Preparation method of curcumin-loaded glycosylated casein high internal phase emulsion based on enzymatic method

Abstract

The invention discloses a method for preparing a high internal phase emulsion and delivering curcumin by using transglutaminase mediated casein-carboxymethyl chitosan covalent substances, aims to improve the stability and bioaccessibility of curcumin, and belongs to the field of food processing. The method comprises the following steps: firstly, carrying out ultrasonic treatment on sodium caseinate for a certain time, and carrying out transglutaminase cross-linking reaction on sodium caseinate and carboxymethyl chitosan to obtain a casein-carboxymethyl chitosan covalent substance; and then adding the mixture into the curcumin-loaded corn oil according to a certain proportion, and homogenizing to obtain the curcumin-loaded high internal phase emulsion. The emulsion has good interface characteristics and can effectively encapsulate curcumin. The preparation process is simple, efficient, green and environmentally friendly, the prepared high internal phase emulsion has high stability and good biological accessibility, a new technical means is provided for delivery of curcumin, and the high internal phase emulsion has wide application prospects in the field of food.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for preparing a high internal phase emulsion of curcumin loaded with enzymatic glycosylated casein. Background Art

[0002] Curcumin is a low molecular weight polyphenol extracted from turmeric. It is rich in biological activity and has the potential to be a natural antioxidant in beverages and functional foods. It also has rich pharmacological activities such as anti-inflammatory, anti-tumor, hypolipidemic, anti-cancer, antibacterial, and dementia relief. However, free curcumin is poorly soluble in water, not easily absorbed in the body, has low bioavailability, and has the characteristics of rapid metabolism and short half-life, which greatly limits its application. Using encapsulation technology to embed and deliver polyphenols such as curcumin to improve their processing and storage stability and human bioavailability is an urgent problem to be solved.

[0003] High internal phase emulsion refers to a type of emulsion with a dispersed phase volume of more than 74%. Due to the high desorption energy at the interface, the emulsion system is not prone to aggregation, Ostwald ripening and demulsification, resulting in excellent emulsion stability. At the same time, high internal phase emulsions form a dense interface layer to provide steric hindrance around the droplets, thereby improving the stability and bioavailability of active ingredients. Currently, high internal phase emulsions stabilized by protein as an emulsifier occupy an important position in food-grade high internal phase emulsions.

[0004] Sodium caseinate (SC) is the sodium salt of casein, the main protein in milk. It has good emulsifying properties and thermal stability and is a high-quality natural raw material for food processing. However, because casein is rich in proline and does not have too many regular secondary or tertiary structures, this open structure makes them easily hydrolyzed by pepsin, causing the embedded active substances to be destroyed or degraded in a strong acidic environment, and cannot achieve the purpose of improving the bioavailability of curcumin. Therefore, it is necessary to modify sodium caseinate to improve the stability of the emulsion and the ability to deliver curcumin. Carboxymethyl chitosan (CMCS) is a macromolecular anionic polysaccharide generated by methylation of chitosan. In addition to having excellent water solubility and biocompatibility, it also has strong hydrophilicity and poor amphiphilicity. Transglutaminase (TGase) is an enzyme that can catalyze the transamidation reaction, mediate the combination of the amino group on the sugar and the amide group on the glutamine in the protein molecule to form a covalent complex of protein-sugar, and at the same time cause the protein to undergo a certain degree of cross-linking to improve the functional properties of the protein. It has the advantages of high safety, non-toxicity and pollution-free. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a high internal phase emulsion of curcumin loaded with enzymatic glycosylated casein, wherein the raw materials of the method are all food grade, and the processing process is safe and green. The prepared high internal phase emulsion has high stability and good bioaccessibility.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing a high internal phase emulsion of curcumin loaded with enzymatic glycosylated casein, wherein the steps of the method are as follows: (1) Preparation of casein solution: Sodium caseinate was completely dissolved in 0.01 M phosphate buffer and hydrated at 4°C for a certain period of time. An ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment, which was continuously adjustable (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10°C. The sample was stored in a refrigerator at 4°C before further processing to obtain a casein solution. (2) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was completely dissolved in 0.01 M phosphate buffer and hydrated at 4° C. for a certain period of time to obtain a carboxymethyl chitosan solution. (3) Preparation of enzymatic glycosylated casein: The sodium caseinate solution after ultrasound was evenly mixed with the carboxymethyl chitosan solution, and transglutaminase (TGase) was added. The mixture was reacted at 37°C for a certain period of time. After the reaction was completed, the enzyme was inactivated at high temperature in a water bath, and the mixture was taken out and quickly cooled to obtain an enzymatic glycosylated casein solution. (4) Preparation of corn oil loaded with curcumin: Curcumin was dissolved in corn oil, stirred at high temperature, and ultrasonically treated for a certain period of time until the curcumin was completely dissolved to obtain corn oil loaded with curcumin. (5) Preparation of enzymatically glycosylated casein high internal phase emulsion loaded with curcumin: The enzymatically glycosylated casein solution and the corn oil loaded with curcumin were mixed in a certain volume ratio and homogenized to obtain the enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0007] Preferably, in step (1), the concentration of the sodium caseinate solution is 2%-4% (w / v), the pH of the phosphate buffer is 6-8, the hydration time is 4-12h, the ultrasonic treatment time is 0-40min, and the treatment power is 300-500W.

