospholipid and polysaccharide.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: and removing the organic phase after mixing to prepare the beta-carotene nanoparticles, wherein the volume ratio of the organic phase to the water phase is 1-3: 7-9.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: and removing the organic phase after mixing to prepare the beta-carotene nano-particle seeds, and mixing the organic phase and the water phase according to the volume ratio of 1: 9.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: the protein used in the preparation of the protein gel comprises one of whey protein isolate, soy protein isolate, sodium caseinate, fish gelatin, beef gelatin and alpha-lactalbumin.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: in the preparation of the protein gel, the protein adopted is alpha-lactalbumin.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: in the preparation of the protein gel, the hydrated protein and beta-carotene are mixed according to the volume ratio of 1-2: 1-2 by volume ratio.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: in the preparation of the protein gel, the hydrated protein and the beta-carotene are mixed according to the volume ratio of 1: 1.

As a preferable embodiment of the method for preparing the gel-coated β -carotene nanoparticle of the present invention, wherein: homogenizing to obtain gel-coated beta-carotene nanoparticles, wherein homogenizing is carried out for 5 times under 100MPa by using a high-pressure homogenizer.

The invention provides a preparation method of gel-coated beta-carotene nanoparticles, which constructs a beta-carotene carrier preparation with stable stomach and intestine, improves the absorption and utilization characteristics of network beta-carotene, assists the absorption of the beta-carotene by using the form of the nano carrier, inhibits the intracellular metabolism of the beta-carotene, ensures that the absorption of the beta-carotene is easier, and ensures that the functional characteristics of the beta-carotene of diminishing inflammation and losing weight are more fully exerted.

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 schematic diagram of the transport pathway of beta-carotene in conventional carriers and gel-coated nanoparticle carriers;

FIG. 2 shows the accumulation of retinol, a β -carotene metabolite, in the liver of mice in both of the above-described vector forms;

FIG. 3 is the accumulation of β -carotene in the adipose tissues of mice in the two above-described vector forms;

FIG. 4 is a section view of the effect of the two forms of the beta-carotene nanocarrier on mouse liver tissue;

in the figure, case 1 is an image obtained by sectioning liver tissue in example 1;

FIG. 5 is a section view of the effect of the two forms of the beta-carotene nanocarrier on mouse adipose tissue;

in the figure, case 1 is an image obtained by slicing adipose tissues in example 1;

FIG. 6 is a flow chart of the preparation process of 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.

Example 1

Fully dissolving 0.1% of beta-carotene by mass in ethyl acetate to obtain an organic phase, fully dissolving 1% of whey protein isolate by mass in ultrapure water to obtain an aqueous phase, mixing the ethyl acetate solution containing the beta-carotene with the aqueous phase according to the volume ratio of 1:9, dispersing at a high speed of 20000rpm for 10min, and homogenizing for 1 time under 70MPa by a homogenizer; and (3) carrying out rotary evaporation on the homogenized mixed suspension for 15min at 250rpm by using a rotary evaporator to remove an organic phase in the suspension to obtain the beta-carotene nanoparticles.

10g of alpha-lactalbumin was dissolved in 50mL of deionized water and hydrated overnight at 4 ℃ at pH 7. And then heating the protein solution at 90 ℃ for 40min, gradually cooling at room temperature, adding the beta-carotene nanoparticles according to the volume ratio of 1:1 when the temperature reaches 55 ℃, and fully stirring. The mixture was allowed to cool to 4 ℃ overnight to form a protein gel. The resulting protein gel was homogenized for 5min at 10000rpm by high speed dispersion, and then diluted 10-fold with deionized water. And finally homogenizing for 5 times under 100MPa by using a high-pressure homogenizer to obtain the beta-carotene nano-particles wrapped by the gel.

Example 2

Fully dissolving 0.1% of beta-carotene by mass volume fraction in ethyl acetate to obtain an organic phase, fully dissolving 1% of soybean protein isolate by mass volume fraction in ultrapure water to obtain an aqueous phase as an aqueous phase wall material, mixing the beta-carotene-containing ethyl acetate solution and the aqueous phase according to the volume ratio of 1:9, dispersing at a high speed of 20000rpm for 10min, and homogenizing for 1 time by a homogenizer under the condition of 70 MPa; and (3) carrying out rotary evaporation on the homogenized mixed suspension for 15min at 250rpm by using a rotary evaporator to remove an organic phase in the suspension to obtain the beta-carotene nanoparticles.

