Method for preparing lutein-loaded nano-carrier by using one-step method for regulating cow milk concentrated protein micelles by using decalcification rate

Abstract

The invention discloses a method for preparing a lutein-loaded nano-carrier by using a one-step method for regulating cow milk concentrated protein micelles with a decalcification rate, which comprises the following steps: dissolving a cow milk concentrated protein powder sample in deionized water, and uniformly mixing to obtain a cow milk concentrated protein solution; adding cation exchange resin into the cow milk concentrated protein solution to obtain a first protein solution; dissolving a powder sample obtained by freeze-drying the first protein solution in water to prepare a second protein solution; the preparation method comprises the following steps: adding xanthophyll into a mixed solution, magnetically stirring to obtain a mixed solution, dialyzing the mixed solution, and freeze-drying to obtain xanthophyll-loaded nano-carrier particles. According to the method, the cation exchange resin is used for controlling the calcium content of the milk concentrated protein, the micelle structure of the milk concentrated protein is remarkably changed, a stable nano shell structure with an internal cavity and a specific function is formed, and efficient embedding and intestinal microenvironment response delivery of hydrophobic substances can be achieved; compared with the prior art, the milk concentrated protein has the advantages of higher encapsulation efficiency and loading capacity, small hydrodynamic diameter and capabilities of improving the delivery efficiency and reducing the nutrient consumption, and is favorable for reducing the use cost of the nutrients, expanding the application field of milk concentrated protein of dairy product processing byproducts and realizing high-valued utilization.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for preparing a lutein-loaded nanocarrier by a one-step method by regulating milk protein concentrate micelles by utilizing decalcification rate. Background Art

[0002] Milk protein concentrate (MPC) is a high-protein milk powder obtained by ultrafiltration to remove most of the lactose in skim milk. It has good functional properties and nutritional value. Compared with traditional milk proteins, such as whey protein concentrates / isolates (WPC / WPI) and sodium caseinate (SC), the preparation process of MPC is simpler and the production cost is lower. MPC includes casein and whey protein, and the functional properties of MPC are mainly affected by the structure of the casein micelles (i.e., the aggregation state of casein), and the structure of casein micelles is mainly maintained by calcium ion bridging and hydrophobic interactions. Removing calcium ions from casein micelles and changing the balance between electrostatic repulsion and hydrophobic interactions in their internal structure can directionally regulate the structure of casein micelles and thus achieve the improvement of MPC functional properties.

[0003] Lutein is a natural substance widely found in vegetables, flowers, fruits and other plants. It plays a variety of important physiological functions in the human body, such as anti-aging and anti-cancer effects. It is also widely used in the field of food and medicine. However, due to the presence of multiple conjugated double bonds in the molecular structure of lutein, its chemical stability is poor. It is easily affected by acidic conditions, oxygen, temperature and light, and isomerization, degradation and oxidation occur. The existing encapsulation and delivery system cannot achieve effective encapsulation and loading of lutein. Therefore, it is necessary to provide a method for effectively encapsulating lutein by regulating the structure of casein micelles to increase the encapsulation rate, loading rate and drug loading. Summary of the invention

[0004] The present invention provides a preparation method for constructing a lutein-loaded nanocarrier in one step by directly regulating the structure of milk protein concentrate micelles by utilizing the decalcification rate, so as to solve the above problems.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] A method for preparing a lutein-loaded nanocarrier by one-step method using milk protein concentrate micelles to regulate decalcification rate comprises the following steps:

[0007] S1: dissolving a milk protein concentrate powder sample in deionized water and mixing well to obtain a milk protein concentrate solution;

[0008] S2: adding a cation exchange resin to the milk protein concentrate solution for decalcification to obtain a first protein solution;

[0009] S3: dissolving the powder sample obtained by freeze-drying the first protein solution in water to prepare a second protein solution;

[0010] S4: adding lutein to the second protein solution, stirring the mixture by magnetic force to obtain a mixed solution, and dialyzing and freeze-drying the mixed solution to obtain lutein-loaded nanocarrier particles.

[0011] Furthermore, the mass ratio of the cation exchange resin to the concentrated milk protein powder is 4-20g:120g.

[0012] Furthermore, the concentration of milk protein concentrate in the second protein solution is 0.5 mg / mL.

[0013] Furthermore, the mass of the lutein is 5% of the mass of the second protein solution.

