Alkali-induced glycosylation of rohu protein hydrolysate loaded with fat-soluble polyphenols, and preparation method and application thereof

Abstract

The present application relates to the technical field of food processing, and particularly relates to a base-induced glycosylated tilapia protein hydrolysate loaded with fat-soluble polyphenols and a preparation method and application thereof. The present application adopts flavor protease with mild action conditions for moderate hydrolysis, and combines with a green, efficient and controllable reaction degree base-induced propylene glycol alginate (PGA) glycosylation strategy to prepare glycosylated tilapia protein hydrolysate, effectively improves the water solubility and emulsifying property of tilapia protein, and develops an oral delivery system of fat-soluble polyphenols such as quercetin by using the glycosylated protein hydrolysate (modified tilapia protein) as a carrier, improves the bioavailability of fat-soluble polyphenols, can be developed as a functional food ingredient to improve the health status of special groups, and promotes the high-value utilization of tilapia protein in protein-based polyphenol functional food ingredients.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to an alkaline-induced glycosylated tilapia protein hydrolysate loaded with fat-soluble polyphenols, its preparation method, and its application. Background Technology

[0002] The continued population growth has led to an increasing demand for high-quality protein. Tilapia is a highly prolific fish. Tilapia protein can be effectively extracted from tilapia meat, and this protein is highly nutritious and easily accessible, making it an ideal alternative to high-quality animal protein. Population aging has led to a high incidence of chronic diseases, prompting increased demand for functional foods. Protein-based polyphenol formulations are a type of functional food ingredient that effectively improves human health. However, tilapia protein has poor water solubility and emulsifying properties, and insufficient binding capacity with polyphenols, greatly limiting its widespread use in functional food processing. Therefore, effectively improving the water solubility and emulsifying properties of tilapia protein and preparing it as a polyphenol-functionalized protein-based food ingredient is a key technical challenge facing the tilapia deep processing industry. Currently, enzymatic hydrolysis and glycosylation are commonly used techniques to improve the water solubility of tilapia protein. However, enzymatic hydrolysis products produce bitter peptides, affecting the acceptability of the protein. Glycosylation reactions suffer from uncontrollable reaction rates and toxic end products.

[0003] Chinese patent CN118576549A, entitled "A Method for Preparing a Quercetin-Loaded Tilapia Protein-Hyaluronic Acid Composite Emulsion," utilizes pH-driven induction to form a protein-polysaccharide complex between tilapia protein and hyaluronic acid via non-covalent bonds, thereby improving the water solubility and emulsifying properties of tilapia protein. A stable emulsion system is then prepared using high-pressure homogenization as a quercetin delivery carrier. However, this method results in weak non-covalent binding between the protein and polysaccharide, leading to low stability of the formed complex and consequently poor stability of the emulsion delivery carrier, making it difficult to effectively improve the bioavailability of quercetin.

[0004] Therefore, there is an urgent need to find a method that can effectively improve the water solubility, emulsifying properties, and quercetin bioavailability of tilapia protein. Summary of the Invention

[0005] The purpose of this invention is to provide an alkaline-induced glycosylated tilapia protein hydrolysate loaded with fat-soluble polyphenols, its preparation method and application, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing base-induced glycosylated tilapia protein hydrolysate loaded with lipid-soluble polyphenols, comprising the following steps:

[0008] Tilapia protein and flavor protease were mixed and then subjected to hydrolysis and enzyme inactivation treatments in sequence to obtain tilapia protein hydrolysate.

[0009] The tilapia protein hydrolysate and propylene glycol alginate were mixed and subjected to an alkali-induced glycosylation reaction to obtain glycosylated tilapia protein hydrolysate.

[0010] The glycosylated tilapia protein hydrolysate was mixed with a fat-soluble polyphenol to obtain the alkali-induced glycosylated tilapia protein hydrolysate loaded with the fat-soluble polyphenol.

[0011] Optionally, the amount of flavor protease added is 6000 U / g-72000 U / g.

[0012] Further optionally, the amount of flavor protease added is 12000 U / g-60000 U / g.

