Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

Abstract

The present invention discloses a method for preparing a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion, and belongs to the technical field of functional factor stabilization processing. The present invention first uses an alkali dissolution and acid precipitation method to extract tilapia protein isolate, and then uses a pH shift method to induce tilapia protein isolate and hyaluronic acid to form a hydrophilic complex. Quercetin of different masses is added to soybean oil in advance and heated in a 50°C water bath to aid dissolution, and then the aqueous phase and the oil phase are mixed and sheared and homogenized, and then homogenized using a high-pressure homogenizer to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion. The composite emulsion prepared by the present invention can increase the bioavailability of quercetin from 19.02% to 58.82%, while increasing the added value of tilapia protein isolate, which is of great significance for promoting the application of quercetin in the fields of food, medicine, etc.

Description

Technical Field

[0001] The invention relates to the technical field of functional factor stabilization processing, and in particular to a method for preparing a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion. Background Art

[0002] Quercetin is a naturally occurring lipophilic bioactive substance and an important member of the flavonoids. It is widely found in many vegetables, fruits, red wine and herbs. A large number of studies have shown that quercetin, due to its polyhydroxyl molecular structure, can play a key role in biological and pharmacological activities, mainly including antioxidant, free radical scavenging, antiviral and anticancer activities. However, due to its low water solubility, poor stability in artificial gastric juice and artificial intestinal juice, and low utilization rate, its application in the pharmaceutical and food fields is greatly limited. Therefore, it is necessary to develop an effective loading system to improve the accessibility of quercetin in the human body.

[0003] A variety of encapsulation technologies have been developed to deliver quercetin, including liposomes, nanoemulsions, biopolymer colloidal particles, solid lipid nanoparticles, and nanostructured lipid carriers, among which nanoemulsions are the most widely used loading system. Oil-in-water nanoemulsions contain small oil droplets dispersed in a continuous aqueous phase, each of which is coated with protective emulsifier molecules and is not prone to particle aggregation and gravity separation, which can greatly improve the bioavailability of hydrophobic compounds.

[0004] Food proteins have functional properties such as emulsification and gelation, and are effective stabilizers and emulsifiers for oil-in-water (O / W) nanoemulsions. Proteins can interact with low molecular weight ligands and polysaccharides, and are therefore widely used as carrier materials for encapsulation and protection of bioactive ingredients. Tilapia protein isolate (TPI) is isolated from tilapia, and has the advantages of high nutrition, easy access, and sustainability, making it a potential high-quality substitute for animal protein. However, tilapia processing in my country faces industry problems such as product scarcity and highly insufficient processing and utilization. Hyaluronic acid (HA), as a multifunctional matrix in the body, has the advantages of high biocompatibility and biodegradability, as well as multiple physiological functions such as anti-oxidation and anti-aging, and is widely used in drug delivery systems, medical cosmetology, tumor treatment and other fields. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion to solve the problems existing in the above-mentioned prior art. By embedding quercetin in an emulsion prepared by tilapia protein isolate and hyaluronic acid, the solubility and stability of quercetin can be improved, and the processing and utilization of tilapia protein isolate can be promoted, thereby increasing its added value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion, comprising the following steps:

[0008] The pH shift method was used to induce tilapia protein isolate and hyaluronic acid to form a hydrophilic tilapia protein isolate-hyaluronic acid complex;

[0009] The quercetin and oil are mixed and heated to prepare an oil phase, and then the oil phase is mixed with the tilapia protein isolate-hyaluronic acid complex, sheared and homogenized, and high-pressure homogenized to obtain a tilapia protein isolate-hyaluronic acid composite emulsion loaded with quercetin.

[0010] Preferably, the method for preparing the tilapia protein isolate-hyaluronic acid complex comprises the following steps:

[0011] The tilapia protein isolate is dispersed in deionized water, the pH value of the dispersion is adjusted to be alkaline, and the dispersion is stirred; then the hyaluronic acid is added, and the stirring is continued to obtain a mixed solution of the tilapia protein isolate and the hyaluronic acid, and the pH value of the mixed solution is adjusted to neutral, and the stirring is continued to obtain the tilapia protein isolate-hyaluronic acid complex.

[0012] Preferably, the mass volume ratio of the tilapia protein isolate to the deionized water is 1:(75-110), and the mass ratio of the tilapia protein isolate to the hyaluronic acid is 1:(0.5-1).