Example 3

10g of whey protein isolate was dissolved in 50mL of deionized water and hydrated overnight at 4 ℃ and pH 7. And then heating the protein solution at 90 ℃ for 40min, gradually cooling at room temperature, adding the beta-carotene nanoparticles according to the volume ratio of 1:1 when the temperature reaches 55 ℃, and fully stirring. The mixture was allowed to cool to 4 ℃ overnight to form a protein gel. The resulting protein gel was homogenized for 5min at 10000rpm by high speed dispersion, and then diluted 10-fold with deionized water. And finally homogenizing for 5 times under 100MPa by using a high-pressure homogenizer to obtain the beta-carotene nano-particles wrapped by the gel.

Example 4

Fully dissolving 0.1% of beta-carotene by mass in ethyl acetate to obtain an organic phase, fully dissolving 1% of whey protein isolate by mass in ultrapure water to obtain an aqueous phase, mixing the organic phase containing beta-carotene ethyl acetate solution nutrient and the aqueous phase according to the volume ratio of 1:9, dispersing at a high speed of 20000rpm for 10min, and homogenizing for 1 time under 70MPa by a homogenizer; and (3) carrying out rotary evaporation on the homogenized mixed suspension for 15min at 250rpm by using a rotary evaporator to remove an organic phase in the suspension to obtain the beta-carotene nanoparticles.

10g of alpha-lactalbumin was dissolved in 50mL of deionized water and hydrated overnight at 4 ℃ at pH 7. And then heating the protein solution at 90 ℃ for 40min, gradually cooling at room temperature, adding the beta-carotene nanoparticles according to the volume ratio of 2:1 when the temperature reaches 55 ℃, and fully stirring. The mixture was allowed to cool to 4 ℃ overnight to form a protein gel. The resulting protein gel was homogenized for 5min at 10000rpm by high speed dispersion, and then diluted 10-fold with deionized water. And finally homogenizing for 5 times under 100MPa by using a high-pressure homogenizer to obtain the beta-carotene nano-particles wrapped by the gel.

Example 5

10g of alpha-lactalbumin was dissolved in 50mL of deionized water and hydrated overnight at 4 ℃ at pH 7. And then heating the protein solution at 90 ℃ for 40min, gradually cooling at room temperature, adding the beta-carotene nanoparticles according to the volume ratio of 1:2 when the temperature reaches 55 ℃, and fully stirring. The mixture was allowed to cool to 4 ℃ overnight to form a protein gel. The resulting protein gel was homogenized for 5min at 10000rpm by high speed dispersion, and then diluted 10-fold with deionized water. And finally homogenizing for 5 times under 100MPa by using a high-pressure homogenizer to obtain the beta-carotene nano-particles wrapped by the gel.

Comparative example 1

Beta-carotene with the mass fraction of 0.1 percent is dissolved in corn oil to be used as an oil phase, and oil with the volume fraction of 10 percent is dispersed in ultrapure water by high-speed dispersion to prepare beta-carotene oil suspension.

Comparative example 2

Fully dissolving 0.1% of beta-carotene by mass volume fraction in ethyl acetate to obtain an organic phase, fully dissolving whey protein isolate in ultrapure water to obtain an aqueous phase, fully dissolving the aqueous phase in the ultrapure water to obtain an aqueous phase, mixing the organic phase containing nutrients and the aqueous phase according to the volume ratio of 1:9, dispersing at a high speed of 20000rpm for 10min, and homogenizing for 1 time by a homogenizer under the condition of 70 MPa; and (3) carrying out rotary evaporation on the homogenized mixed suspension for 15min at 250rpm by using a rotary evaporator to remove an organic phase in the suspension to obtain the beta-carotene nanoparticles.

Comparative example 3

Fully dissolving 0.1% of beta-carotene by mass and volume in corn oil to obtain an organic phase, fully dissolving whey protein isolate in ultrapure water to obtain a water phase wall material, fully dissolving the water phase wall material in the ultrapure water to obtain a water phase, mixing an oil phase containing nutrients and the water phase according to the volume ratio of 1:9, dispersing at a high speed of 20000rpm for 2min, and homogenizing for 7 times at 70MPa by a homogenizer; to obtain the beta-carotene nanoemulsion.