[0013] Optionally, the mass ratio of the tilapia protein hydrolysate to the propylene glycol alginate is (1.50-2.50):1.

[0014] Further optionally, the mass ratio of the tilapia protein hydrolysate to the propylene glycol alginate is (1.75-2.25):1.

[0015] Optionally, the mass ratio of the fat-soluble polyphenol to the glycosylated tilapia protein hydrolysate is 1:(5-100).

[0016] The fat-soluble polyphenols include one or more of quercetin, curcumin, and tea polyphenols.

[0017] Further optionally, the mass ratio of the fat-soluble polyphenols to the glycosylated tilapia protein hydrolysate is 1:(20-100).

[0018] The fat-soluble polyphenol is quercetin.

[0019] Optionally, the mass ratio of the fat-soluble polyphenol to the glycosylated tilapia protein hydrolysate is 1:(40-100).

[0020] Optionally, the mass ratio of the fat-soluble polyphenol to the glycosylated tilapia protein hydrolysate is 1:(60-100).

[0021] Optionally, the alkali-induced glycosylation reaction is carried out for 3 hours at a pH of 11.0.

[0022] Optionally, the hydrolysis treatment takes 90 minutes, is performed at a temperature of 45°C, and has a pH of 7.0.

[0023] The enzyme inactivation treatment lasted for 10 minutes at a temperature of 90°C.

[0024] Optionally, the mixture of the glycosylated tilapia protein hydrolysate and the fat-soluble polyphenol further includes adjusting the pH of the resulting mixture to 12.0 and reacting for 30 min, followed by adjusting the pH of the resulting reaction solution to 7.0 to obtain the alkali-induced glycosylated tilapia protein hydrolysate loaded with fat-soluble polyphenols.

[0025] Optionally, the method for preparing the tilapia protein includes mixing tilapia meat and water at a mass ratio of 1:9, adjusting the pH value to 11.0, stirring evenly, centrifuging, taking the supernatant, adjusting the pH value to 5.5, centrifuging again, collecting the precipitate, dispersing the precipitate with water, adjusting the pH value of the dispersion to 7, dialyzing, and freeze-drying to obtain the tilapia protein.

[0026] This invention provides a base-induced glycosylated tilapia protein hydrolysate loaded with lipophilic polyphenols prepared by the above method.

[0027] This invention provides the application of the above-mentioned base-induced glycosylated tilapia protein hydrolysate loaded with lipophilic polyphenols in the preparation of delivery systems that improve the bioaccessibility of lipophilic polyphenols.

[0028] The present invention discloses the following technical effects:

[0029] This invention proposes a method to effectively improve the water solubility and emulsifying properties of tilapia protein by moderate hydrolysis with flavor protease combined with alkali-induced propylene glycol alginate (PGA) glycosylation. The flavor protease effectively hydrolyzes tilapia protein, exposing glycosylation sites and reducing the production of bitter peptides. Under alkaline conditions, the carboxylic acid functional groups generated from the hydrolysis of PGA ester bonds undergo acyl transfer reactions with the amino or hydrogen bonds of the protein, thereby achieving glycosylation of the protein hydrolysate and effectively improving its water solubility and emulsifying properties. Using the glycosylated tilapia protein hydrolysate as a carrier, an oral delivery system for fat-soluble polyphenols such as quercetin is developed, effectively improving the bioavailability of quercetin and preparing it as a functional food ingredient to increase the added value of tilapia protein. Therefore, this invention employs a mild-condition flavor protease for appropriate hydrolysis, combined with a green, efficient, and controllable alkali-induced PGA glycosylation strategy to prepare glycosylated tilapia protein hydrolysates. This effectively improves the water solubility and emulsifying properties of tilapia protein. Furthermore, using the glycosylated protein hydrolysates (modified tilapia protein) as a carrier, an oral delivery system for fat-soluble polyphenols such as quercetin is developed, improving the bioavailability of fat-soluble polyphenols. This system can be used as a functional food ingredient to improve the health of specific groups and promote the high-value utilization of tilapia protein in protein-based polyphenol functional food ingredients. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The effect of flavor protease addition on the degree of tilapia protein hydrolysis;

[0032] Figure 2 The effect of flavor protease addition on tilapia protein solubility was investigated; TPI is tilapia protein isolate.