[0014] Further, in S2, the magnetic stirring speed is 30-40 rpm / min, and the time is 4 hours;

[0015] In S4, the magnetic stirring frequency is 30-40 rpm / min, the time is 12 hours, and the temperature is 25°C.

[0016] Furthermore, the dialysis method is to use a dialysis bag to dialyze in deionized water for 3 days, and the retention capacity of the dialysis bag is 3500 Daltons.

[0017] Furthermore, the freeze-drying time is 48 hours.

[0018] The beneficial effects of the present invention are:

[0019] The present invention discloses a method for preparing a lutein-loaded nanocarrier by one-step method using decalcification rate to regulate milk concentrate protein micelles, using cation exchange resin to control the calcium content of milk concentrate protein, significantly changing the structure of milk concentrate protein micelles, forming a stable nanoshell structure with an internal cavity and specific functions, which can efficiently embed hydrophobic substances and deliver intestinal microenvironment response; the lutein-loaded milk concentrate protein delivery system prepared by this method has a nanometer particle size and a larger specific surface area, so that it has a higher encapsulation rate and loading capacity, and has the advantage of a small hydrodynamic diameter, which can improve the delivery efficiency and reduce the amount of nutrients, and the present invention will be conducive to reducing the use cost of nutrients, expanding the application field of milk concentrate protein, a by-product of dairy processing, and realizing high-value utilization. This method does not require pressure homogenization and the homogenization steps of a high-speed shear disperser, and the method is simple and convenient for industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a comparison chart of the decalcification rate and calcium content after improvement of Examples 1-5 of the present invention and Comparative Example 1;

[0022] Figure 2 The graph of the fluorescence intensity changes after improvement of Examples 1-5 of the present invention and Comparative Example 1;

[0023] Figure 3 Schematic diagram of the improved gel electrophoresis molecular size of Examples 1-5 of the present invention and Comparative Example 1;

[0024] Figure 4 Schematic diagram of the digestibility after improvement of Examples 1-5 of the present invention and Comparative Example 1;

[0025] Figure 5 Schematic diagram of the improved encapsulation efficiency and drug loading of Examples 1-5 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example

[0028] Embodiment 1:

[0029] A method for preparing a lutein-loaded nanocarrier by one-step method using milk protein concentrate micelles to regulate decalcification rate comprises the following steps:

[0030] S1: Dissolve 120 g of milk protein concentrate powder sample in 880 g of deionized water, and mix well to obtain a milk protein concentrate solution;

[0031] S2: adding 4 g of cation exchange resin to the milk protein concentrate solution, and magnetically stirring for 4 hours at a stirring speed of 30-40 rpm / min to obtain a first protein solution;

[0032] S3: After the freeze-drying step, the first protein solution is subjected to a powder sample MPC-4, and the powder sample is dissolved in deionized water to obtain a second protein solution of MPC-4, wherein the concentration of the milk protein concentrate in the second protein solution is 0.5 mg / mL;

[0033] S5: Add lutein to the second protein solution of MPC-4, the mass of lutein is 5% of the mass of MPC-4 protein solution II, and magnetically stir at 25°C for 12 hours to obtain a mixed solution, and the stirring speed is 30-40rpm / min; the mixed solution is dialyzed in deionized water for 3 days through a dialysis bag with a retention capacity of 3500 Daltons, and then freeze-dried for 48 hours to obtain milk concentrate protein micelle nanoparticles MPC-4 loaded with lutein, and the preparation of the delivery system (lutein-loaded nanocarrier) is completed.

[0034] Embodiment 2:

[0035] A method for preparing a lutein-loaded nanocarrier by one-step method using milk protein concentrate micelles to regulate decalcification rate comprises the following steps:

[0036] S1: Dissolve 120 g of milk protein concentrate powder sample in 880 g of deionized water, and mix well to obtain a milk protein concentrate solution;

[0037] S2: adding 8 g of cation exchange resin to the milk protein concentrate solution, and magnetically stirring for 4 hours at a stirring speed of 30-40 rpm / min to obtain a first protein solution;

[0038] S3: After the freeze-drying step, the first protein solution is subjected to a powder sample MPC-8, and the powder sample is dissolved in deionized water to obtain a second protein solution of MPC-8, wherein the concentration of the milk protein concentrate in the second protein solution is 0.5 mg / mL;

[0039] S5: Add lutein to the MPC-4 second protein solution, the mass of lutein is 5% of the mass of the MPC-8 second protein solution, and magnetically stir at 25°C for 12 hours to obtain a mixed solution, and the stirring speed is 30-40rpm / min; the mixed solution is dialyzed in deionized water for 3 days through a dialysis bag with a retention capacity of 3500 Daltons, and then freeze-dried for 48 hours to obtain milk concentrate protein micelle nanoparticles MPC-8 loaded with lutein, and the preparation of the delivery system (lutein-loaded nanocarrier) is completed.