[0008] Preferably, in step (2), the concentration of the carboxymethyl chitosan solution is 1%-3% (w / v), the pH of the phosphate buffer is 6-8, and the hydration time is 4-12h.

[0009] Preferably, in step (3), the amount of TGase added is 10-40 U / g (based on protein content), the reaction conditions are pH = 6-8, the reaction time is 3-5 h, the enzyme inactivation temperature is 80-90°C water bath for 5-10 min, and the protein content of the resulting enzymatically glycosylated casein solution is 0.5%-1.5%.

[0010] Preferably, in step (4), the amount of curcumin added is 0.1%-0.2% (w / v), the stirring time is 0.5-2h, and the temperature is 60-70°C.

[0011] Preferably, in step (4), the ultrasonic treatment time is 0.5-1 h, and the volume ratio of the corn oil loaded with curcumin is 70%-85%.

[0012] Preferably, in step (5), the homogenization treatment conditions are: centrifugation at 10000-15000 rpm for 2-3 min.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The production process adopted by the invention is simple, green and environmentally friendly, and has wide application. The invention can be used in the fields of food, medicine, etc., and is conducive to industrialized production.

[0015] The invention utilizes enzymatic glycosylation of casein to prepare a high internal phase emulsion and carry curcumin. The obtained high internal phase emulsion has good stability and high encapsulation efficiency, and can effectively improve the bioaccessibility of curcumin.

[0016] The present invention provides a new technical means for delivering curcumin, and has broad application prospects in the field of functional foods. Meanwhile, the prepared high internal phase emulsion can be used as a saturated fat substitute in the processing of low-fat healthy foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention is a flow chart of a high internal phase emulsion based on enzymatic glycosylation of casein loaded with curcumin.

[0018] Figure 2 It is a comparison chart of the interfacial adsorbed protein content of the high internal phase emulsions in Examples 1-3 of the present invention and Comparative Examples 1-3;

[0019] Figure 3 The particle size (D 4,3 )Comparison chart;

[0020] Figure 4 It is a comparison diagram of the primary oxidation products (POV) of the high internal phase emulsions in Examples 1-3 of the present invention and Comparative Examples 1-3;

[0021] Figure 5 It is a comparison diagram of secondary oxidation products (TBARS) of high internal phase emulsions in Examples 1-3 of the present invention and Comparative Examples 1-3;

[0022] Figure 6 It is a comparison chart of the curcumin encapsulation efficiency of the high internal phase emulsion in Examples 1-3 of the present invention and Comparative Examples 1-3;

[0023] Figure 7 It is a comparison chart of the bioaccessibility of curcumin in the high internal phase emulsion in Examples 1-3 of the present invention and Comparative Examples 1-3. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0025] Example 1

[0026] (1) Preparation of casein solution: 3% (w / v) sodium caseinate was completely dissolved in 0.01 M phosphate buffer at pH 7, and hydrated at 4°C for 12 h. An ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment at 400 W for 10 min, with continuous adjustment (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10-20°C. The sample was stored in a refrigerator at 4°C before further treatment to obtain a casein solution.

