Lutein-loaded ternary complex and preparation method thereof

Abstract

The invention discloses a lutein-loaded ternary complex, which is a nano-particle obtained by ultrasonic coupling of ovalbumin, fatty acid and lutein under an alkaline condition. The invention also discloses a preparation method of the ternary complex, ultrasonic coupling is carried out under an alkaline pH condition, embedding of xanthophyll is realized, the ternary complex with good solubility and stability is successfully prepared, and under the action of gastrointestinal tract digestive enzyme, the complex can be used for preparing the xanthophyll-containing ternary complex with good solubility and stability. The lutein is released from the compound and forms a mixed micelle with the lipid, so that the micellization effect of the lutein is effectively promoted, the biological accessibility of the lutein is also improved, and the lutein serving as a functional nutrient plays a better role in the fields of foods, medicines and the like.

Description

Technical Field

[0001] The invention relates to a ternary complex consisting of ovalbumin, fatty acid and lutein, belonging to the field of medicine and health food. Background Art

[0002] Lutein (Lutein, LUT) is also known as "plant lutein", chemical formula C 40 H 56 O2, molecular weight 568.871, is a natural oxygen-containing carotenoid, the main component of plant pigments, and the main pigment of the macula of the human retina, which can protect vision and prevent age-related macular degeneration. It is also an excellent antioxidant that can resist cell and organ damage caused by free radicals in the human body, thereby preventing cardiovascular sclerosis, coronary heart disease, tumors and other diseases.

[0003]

[0004] However, the molecular structure of LUT has a long carbon chain with multiple alternating single and double carbon bonds, a connected methyl side group and two free hydroxyl groups at the end. Exposure to light, oxygen, heat, acid and alkali during processing can lead to degradation or isomerization of the long chain of conjugated double bonds in the structure, reducing its stability during processing, storage and utilization. In addition, due to the poor water solubility of LUT due to its crystalline structure in aqueous solution, only a small part of LUT can combine with lipid compounds after gastrointestinal digestion to form mixed micelles during digestion, thereby affecting its intestinal epithelial absorption. This largely limits the application of LUT as a functional nutrient in the fields of food and medicine (Zhu Haixia et al. Structure, distribution, physicochemical properties and physiological functions of lutein [J]. Chinese Food Additives, 2005, 2005(5): 48-55.).

[0005] In recent years, proteins have attracted more and more attention due to their advantages such as amphiphilicity, biocompatibility and safety. In addition, proteins can be induced to self-assemble under acid-base conditions, which is suitable for encapsulating drug ingredients, especially improving the water solubility and stability of poorly soluble and unstable drugs. However, since carotenoids must form mixed micelles with lipids in the intestinal cavity to be effectively absorbed, the role of a simple protein carrier system in promoting the bioavailability of carotenoids is very limited.

[0006] Previous studies have shown that as a type of lipid, small molecule fatty acids not only have a molecular solubilizing effect on carotenoids, but also promote their micellization, thereby affecting the in vitro / in vivo absorption of carotenoids (GUO Z, LIU Y, LUOY. Mechanisms of carotenoid intestinal absorption and the regulation of dietary lipids: lipid transporter-mediated transintestinal epithelial pathways [J]. Critical Reviews in Food Science and Nutrition, 2024, 64(7):1791-816.). Ovalbumin (OVA) is a typical high-quality globular protein with high content and wide source. It contains a hydrophobic pocket structure and has amphiphilicity and self-assembly. It can be used as a carrier material for fat-soluble nutrients in the food industry (FU X, BELWAL T, HE Y, et al. Interaction and binding mechanism ofcyanidin-3-O-glucoside to ovalbumin in varying pH conditions: A spectroscopicand molecular docking study [J]. Food Chemistry, 2020, 320: 126616.). Based on this, the present invention uses fatty acid-modified proteins or forms fatty acid-protein complexes as drug carriers for lutein, and uses alkaline pH ultrasonic coupling technology to prepare a self-assembled protein-fatty acid-lutein ternary complex to improve the water solubility, stability and bioaccessibility of lutein. Summary of the invention

[0007] The purpose of the present invention is to provide a ternary complex with self-assembled protein-fatty acid as carrier and loaded with lutein, aiming to solve the problems of unstable properties and low bioavailability of lutein.

