Preparation method and application of beta-lactoglobulin-flavone covalent complex system
The β-lactoglobulin-flavonoid covalent complex system was prepared by ultrasound-assisted alkali treatment, which solved the stability and solubility problems of fat-soluble functional factors in the food industry and achieved efficient delivery and sustained release of curcumin and lycopene.
Patent Information
- Application Number
- CN202510890719.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-11-07
AI Technical Summary
In the existing technology, fat-soluble functional factors such as curcumin and lycopene have limited their application in the food industry due to their poor stability and poor solubility. Single protein delivery systems are easily affected by factors such as temperature, ion concentration and pH.
A covalent complex system of β-lactoglobulin-flavonoids was prepared by ultrasound-assisted alkaline treatment. β-lactoglobulin and flavonoid compounds were ultrasonically treated and mixed under alkaline conditions to form a covalent complex. Subsequently, dialysis and freeze-drying were performed to enhance its structural and functional properties.
It improved the loading rate and antioxidant properties of fat-soluble functional factors, enhanced thermal stability, and demonstrated good sustained-release effect in in vitro simulated digestion, thereby improving the delivery efficiency of curcumin and lycopene.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a preparation method and application of a beta-lactoglobulin-flavonoid covalent complex system, in particular to a preparation method and application of a beta-lactoglobulin-flavonoid covalent complex system based on an ultrasonic-assisted alkali treatment method. BACKGROUND
[0002] Fat-soluble functional factors such as curcumin (Cur) and lycopene (Lyc) have effects of anti-inflammation, anti-oxidation, and protection of cardiovascular health, but their wide application is limited due to poor stability and poor solubility. Proteins are often used to construct functional factor delivery systems due to good biocompatibility, but single proteins are easily affected by factors such as temperature, ion concentration, and pH, which limits their application in the food industry. Therefore, a delivery system capable of effectively delivering fat-soluble functional factors such as curcumin and lycopene is needed. SUMMARY
[0003] The present application aims to provide a preparation method and application of a beta-lactoglobulin-flavonoid covalent complex system. The beta-lactoglobulin-flavonoid covalent complex system prepared by the method has good structural and functional properties, and can effectively deliver fat-soluble functional factors.
[0004] The technical solution of the present application is a preparation method of a beta-lactoglobulin-flavonoid covalent complex system, comprising the following steps: S1. 1 g of beta-Lg is dissolved in 50 mL of deionized water, the pH value of the solution is adjusted to 9.0 with 0.1 mol / L NaOH, and ultrasonic treatment is performed at room temperature to obtain product A; S2. A certain amount of flavonoid compound is dissolved in an appropriate amount of ethanol, then diluted with deionized water to 50 mL to obtain a final concentration of 0.35 mmol / L, and the pH value is adjusted to 9.0 to obtain product B; S3. Product A and product B are mixed and stirred for 12 h, and reacted at room temperature for 24 h to obtain product C; S4. Product C is continuously reacted for 48 h in a 3500 Da dialysis bag, finally, the ethanol in the solution is removed by a rotary evaporator, and then freeze-drying treatment is performed to obtain the finished product.
[0005] In the aforementioned preparation method of a beta-lactoglobulin-flavonoid covalent complex system, the ultrasonic treatment in S1 is performed at a power of 400 W for 15 min.
[0006] In the aforementioned preparation method of a beta-lactoglobulin-flavonoid covalent complex system, the flavonoid compound in S2 can be apigenin, luteolin, myricetin, apigenin-7-O-glucoside, luteoloside, or myricitrin.
[0007] The preparation method of the beta-lactoglobulin-flavonoid covalent complex system, wherein the flavonoid compound in S2 is myricetin.
[0008] The preparation method of the beta-lactoglobulin-flavonoid covalent complex system, wherein the volume ratio of A to B is 1:1.
[0009] The application of the beta-lactoglobulin-flavonoid covalent complex system, comprising the following steps: Q1. Dissolve the fat-soluble functional factor in anhydrous ethanol to obtain D, wherein the concentration of the fat-soluble functional factor is 0.5 mg / mL; Q2. Dissolve the product prepared in any one of claims 1-5 in deionized water to obtain E, wherein the concentration of the product is 0.5 mg / mL; Q3. Mix D and E in a volume ratio of 1:3, and then perform magnetic stirring on the mixture for 1 h, and then perform centrifugation on the mixture, and then perform vacuum freeze-drying on the supernatant to obtain the beta-lactoglobulin-flavonoid covalent complex carrying the fat-soluble functional factor.
