Dual emulsions based on protein microgel-polysaccharide covalent complexes, their preparation and applications

Abstract

This invention discloses a dual emulsion based on a protein microgel-polysaccharide covalent complex, its preparation method, and its application. The dual emulsion comprises an inner aqueous phase, an oil phase, and an outer aqueous phase; wherein the inner aqueous phase comprises NaCl, hydrophilic functional factor tea polyphenols, and water; the oil phase comprises polyglycerol ricinoleate, hydrophobic functional factor curcumin, and medium-chain triglycerides; and the outer aqueous phase comprises the protein microgel-polysaccharide covalent complex and water. The dual emulsion provided by this invention exhibits good environmental stability, significantly enhances the photothermal stability and bioavailability of tea polyphenols and curcumin, and can be used to improve neuroinflammation and cognitive memory impairment.

Description

Technical Field

[0001] This invention specifically relates to a dual emulsion based on a protein microgel-polysaccharide covalent complex, its preparation method, and its application. Background Technology

[0002] Curcumin and tea polyphenols are two plant polyphenols that are widely available in nature and have shown outstanding effects in preventing and improving neuroinflammation. However, hydrophobic curcumin and hydrophilic tea polyphenols are sensitive to environmental factors such as light, heat, oxygen, and pH, and are extremely unstable during processing, storage, and digestion, resulting in low oral bioavailability.

[0003] Encapsulating and protecting functional agents using biocompatible, food-grade delivery systems is an effective strategy for improving their stability and bioavailability. However, traditional delivery systems (such as nanoparticles, micelles, or emulsions) struggle to co-encapsulate functional agents with varying hydrophilicity and hydrophobicity. Recent studies have found that water-in-oil-in-water (W / O / W) dual emulsions can address these issues; however, due to their structural complexity, dual emulsions typically exhibit poor stability, making it difficult to achieve long-term protection of the encapsulated functional agents. Summary of the Invention

[0004] The main objective of this invention is to provide a dual emulsion based on a protein microgel-polysaccharide covalent complex, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] This invention provides a dual emulsion based on a protein microgel-polysaccharide covalent complex, comprising an inner aqueous phase, an oil phase, and an outer aqueous phase. The inner aqueous phase comprises NaCl, hydrophilic functional factors such as tea polyphenols (e.g., epigallocatechin gallate ester EGCG, epicatechin gallate ester ECG), and water. The oil phase comprises polyglycerol ricinoleate (PGPR), hydrophobic functional factors such as curcumin, and medium-chain triglycerides. The outer aqueous phase comprises the protein microgel-polysaccharide covalent complex and water.

[0007] This invention also provides a method for preparing the aforementioned dual emulsion based on a protein microgel-polysaccharide covalent complex, comprising:

[0008] It provides an inner aqueous phase containing NaCl, hydrophilic functional factor tea polyphenols and water, an oil phase containing PGPR, hydrophobic functional factor curcumin and medium-chain triglycerides, and an outer aqueous phase containing protein microgel-polysaccharide covalent complex and water.

[0009] The aqueous phase and oil phase are mixed and subjected to a first shear homogenization process to obtain a W / O emulsion.

[0010] Furthermore, the W / O emulsion is mixed with an external aqueous phase and subjected to a second shear homogenization treatment to obtain a W / O / W dual emulsion, namely a dual emulsion based on a protein microgel-polysaccharide covalent complex.

[0011] This invention also provides the use of the aforementioned dual emulsion based on a protein microgel-polysaccharide covalent complex in the preparation of products for the prevention and / or improvement of cognitive memory impairment or neuroinflammation.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] (1) Compared with commercial single proteins, the present invention utilizes protein microgel-polysaccharide covalent complex as oil-water interface emulsifier to significantly enhance the stability of emulsion. The emulsifier raw material is not only inexpensive and readily available, but also has good biosafety and biodegradability. Moreover, the preparation method is green, safe and reproducible.