[0008] To achieve the above object, the technical solution of the present invention is as follows: A ternary complex loaded with lutein is a nanoparticle obtained by ultrasonic coupling of ovalbumin, fatty acid and lutein under alkaline conditions, wherein the mass ratio of the ovalbumin, fatty acid and lutein is 10-100:1-10:0.1-1.

[0009] Preferably, the mass ratio of ovalbumin, fatty acid and lutein is 20-60:2-5:0.1-0.5.

[0010] More preferably, the mass ratio of ovalbumin, fatty acid and lutein is 30-50:2-3:0.3-0.5.

[0011] Preferably, the fatty acid is oleic acid.

[0012] A method for preparing the ternary complex comprises the following steps: 1) dissolving ovalbumin in water to prepare an ovalbumin solution with a concentration of 1-10 mg / mL and adjusting the pH to 7-11; 2) Dissolve lutein in anhydrous ethanol to prepare a lutein solution with a concentration of 1-5 mg / mL; 3) Dissolve the fatty acid in anhydrous ethanol to prepare a fatty acid solution with a concentration of 125-250 mg / mL; 4) The ovalbumin alkaline solution, the lutein solution and the fatty acid solution are mixed and ultrasonically coupled to form nanoparticles, and finally the pH of the solution is adjusted to neutral to obtain a solution containing nanoparticles.

[0013] Preferably, the concentration of the ovalbumin solution is 3-5 mg / mL.

[0014] Preferably, the pH of the ovalbumin solution is 8-10.

[0015] Preferably, the power of the ultrasonic coupling is 100-500 W, and the time is 1-5 min.

[0016] The beneficial effects of the present invention are: The present invention realizes the self-assembly of ovalbumin by ultrasonic coupling under alkaline pH conditions, forming a nanostructure with a certain shape and strength, and promotes the non-covalent binding of ovalbumin with fatty acids and lutein, and finally achieves the embedding of lutein, and successfully prepares a ternary complex with good solubility and stability. Under the action of digestive enzymes in the gastrointestinal tract, lutein is released from the complex and forms mixed micelles with lipids, which effectively promotes the micellization of lutein. Therefore, the present invention also improves the bioaccessibility of lutein, so that it can play a better role as a functional nutrient in the fields of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the FTIR and TEM images of the LUT-OA-OVA ternary complex. DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are only used to explain the present invention and are not intended to limit the present invention. In addition, the various experimental operations involved in the embodiments are routine techniques in the art, and those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature or related specifications, manuals, etc. before the application date of the present invention for implementation without special instructions.

[0019] Example 1 Fatty acid screening test Fatty acids (FA) were screened by simulating gastrointestinal digestion in vitro. Nine common dietary fatty acids were selected: palmitic acid (PA), stearic acid (SA), arachidic acid (ARA), behenic acid (BEA), oleic acid (OA), linoleic acid (LA), linolenic acid (LOA), arachidonic acid (AA) and docosahexaenoic acid (DHA), which were dissolved in anhydrous ethanol to obtain 125 mg / mL FA alcohol solution, and LUT powder was dissolved in anhydrous ethanol to obtain 5 mg / mL LUT solution. 20 μL of FA alcohol solution and 100 μL of LUT solution were added to 10 mL of deionized water and fully dissolved to obtain LUT-FA solution. Another 100 μL of LUT solution was added to 10 mL of deionized water and fully dissolved to obtain free LUT solution (blank control).