[0027] (2) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was completely dissolved in 0.01 M phosphate buffer at pH 7 and hydrated at 4° C. for 12 h to obtain a carboxymethyl chitosan solution.

[0028] (3) Preparation of enzymatic glycosylated casein: The sodium caseinate solution after ultrasound was uniformly mixed with a 1% (w / v) carboxymethyl chitosan solution, and 20 U / g (based on protein content) TGase was added, pH = 7.5, and the reaction was carried out at 37°C for 4 h. After the reaction was completed, the enzyme was inactivated in a water bath at 85°C for 5 min, and the solution was taken out and quickly cooled, and diluted to obtain an enzymatic glycosylated casein solution with a protein content of 0.5%.

[0029] (4) Preparation of corn oil loaded with curcumin: 0.1% (w / v) curcumin was dissolved in corn oil, stirred at 60° C. for 1 h, and ultrasonically treated for 30 min until the curcumin was completely dissolved to obtain corn oil loaded with curcumin.

[0030] (5) Preparation of enzymatically glycosylated casein high internal phase emulsion loaded with curcumin: The enzymatically glycosylated casein solution was mixed with corn oil loaded with curcumin, wherein the oil phase volume was 75%, and homogenized at 12000 rpm for 3 min to obtain an enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0031] Example 2

[0032] (1) Preparation of casein solution: 3% (w / v) sodium caseinate was completely dissolved in 0.01 M phosphate buffer at pH 7, and hydrated at 4°C for 12 h. An ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment at 400 W for 10 min, with continuous adjustment (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10-20°C. The sample was stored in a refrigerator at 4°C before further treatment to obtain a casein solution.

[0033] (2) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was completely dissolved in 0.01 M phosphate buffer at pH 7 and hydrated at 4° C. for 12 h to obtain a carboxymethyl chitosan solution.

[0034] (3) Preparation of enzymatic glycosylated casein: The sodium caseinate solution after ultrasound was uniformly mixed with a 1% (w / v) carboxymethyl chitosan solution, and 20 U / g (based on protein content) TGase was added, pH = 7.5, and the reaction was carried out at 37°C for 4 h. After the reaction was completed, the enzyme was inactivated in a water bath at 85°C for 5 min, and the solution was taken out and quickly cooled, and diluted to obtain an enzymatic glycosylated casein solution with a protein content of 1%.

[0035] (4) Preparation of corn oil loaded with curcumin: 0.1% (w / v) curcumin was dissolved in corn oil, stirred at 60° C. for 1 h, and ultrasonically treated for 30 min until the curcumin was completely dissolved to obtain corn oil loaded with curcumin.

[0036] (5) Preparation of enzymatically glycosylated casein high internal phase emulsion loaded with curcumin: The enzymatically glycosylated casein solution was mixed with corn oil loaded with curcumin, wherein the oil phase volume was 75%, and homogenized at 12000 rpm for 3 min to obtain an enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0037] Example 3

[0038] (1) Preparation of casein solution: 3% (w / v) sodium caseinate was completely dissolved in 0.01 M phosphate buffer at pH 7, and hydrated at 4°C for 12 h. An ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment at 400 W for 10 min, with continuous adjustment (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10-20°C. The sample was stored in a refrigerator at 4°C before further treatment to obtain a casein solution.

[0039] (2) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was completely dissolved in 0.01 M phosphate buffer at pH 7 and hydrated at 4° C. for 12 h to obtain a carboxymethyl chitosan solution.

[0040] (3) Preparation of enzymatic glycosylated casein: The sodium caseinate solution after ultrasound was uniformly mixed with a 1% (w / v) carboxymethyl chitosan solution, and 20 U / g (based on protein content) TGase was added, pH = 7.5, and the reaction was carried out at 37°C for 4 h. After the reaction was completed, the enzyme was inactivated in a water bath at 85°C for 5 min, and the solution was taken out and quickly cooled to obtain an enzymatic glycosylated casein solution with a protein content of 1.5%.

[0041] (4) Preparation of corn oil loaded with curcumin: 0.1% (w / v) curcumin was dissolved in corn oil, stirred at 60° C. for 1 h, and ultrasonically treated for 30 min until the curcumin was completely dissolved to obtain corn oil loaded with curcumin.