[0014] (2) Compared with free curcumin or tea polyphenols, the curcumin / tea polyphenol dual emulsion provided by the present invention not only solves the problem of curcumin being insoluble in water, but also effectively improves the photothermal stability and bioavailability of curcumin and tea polyphenols, and effectively alleviates cognitive dysfunction and neuroinflammation induced by lipopolysaccharide (LPS). Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figures 1a-1b The present invention describes the interfacial layer thickness, interfacial protein adsorption rate (%), and protein surface load (mg / m³) of untreated pea protein isolate (PPI), pea protein isolate microgel (hereinafter referred to as "microgel"), pea protein isolate microgel-sodium alginate physical mixture (hereinafter referred to as "mixture"), and pea protein isolate microgel-sodium alginate covalent complex (hereinafter referred to as "covalent complex") at the oil-water interface. 2 The figure shows that different uppercase and lowercase letters at the top of the bars represent significant differences between samples (p < 0.05).

[0017] Figure 2These are optical microscopic images of pea protein isolate, pea protein isolate microgel, pea protein isolate microgel-sodium alginate physical mixture, and pea protein isolate microgel-sodium alginate covalent complex stable O / W emulsions (PPI-emulsion, microgel-emulsion, mixture-emulsion, covalent complex-emulsion) under different environmental stimuli (light, heat, freeze-thaw) in this invention.

[0018] Figures 3a-3b In this invention, curcumin and EGCG, a tea polyphenol, are present in a W / O / W dual emulsion under light irradiation (0.35 W / m²). 2 120 min Figure 3a ) and heating (90 ℃, 120 min, Figure 3b Retention rate plot under ) treatment;

[0019] Figure 4 This is a diagram showing the bioavailability of curcumin and EGCG in the W / O / W dual emulsion of this invention. Different uppercase and lowercase letters at the top of the columns in the diagram represent significant differences between samples (p < 0.05).

[0020] Figures 5a-5d This is a diagram illustrating the effects of the curcumin / EGCG dual emulsion of this invention on cognitive memory in LPS-inflamed mice, including the mice's spontaneous activity ability in the open field test. Figure 5a ), the total number of arm advances in the Y-maze experiment ( Figure 5b ) and the percentage of spontaneous alternation ( Figure 5c ), the recognition index in the new object recognition experiment ( Figure 5d In the figure, different letters at the top of the columns represent significant differences between samples (p < 0.05).

[0021] Figures 6a-6f The curcumin / EGCG dual emulsion in this invention has an effect on the inflammatory factors in the brains of LPS-inflamed mice ( Figure 6a IL-1β; Figure 6b IL-6; Figure 6c TNF-α; Figure 6d IL-10) and inflammatory proteins ( Figure 6e iNOS; Figure 6f The graph shows the effect of COX-2 mRNA expression levels on the samples. Different letters at the top of the bars represent significant differences between samples (p < 0.05).

[0022] Figure 7 This is a schematic diagram of the preparation process of a dual emulsion based on a protein microgel-polysaccharide covalent complex in a typical embodiment of the present invention. Detailed Implementation

[0023] In view of the deficiencies of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention, which mainly utilizes a protein microgel-polysaccharide covalent complex-stabilized W / O / W dual emulsion to co-encapsulate curcumin and tea polyphenols, so as to improve their stability, oral bioavailability and bioactivity.

[0024] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Specifically, as one aspect of the technical solution of the present invention, a dual emulsion based on a protein microgel-polysaccharide covalent complex includes an inner aqueous phase, an oil phase, and an outer aqueous phase; wherein, the inner aqueous phase includes NaCl, hydrophilic functional factors tea polyphenols (EGCG, ECG, etc.) and water; the oil phase includes PGPR, hydrophobic functional factors curcumin and medium-chain triglycerides; and the outer aqueous phase includes the protein microgel-polysaccharide covalent complex and water.

[0026] In some preferred embodiments, the NaCl content in the internal aqueous phase is 0.5~2 wt%.

[0027] Furthermore, the NaCl content in the internal aqueous phase is 1 wt%.

[0028] In some preferred embodiments, the concentration of the hydrophilic functional factor tea polyphenols in the internal aqueous phase is 1.0~2.0 mg / mL.

[0029] Furthermore, the concentration of the hydrophilic functional factor tea polyphenols in the internal aqueous phase is 1.4 mg / mL.

[0030] In some preferred embodiments, the volume fraction of PGPR in the oil phase is 2-8%.

[0031] Furthermore, the volume fraction of PGPR in the oil phase is 5%.

[0032] In some preferred embodiments, the concentration of the hydrophobic functional factor curcumin in the oil phase is 0.6 mg / mL.