Example 6

The properties of the finished products obtained in examples 1 to 5 and comparative examples 1 to 3 were measured, and the results are shown in table 1 and fig. 2, 3, 4 and 5, and the results of the measurements were recorded in table 1, fig. 2 and fig. 3, for the VA liver tissue content and the VA adipose tissue content, respectively.

The measurement mode of the VA liver tissue content and the VA fat tissue content is as follows: mice were gavaged with samples of beta-carotene protein nanoemulsion, protein nanoparticles, protein macroemulsion, and beta-carotene dispersion, respectively. A mouse perfused with PBS is used as a blank control group, the mouse is anesthetized after 30min to take the liver and epididymis fat, the tissue is homogenized in 0.9% sodium chloride solution, then a mixed organic solvent of methanol and n-hexane (containing 0.1% BHT) is added according to the proportion of 2:3, vortex oscillation is carried out for 30s, and the upper organic phase is collected after centrifugation for 4min at 2000 g. This extraction process was repeated three times. After this time, the organic solvent was blown dry with nitrogen and redissolved in 200. mu.L of dichloromethane for HPLC quantitative analysis.

HPLC quantitative analysis method: the content of β -carotene (BC) and its metabolites Retinol (ROL), Retinol Palmitate (RP) in the samples was quantified using an Alliance HPLC system equipped with a Waters 2998PDA detector. The column was a COSMOSIL Cholester column (250X 4.6mm, 5 μm diameter) with a pre-column. The HPLC chromatographic conditions are as follows: flow rate: 1 mL/min; column temperature: 10 ℃; sample introduction amount: 20 mu L of the solution; detection wavelength: 450nm (for BC) and 340nm (for ROL and RP); analysis time: 28 min; all samples were placed at 4 ℃; mobile phase: phase A (84% methanol: 14% acetonitrile: 2% ultrapure water, v/v/v) and phase B (dichloromethane) were mixed; the gradient elution procedure was: changing from 80% A, 20% B linearity to 45% A, 55% B linearity at 0-15 min; maintaining stability under conditions of 45% A and 55% B for 15-20 min; changing from 45% A, 55% B linearity to 80% A, 20% B linearity for 20-25 min; keeping the ratio of 80% A and 20% B stable for 3min after 25-28 min. The standard is linear in the range of 0.1-100 mug/mL. Calculating a regression equation of the calibration curve from the peak area to the standard concentration, R²>0.99, the data obtained are recorded in Table 1, and the images are drawn according to the data in Table 1Recorded in fig. 2 and 3.

TABLE 1 Loading and Retention of beta-Carotene Carriers prepared in examples 1-5 and comparative examples 1-3

Examples	VA liver tissue content (ng/g tissue)	VA adipose tissue content (ng/g tissue)
			Example 1	1835±653	223±37
Example 2	1986±743	183±25
			Example 3	2004±589	192±43
Example 4	2169±230	201±17
			Example 5	2057±208	187±26
Comparative example 1	1668±683	7±9
			Comparative example 2	1503±471	97±31
Comparative example 3	5634±1026	82±26

As can be seen from table 1, the liver tissue content of VA is the least and the adipose tissue content of VA is the most in example 1, which indicates that the gel-coated β -carotene nanoparticles prepared in example 1 are most effective in reducing the content of the target substance in the liver and increasing the content of the target substance in the adipose tissue; the aqueous wall material used preferably contains whey protein isolate, and the protein used in the gel structure preferably contains alpha-lactalbumin, according to the VA liver tissue content and VA adipose tissue content of the gel-encapsulated beta-carotene nanoparticles prepared in examples 1, 2 and 3; according to the VA liver tissue content and VA adipose tissue content of the gel-coated β -carotene nanoparticles prepared in examples 1, 4, and 5, a preferred ratio of the protein solution to the β -carotene nanoparticles when mixed is 1: 1.

example 7

The beta-carotene protein nanoemulsion, the protein nanoparticles, the protein coarse emulsion, the beta-carotene dispersion liquid and PBS are used for carrying out gastric lavage on the mouse, the mouse is anesthetized after 30min to take the liver and epididymis fat, the obtained sample is sliced and subjected to HE dyeing treatment, and the obtained image is shown in fig. 4 and fig. 5. In fig. 4, nine pictures show that blue dyeing objects are doped with a large amount of red dyeing substances, wherein the red dyeing substances of three pictures in the column of case 1 are the least, and a large amount of red dyeing substances are shown in the columns of comparative example 1 and comparative example 3; the purple staining was less and less intense in the columns of comparative example 1 and comparative example 3 in the three pictures in the column in which case 1 was located in fig. 5, more staining occurred in the column of case 1 and densely stained areas in the pictures below the column of case 1.