[0033] Figure 3 The effect of flavor protease addition on the emulsifying properties of tilapia protein was investigated; TPI was tilapia protein isolate.

[0034] Figure 4 The effect of the mass ratio of tilapia protein hydrolysate to PGA on the grafting degree of glycosylated products;

[0035] Figure 5 The effect of the mass ratio of tilapia protein hydrolysate to PGA on the solubility of glycosylated products;

[0036] Figure 6 The effect of the mass ratio of tilapia protein hydrolysate to PGA on the emulsifying properties of glycosylated products;

[0037] Figure 7 The effect of the mass ratio of quercetin to glycosylated tilapia protein hydrolysate on the encapsulation efficiency of quercetin;

[0038] Figure 8 The effect of the mass ratio of quercetin to glycosylated tilapia protein hydrolysate on the bioavailability of quercetin during simulated gastrointestinal digestion. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] Unless otherwise specified, all components used in this invention are commonly purchased by those skilled in the art, and all methods used in this invention are well known to those skilled in the art.

[0045] Example 1: Preparation of a moderately hydrolyzed tilapia protein

[0046] 1. Tilapia protein extraction:

[0047] Fresh tilapia back meat was minced and homogenized with ice water at a ratio of 1:9 (m / m). The pH was adjusted to 11.0 with 1 mol / L NaOH, and the mixture was extracted magnetically for 30 min (450 rpm) followed by centrifugation (4℃, 10,000 r / min, 20 min). The supernatant was filtered through 8 layers of gauze, and the pH of the filtrate was adjusted to 5.5 with 1 mol / L HCl. The mixture was centrifuged again (4℃, 10,000 r / min, 20 min), and the precipitate was homogenized with an appropriate amount of ice water. The pH was adjusted to 7.0, and the mixture was dialyzed using a dialysis bag (8,000~14,000 Da) for 48 h. The resulting tilapia protein powder was then freeze-dried.

[0048] 2. Preparation of tilapia protein hydrolysate:

[0049] 2.0 g of tilapia protein was dispersed in 100 mL of deionized water, and flavor protease (12000 U / g, 100000 U / g activity) was added. The pH of the mixture was adjusted to 7.0 with 1.0 M NaOH or HCl solution, and the mixture was placed in a 45℃ water bath for 90 min to react. After hydrolysis, the mixture was placed in a 90℃ water bath for 10 min to inactivate the enzyme, and then cooled to room temperature in an ice bath to obtain tilapia protein hydrolysate.

[0050] 3. Determine the dosage of flavor protease:

[0051] Repeat the above experimental steps, and adjust the amount of flavor protease (manufacturer: McLean, catalog number: F768979) to 24000 U / g, 36000 U / g, 48000 U / g, and 60000 U / g respectively to prepare tilapia protein with different degrees of hydrolysis.

[0052] 4. Indicator Testing:

[0053] (1) Degree of protein hydrolysis (DH) detection: 5 mL of enzymatic hydrolysate was measured and diluted to 100 mL with distilled water to obtain a diluted solution. Then, 20 mL of the diluted solution was measured, 60 mL of distilled water was added, and the pH of the mixture was adjusted to 8.2 with 0.05 M NaOH solution. 10 mL of 36 wt% formaldehyde solution was added to the system, and the pH was adjusted to 9.2 again with 0.05 M NaOH solution. The volume (V) of NaOH solution consumed after adding formaldehyde solution was recorded.

[0054] The blank experiment uses distilled water instead of the sample solution, following the same steps, and records the volume (V0) of NaOH solution consumed in the blank experiment. The degree of hydrolysis of tilapia protein is calculated using the following formula:

[0055] ;

[0056] Where V is the volume of standard sodium hydroxide (NaOH) solution consumed after adding formaldehyde; V0 is the volume of standard sodium hydroxide (NaOH) solution consumed after adding formaldehyde in the reagent blank experiment; V1 is the total volume of the enzyme hydrolysate; C is the concentration of the standard sodium hydroxide (NaOH) solution; m is the mass of protein in the substrate; w is the protein content in tilapia; h tot The number of millimoles of peptide bonds per gram of protein substrate (7.2).