[0010] The application of the beta-lactoglobulin-flavonoid covalent complex system, wherein the centrifuge parameters in Q3 are set as 8000 r / min for 10 min.
[0011] The application of the beta-lactoglobulin-flavonoid covalent complex system, wherein the fat-soluble functional factor is curcumin or lycopene.
[0012] The application of the beta-lactoglobulin-flavonoid covalent complex system, wherein the product is the product prepared by using myricetin as the flavonoid compound in S2 and by performing S3 and S4.
[0013] Compared with the prior art, the beta-lactoglobulin-flavonoid complex prepared by the method has the advantages that the antioxidant property and the thermal stability of the beta-lactoglobulin-flavonoid complex are enhanced, the surface hydrophobicity is low, the loading rate of the beta-lactoglobulin-flavonoid complex in the delivery of fat-soluble functional factors such as curcumin and lycopene can be effectively improved, and the beta-lactoglobulin-flavonoid complex exhibits good sustained-release effect in in-vitro simulated digestion, and the loading rate of the beta-lactoglobulin-flavonoid complex in the delivery of curcumin and lycopene can be increased by 21.32% and 20.68% respectively compared with the beta-lactoglobulin prepared by ultrasonic-assisted preparation, so that the fat-soluble functional factors such as curcumin and lycopene can be effectively applied in the food industry. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 is the ability diagram of U-beta-Lg, U-beta-Lg-MY covalent complex carrying Cur and Lyc of the application. Figure 2 is a release rate diagram of Cur, Lyc in simulated oral, gastric, intestinal fluid. DETAILED DESCRIPTION
[0015] The application will be further described in conjunction with the accompanying drawings and examples, but not as the basis for limiting the application. The applicant has conducted a series of experiments, which can prove that the method provided by the application is effective and the β-lactoglobulin-flavonoid covalent complex system prepared has good structural and functional properties, and can effectively realize the delivery of fat-soluble functional factors.
[0016] Table 1 Materials and reagents:
[0017] Table 2 Equipment used:
[0018] Example. A preparation method of amyloid β-lactoglobulin fiber-fucoidan self-assembly complex for lycopene delivery, comprising the following steps: S1. 1 g of β-Lg was dissolved in 50 mL of deionized water, the solution pH was adjusted to 9.0 with 0.1 mol / L NaOH, and ultrasonic treatment was carried out at room temperature, the ultrasonic treatment conditions were power setting of 400 W and time duration of 15 min, to obtain product A; S2. Myricetin (MY) was dissolved in an appropriate amount of ethanol, then diluted with deionized water to 50 mL to make its final concentration 0.35 mmol / L, and the pH was adjusted to 9.0 to obtain product B; S3. Product A and product B in a volume ratio of 1:1 were mixed, stirred for 12 h, and reacted at room temperature for 24 h to obtain product C; S4. Product C was continuously reacted for 48 h with a 3500 Da dialysis bag, finally, ethanol in the solution was removed by a rotary evaporator, followed by freeze-drying treatment to obtain finished product U-β-Lg-MY.
[0019] Comparative Example 1. β-Lg was dissolved in deionized water, the solution pH was adjusted to 9.0, and then freeze-dried to obtain finished product β-Lg.
[0020] Comparative Example 2. On the basis of the example, β-Lg was dissolved in deionized water, the solution pH was adjusted to 9.0, then directly mixed with a myricetin solution with pH of 9.0 without ultrasonic treatment, the rest of the steps were consistent with the example, product A and product B were mixed and stirred for 12 h, and reacted at room temperature for 24 h to obtain product C, product C was continuously reacted for 48 h with a 3500 Da dialysis bag, finally, ethanol in the solution was removed by a rotary evaporator, followed by freeze-drying treatment to obtain finished product β-Lg-MY.
[0021] Comparative Example 3, on the basis of Comparative Example 2, the myricetin (MY) solution was replaced by the apigenin (API) solution, and the rest was consistent with Comparative Example 2, to prepare the finished product β-Lg-API.
[0022] Comparative Example 4, on the basis of Comparative Example 2, the myricetin (MY) solution was replaced by the luteolin (LUT) solution, and the rest was consistent with Comparative Example 2, to prepare the finished product β-Lg-LUT.
[0023] Comparative Example 5, on the basis of Comparative Example 2, the myricetin (MY) solution was replaced by the apigenin-7-O-glucoside (AGL) solution, and the rest was consistent with Comparative Example 2, to prepare the finished product β-Lg-AGL.