[0033] In some preferred embodiments, the content of the protein microgel-polysaccharide covalent complex in the external aqueous phase is 1~4 wt%.

[0034] Furthermore, the content of the protein microgel-polysaccharide covalent complex in the external aqueous phase is 2 wt%.

[0035] In some preferred embodiments, the protein microgel-polysaccharide covalent complex includes a pea protein isolate microgel-sodium alginate covalent complex.

[0036] In some preferred embodiments, the method for preparing the pea protein isolate microgel-sodium alginate covalent complex includes:

[0037] Step (1): Disperse pea protein isolate in distilled water, heat in an 85 ℃ water bath for 30 min, add transglutaminase (TG enzyme, 20 U / g protein), heat in a 45 ℃ water bath for 4 h, cool, and prepare pea protein isolate gel; place the pea protein isolate gel in a certain volume of distilled water (pH=7), shear and break at 10000 rpm for 2 min, homogenize at 50 MPa for 3 cycles, and prepare pea protein isolate microgel.

[0038] Step (2): Mix pea protein isolate microgel dispersion (4 wt%) and sodium alginate dispersion (0.2 wt%) in equal volumes, adjust pH to 10, sonicate at 195 W for 20 min, heat in a 95 ℃ water bath for 6 h, cool at room temperature, and terminate the reaction to obtain pea protein isolate microgel-sodium alginate covalent complex.

[0039] Another aspect of the present invention provides a method for preparing the aforementioned dual emulsion based on a protein microgel-polysaccharide covalent complex, comprising:

[0040] It provides an inner aqueous phase containing NaCl, hydrophilic functional factor tea polyphenols and water, an oil phase containing PGPR, hydrophobic functional factor curcumin and medium-chain triglycerides, and an outer aqueous phase containing protein microgel-polysaccharide covalent complex and water.

[0041] The aqueous phase and oil phase are mixed and subjected to a first shear homogenization process to obtain a W / O emulsion.

[0042] Furthermore, the W / O emulsion is mixed with an external aqueous phase and subjected to a second shear homogenization treatment to obtain a W / O / W dual emulsion, namely a dual emulsion based on a protein microgel-polysaccharide covalent complex.

[0043] In some preferred embodiments, the preparation method further includes:

[0044] Proteins are dispersed in water and heated, then TG enzyme is added and heated and cooled to obtain a protein gel. The protein gel is then sheared, broken up and homogenized to obtain a protein microgel.

[0045] Furthermore, the protein microgel is mixed with polysaccharide and subjected to Maillard reaction to obtain a protein microgel-polysaccharide covalent complex.

[0046] Furthermore, the protein includes any one or more combinations of pea protein isolate, soy protein, and whey protein, but is not limited thereto.

[0047] Furthermore, the polysaccharide includes, but is not limited to, any one or more combinations of sodium alginate, sodium hyaluronate, and pectin.

[0048] In some preferred embodiments, the volume ratio of the internal aqueous phase to the oil phase is 2:8 to 4:6.

[0049] Furthermore, the volume ratio of the internal aqueous phase to the oil phase is 3:7.

[0050] In some preferred embodiments, the volume ratio of the W / O emulsion to the external aqueous phase is 2:8 to 5:5.

[0051] Furthermore, the volume ratio of the W / O emulsion to the external aqueous phase is 5:5.

[0052] In some preferred embodiments, the preparation method specifically includes: performing a first shearing and homogenization treatment at 10,000 to 15,000 rpm for 3 to 5 minutes.

[0053] In some preferred embodiments, the preparation method specifically includes: performing a second shearing and homogenization treatment at 7000~10000 rpm for 3~5 min.

[0054] In some more specific embodiments, the preparation method of the dual emulsion based on the protein microgel-polysaccharide covalent complex includes the following steps: mixing the inner aqueous phase (containing 1 wt% NaCl and 1.4 mg / mL EGCG) and the oil phase (containing 5% v / v PGPR and 0.6 mg / mL curcumin) at a volume ratio of 3:7, and shearing at 10000 rpm for 3 min to obtain a W / O emulsion; then mixing the W / O emulsion with the outer aqueous phase (containing 2 wt% pea protein isolate microgel-sodium alginate covalent complex, with a physical mixture of 2 wt% pea protein isolate, pea protein isolate microgel, and pea protein isolate microgel-sodium alginate as a control group) at a volume ratio of 5:5, and shearing at 7000 rpm for 3 min to prepare a curcumin / EGCG dual emulsion.