As can be seen from fig. 2 and 3, the β -carotene nanoparticles can significantly increase the ratio of β -carotene in fat and decrease the ratio of β -carotene in mouse liver, and fig. 4 and 5 verify the above results, and the distribution of β -carotene in mouse liver tissue is significantly decreased, while the content of β -carotene in fat tissue is significantly increased.

According to the fig. 1-4, the beta-carotene oil suspension, the beta-carotene nanoparticles and the beta-carotene nanoemulsion prepared in the comparative example have no obvious effect on the content of the beta-carotene in the liver and fat cells by the beta-carotene entering the liver and the fat.

The distribution of gel-encapsulated beta-carotene nanoparticles prepared in example 1 of fig. 5 in liver and fat cells was obtained, with the best performance in example 1, and the aqueous wall material was preferably whey protein isolate, and the protein used in the gel structure was preferably alpha-lactalbumin.

According to the data of the content of the VA liver tissue and the VA adipose tissue images of the finished products prepared in the example 1 and the comparative examples 1 and 3 in the figure 5, the finished products prepared in the comparative examples 1 to 3 have poor guiding capability for the beta-carotene to enter the adipose cells, and the capability of leading the beta-carotene to enter the adipose cells in a large amount is in sharp contrast with the capability of leading the beta-carotene to enter the adipose cells in the invention.

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

1. A method for preparing gel-coated beta-carotene nanoparticles, which is characterized by comprising the following steps: the method comprises the following steps:

preparing an aqueous phase: selecting a water phase wall material to be dissolved in ultrapure water as a water phase;

preparing protein gel: taking protein for hydration, heating at high temperature for a period of time after hydration, cooling, mixing the cooled protein solution with beta-carotene nanoparticles, cooling and forming protein gel;

homogenizing to obtain gel-coated beta-carotene nanoparticles: and dispersing the protein gel at a high speed, diluting the protein gel with deionized water, and homogenizing the protein gel to obtain the beta-carotene nano-particles wrapped by the gel.

2. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1, wherein: the organic solvent in the organic phase comprises one or more of ethyl acetate, dichloromethane, chloroform, methanol, acetonitrile and ethanol.

3. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1, wherein: the water phase wall material adopted in the preparation of the water phase comprises one or more of protein, modified starch, phospholipid and polysaccharide.

4. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1, wherein: and in the preparation of the beta-carotene nanoparticles by removing the organic phase after mixing, the volume ratio of the organic phase to the water phase is 1-3: 7-9.

5. The method for preparing gel-encapsulated β -carotene nanoparticles according to example 1 or 4, characterized in that: and removing the organic phase after mixing to prepare the beta-carotene nano-particle seeds, and mixing the organic phase and the water phase according to the volume ratio of 1: 9.

6. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1, wherein: in the preparation of the protein gel, the adopted protein comprises one of whey protein isolate, soy protein isolate, sodium caseinate, fish gelatin, beef gelatin and alpha-lactalbumin.

7. The method for preparing gel-encapsulated β -carotene nanoparticles according to claim 1 or 6, characterized in that: in the preparation of the protein gel, the adopted protein is alpha-lactalbumin.

8. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1, characterized in that: in the preparation of the protein gel, the hydrated protein and beta-carotene are mixed according to the volume ratio of 1-2: 1-2 by volume ratio.

9. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1 or 8, characterized in that: in the preparation of the protein gel, hydrated protein and beta-carotene are mixed according to the volume ratio of 1: 1.

10. The method of preparing gel-encapsulated β -carotene nanoparticles according to claim 1, characterized in that: and homogenizing for 5 times under 100MPa by using a high-pressure homogenizer in the beta-carotene nanoparticles wrapped by the gel obtained by homogenizing.