[0013] Preferably, the pH of the dispersion is adjusted to 12, and stirred at 400-500 rpm for 20-30 min;

[0014] Add the hyaluronic acid and continue stirring at 400-500 rpm for 1 h;

[0015] The pH value of the mixed solution was adjusted to 7.0, and stirring was continued at 400-500 rpm for 20-30 min.

[0016] Preferably, the mass volume ratio of the quercetin to the oil is (0.01-0.2) g:25 mL, and the volume ratio of the oil phase to the tilapia protein isolate-hyaluronic acid complex is 1:4.

[0017] Preferably, the concentration of quercetin in the system of the quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion is 0.08-1.6 mg / mL.

[0018] Preferably, the oil phase and the tilapia protein isolate-hyaluronic acid complex are sheared and homogenized to obtain a crude emulsion, and then a high-pressure homogenizer is used for high-pressure homogenization at 60 MPa to obtain the quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0019] Preferably, the method for preparing the tilapia protein isolate comprises the following steps:

[0020] Tilapia meat and water are mixed in a mass ratio of 1:9, homogenized, and then the pH value is adjusted to 12, stirred evenly, centrifuged, the supernatant is adjusted to a pH value of 5.5, the centrifugation is continued, the precipitate is collected, and the precipitate is dispersed with water, the pH value of the dispersion is adjusted to 7, dialyzed, and freeze-dried to obtain the tilapia protein isolate.

[0021] The invention also provides a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion, which is prepared by the preparation method.

[0022] The present invention also provides the use of the quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion in preparing a delivery system for improving the bioaccessibility of quercetin.

[0023] The present invention discloses the following technical effects:

[0024] (1) The present invention prepares a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion, adopts a pH shift method to make tilapia protein isolate and hyaluronic acid form a soluble tilapia protein isolate-hyaluronic acid complex, and realizes the regulation of the quercetin-loaded emulsion. Compared with the traditional method, the tilapia protein isolate-hyaluronic acid complex prepared by the present invention has better solubility, which is conducive to loading more quercetin. Thereby helping to improve the bioaccessibility of quercetin and providing new possibilities for the application of quercetin in the fields of food and medicine.

[0025] (2) The average particle size of the quercetin-loaded tilapia protein isolate-hyaluronic acid emulsion prepared by the present invention is 1.9-3.0 μm, the embedding efficiency is 79.6-96.85%, and the bioaccessibility is increased from 19.02% to 58.82%.

[0026] (3) The preparation method provided by the present invention will improve the water solubility and bioaccessibility of quercetin while realizing the green and high value-added utilization of tilapia protein isolate. On the premise of retaining its original function, its added value and deep processing level will be improved, thus creating greater economic benefits for my country's food and biopharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 A flow chart of the quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion prepared by the present invention;

[0029] Figure 2 The average particle size of the tilapia protein isolate-hyaluronic acid composite emulsion loaded with different concentrations of quercetin prepared in Examples 1-5 of the present invention;

[0030] Figure 3 The apparent viscosity diagram of the tilapia protein isolate-hyaluronic acid composite emulsion loaded with different concentrations of quercetin prepared in Examples 1-5 of the present invention;

[0031] Figure 4 The frequency scanning diagram of the tilapia protein isolate-hyaluronic acid composite emulsion loaded with different concentrations of quercetin prepared in Examples 1-5 of the present invention;

[0032] Figure 5 The embedding efficiency of quercetin with different concentrations loaded in the tilapia protein isolate-hyaluronic acid composite emulsion prepared in Examples 1-5 of the present invention;

[0033] Figure 6 The bioaccessibility of quercetin at different concentrations loaded in the tilapia protein isolate-hyaluronic acid composite emulsion prepared in Examples 1-5 of the present invention. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The invention firstly utilizes an alkali dissolution and acid precipitation method to extract tilapia protein isolate, and then uses a pH shift method to induce the tilapia protein isolate and hyaluronic acid to form a hydrophilic complex, and adds different masses of quercetin to soybean oil in advance, and heats the soybean oil in a 50° C. water bath to promote dissolution, and then mixes the aqueous phase and the oil phase for shear homogenization, and then uses a high-pressure homogenizer for homogenization, so as to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion. The flow chart thereof is shown in FIG. Figure 1 .

[0040] The following examples involve the experimental method for verifying the effect of the prepared quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0041] (1) Detection of average particle size: The emulsion was diluted 300 times with deionized water and analyzed using a laser particle size analyzer.