[0055] Further, the preparation method of the pea protein isolate microgel includes the following steps: dispersing 10 wt% pea protein isolate in distilled water, heating in an 85 ℃ water bath for 30 min, adding TG enzyme (20 U / g protein), heating in a 45 ℃ water bath for 4 h, cooling, and obtaining pea protein isolate gel; placing the pea protein isolate gel in a certain volume of distilled water (pH=7), shearing and breaking at 10000 rpm for 2 min, homogenizing at 50 MPa for 3 cycles, and obtaining pea protein isolate microgel.

[0056] Furthermore, the preparation method of the pea protein isolate microgel-sodium alginate physical mixture includes the following steps: mixing 4 wt% pea protein isolate microgel dispersion and 0.2 wt% sodium alginate dispersion in equal volumes, adjusting the pH to 7, and obtaining a pea protein isolate microgel-sodium alginate physical mixture dispersion with a concentration of 2 wt%.

[0057] Further, the preparation method of the pea protein isolate microgel-sodium alginate covalent complex includes the following steps: mixing 4 wt% pea protein isolate microgel dispersion and 0.2 wt% sodium alginate dispersion in equal volumes, adjusting the pH to 10, pretreating with ultrasound at 195 W for 20 min, heating in a water bath at 95 ℃ for 6 h, cooling at room temperature, terminating the reaction, and obtaining a pea protein isolate microgel-sodium alginate covalent complex dispersion with a concentration of 2 wt%.

[0058] In some preferred embodiments, a schematic diagram of the preparation process of the dual emulsion based on the protein microgel-polysaccharide covalent complex of the present invention is shown below. Figure 7 As shown.

[0059] Another aspect of the present invention provides the use of a dual emulsion based on a protein microgel-polysaccharide covalent complex in the preparation of products for the prevention and / or improvement of cognitive memory impairment or neuroinflammation.

[0060] For example, the dual emulsion is used as a functional factor / drug carrier in the preparation of products for the prevention and / or improvement of cognitive memory impairment or neuroinflammation.

[0061] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0062] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0063] Example 1

[0064] Different oil-water interface emulsifiers were prepared according to the following methods, specifically including:

[0065] Preparation method of pea protein isolate dispersion: Disperse commercially available pea protein isolate powder in a certain volume of distilled water, stir evenly, and prepare a pea protein isolate dispersion with a mass fraction of 2%.

[0066] Preparation method of pea protein isolate microgel: Commercially available pea protein isolate powder was dispersed in a certain volume of distilled water and stirred evenly to obtain a 10% (w / w) pea protein isolate dispersion. The dispersion was then heated in an 85 ℃ water bath for 30 min, cooled, and TG enzyme (20 U / g protein) was added. The mixture was then heated in a 45 ℃ water bath for 4 h, and cooled to obtain a pea protein isolate gel. The pea protein isolate gel was placed in a certain volume of distilled water (pH=7) and subjected to shearing (10000 rpm, 2 min) and high-pressure homogenization (50 MPa, 3 cycles) to prepare a 2% (w / w) pea protein isolate microgel dispersion.

[0067] Preparation method of pea protein isolate microgel-sodium alginate physical mixture: Prepare a pea protein isolate microgel dispersion with a mass fraction of 4% according to the above method, and then mix it with an equal volume of sodium alginate dispersion with a mass fraction of 0.2%, adjust the pH to 7, and prepare a pea protein isolate microgel-sodium alginate physical mixture dispersion with a protein concentration of 2 wt%.

[0068] Preparation method of pea protein isolate microgel-sodium alginate covalent complex: A 4% (w / w) pea protein isolate microgel dispersion was prepared according to the above method. Then, it was mixed with an equal volume of a 0.2% (w / w) sodium alginate dispersion. The pH was adjusted to 10, and the mixture was pretreated by ultrasonication at 195 W for 20 min. The mixture was then heated in a water bath at 95 ℃ for 6 h and cooled to room temperature to terminate the reaction, thus obtaining a 2 wt% pea protein isolate microgel-sodium alginate covalent complex dispersion.