[0042] (5) Preparation of enzymatically glycosylated casein high internal phase emulsion loaded with curcumin: The enzymatically glycosylated casein solution was mixed with corn oil loaded with curcumin, wherein the oil phase volume was 75%, and homogenized at 12000 rpm for 3 min to obtain an enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0043] Comparative Example 1

[0044] (1) Preparation of casein solution: 3% (w / v) sodium caseinate was completely dissolved in 0.01 M, pH = 7 phosphate buffer, hydrated at 4°C for 12 h, and ultrasonic pretreatment was performed by inserting an ultrasonic probe 2 cm below the surface of the dispersion. The treatment was performed at 400 W for 10 min, with continuous adjustment (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10-20°C. The sample was stored in a refrigerator at 4°C before further processing and diluted to obtain a casein solution with a protein content of 0.5%.

[0045] (2) Preparation of aqueous phase: A casein solution having a protein content of 0.5% after ultrasound was uniformly mixed with a 0.01 M phosphate buffer solution having a pH of 7 to obtain an aqueous phase.

[0046] (3) Preparation of corn oil loaded with curcumin: 0.1% (w / v) curcumin was dissolved in corn oil, stirred at 60° C. for 1 h, and ultrasonically treated for 30 min until the curcumin was completely dissolved to obtain corn oil loaded with curcumin.

[0047] (4) Preparation of curcumin-loaded casein high internal phase emulsion: The aqueous phase was mixed with corn oil loaded with curcumin, wherein the volume of the oil phase was 75%, and homogenized at 12000 rpm for 3 min to obtain an enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0048] Comparative Example 2

[0049] (1) Preparation of casein solution: 3% (w / v) sodium caseinate was completely dissolved in 0.01 M phosphate buffer at pH 7, and hydrated at 4°C for 12 h. The ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment at 400 W for 10 min, with continuous adjustment (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10-20°C. The sample was stored in a refrigerator at 4°C before further processing and diluted to obtain a casein solution with a protein content of 1%.

[0050] (2) Preparation of aqueous phase: A casein solution with a protein content of 1% after ultrasound was uniformly mixed with a 0.01 M phosphate buffer solution with a pH of 7 to obtain an aqueous phase.

[0051] (3) Preparation of corn oil loaded with curcumin: 0.1% (w / v) curcumin was dissolved in corn oil, stirred at 60° C. for 1 h, and ultrasonically treated for 30 min until the curcumin was completely dissolved to obtain corn oil loaded with curcumin.

[0052] (4) Preparation of curcumin-loaded casein high internal phase emulsion: The aqueous phase was mixed with corn oil loaded with curcumin, wherein the volume of the oil phase was 75%, and homogenized at 12000 rpm for 3 min to obtain an enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0053] Comparative Example 3

[0054] (1) Preparation of casein solution: 3% (w / v) sodium caseinate was completely dissolved in 0.01 M phosphate buffer at pH 7, and hydrated at 4°C for 12 h. An ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment at 400 W for 10 min, with continuous adjustment (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10-20°C. The sample was stored in a refrigerator at 4°C before further treatment to obtain a casein solution with a protein content of 1.5%.

[0055] (2) Preparation of aqueous phase: A casein solution having a protein content of 1.5% after ultrasound was uniformly mixed with a 0.01 M phosphate buffer solution having a pH of 7 to obtain an aqueous phase.

[0056] (3) Preparation of corn oil loaded with curcumin: 0.1% (w / v) curcumin was dissolved in corn oil, stirred at 60° C. for 1 h, and ultrasonically treated for 30 min until the curcumin was completely dissolved to obtain corn oil loaded with curcumin.

[0057] (4) Preparation of curcumin-loaded casein high internal phase emulsion: The aqueous phase was mixed with corn oil loaded with curcumin, wherein the volume of the oil phase was 75%, and homogenized at 12000 rpm for 3 min to obtain an enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

[0058] The physical and chemical properties and stability of the high internal phase emulsions prepared in Examples 1-3 and Comparative Examples 1-3 were tested

[0059] 1. Test method for the percentage of interfacial protein adsorption The aqueous phase of the high internal phase emulsion was obtained by centrifugation (13000 g, 30 min) at room temperature. Subsequently, the aqueous phase was filtered using a 0.45 μm filter and the protein concentration was determined using a BCA protein assay kit.