[0040] Embodiment 3:

[0041] A method for preparing a lutein-loaded nanocarrier by one-step method using milk protein concentrate micelles to regulate decalcification rate comprises the following steps:

[0042] S1: Dissolve 120 g of milk protein concentrate powder sample in 880 g of deionized water, and mix well to obtain a milk protein concentrate solution;

[0043] S2: adding 12 g of cation exchange resin to the milk protein concentrate solution, and magnetically stirring for 4 hours at a stirring speed of 30-40 rpm / min to obtain a first protein solution;

[0044] S3: After the freeze-drying step, the first protein solution is subjected to a powder sample MPC-12, and the powder sample is dissolved in deionized water to obtain a second protein solution of MPC-12, wherein the concentration of the milk protein concentrate in the second protein solution is 0.5 mg / mL;

[0045] S5: Add lutein to the MPC-12 second protein solution. The mass of lutein is

[0046] The mixture was dialyzed in deionized water for 3 days through a dialysis bag with a cutoff of 3500 Daltons, and then freeze-dried for 48 hours to obtain milk concentrate protein micelle nanoparticles MPC-12 loaded with lutein, and the preparation of the (lutein-loaded nanocarrier) delivery system was completed.

[0047] Embodiment 4:

[0048] A method for preparing a lutein-loaded nanocarrier by one-step method using milk protein concentrate micelles to regulate decalcification rate comprises the following steps:

[0049] S1: Dissolve 120 g of milk protein concentrate powder sample in 880 g of deionized water, and mix well to obtain a milk protein concentrate solution;

[0050] S2: adding 16 g of cation exchange resin to the milk protein concentrate solution, and magnetically stirring for 4 hours at a stirring speed of 30-40 rpm / min to obtain a first protein solution;

[0051] S3: After the freeze-drying step, the first protein solution is subjected to a powder sample MPC-16, and the powder sample is dissolved in deionized water to obtain a second protein solution of MPC-16, wherein the concentration of the milk protein concentrate in the second protein solution is 0.5 mg / mL;

[0052] S5: Add lutein to the MPC-16 second protein solution, the mass of lutein is 5% of the mass of the MPC-16 second protein solution, and magnetically stir at 25°C for 12 hours to obtain a mixed solution, and the stirring speed is 30-40rpm / min; the mixed solution is dialyzed in deionized water for 3 days through a dialysis bag with a retention capacity of 3500 Daltons, and then freeze-dried for 48 hours to obtain milk concentrate protein micelle nanoparticles MPC-16 loaded with lutein, and the preparation of the delivery system (lutein-loaded nanocarrier) is completed.

[0053] Embodiment 5:

[0054] A method for preparing a lutein-loaded nanocarrier by one-step method using milk protein concentrate micelles to regulate decalcification rate comprises the following steps:

[0055] S1: Dissolve 120 g of milk protein concentrate powder sample in 880 g of deionized water, and mix well to obtain a milk protein concentrate solution;

[0056] S2: adding 20 g of cation exchange resin to the milk protein concentrate solution, and magnetically stirring for 4 hours at a stirring speed of 30-40 rpm / min to obtain a first protein solution;

[0057] S3: After the freeze-drying step, the first protein solution is subjected to a powder sample MPC-20, and the powder sample is dissolved in deionized water to obtain a second protein solution MPC-20, wherein the concentration of the milk protein concentrate in the second protein solution is 0.5 mg / mL;

[0058] S5: Add lutein to the MPC-20 second protein solution, the mass of lutein is 5% of the mass of the MPC-20 second protein solution, and magnetically stir at 25°C for 12 hours to obtain a mixed solution, and the stirring speed is 30-40rpm / min; the mixed solution is dialyzed in deionized water for 3 days through a dialysis bag with a retention capacity of 3500 Daltons, and then freeze-dried for 48 hours to obtain milk concentrate protein micelle nanoparticles MPC-20 loaded with lutein, and the preparation of the delivery system (lutein-loaded nanocarrier) is completed.