[0020] First, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared. Various required substances were accurately weighed and dissolved in deionized water. SGF: 3.2 mg / mL pepsin and 0.15 mol / L sodium chloride solution. SIF: 1.0 mg / mL pancreatin, 20.0 mg / mL bile salt and 1.0 mmol / L calcium chloride solution. Simulated gastric digestion process: 4.0 mL of LUT-FA solution and free LUT solution were mixed evenly with 4.0 mL SGF, and the pH of the mixed solution was adjusted to 2.0 with 1.0 mol / L HCl solution. The shaker temperature was set to 37℃ and the speed was 100 rpm / min. It was preheated first, and then the simulated gastric digestion was performed in the dark for 2 h. After the end, the sample was immediately taken out and treated with an ice bath, and then the pH of the mixed solution was adjusted to 7.0 to terminate the gastric digestion stage and obtain the simulated gastric digestion termination solution. Simulated intestinal digestion stage: 4.0 mL of simulated gastric digestion termination solution was mixed with 4.0 mL of SIF, and the pH of the mixture was adjusted to 7.4 with 1.0 mol / L NaOH. The simulated intestinal digestion was carried out in a shaker at 100 rpm / min and 37°C in the dark for 2 h. After the end, the sample was immediately taken out and treated with an ice bath, and then the pH of the solution was adjusted to 7.0 to obtain the simulated intestinal digestion termination solution.

[0021] Determination of retention rate, micellization rate and bioavailability of LUT in simulated gastrointestinal digestion process: 4.0 mL of simulated intestinal digestion termination solution was taken and centrifuged at 5000 rpm / min for 10 min to separate into an upper transparent micelle phase and a lower precipitate phase. 0.5 mL of simulated digestion termination solution or micelle phase was mixed with 2.5 mL of dichloromethane and 1.0 mL of methanol, vortexed for 30 s, centrifuged at 8000 rpm / min for 10 min, and the dichloromethane layer was carefully collected. The absorbance at 445 nm was measured using an ELISA reader. Based on the standard curve (y=0.06577x+0.04232, R 2 =0.99977) to calculate the content of LUT in the stop solution and micelle phase, and the retention rate, micellization rate and bioavailability of LUT in the digestion stage were calculated according to the following equations: .

[0022] The results are shown in Table 1. Considering the retention rate, micellization rate and bioavailability of LUT, oleic acid (OA) was selected for the preparation of the ternary complex.

[0023] Table 1 Results of simulated gastrointestinal digestion of LUT-FA complex

[0024] Example 2 Ingredients: Egg white protein 40mg; Oleic acid 2.5mg; Lutein 0.5mg (1) Weigh OVA powder and dissolve it in deionized water. Stir for 30 min to obtain a 4 mg / mL OVA solution. Hydrate it in a refrigerator at 4°C overnight to fully dissolve it. Adjust the pH value of the OVA solution to 9.0 with 1.0 mol / L NaOH solution. (2) Weigh LUT powder and dissolve it in anhydrous ethanol to obtain a 5 mg / mL LUT solution; (3) Dissolve OA in anhydrous ethanol to prepare a 125 mg / mL OA alcohol solution; (4) Add 20 μL of OA alcohol solution and 100 μL of LUT solution to 10 mL of alkaline OVA solution and vortex for 30 seconds; (5) The mixed solution was ultrasonically treated at 200 W for 2 min and allowed to stand in the dark at room temperature for 30 min to complete the reaction. Finally, the mixture was adjusted to pH 7.0 with 1.0 mol / L HCl solution to obtain a LUT-OA-OVA ternary complex aqueous solution system to achieve the loading of the hydrophobic LUT.