[0060] Interface adsorbed protein content (%) = (initial protein concentration - protein concentration in filtrate) / initial protein concentration

[0061] The results are as follows Figure 2 As shown, in the high internal phase emulsion prepared by the present invention, the content of interfacial adsorbed protein increases with the increase of protein concentration, and at the same protein concentration, the interfacial adsorption content of the enzymatically glycosylated casein high internal phase emulsion is significantly greater than that of the single casein high internal phase emulsion. The high internal phase emulsion containing 1.5% glycosylated casein has the highest content of interfacial adsorbed protein (80.78%). The covalent insertion of sugar enhances the electrostatic interaction and steric hindrance between oil droplets, making it easier for protein to adsorb to the oil-water interface.

[0062] 2. Storage stability test method 0.1 g of the high internal phase emulsion in Examples 1-3 and Comparative Examples 1-3 after storage at 0 d and 4 °C for 14 d, respectively, was taken, diluted 100 times with deionized water, and oscillated on a vortex oscillator for 2 min to disperse the emulsion. The average particle size of the emulsion was measured by a laser particle size analyzer to record the volume mass equivalent average diameter of the oil droplets in the emulsion (D 4，3 ), the analysis mode was wet injection, the refractive index was 1.450, and the dispersion medium was water, with a refractive index of 1.330.

[0063] The results are as follows Figure 3As shown in the figure, after 14 days of storage, the particle size of all samples increased, which may be due to the partial aggregation of emulsion droplets due to long-term storage; the particle size decreased with the increase of protein concentration. As the protein concentration increased, the protein adsorbed on the oil-water interface increased, preventing the droplets from agglomerating, resulting in smaller droplet size; at the same protein concentration, the particle size of the glycosylated casein high internal phase emulsion was smaller than that of the casein high internal phase emulsion. The enzymatic glycosylated casein was connected with carboxymethyl chitosan, which provided higher viscosity and steric hindrance and could effectively prevent the aggregation of droplets; and 1.5% enzymatic glycosylated casein had the best storage stability. On the 14th day, the particle size increased from 11.667±0.01μm to 16.51±0.28μm, with the smallest increase. Enzymatic glycosylation gave the emulsion a stronger viscosity and three-dimensional network structure, thereby improving stability.

[0064] 3. Test method for oxidative stability of oils and fats The newly prepared high internal phase emulsion was placed in a sealed cork bottle and stored in a dark incubator at 50 °C for 12 days, and samples were taken at 0, 3, 6, 9, and 12 days; Primary oxidation product (POV) determination: First, 0.1 g of the emulsion was added to 1.5 mL of a mixture of isooctane and isopropanol (3:1, v / v), mixed well and centrifuged at 10000 g for 2 min; then 200 μL of the supernatant was taken out and added to 2.8 mL of a mixture of methanol and n-butanol (2:1, v / v); after mixing, 15 μL of the prepared 3.94 M ammonium thiocyanate solution and 15 μL of 0.132 M ferrous solution (prepared by mixing equal amounts of 0.132 M BaCl2 and 0.144 M FeSO4) were added; after reacting in the dark for 20 min, the absorbance of the mixture was measured at a wavelength of 510 nm using a UV-visible spectrophotometer; Secondary oxidation products (TBARS) determination: First, 0.1 g of the emulsion was added to 1.8 mL of deionized water, and then 4 mL of TBA reagent was added to the mixture, and the mixture was heated in boiling water at 100 ° C for 15 min; TBA consisted of 15 g of TCA (trichloroacetic acid), 0.375 g of TBA, and 100 mL of 4 M hydrochloric acid; Next, the cooled solution was centrifuged at 10000 g for 5 min, and then the supernatant was taken and the absorbance was measured at a wavelength of 532 nm using a UV-visible spectrophotometer. A standard curve was obtained based on 1,1,3,3-tetraethoxypropane. (y = 1.667x - 0.1397, R 2 =0.9991)

[0065] The results are as follows Figure 4-5As shown in the figure, the increase of protein concentration can inhibit the oil oxidation ability of high internal phase emulsion and reduce the generation of primary oxidation products and secondary oxidation products; this is because the high concentration of protein content promotes interfacial adsorption and forms a thicker interfacial film; the antioxidant capacity of high internal phase emulsion of enzymatic glycosylated casein is significantly improved, and it is the highest when the protein content is 1.5%. This is because the covalently grafted sugar chains form a physical barrier around the oil droplets, effectively preventing the outside from contacting the oil droplets.