[0042] (2) Apparent viscosity and frequency sweep test: A shear test was performed at 25°C using parallel plates (60 mm diameter, 1 mm gap) with a shear rate ranging from 0.1 to 100 s -1 The apparent viscosity was measured within a range of 0.1 Hz to 10.0 Hz, and the storage modulus (G') of the sample was recorded between the scanning frequencies of 0.1 and 10.0 Hz.

[0043] (3) Detection of quercetin embedding efficiency: Mix the sample with dichloromethane and vortex to extract quercetin. Transfer the organic phase to a centrifuge tube. Repeat the extraction until the aqueous phase is transparent. Remove the organic phase by nitrogen blowing, and then use dimethyl sulfoxide to redissolve quercetin. Measure the absorbance at 374nm by ultraviolet spectrophotometer. Calculate the quercetin content according to the standard curve (Y = 0.076X, R 2 =0.999).

[0044] (4) Detection of quercetin bioaccessibility: Free and emulsion-encapsulated quercetin samples were subjected to continuous simulated gastric and intestinal digestion. The conditions for the simulated gastric digestion stage were as follows: 10 mL of the emulsion sample was mixed evenly with 20 mL of gastric simulated digestion fluid (3.2 mg / mL pepsin, 0.7% HCl, 2 mg / mL NaCL), the pH was adjusted to 2.0, and the mixture was shaken and incubated at 37°C for 1 hour. The conditions for simulated intestinal digestion were as follows: 20 mL of gastric digestion fluid was mixed with 20 mL of small intestinal digestion simulated fluid (5 mg / mL bile salts, 0.4 mg / mL pancreatin, 3.2 mg / mL lipase), the pH was adjusted to 7.0, and the mixture was shaken and incubated at 37°C for 2 hours. 2 mL of the intestinal digestion sample was taken and centrifuged (5000 g, 30 min) to obtain the mixed micelle phase in the supernatant. Quercetin before centrifugation and in the mixed micelle phase was extracted with dimethyl sulfoxide, and the absorbance at 374 nm was measured by UV spectrophotometer. 2 =0.999) to calculate the quercetin content, and the bioaccessibility calculation formula of quercetin is as follows:

[0045]

[0046] Wherein, C is the quercetin content in the mixed micelle phase, and C0 is the quercetin content in the sample before centrifugation.

[0047] Example 1 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0048] (1) Preparation of Tilapia Protein Isolate:

[0049] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0050] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0051] 1 g of tilapia protein isolate was dispersed in 100 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 30 min (450 rpm). 0.8 g of hyaluronic acid was added, and stirred at 450 rpm for 1 h (1.0 M NaOH was needed to be supplemented during this period to maintain the pH at 12), to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 30 min (450 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0052] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0053] 0.0125 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0054] At this time, the concentration of quercetin in the composite emulsion system was 0.1 mg / mL, and the average particle size of the emulsion system was 1950.14 nm (see Figure 2 ); shear rate is 100s -1 The apparent viscosity is 192.37 mPa·s (see Figure 3 ); the elastic modulus at a frequency of 10 Hz is 31.54 Pa (see Figure 4 ); the embedding efficiency of quercetin was 79.60% (see Figure 5 ) ; The bioaccessibility of quercetin increased from 19.02% in free form to 29.80% (see Figure 6 ).

[0055] Example 2 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0056] (1) Preparation of Tilapia Protein Isolate:

[0057] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0058] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0059] 1 g of tilapia protein isolate was dispersed in 100 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 30 min (450 rpm). 0.8 g of hyaluronic acid was added, and stirred at 450 rpm for 1 h (1.0 M NaOH was needed to maintain the pH at 12) to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 30 min (450 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0060] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0061] 0.025 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0062] At this time, the concentration of quercetin in the composite emulsion system was 0.2 mg / mL, and the average particle size of the emulsion system was 2066.45 nm (see Figure 2 ); shear rate is 100s -1 The apparent viscosity is 198.84 mPa·s (see Figure 3 ); the elastic modulus at a frequency of 10 Hz is 44.57 Pa (see Figure 4 ); the embedding efficiency of quercetin was 87.85% (see Figure 5 ); the bioaccessibility of quercetin increased from 19.02% in free form to 40.83% (see Figure 6 ).