[0025] Example 3 Ingredients: egg white protein 30mg; oleic acid 3mg; lutein 0.2mg (1) Weigh OVA powder and dissolve it in deionized water. Stir for 30 min to obtain a 3 mg / mL OVA solution. Hydrate it in a refrigerator at 4°C overnight to fully dissolve it. Adjust the pH value of the OVA solution to 10.0 with 1.0 mol / L NaOH solution. (2) Weigh LUT powder and dissolve it in anhydrous ethanol to obtain a 2 mg / mL LUT solution; (3) Dissolve OA in anhydrous ethanol to prepare a 150 mg / mL OA alcohol solution; (4) Add 20 μL of OA alcohol solution and 100 μL of LUT solution to 10 mL of alkaline OVA solution and vortex for 30 seconds; (5) The mixed solution was ultrasonically treated at 200 W for 2 min and allowed to stand in the dark at room temperature for 30 min to complete the reaction. Finally, the mixture was adjusted to pH 7.0 with 1.0 mol / L HCl solution to obtain a LUT-OA-OVA ternary complex aqueous solution system to achieve the loading of the hydrophobic LUT.

[0026] Example 4 Ingredients: Egg white protein 60mg; Oleic acid 4mg; Lutein 0.4mg (1) Weigh OVA powder and dissolve it in deionized water. Stir for 30 min to obtain a 6 mg / mL OVA solution. Hydrate it in a refrigerator at 4°C overnight to fully dissolve it. Adjust the pH value of the OVA solution to 7.0 with 1.0 mol / L NaOH solution. (2) Weigh LUT powder and dissolve it in anhydrous ethanol to obtain a 4 mg / mL LUT solution; (3) Dissolve OA in anhydrous ethanol to prepare a 200 mg / mL OA alcohol solution; (4) Add 20 μL of OA alcohol solution and 100 μL of LUT solution to 10 mL of alkaline OVA solution and vortex for 30 seconds; (5) The mixed solution was ultrasonically treated at 200 W for 2 min and allowed to stand in the dark at room temperature for 30 min to complete the reaction. Finally, the mixture was adjusted to pH 7.0 with 1.0 mol / L HCl solution to obtain a LUT-OA-OVA ternary complex aqueous solution system to achieve the loading of the hydrophobic LUT.

[0027] Example 5 Ingredients: Egg white protein 50mg; Oleic acid 2mg; Lutein 0.3mg (1) Weigh OVA powder and dissolve it in deionized water. Stir for 30 min to obtain a 5 mg / mL OVA solution. Hydrate it in a refrigerator at 4°C overnight to fully dissolve it. Adjust the pH value of the OVA solution to 8.0 with 1.0 mol / L NaOH solution. (2) Weigh LUT powder and dissolve it in anhydrous ethanol to obtain a 3 mg / mL LUT solution; (3) Dissolve OA in anhydrous ethanol to prepare a 100 mg / mL OA alcohol solution; (4) Add 20 μL of OA alcohol solution and 100 μL of LUT solution to 10 mL of alkaline OVA solution and vortex for 30 seconds; (5) The mixed solution was ultrasonically treated at 200 W for 2 min and allowed to stand in the dark at room temperature for 30 min to complete the reaction. Finally, the mixture was adjusted to pH 7.0 with 1.0 mol / L HCl solution to obtain a LUT-OA-OVA ternary complex aqueous solution system to achieve the loading of the hydrophobic LUT.

[0028] Example 6 Ingredients: Egg white protein 20mg; Oleic acid 5mg; Lutein 0.1mg (1) Weigh OVA powder and dissolve it in deionized water. Stir for 30 min to obtain a 2 mg / mL OVA solution. Hydrate it in a refrigerator at 4°C overnight to fully dissolve it. Adjust the pH value of the OVA solution to 11.0 with 1.0 mol / L NaOH solution. (2) Weigh LUT powder and dissolve it in anhydrous ethanol to obtain a 1 mg / mL LUT solution; (3) Dissolve OA in anhydrous ethanol to prepare a 250 mg / mL OA alcohol solution; (4) Add 20 μL of OA alcohol solution and 100 μL of LUT solution to 10 mL of alkaline OVA solution and vortex for 30 seconds; (5) The mixed solution was ultrasonically treated at 200 W for 2 min and allowed to stand in the dark at room temperature for 30 min to complete the reaction. Finally, the mixture was adjusted to pH 7.0 with 1.0 mol / L HCl solution to obtain a LUT-OA-OVA ternary complex aqueous solution system to achieve the loading of the hydrophobic LUT.