[0066] 4. Test method for curcumin encapsulation efficiency The curcumin concentration standard curve was established by dissolving 3 mg curcumin in 50 mL of 95% ethanol, taking 0.2, 0.5, 1.0, 2.0 and 3.0 mL of the solution, and diluting to 10 mL with 95% ethanol in sequence (the curcumin concentration ranged from 1.2 mg / L to 18 mg / L, using 95% ethanol as a blank), and measuring the absorbance at 425 nm; taking the absorbance as the Y axis and the curcumin concentration as the X axis, a standard curve of the curcumin content was obtained; (y=0.1365x+0.074, R 2 =0.9993) The sample was centrifuged at 10000 rpm for 20 min; the free curcumin in the precipitate was dissolved in anhydrous ethanol, and the absorbance of the free curcumin was recorded at 425 nm; and the amount of free curcumin was then determined based on a pre-determined standard curve.

[0067] Encapsulation efficiency of curcumin (%) = (total amount of curcumin in sample - amount of free curcumin) / total amount of curcumin in sample

[0068] The results are as follows Figure 6 As shown in the figure, the encapsulation efficiency of 0.5%, 1% and 1.5% enzymatically glycosylated casein high internal phase emulsions were 63.83%, 84.22% and 93.24%, respectively, which were significantly higher than the encapsulation efficiency of casein high internal phase emulsions at the same concentration, and increased with the increase of protein concentration; this may be due to the high concentration of protein and enzymatically glycosylated casein-curcumin system forming a dense interface layer and strong stability, which inhibited the release of curcumin; in general, the high internal phase emulsion prepared by high concentration enzymatically glycosylated casein has an excellent encapsulation effect on polyphenols.

[0069] 5. Test methods for bioaccessibility of curcumin The digestive behavior of the emulsion and the bioaccessibility of curcumin were evaluated using a simulated gastrointestinal tract (GIT) model; Gastric stage: The pH of simulated gastric fluid (SGF) containing 32 mg / mL pepsin was adjusted to 1.2 with 1.0 mol / L HCl; subsequently, the pH of the high internal phase emulsion sample (10 g) was adjusted to 2.0 with 0.1 mol / L HCl and then mixed with SGF at 37 °C with continuous shaking for 120 min; Intestinal stage: The pH of the solution was adjusted to 7.0 using 1.5 mol / L NaHCO3 to inactivate pepsin; the emulsion sample (10 g) was mixed with simulated intestinal fluid (SIF) containing 20 mg / mL bile salts, 10 mg / mL pancreatin, and 0.75 mol / L CaCl2 at a ratio of 1:2 (v / v); then continuously shaken at 37 °C for 120 min; finally, the sample was immersed in an ice bath for 10 min to stop the reaction; After the in vitro digestion process of the small intestine stage was completed, a portion of the total sample after digestion was transferred to a centrifuge tube, and the supernatant obtained from the middle layer was collected as a mixed micelle sample after centrifugation at 10,000 rpm for 30 min; curcumin was extracted using anhydrous ethanol in a volume ratio of 3:1: 3500 g for 10 min; the concentration of curcumin was then determined using a UV-visible spectrophotometer at a wavelength of 425 nm, and the concentration of curcumin in the initial sample before digestion and the concentration of curcumin in the mixed micelle sample were calculated based on the standard curve;

[0070] Bioaccessibility of curcumin = (concentration of curcumin in mixed micelles / concentration of curcumin in initial sample) x 100%.

[0071] The results are as follows Figure 7 As shown, during the digestion of the emulsion, the internal oil phase is converted into free fatty acids and monoacylglycerol, which then form mixed micelles. As lipolysis proceeds, curcumin originally dissolved in the oil phase is transferred to the mixed micelles and becomes the core of the micelles. The curcumin in the micelle core is considered to be bioaccessible; a higher lipolysis rate leads to more free fatty acids, which is conducive to the formation of more micelles. Since hydrophobic curcumin is bioaccessible after dissolving into the micelle core, the more micelles there are, the higher the bioaccessibility of curcumin; with the increase of protein content and the production of enzymatic glycosylated casein, the emulsion has a smaller droplet size, giving the emulsion a larger interface area. Since lipid digestion is an interfacial phenomenon, a larger interface area can promote sufficient contact between lipase and oil, resulting in a higher lipolysis rate to achieve a higher bioaccessibility rate; the curcumin bioaccessibility of Example 3 is the highest, at 65.06%, so the 1.5% enzymatic glycosylated casein high internal phase emulsion can achieve effective encapsulation of curcumin and improve bioaccessibility.