[0057] (2) Protein solubility: Tilapia protein hydrolysate was appropriately diluted with deionized water and centrifuged at 10000×g (relative centrifugal force) for 25 min. The protein content in the supernatant and the original sample was determined by the BCA method (diquinoline carboxylic acid method), and the protein solubility was calculated using the following formula:

[0058] .

[0059] (3) Emulsification test: The prepared sample solution was diluted with deionized water to a protein concentration of 5 mg / mL and stirred at 450 rpm for 30 min. The diluted sample was mixed with soybean oil at an oil-water volume ratio of 1:4 and then homogenized at 20,000 rpm for 1 min to prepare an emulsion.

[0060] Immediately aspirate 50 μL of the emulsion from the bottom and add it to 5 mL of 0.1% sodium dodecyl sulfate (SDS) solution. Immediately read the absorbance value A0 at 500 nm using a UV spectrophotometer. After allowing the emulsion to stand for 10 min, repeat the sampling procedure to measure the absorbance value A0. 10 The formulas for calculating the Emulsifying Activity Index (EAI) and the Emulsifying Stability Index (ESI) are as follows:

[0061] ;

[0062] ;

[0063] Where DF is the dilution factor (100), C is the protein concentration (g / mL), φ is the oil phase volume fraction (0.20), and A0 and A 10 These are the absorbance values ​​at 0 and 10 min, respectively.

[0064] The test results for protein hydrolysis degree, solubility, and emulsifying properties are shown below. Figure 1 , Figure 2 and Figure 3 Based on these three indicators, tilapia protein hydrolysate prepared under the condition of adding 24000 U / g of flavor protease was selected for subsequent experiments.

[0065] Example 2: Preparation of an alkaline-induced glycosylated tilapia protein hydrolysate

[0066] The experiment was conducted using the tilapia protein hydrolysate prepared in Example 1 (with 24,000 U / g of flavor protease added).

[0067] 1. Preparation of glycosylated tilapia protein hydrolysate using base-induced PGA acyl transfer technology:

[0068] A certain mass of PGA was added to 100 mL of tilapia protein hydrolysate (the mass ratio of tilapia protein hydrolysate to PGA was 2.25:1). The pH was adjusted to 11.0 using NaOH, and the mixture was reacted for 3 h with magnetic stirring (300 rpm). After the reaction was completed, the pH of the mixture was adjusted to 7, and the mixture was dialyzed for 48 h using a dialysis bag (8,000~14,000 Da) to remove salt. The mixture was then freeze-dried for 48 h to obtain glycosylated protein hydrolysate powder.

[0069] 2. Determine the dosage of PGA:

[0070] Repeat the above experimental steps, and adjust the mass ratio of tilapia protein hydrolysate to PGA to 2:1, 1.75:1, 1.5:1 and 1.25:1, respectively, to prepare tilapia protein hydrolysate with different grafting degrees.

[0071] 3. Indicator Testing:

[0072] (1) Grafting degree analysis: The free amino groups were determined and the grafting degree was calculated using the o-phthalaldehyde (OPA) method. 4.0 mL of OPA reagent (accurately weigh 40.0 mg of OPA, dissolve it in 1.0 mL of methanol, then add 5 mL of 10% (w / w) sodium dodecyl sulfate (SDS), 25 mL of borax solution (0.1 mol / L), 100 μL of β-mercaptoethanol, and finally dilute to 50 mL with distilled water) was placed in a test tube. 200 μL of sample was added, mixed thoroughly, and reacted in a 35℃ water bath for 2 min. The absorbance value A was measured at 340 nm. Another 4.0 mL of OPA reagent was placed in a test tube, and 200 µL of water was added as a blank control. The grafting degree was calculated based on the change in absorbance. The specific calculation formula is shown below:

[0073] ;

[0074] In the formula, A0 and A1 are the absorbance values ​​of the solution before and after the grafting reaction, respectively.