[0069] The interfacial layer thickness, interfacial protein adsorption rate, and protein surface load of the different emulsifiers at the oil-water interface are shown in the figure. Figures 1a-1b .Depend on Figures 1a-1b It can be seen that, compared with untreated pea protein isolate, pea protein isolate microgel, and pea protein isolate microgel-sodium alginate physical mixture, the pea protein isolate microgel-sodium alginate covalent complex formed the thickest interfacial layer at the oil-water interface (40.68±4.63 nm) and had the highest interfacial protein adsorption rate (46.92±1.17%).

[0070] Example 2

[0071] O / W emulsions with different interfacial compositions and structures were prepared according to the following method, and the stability of the emulsions under different environmental stimuli (heat, light, freeze-thaw) was investigated.

[0072] Methods for preparing O / W emulsions with different interfacial compositions and structures: Dispersions of pea protein isolate (2% by mass), pea protein isolate microgel, a physical mixture of pea protein isolate microgel and sodium alginate, and a covalent complex of pea protein isolate microgel and sodium alginate were used as the aqueous phase. These were then mixed with medium-chain triglycerides at a volume ratio of 8:2 and sheared at 10,000 rpm for 3 min to prepare different O / W emulsions.

[0073] Thermal stability test: The freshly prepared emulsion was heated in a water bath at 90 °C for 3 h, and its optical microscopic image was taken.

[0074] Photostability test: The freshly prepared emulsion was placed in a xenon lamp aging chamber at 0.35 W / cm². 2 Irradiate the sample under light intensity for 3 hours and then take optical microscopic images.

[0075] Freeze-thaw stability test: The freshly prepared emulsion was frozen at -80 ℃ for 24 h. After the sample thawed at room temperature, its optical microscopic image was taken.

[0076] The microstructure changes of different emulsions after heating, light exposure, or freeze-thaw treatment are as follows: Figure 2 As shown. By Figure 2 It can be seen that, compared with untreated pea protein isolate stabilized emulsions, pea protein isolate microgels and pea protein isolate microgel-sodium alginate system stabilized emulsions exhibit better environmental stability.

[0077] Example 3

[0078] Pea protein isolate microgel-sodium alginate covalent complex with excellent interfacial properties was selected as the external aqueous phase emulsifier, and a W / O / W dual emulsion co-encapsulating curcumin and EGCG was prepared according to the following method.

[0079] Hydrophilic EGCG was dissolved in a 1 wt% NaCl aqueous solution to prepare an inner aqueous phase with an EGCG concentration of 1.4 mg / mL; lipophilic curcumin was dispersed in a medium-chain triglyceride containing 5% v / v PGPR to prepare an oil phase with a curcumin concentration of 0.6 mg / mL; a 2 wt% pea protein isolate microgel-sodium alginate covalent complex dispersion was used as the outer aqueous phase; the inner aqueous phase and oil phase were mixed at a volume ratio of 3:7 and sheared at 10000 rpm for 3 min to obtain a W / O primary emulsion; then the W / O primary emulsion and the outer aqueous phase were mixed at a volume ratio of 5:5 and sheared at 7000 rpm for 3 min to prepare a W / O / W dual emulsion co-encapsulated with curcumin and EGCG.

[0080] Free curcumin and EGCG dispersed in distilled water were used as the control group, while encapsulated curcumin and EGCG were subjected to light (0.35 W / m²). 2 Retention rates under heating (90 °C, 120 min) and heating (90 °C, 120 min) conditions are as follows: Figures 3a-3b As shown. By Figures 3a-3b It can be seen that, compared with free curcumin and EGCG, the photothermal stability of encapsulated curcumin and EGCG is significantly improved, and the dual emulsion stabilized by pea protein isolate microgel-sodium alginate covalent complex exhibits a more prominent protective effect on curcumin and EGCG.

[0081] Example 4

[0082] The bioavailability of curcumin and EGCG in double emulsions was evaluated using an in vitro digestion model to simulate different stages of digestion (oral cavity, stomach, intestine).

[0083] Before the experiment, 1.25× simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF) were prepared according to the INFOGEST method.