[0059] Comparative Example:

[0060] Comparative Example 1:

[0061] The only difference from Example 1 is that in this comparative example, no cation exchange resin is added in step S2.

[0062] Performance Test:

[0063] The structure, morphological characteristics and loading characteristics of the calcium decalcification rate-controlled milk protein concentrate micelles prepared in Examples 1-5 were analyzed:

[0064] (1) In Examples 1-5, the calcium content and decalcification rate after calcium ions in milk protein concentrate were removed using different amounts of ion exchange resin were measured. Figure 1 As shown, the calcium content of MPC in comparative example 1 was measured to be 92.8% (w / w); for MPC-4, MPC-8, MPC-12, MPC-16 and MPC-20 to which 4, 8, 12, 16 and 20 g of ion exchange resin were added, the remaining calcium contents at the end of the ion exchange treatment were 79.5%, 69.3%, 53.4%, 40.6% and 39.9% (w / w), respectively; as the intensity of the interaction between the cation exchange resin and casein increased, the fluorescence intensity decreased, indicating that in the later stage of ion exchange, as the calcium ions in casein decreased, the micelle structure changed, and the binding effect with calcium ions decreased.

[0065] (2) The fluorescence intensity of the milk protein concentrates with different decalcification rates prepared in Examples 1-5 was analyzed using a fluorescence spectrometer. Figure 2 As shown in the figure, when excited at 290nm, tryptophan was observed at a wavelength of 350nm, and the fluorescence intensity at about 350nm decreased with the increase of decalcification rate. The results show that the fluorescence intensity changes differently with the different degrees of decalcification of milk protein concentrate; as the intensity of the interaction between the cation exchange resin and casein increases, the fluorescence intensity decreases, indicating that in the later stage of ion exchange, as the calcium ions in casein decrease, the micelle structure changes, and the binding effect with calcium ions decreases.

[0066] (3) The milk protein concentrates with different calcium contents prepared in Examples 1-5 were measured by gel electrophoresis. The samples with different decalcification ratios were mainly composed of α-casein (α-CN), β-casein (β-CN) and κ-casein (κ-CN). Figure 3 In the MPC, the bands of 18 kDa and 16 kDa indicated that β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) may be the main components. Compared with the comparative example 1 MPC, all examples 1-5 had the same band type and intensity, indicating that decalcification had no effect on the type of protein components. Figure 3 As shown; compared with Comparative Example 1, the molecular size of casein was slightly reduced by adding different proportions of cation exchange resin (Examples 1-5); at the same time, the molecular weight and content of each example were similar.

[0067] (5) The lutein-loaded milk protein concentrate micelles prepared in Examples 1-5 and Comparative Example 1 were subjected to a simulated gastrointestinal digestion test:

[0068] The specific steps of the simulated gastrointestinal digestion test are as follows:

[0069] Step 1: Simulating the Stomach Stage

[0070] First, 60 mg of the lutein-loaded milk protein concentrate micelles of Examples 1-5 and Comparative Example 1 were dissolved in 30 ml of deionized water, and then 30 ml of simulated gastric fluid (3.2 g / L pepsin solution, pH 2.5) was added and mixed, and the mixture was continuously stirred at 37° C. and 30-40 rpm / min for 2 hours;

[0071] Step 2: Simulating the Intestinal Stage

[0072] The simulated intestinal fluid contained 1.5 ml of saline solution (50 mM NaCl, 10 mM CaCl2), 4 ml of lipase solution (187.5 mg in PBS, pH 7.0) and 4 ml of bile salt solution (187.5 mg in PBS, pH 7.0). 30 ml of the sample from the gastric stage was added to the simulated intestinal fluid, the pH of the mixture was adjusted to 7.0, and stirring was continued at 37°C at 30-40 rpm / min for 2 hours.

[0073] Samples were taken at different time periods during the simulated gastrointestinal digestion process and passed through a syringe filter to obtain a solution containing lutein. The absorbance was measured using an ultraviolet spectrophotometer, and the concentration of lutein in the solution was calculated using a standard curve.