[0075] (2) Protein solubility detection: Same as in Example 1.

[0076] (3) Emulsification test: Same as in Example 1.

[0077] The effects of the mass ratio of tilapia protein hydrolysate to PGA on the grafting degree, solubility, and emulsifying properties of tilapia protein hydrolysate are as follows: Figure 4 , Figure 5 and Figure 6 As shown, considering these three indicators, the glycosylated tilapia protein hydrolysate prepared under the condition of a mass ratio of tilapia protein hydrolysate to PGA of 2:1 was selected for subsequent experiments.

[0078] Example 3: Preparation of a quercetin-loaded glycosylated tilapia protein hydrolysate

[0079] The experiment was conducted using the glycosylated tilapia protein hydrolysate prepared in Example 2 (the mass ratio of tilapia protein hydrolysate to PGA was 2:1).

[0080] 1. Preparation of quercetin-loaded glycosylated tilapia protein hydrolysate: A certain mass of glycosylated tilapia protein hydrolysate was dispersed in deionized water, and different masses of quercetin were added (the mass ratio of quercetin to glycosylated tilapia protein hydrolysate was 1:100). The pH of the mixture was adjusted to 12 and reacted for 30 min. Then, the pH was adjusted back to 7 to obtain a quercetin-loaded glycosylated tilapia protein hydrolysate dispersion. The obtained dispersion was dialyzed for 48 h using a dialysis bag (8,000~14,000 Da) to remove salt and then freeze-dried to obtain a powder of the quercetin-loaded glycosylated product.

[0081] 2. Indicator Testing:

[0082] (1) Determination of encapsulation efficiency: The prepared dispersion of glycosylated tilapia protein hydrolysate loaded with quercetin was centrifuged at 5000 rpm for 15 min. The supernatant was appropriately diluted with anhydrous ethanol to extract quercetin. The absorbance at 374 nm was measured by ultraviolet spectrophotometer, and the content of quercetin was calculated according to the standard curve (Y=0.076X, R). 2 =0.999). The formula for calculating the encapsulation efficiency of quercetin is as follows:

[0083] .

[0084] (2) Detection of quercetin bioavailability: Free quercetin samples and quercetin-loaded glycosylated tilapia protein hydrolysate dispersions were subjected to continuous simulated gastric and intestinal digestion. The simulated gastric digestion conditions were as follows: 10 mL of quercetin-loaded glycosylated tilapia protein hydrolysate dispersion was mixed with 20 mL of simulated gastric digestion solution (3.2 mg / mL pepsin, 0.7% HCl, and 2 mg / mL NaCl), the pH was adjusted to 2.0, and the mixture was incubated at 37°C with shaking for 1 h. The simulated intestinal digestion conditions were as follows: 20 mL of simulated gastric digestion solution was mixed with 20 mL of simulated small intestinal digestion solution (5 mg / mL bile salts, 0.4 mg / mL pancreatin, and 3.2 mg / mL lipase), the pH was adjusted to 7.0, and the mixture was incubated at 37°C with shaking for 2 h. Take 2 mL of the digested sample and centrifuge (5000g, 30 min) to obtain the mixed micelle phase in the supernatant. Extract quercetin from the undigested sample and the mixed micelle phase using anhydrous ethanol. Measure the absorbance at 374 nm using a UV spectrophotometer. Analyze the results according to the standard curve (y=0.076X, R0).2 Quercetin content was calculated using the formula (=0.999). The formula for calculating the bioavailability of quercetin is shown below:

[0085] ;

[0086] Where C represents the quercetin content in the mixed micelle phase, and C0 represents the quercetin content in the sample before centrifugation.

[0087] Under these conditions, the encapsulation rate of quercetin in glycosylated tilapia protein hydrolysate was 88.23%, and the bioavailability of the encapsulated quercetin after in vitro gastrointestinal digestion was 75.63%, which was 3.91 times higher than that of free quercetin (15.40%). Figure 7 and Figure 8 ).

[0088] Example 4: Preparation of a quercetin-loaded glycosylated tilapia protein hydrolysate

[0089] The difference from Example 3 is that the mass ratio of quercetin to the glycosylated product is 1:80.