[0084] Simulated oral digestion: 10 mL of emulsion was mixed with 8 mL of 1.25× SSF, and 50 μL of 0.3 M CaCl2·(H2O)2 solution was added. The pH of the mixture was adjusted to 7 using 2 M HCl and 2 M NaOH, and the final volume was made up to 20 mL with distilled water. The sample was then incubated in a 37 °C water bath with a magnetic stirrer for 2 min.

[0085] Simulated gastric digestion: 15 mL of the oral digestion fluid was mixed with 12 mL of 1.25× SGF, 7.5 μL of 0.3 M CaCl2·(H2O)2 solution and 1 mL of pepsin solution (final concentration 2000 U / mL) were added, the pH was adjusted to 3 with 2 M HCl, and the final volume was made up to 30 mL with distilled water. The sample was then incubated in a 37 °C water bath with a magnetic stirrer for 2 h.

[0086] Simulated intestinal digestion: 20 mL of the above-mentioned gastric digestive fluid was mixed with 8 mL of 1.25× SIF solution, and 40 μL of 0.3M CaCl2·(H2O)2 solution, 4 mL of bile salt solution, 2.5 mL of lipase solution, and 2.5 mL of trypsin solution were added. Before the experiment, the bile salts and digestive enzymes were pre-dissolved in 1.25× SIF solution. The pH of the mixture was adjusted to 7 using 2 M HCl and 2 M NaOH, and the final volume was brought to 40 mL with distilled water. The sample was then incubated in a 37 °C water bath with a magnetic stirrer for 2 h, maintaining the pH at 7 continuously with 1 M NaOH solution. In the final system, the concentration of bile salts was 10 mM, the activity of trypsin was 100 U / mL, and the activity of lipase was 2000 U / mL.

[0087] After simulated intestinal digestion, the digestive fluid was collected and centrifuged at 4000 rpm for 40 min. The intermediate micelle phase was collected and mixed with an equal volume of anhydrous ethanol, stored overnight at 4 °C, and centrifuged at 8000 rpm for 20 min. The supernatant was collected and filtered through an aqueous membrane with a pore size of 0.45 μm. The absorbance of curcumin and EGCG in the filtrate was then measured at 424.5 nm and 276 nm, respectively. The content of functional factors in the micelle phase was determined according to their corresponding standard curves. Finally, the bioavailability of curcumin and EGCG was calculated according to the following formula:

[0088] Bioaccessibility (%) = Content of functional factors in micelle phase / Total content of functional factors × 100

[0089] Bioavailability of curcumin and EGCG in different double emulsions, such as Figure 4 As shown, the bioavailability of encapsulated curcumin and EGCG was significantly enhanced compared to their free forms. In a dual emulsion stabilized by a pea protein isolate microgel-sodium alginate covalent complex, the bioavailability of curcumin and EGCG reached 55.88% and 98.98%, respectively, which are approximately 14.66 times and 2.59 times that of their free forms.

[0090] Example 5

[0091] A curcumin / EGCG dual emulsion, stabilized by a pea protein isolate microgel-sodium alginate covalent complex, was used to conduct dietary intervention on LPS-inflamed mice. The effects of the curcumin / EGCG dual emulsion on cognitive memory in LPS-inflamed mice were then assessed through behavioral tests (including open field, novel object recognition, and Y maze).

[0092] Animal grouping and treatment: Eighty-four 8-week-old male C57BL / 6J mice were randomly divided into six groups (n=14 per group) after 7 days of acclimatization: control group, LPS model group, LPS + curcumin group, LPS + EGCG group, LPS + curcumin / EGCG double emulsion group, and empty double emulsion group. Mice were administered free curcumin, free EGCG, curcumin / EGCG double emulsion, and empty double emulsion by gavage for five consecutive weeks, according to their group. The control and model groups were administered physiological saline by gavage. On day 29, except for the control group, mice in the other groups were intraperitoneally injected with LPS (250 μg / kg / d) 30 minutes after gavage, for 7 consecutive days. The control group received an equal volume of physiological saline intraperitoneally. Behavioral tests were performed on day 32. On day 36, mice were sacrificed, and their brain tissue was collected and stored at -80 ℃ for later use.