[0029] Test example 1. Determination of Encapsulation Efficiency and Loading Amount Take 0.5 mL of freshly prepared LUT-OA-OVA solution in a centrifuge tube, add 3.5 mL of n-hexane, vortex mix for 1 min, and centrifuge at 8000 rpm / min for 5 min. After centrifugation, carefully aspirate the supernatant and measure the absorbance of each sample at 445 nm using an ELISA reader. According to the standard curve (y=0.06577x+0.04232, R 2 =0.99977) to calculate the mass of LUT not embedded in the complex. The embedding rate and loading amount of LUT in the complex were calculated according to the following formula: .

[0030] 2. Particle size, PDI, Zeta potential and transmittance determination The measurements were performed using a Zetasizer Nano-ZS90 light scattering particle size analyzer. The parameters for the measurement conditions were set as follows: equilibrium time 120 s, fixed scattering angle 90.0°, repeated measurements three times at 25°C, and particle size data were reported as density-weighted average diameter.

[0031] The physicochemical properties of the constructed ternary complex and the embedding rate and loading amount of LUT are shown in Table 2. The LUT-OA-OVA ternary complex prepared in Examples 2-6 has good physicochemical properties and is a stable monodisperse system, among which Example 2 has the highest embedding rate and loading amount.

[0032] Table 2 Particle size and encapsulation efficiency of LUT-OA-OVA ternary complex

[0033] 3. Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) determination The interaction between LUT, OA and OVA was determined by Fourier transform infrared spectroscopy (FTIR). In the FTIR graph, the amide I band (1600 cm -1 ~1700cm -1 ) and band II (1550 cm -1 The amide A band (3100 cm −1 ~ 3500 cm −1 ) is related to the hydrogen bonds between molecules. The shift of the characteristic peaks indicates that the introduction of OA promotes the non-covalent interactions between OVA and LUT, including electrostatic, hydrogen bonding and hydrophobic interactions. Transmission electron microscopy (TEM) images show that the LUT-OA-OVA ternary complex is a smooth spherical particle. The results are shown in Figure 1 .

[0034] 4. Stability test (1) Improvement effect of thermal processing stability In order to explore the effect of oleic acid-protein carrier on the thermal processing stability of LUT, pasteurization and hot cooking sterilization in food processing were simulated by heating at 65℃ for 30min and boiling at 100℃ for 10min, respectively. After the freshly prepared complex solution was heated under the above heating conditions, the sample was immediately cooled to room temperature with running water in the dark to end the heating. The free LUT solution was used as a control. The particle size, PDI, Zeta-potential and retention rate of LUT of the heated samples were measured.

[0035] Determination of the retention rate of LUT in samples after heat treatment: Take 0.5 mL of the heated sample and mix it with 2.5 mL of dichloromethane and 1.0 mL of methanol solution, vortex for 30 s, and then centrifuge at 5000 rpm / min for 10 min at a low temperature of 4°C. Collect the dichloromethane phase and measure its absorbance at a wavelength of 445 nm using an ELISA reader. Calculate the LUT content according to the standard curve, and calculate the retention rate of LUT after heat treatment according to the equation.

[0036] .

[0037] It can be found from Table 3 that after being embedded in the OA-OVA carrier, the retention rate of LUT increased by about 20% under the conditions of heating at 65°C for 30 min and boiling at 100°C for 10 min, and it still had good physical and chemical properties, indicating that the carrier improved the thermal processing stability of LUT.