[0090] Under these conditions, the encapsulation rate of quercetin in glycosylated tilapia protein hydrolysate was 82.24%, and the bioavailability of the encapsulated quercetin after in vitro gastrointestinal digestion was 68.70%, which was 3.61 times higher than that of free quercetin (14.90%). Figure 7 and Figure 8 ).

[0091] Example 5: Preparation of a quercetin-loaded glycosylated tilapia protein hydrolysate

[0092] The difference from Example 3 is that the mass ratio of quercetin to the glycosylated product is 1:60.

[0093] Under these conditions, the encapsulation rate of quercetin in glycosylated tilapia protein hydrolysate was 75.73%, and the bioavailability of the encapsulated quercetin after in vitro gastrointestinal digestion was 64.27%, which was 3.40 times higher than that of free quercetin (14.62%). Figure 7 and Figure 8 ).

[0094] Example 6 Preparation of a quercetin-loaded glycosylated tilapia protein hydrolysate

[0095] The difference from Example 3 is that the mass ratio of quercetin to the glycosylated product is 1:40.

[0096] Under these conditions, the encapsulation rate of quercetin in glycosylated tilapia protein hydrolysate was 72.77%, and the bioavailability of the encapsulated quercetin after in vitro gastrointestinal digestion was 56.83%, which was 2.76 times higher than that of free quercetin (15.13%). Figure 7 and Figure 8 ).

[0097] Example 7 Preparation of a quercetin-loaded glycosylated tilapia protein hydrolysate

[0098] The difference from Example 3 is that the mass ratio of quercetin to the glycosylated product is 1:20.

[0099] Under these conditions, the encapsulation rate of quercetin in glycosylated tilapia protein hydrolysate was 65.22%, and the bioavailability of the encapsulated quercetin after in vitro gastrointestinal digestion was 48.75%, which was 2.17 times higher than that of free quercetin (15.36%). Figure 7 and Figure 8 ).

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of quercetin-loaded alkali-induced glycosylated tilapia protein hydrolysate in the preparation of a delivery system that improves the bioavailability of quercetin, characterized in that, The method for preparing the quercetin-loaded alkali-induced glycosylated tilapia protein hydrolysate comprises the following steps: Tilapia protein and flavor protease were mixed and then subjected to hydrolysis and enzyme inactivation treatments in sequence to obtain tilapia protein hydrolysate. The tilapia protein hydrolysate and propylene glycol alginate were mixed and subjected to an alkali-induced glycosylation reaction to obtain glycosylated tilapia protein hydrolysate. The glycosylated tilapia protein hydrolysate was mixed with quercetin to obtain the quercetin-loaded alkali-induced glycosylated tilapia protein hydrolysate. The amount of flavor protease added is 6000 U / g-72000 U / g; The mass ratio of the tilapia protein hydrolysate to the propylene glycol alginate is (1.50-2.50):1; The mass ratio of quercetin to glycosylated tilapia protein hydrolysate is 1:(5-100). The alkali-induced glycosylation reaction was carried out over a period of 3 hours at a pH of 11.

0. The hydrolysis treatment lasted for 90 minutes, at a temperature of 45°C, and at a pH of 7.

0. The enzyme inactivation treatment lasted for 10 minutes at a temperature of 90°C.

2. The application according to claim 1, characterized in that, The process of mixing the glycosylated tilapia protein hydrolysate and the quercetin further includes adjusting the pH of the resulting mixture to 12.0 and reacting for 30 min, followed by adjusting the pH of the resulting reaction solution to 7.0 to obtain the quercetin-loaded alkali-induced glycosylated tilapia protein hydrolysate.

3. The application according to claim 1, characterized in that, The method for preparing the tilapia protein includes mixing tilapia meat and water at a mass ratio of 1:9, adjusting the pH value to 11.0, stirring evenly, centrifuging, taking the supernatant, adjusting the pH value to 5.5, centrifuging again, collecting the precipitate, dispersing the precipitate with water, adjusting the pH value of the dispersion to 7, dialyzing, and freeze-drying to obtain the tilapia protein.

Images