[0063] Example 3 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0064] (1) Preparation of Tilapia Protein Isolate:

[0065] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0066] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0067] 1 g of tilapia protein isolate was dispersed in 100 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 30 min (450 rpm). 0.8 g of hyaluronic acid was added, and stirred at 450 rpm for 1 h (1.0 M NaOH was needed to maintain the pH at 12) to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 30 min (450 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0068] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0069] 0.05 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0070] At this time, the quercetin concentration in the composite emulsion system was 0.4 mg / mL, and the average particle size of the emulsion system was 2213.42 nm (see Figure 2 ); shear rate is 100s -1The apparent viscosity is 153.24 mPa·s (see Figure 3 ); the elastic modulus at a frequency of 10 Hz is 65.63 Pa (see Figure 4 ); the embedding efficiency of quercetin was 94.32% (see Figure 5 ) ; the bioaccessibility of quercetin increased from 19.02% in free form to 50.20% (see Figure 6 ).

[0071] Example 4 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0072] (1) Preparation of Tilapia Protein Isolate:

[0073] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0074] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0075] 1 g of tilapia protein isolate was dispersed in 100 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 30 min (450 rpm). 0.8 g of hyaluronic acid was added, and stirred at 450 rpm for 1 h (1.0 M NaOH was needed to maintain the pH at 12) to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 30 min (450 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0076] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0077] 0.075 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0078] At this time, the concentration of quercetin in the composite emulsion system was 0.6 mg / mL, and the average particle size of the emulsion system was 2724.42 nm (see Figure 2 ); shear rate is 100s -1 The apparent viscosity is 237.64 mPa·s (see Figure 3 ); the elastic modulus at a frequency of 10 Hz is 81.08 Pa (see Figure 4 ); the embedding efficiency of quercetin was 96.06% (see Figure 5 ); the bioaccessibility of quercetin increased from 19.02% in free form to 58.82% (see Figure 6 ).

[0079] Example 5 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0080] (1) Preparation of Tilapia Protein Isolate:

[0081] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0082] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0083] 1 g of tilapia protein isolate was dispersed in 100 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 30 min (450 rpm). 0.8 g of hyaluronic acid was added, and stirred at 450 rpm for 1 h (1.0 M NaOH was needed to maintain the pH at 12) to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 30 min (450 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0084] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0085] 0.1 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0086] At this time, the quercetin concentration in the composite emulsion system was 0.8 mg / mL, and the average particle size of the emulsion system was 2879.65 nm (see Figure 2 ); shear rate is 100s -1 The apparent viscosity is 195.51 mPa·s (see Figure 3 ); the elastic modulus at a frequency of 10 Hz is 72.25 Pa (see Figure 4 ); the embedding efficiency of quercetin was 86.79% (see Figure 5 ) ; the bioaccessibility of quercetin increased from 19.02% in free form to 49.23% (see Figure 6 ).

[0087] Example 6 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0088] (1) Preparation of Tilapia Protein Isolate:

[0089] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0090] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0091] 1 g of tilapia protein isolate was dispersed in 75 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 20 min (500 rpm). 0.5 g of hyaluronic acid was added, and stirred at 400 rpm for 1 h (1.0 M NaOH was needed to maintain the pH at 12) to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 30 min (400 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0092] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0093] 0.01 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0094] At this time, the concentration of quercetin in the composite emulsion system was 0.08 mg / mL, and the average particle size of the emulsion system was 2013.07 nm; the shear rate was 100 s -1 The apparent viscosity was 198.15 mPa·s; the elastic modulus was 38.31 Pa at a frequency of 10 Hz; the quercetin encapsulation rate was 76.12%; and the bioaccessibility of quercetin was increased from 19.02% in the free state to 27.08%.

[0095] Example 7 Preparation method of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion

[0096] (1) Preparation of Tilapia Protein Isolate:

[0097] A certain amount of tilapia meat was thawed at 4°C, the fish meat was minced and ice distilled water was added, and the ratio of fish meat to water was 1:9 (w / w). A shear homogenizer was used to homogenize at 10000rpm for 2min, and the solution pH was adjusted to 12 with 2.0M NaOH and stirred for 30min (750rpm), followed by centrifugation for 20min (9000rpm, 4°C). The supernatant was filtered with cotton gauze and the pH was adjusted to 5.5 with 2.0M HCl, and then centrifuged for 20min (9000rpm, 4°C). The precipitate was dispersed with a small amount of ice water, and the pH value of the dispersion was adjusted to 7. The dialysis bag (8000-14000Da) was used for 48h. The dialyzed protein solution was freeze-dried using a freeze dryer.