[0093] Open field test: This test is typically used to assess exploratory behavior and anxiety levels in mice, as well as their ability to adapt to new environments. Mice are placed in a white motion monitoring box (40 cm × 40 cm × 40 cm) for 5 minutes, allowing them free movement. The total distance moved (mm) is recorded using a computer video tracking system. Before each test, all mice must be acclimatized to the laboratory environment for at least 1 hour, and the monitoring box is cleaned with 75% ethanol each time to minimize interference with the results.

[0094] Y-maze test: Widely used to study learning, memory, and navigation in rodents. The Y-maze consists of isometric arms (length:height:width = 35 cm:15 cm:5 cm). Before each test, mice are placed in an empty box and allowed to move around for 5 minutes. Then, the mice are allowed to explore from the center of the Y-maze for 5 minutes, and their movement paths and the order in which they enter the arms are recorded. The percentage of spontaneous alternation (%) is calculated using the following formula:

[0095] Spontaneous alternation percentage (%) = Number of consecutive entries into three different arms / (Total number of arm entries - 2) × 100

[0096] Novel object recognition test: A common method for assessing short-term non-spatial recognition memory in mice, consisting of three phases: adaptation period (day 1), familiarization period (day 2), and testing period (day 3). The procedure for the adaptation period is basically the same as the open field test. During the familiarization period, two objects with the same appearance, color, and texture are placed diagonally in a white motion monitoring box, and the mice are allowed to explore freely for 10 minutes. During the testing period, one of the old objects from the familiarization period is replaced with a new object that is similar to it, and the mice are allowed to explore freely from the center of the testing box for 5 minutes. The duration for which the mice smell the old and new objects is recorded, and the recognition index (%) is calculated according to the following formula:

[0097] Recognition Index (%) = (Duration of exploring new objects / Duration of exploring old objects) × 100

[0098] Effects of curcumin / EGCG dual emulsion on cognitive memory in LPS-inflamed mice: Figures 5a-5d As shown. By Figure 5a and 5b It was found that there was no significant difference in the spontaneous activity ability among the groups of mice, indicating that LPS, curcumin, or EGCG intervention did not alter the spontaneous activity ability of mice. The percentage of alternating mice in the Y-maze test showed ( Figure 5c Compared to the control group, the proportion of mice in the LPS group who received consecutive injections into three different arms was significantly lower, indicating that intraperitoneal injection of LPS significantly reduced working memory in mice. There were no significant differences between the curcumin, EGCG, and empty-load dual emulsion groups and the LPS group; however, the EGCG / curcumin dual emulsion significantly increased this proportion, alleviating LPS-induced working memory impairment. Furthermore... Figure 5d The results of the new object recognition experiment showed that the EGCG / curcumin dual emulsion could significantly improve the learning and memory abilities of LPS-inflamed mice, and its effect was higher than that of free curcumin and EGCG.

[0099] Example 6

[0100] The relative mRNA expression levels of inflammatory factors (IL-1β, IL-6, TNF-α, and IL-10) and inflammatory proteins (iNOS and COX-2) in the brains of mice in each group were investigated using the following method. Specific procedures included:

[0101] RNA extraction and reverse transcription: Weigh approximately 70 mg of brain tissue and add it to a grinding tube containing 0.7 mL of Trizol RNA extraction reagent. Add two grinding beads and homogenize thoroughly with an electric homogenizer for approximately 1–2 minutes. Then, extract RNA according to the instructions. Assess the purity of the RNA using a Quawell 5000 UV-Vis spectrophotometer and dilute to a uniform concentration. Prepare the reverse transcription system according to the instructions of the reverse transcription kit, gently mix, and perform the reverse transcription reaction. After the reaction, store the cDNA at -80°C for later use.

[0102] Real-time quantitative PCR: Fluorescent quantification was performed using SYBR Green PCR Mater mix. PCR reaction solutions were prepared according to Table 1, and two-step PCR amplification was performed according to Table 2, following the kit instructions.

[0103] Table 1 Real-time quantitative PCR reaction system

[0104] Table 2 Real-time quantitative PCR reaction program design

[0105] The primer information for the target gene in real-time quantitative PCR is shown in Table 3. GAPDH was used as an internal control, and 2... -∆∆Ct The method calculates the relative expression level of genes.