[0074] The experimental data of each group of samples were statistically analyzed and the lutein release amount was calculated. The results are as follows: Figure 4 As shown in the figure, in the gastric phase, compared with the comparative example 1, the digestibility of Examples 1-5 did not change significantly, and was almost the same as that of the comparative example 1; while in the small intestine phase, there was some change in Examples 1-5 after decalcification, and the digestibility was improved; under the simulated gastric digestion conditions, the absorbance of all the milk protein concentrate micelle samples loaded with lutein was zero, that is, no release occurred, indicating that the milk protein concentrate micelle loaded with lutein had acid stability; due to the stronger intramolecular and intermolecular hydrophobic interactions between the milk protein concentrate micelles loaded with lutein, the lutein-containing The hydrophobic chains of lutein-loaded milk concentrate micelles can self-assemble into a denser structure in aqueous solution and maintain a tight structure; when entering the simulated intestinal digestion process, the pH rises to 7.0, and the ionization of the carboxylic acid groups in the lutein-loaded milk concentrate micelles and the electrostatic repulsion lead to the expansion, extension and loosening of the overall structure. The absorbance of all sodium alginate derivative samples encapsulating fucoxanthin increases, and lutein begins to be released. This shows that the lutein-loaded milk concentrate micelles are responsive to the intestinal microenvironment.

[0075] (6) The encapsulation efficiency and drug loading of Examples 1-5 and Comparative Example 1 were tested. First, a calibration curve was drawn using lutein ethanol at different concentrations (0-20.0 μg / mL). The formula is y=0.1367x+0.0047, R 2=0.9999. Then, 1 mg of the prepared freeze-dried lutein milk protein concentrate micelles (loaded with lutein nanocarriers) was added to 10 mL of anhydrous ethanol and ultrasonicated for 30 minutes. The lutein concentration was determined by measuring the ultraviolet absorbance at 445 nm. The encapsulation efficiency (EE) and drug loading (LC) were calculated according to the following formula:

[0076]

[0077] Table 1 Results of calcium content, digestibility (small intestine phase), encapsulation efficiency and drug loading of different samples

[0078]

[0079] The results are as follows Figure 5 As shown in Table 1, compared with Comparative Example 1, Examples 1-5 have a trend of change in both encapsulation efficiency and drug loading, either increasing or decreasing, among which the encapsulation efficiency and drug loading of Example 2 are enhanced compared with Comparative Example 1, that is, Example 2 is the best preparation condition.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing lutein-loaded nanocarriers by one-step method using milk protein concentrate micelles to regulate decalcification rate, characterized in that: The following steps are involved: S1: dissolving a milk protein concentrate powder sample in deionized water and mixing well to obtain a milk protein concentrate solution; S2: adding a cation exchange resin to the milk protein concentrate solution for decalcification, and obtaining a first protein solution by magnetic stirring; S3: dissolving the powder sample obtained by freeze-drying the first protein solution in water to prepare a second protein solution; S4: adding lutein to the second protein solution, stirring the mixture by magnetic force to obtain a mixed solution, and dialyzing and freeze-drying the mixed solution to obtain lutein-loaded nanocarrier particles.

2. According to the method for preparing lutein-loaded nanocarriers by one-step method using milk protein concentrate micelles to regulate the decalcification rate according to claim 1, the mass ratio of cation exchange resin to milk protein concentrate powder is 4-20g:120g.

3. According to the method for preparing lutein-loaded nanocarriers by one-step method using milk concentrate protein micelles to regulate decalcification rate according to claim 1, the concentration of milk concentrate protein in the second protein solution is 0.5 mg / mL.

4. The method for preparing lutein-loaded nanocarriers by one-step method using milk protein concentrate micelles to regulate decalcification rate according to claim 1, wherein the mass of the lutein is 5% of the mass of the second protein solution.

5. The method for preparing lutein-loaded nanocarriers by one-step method using milk protein concentrate micelles to regulate decalcification rate according to claim 1, wherein in S2, the magnetic stirring speed is 30-40 rpm / min, and the time is 4 hours; In S4, the magnetic stirring speed is 30-40 rpm / min, the time is 12 hours, and the temperature is 25°C.

6. The method for preparing lutein-loaded nanocarriers by one-step method of regulating milk protein concentrate micelles by decalcification rate according to claim 1, wherein the dialysis method is to use a dialysis bag to dialyze in deionized water for 3 days, and the retention capacity of the dialysis bag is 3500 Daltons.

7. The method for preparing lutein-loaded nanocarriers by one-step method using milk protein concentrate micelles to regulate decalcification rate according to claim 1, wherein the freeze-drying time is 48 hours.

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