[0098] (2) Preparation of Tilapia Protein Isolate-Hyaluronic Acid Complex:

[0099] 1 g of tilapia protein isolate was dispersed in 110 mL of deionized water, 1.0 M NaOH was used to adjust the pH of the dispersion to 12, and the tilapia protein isolate was fully dissolved by stirring for 30 min (400 rpm). 1.0 g of hyaluronic acid was added, and stirred at 500 rpm for 1 h (1.0 M NaOH was needed to maintain the pH at 12) to obtain a mixed solution of tilapia protein isolate and hyaluronic acid, 1.0 M HCl was used to adjust the pH of the mixed protein solution to 7, and stirring was continued for 20 min (500 rpm) to obtain a tilapia protein isolate-hyaluronic acid complex.

[0100] (3) Preparation of quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion:

[0101] 0.2 g of quercetin was added to 25 mL of soybean oil in advance, heated in a 50°C water bath for 30 min, the oil phase and the aqueous phase were mixed in a ratio of 1:4, and homogenized at 10,000 rpm for 2 min using a shear homogenizer to obtain a crude emulsion. Subsequently, a high-pressure homogenizer was used to circulate 5 times at 60 MPa to obtain a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion.

[0102] At this time, the concentration of quercetin in the composite emulsion system was 1.6 mg / mL, and the average particle size of the emulsion system was 3456.15 nm; the shear rate was 100 s -1 The apparent viscosity was 262.03 mPa·s; the elastic modulus was 129.71 Pa at a frequency of 10 Hz; the quercetin encapsulation rate was 65.72%; and the bioaccessibility of quercetin was increased from 19.02% in the free state to 23.97%.

[0103] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion, characterized in that: The following steps are involved: The pH shift method was used to induce tilapia protein isolate and hyaluronic acid to form a hydrophilic tilapia protein isolate-hyaluronic acid complex; The quercetin and oil are mixed and heated to prepare an oil phase, and then the oil phase is mixed with the tilapia protein isolate-hyaluronic acid complex and sheared and homogenized to obtain a tilapia protein isolate-hyaluronic acid composite emulsion loaded with quercetin; The mass ratio of the tilapia protein isolate to the hyaluronic acid is 1:(0.5-1); The mass volume ratio of the quercetin to the oil is (0.01-0.2) g:25 mL, and the volume ratio of the oil phase to the tilapia protein isolate-hyaluronic acid complex is 1:4; The concentration of quercetin in the system of the quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion is 0.08-1.6 mg / mL.

2. The preparation method according to claim 1, characterized in that The preparation method of the tilapia protein isolate-hyaluronic acid complex comprises the following steps: The tilapia protein isolate is dispersed in deionized water, the pH value of the dispersion is adjusted to be alkaline and stirred; then the hyaluronic acid is added and stirred continuously to obtain a mixed solution of the tilapia protein isolate and the hyaluronic acid, and the pH value of the mixed solution is adjusted to be neutral and stirred continuously to obtain the tilapia protein isolate-hyaluronic acid complex.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the tilapia protein isolate to the deionized water is 1:(75-110).

4. The preparation method according to claim 2, characterized in that: Adjust the pH of the dispersion to 12, and stir at 400-500 rpm for 20-30 min; Add the hyaluronic acid and continue stirring at 400-500 rpm for 1 h; The pH value of the mixed solution was adjusted to 7.0, and stirring was continued at 400-500 rpm for 20-30 min.

5. The preparation method according to claim 1, characterized in that: The oil phase and the tilapia protein isolate-hyaluronic acid complex are sheared and homogenized to obtain a crude emulsion, and then sheared and homogenized again under a high pressure of 60 MPa to obtain the tilapia protein isolate-hyaluronic acid composite emulsion loaded with quercetin.

6. The preparation method according to claim 1, characterized in that: The preparation method of the tilapia protein isolate comprises the following steps: Tilapia meat and water are mixed in a mass ratio of 1:9, homogenized, and then the pH value is adjusted to 12, stirred evenly, centrifuged, the supernatant is adjusted to a pH value of 5.5, the centrifugation is continued, the precipitate is collected, and the precipitate is dispersed with water, the pH value of the dispersion is adjusted to 7, dialyzed, and freeze-dried to obtain the tilapia protein isolate.

7. A quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the quercetin-loaded tilapia protein isolate-hyaluronic acid composite emulsion according to claim 7 in preparing a delivery system for improving the bioaccessibility of quercetin.

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