[0106] Table 3 Primer sequences (5′-3′) for target genes in real-time quantitative PCR

[0107] like Figures 6a-6e As shown, real-time quantitative PCR results indicated that, compared with the control group, intraperitoneal injection of LPS significantly upregulated the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and inflammatory proteins (iNOS, COX-2) in the brain, while curcumin and EGCG interventions significantly downregulated the high expression of pro-inflammatory mediators induced by LPS, with the curcumin / EGCG dual emulsion exhibiting the best anti-inflammatory effect. Furthermore, the curcumin / EGCG dual emulsion significantly improved the low expression of the LPS-induced anti-inflammatory cytokine IL-10. In contrast, there was no significant difference between the curcumin / EGCG group and the LPS group, indicating that emulsion encapsulation significantly enhanced the bioactivity of curcumin and EGCG in alleviating inflammatory responses in the mouse brain.

[0108] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0109] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A dual emulsion based on a protein microgel-polysaccharide covalent complex, characterized in that: The dual emulsion is composed of an inner aqueous phase, an oil phase, and an outer aqueous phase. The inner aqueous phase is composed of NaCl, hydrophilic functional factor tea polyphenols, and water. The oil phase is composed of polyglycerol ricinoleate, hydrophobic functional factor curcumin, and medium-chain triglycerides. The outer aqueous phase is composed of a protein microgel-polysaccharide covalent complex and water. The NaCl content in the inner aqueous phase is 0.5–2 wt%; the volume fraction of polyglycerol ricinoleate in the oil phase is 2–8%; the content of the protein microgel-polysaccharide covalent complex in the outer aqueous phase is 1–4 wt%; and the volume ratio of the inner aqueous phase to the oil phase is 2:8–4:

6. The inner aqueous phase and oil phase are mixed and homogenized by shearing to obtain a W / O emulsion, and the volume ratio of the W / O emulsion to the outer aqueous phase is 2:8–5:

5. The preparation method of the protein microgel-polysaccharide covalent complex includes: Protein is dispersed in water and heated, then transglutaminase is added and the mixture is heated and cooled to obtain a protein gel. The protein gel is then sheared, broken down, and homogenized to obtain a protein microgel. The protein is pea protein isolate. The polysaccharide includes any one or more combinations of sodium alginate, sodium hyaluronate, and pectin. Furthermore, the protein microgel is mixed with polysaccharide and subjected to Maillard reaction to obtain a protein microgel-polysaccharide covalent complex.

2. The dual emulsion according to claim 1, characterized in that: The tea polyphenols include EGCG and / or ECG.

3. The dual emulsion according to claim 1, characterized in that: The concentration of the hydrophilic functional factor tea polyphenols in the internal aqueous phase is 1.0~2.0 mg / mL.

4. The dual emulsion according to claim 1, characterized in that: The concentration of curcumin, a hydrophobic functional factor, in the oil phase was 0.6 mg / mL.

5. The dual emulsion according to claim 1, characterized in that: The protein microgel-polysaccharide covalent complex includes a pea protein isolate microgel-sodium alginate covalent complex.

6. The method for preparing the dual emulsion based on the protein microgel-polysaccharide covalent complex according to any one of claims 1-5, characterized in that, include: It provides an inner aqueous phase composed of NaCl, hydrophilic functional factor tea polyphenols and water, an oil phase composed of polyglycerol ricinoleate, hydrophobic functional factor curcumin and medium-chain triglycerides, and an outer aqueous phase composed of protein microgel-polysaccharide covalent complex and water; The aqueous phase and oil phase are mixed and subjected to a first shear homogenization process to obtain a W / O emulsion. Furthermore, the W / O emulsion is mixed with an external aqueous phase and subjected to a second shear homogenization treatment to obtain a W / O / W dual emulsion, namely a dual emulsion based on a protein microgel-polysaccharide covalent complex.

7. The preparation method according to claim 6, characterized in that, Specifically, it includes: The first shearing and homogenization process was carried out at 10,000 to 15,000 rpm for 3 to 5 minutes.

8. The preparation method according to claim 6, characterized in that: A second shearing and homogenization process was performed at 7000~10000 rpm for 3~5 min.

9. The use of the dual emulsion based on the protein microgel-polysaccharide covalent complex according to any one of claims 1-5 as a functional factor / drug carrier in the preparation of products for the prevention and / or improvement of cognitive memory impairment or neuroinflammation.

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