[0038] Table 3-1 Retention rate of LUT and LUT-OA-OVA ternary complex after simulated thermal processing

[0039] Table 3-2 Changes in the physicochemical properties of the LUT-OA-OVA ternary complex before and after simulated thermal processing

[0040] (2) Improvement of storage stability The prepared container was autoclaved to effectively evaluate the effect of the OA-OVA carrier on the storage stability of LUT. The freshly prepared LUT-OA-OVA solution was placed in a dark environment at 4°C for 28 days. The particle size and PDI, zeta-potential and remaining LUT content of the system were measured every seven days. The LUT degradation kinetics during storage were calculated according to the following formula:

[0041] in, M t and M0 represent the LUT contents at time t and 0 respectively,k is the degradation rate constant (d -1 ), t is the storage time (d), t 1 / 2 is the half-life of LUT (d), which represents the time required for the initial LUT content to degrade by 50%.

[0042] It can be concluded from Table 4 that the LUT-OA-OVA ternary complex still has good particle stability and uniformity after storage for 28 days, which effectively improves the storage stability of LUT and reduces the degradation of LUT during storage.

[0043] Table 4-1 Degradation of LUT and LUT-OA-OVA ternary complex after 28 days of storage k Value and half-life t 1 / 2

[0044] Table 4-2 Changes in the physicochemical properties of the LUT-OA-OVA ternary complex after 28-day storage

[0045] 5. In vitro gastrointestinal simulated digestion test After being loaded on a protein-based carrier, the ingested LUT is released from the protein-based complex under the action of digestive enzymes in the gastrointestinal tract, and forms mixed micelles with lipids, thereby promoting the intestinal absorption of LUT. This experiment analyzes the micellization rate and bioavailability of LUT in the simulated gastrointestinal digestion process to evaluate the digestion and absorption effect of the prepared LUT-OA-OVA complex. For specific test methods, see Example 1.

[0046] The results in Table 5 show that after being embedded in the OA-OVA carrier, the digestion stability, micellization rate and bioaccessibility of LUT were significantly improved, which proves that the complex prepared by the present invention effectively improves the gastrointestinal stability and bioaccessibility of LUT.

[0047] Table 5 Simulated gastrointestinal digestion results of LUT-OA-OVA ternary complex

Claims

1. A ternary complex loaded with lutein, which is a nanoparticle obtained by ultrasonic coupling of ovalbumin, fatty acids and lutein under alkaline conditions, wherein the mass ratio of the ovalbumin, fatty acids and lutein is 10-100:1-10:0.1-1.

2. The ternary complex according to claim 1, characterized in that: The mass ratio of the ovalbumin, fatty acid and lutein is 20-60:2-5:0.1-0.

5.

3. The ternary complex according to claim 1, characterized in that: The mass ratio of ovalbumin, fatty acid and lutein is 30-50:2-3:0.3-0.

5.

4. The ternary complex according to claim 1, characterized in that: The fatty acid is oleic acid.

5. A method for preparing the ternary complex according to any one of claims 1 to 4, characterized in that The following steps are involved: 1) dissolving ovalbumin in water to prepare an ovalbumin solution with a concentration of 1-10 mg / mL and adjusting the pH to 7-11; 2) Dissolve lutein in anhydrous ethanol to prepare a lutein solution with a concentration of 1-5 mg / mL; 3) Dissolve the fatty acid in anhydrous ethanol to prepare a fatty acid solution with a concentration of 125-250 mg / mL; 4) The ovalbumin solution, the lutein solution and the fatty acid solution are mixed and ultrasonically coupled to form nanoparticles, and finally the pH of the solution is adjusted to neutral to obtain a solution containing nanoparticles.

6. The preparation method according to claim 5, characterized in that: The concentration of the ovalbumin solution is 3-5 mg / mL.

7. The preparation method according to claim 5, characterized in that: The pH of the ovalbumin solution is 8-10.

8. The preparation method according to claim 5, characterized in that: The power of the ultrasonic coupling is 100-500 W, and the time is 1-5 min.

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