[0072] The above embodiments are only exemplary descriptions of the present invention, and the implementation methods of the present invention are not limited to the above embodiments. All changes, modifications, and equivalent substitutions made within the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a high internal phase emulsion of curcumin loaded with enzymatic glycosylated casein, characterized in that: The steps of the method are as follows: (1) Preparation of casein solution: Sodium caseinate was completely dissolved in 0.01 M phosphate buffer, hydrated at 4°C for a certain period of time, and an ultrasonic probe was inserted 2 cm below the surface of the dispersion for ultrasonic pretreatment, which was continuously adjustable (5s-on-5s-off). Ice cubes were placed in the interlayer of the container and the temperature was controlled at 10°C. The sample was stored in a refrigerator at 4°C before further treatment to obtain a casein solution; (2) Preparation of carboxymethyl chitosan solution: completely dissolving carboxymethyl chitosan in 0.01 M phosphate buffer and hydrating at 4° C. for a certain period of time to obtain a carboxymethyl chitosan solution; (3) Preparation of enzymatic glycosylated casein: The sodium caseinate solution after ultrasound was evenly mixed with the carboxymethyl chitosan solution, and transglutaminase (TGase) was added. The mixture was reacted at 37°C for a certain period of time. After the reaction was completed, the enzyme was inactivated at high temperature in a water bath, and the mixture was taken out and quickly cooled to obtain an enzymatic glycosylated casein solution. (4) Preparation of corn oil loaded with curcumin: curcumin is dissolved in corn oil, stirred at high temperature, and ultrasonically treated for a certain period of time until the curcumin is completely dissolved, thereby obtaining corn oil loaded with curcumin; (5) Preparation of enzymatically glycosylated casein high internal phase emulsion loaded with curcumin: The enzymatically glycosylated casein solution and the corn oil loaded with curcumin were mixed in a certain volume ratio and homogenized to obtain the enzymatically glycosylated casein high internal phase emulsion loaded with curcumin.

2. A method for preparing a high internal phase emulsion of curcumin loaded with enzymatic glycosylated casein according to claim 1, characterized in that: In step (1), the concentration of the sodium caseinate solution is 2%-4% (w / v), the pH of the phosphate buffer is 6-8, the hydration time is 4-12h, the ultrasonic treatment time is 0-40min, and the treatment power is 300-500W.

3. A method for preparing nanoparticles for improving the bioavailability of curcumin according to claim 1, characterized in that: In step (2), the concentration of the carboxymethyl chitosan solution is 1%-3% (w / v), the pH of the phosphate buffer is 6-8, and the hydration time is 4-12h.

4. A method for preparing nanoparticles for improving the bioavailability of curcumin according to claim 1, characterized in that: In step (3), the amount of TGase added is 10-40 U / g (based on protein content), the reaction conditions are pH = 6-8, the reaction time is 3-5 hours, the enzyme inactivation temperature is 80-90°C water bath for 5-10 minutes, and the protein content of the obtained enzymatic glycosylated casein solution is 0.5%-1.5%.

5. A method for preparing nanoparticles for improving the bioavailability of curcumin according to claim 1, characterized in that: In step (4), the amount of curcumin added is 0.1%-0.2% (w / v), the stirring time is 0.5-2h, and the temperature is 60-70°C.

6. A method for preparing nanoparticles for improving the bioavailability of curcumin according to claim 1, characterized in that: In step (4), the ultrasonic treatment time is 0.5-1h, and the volume ratio of the corn oil loaded with curcumin is 70%-85%.

7. A method for preparing nanoparticles for improving the bioavailability of curcumin according to claim 1, characterized in that: In step (5), the homogenization treatment conditions are: centrifugation at 10000-15000 rpm for 2-3 min.

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