[0024] Comparative Example 6, on the basis of Comparative Example 2, the myricetin (MY) solution was replaced by the luteolin-7-O-glucoside (LGL) solution, and the rest was consistent with Comparative Example 2, to prepare the finished product β-Lg-LGL.
[0025] Comparative Example 7, on the basis of Comparative Example 2, the myricetin (MY) solution was replaced by the myricitrin (MYR) solution, and the rest was consistent with Comparative Example 2, to prepare the finished product β-Lg-MYR.
[0026] Comparative Example 8, after dissolving β-Lg in deionized water, the solution pH was adjusted to 9.0, and after ultrasonic treatment (power 400 W, time 15 min) at room temperature, freeze-drying was carried out to prepare the finished product U-β-Lg.
[0027] Comparative Example 9, on the basis of the example, the myricetin (MY) solution was replaced by the apigenin (API) solution, and the rest was consistent with the example, to prepare the finished product U-β-Lg-API.
[0028] Comparative Example 10, on the basis of the example, the myricetin (MY) solution was replaced by the luteolin (LUT) solution, and the rest was consistent with the example, to prepare the finished product U-β-Lg-LUT.
[0029] Comparative Example 11, on the basis of the example, the myricetin (MY) solution was replaced by the apigenin-7-O-glucoside (AGL) solution, and the rest was consistent with the example, to prepare the finished product U-β-Lg-AGL.
[0030] Comparative Example 12, on the basis of the example, the myricetin (MY) solution was replaced by the luteolin-7-O-glucoside (LGL) solution, and the rest was consistent with the example, to prepare the finished product U-β-Lg-LGL.
[0031] Comparative Example 13, on the basis of the examples, replace the myricetin (MY) solution with myricitrin (MYR) solution, the rest is consistent with the example, the finished product U-β-Lg-MYR is prepared.
[0032] The structural and functional properties of the finished products prepared in Examples and Comparative Examples 1-13 are analyzed. The structural property analysis includes the detection of reactive groups involved; circular dichroism spectrum analysis, etc. The functional property analysis includes antioxidant activity analysis and thermal stability analysis.
[0033] The detection of reactive groups involved includes the detection of free amino groups, free thiol groups and free tryptophan: The detection method of free amino groups: 8 mg of OPA is dissolved in 5 mL of methanol, 0.5 mL of 20% w / v sodium dodecyl sulfate, 20 μL of β-mercaptoethanol and 0.1 mol / L of 5 mL of borax solution are added, and the volume is made to 10 mL. The finished products of Examples and Comparative Examples 1-13 are reconstituted with deionized water and adjusted to a concentration of 0.5 mg / mL. Take 50 μL of the solution of the finished products of Examples and Comparative Examples 1-13 in a test tube, add 1 mL of OPA reagent, shake well for 10 s, then place in a 35°C water bath for 2 min. Then use the enzyme marker to measure the absorbance at 340 nm, and use the glycine standard curve as the basis for quantification.
[0034] The detection method of free thiol groups: prepare Ellman reagent by fully dissolving 4 mg of DTNB in 1 mL of 50 mmol / L Tris / HCl buffer solution (containing 1 mmol / L EDTA at pH 8.0). Weigh 3 mg of the finished products of Examples and Comparative Examples 1-13, add 1 mL of Tris / HCl buffer solution containing 8.0 mol / L urea, shake well to mix, then add 10 μL of Ellman reagent, react for 1 h at room temperature, and use the enzyme marker to measure the absorbance at 412 nm,
[0035] In the formula, C is the sample concentration (mg / mL), and A412 is the absorbance of the sample at 412 nm.
[0036] The detection method of free tryptophan: reconstitute the freeze-dried sample with deionized water to prepare a 1 mg / mL aqueous solution. Add 0.9 mL of 1 mg / mL of the finished products of Examples and Comparative Examples 1-13 to 1 mL of 16 mol / L HNO3, and react in a 50°C water bath for 15 min. After cooling at room temperature, add 4 mL of 5 mol / L NaOH and 4 mL of absolute ethanol to the solution, mix well, and then use the enzyme marker to measure the absorbance at 360 nm and 430 nm wavelengths,
[0037] wherein A430 is the absorbance of the sample at 430 nm, and A360 is the absorbance of the sample at 360 nm.
[0038] The results are shown in the following table: Table 3 Free amino, sulfhydryl and tryptophan contents of β-Lg-flavonoid covalent complexes before and after ultrasonic treatment
[0039] From the above data, it can be seen that the contents of free amino, sulfhydryl and tryptophan significantly decreased after ultrasonic treatment, and the decrease was most obvious for the product U-β-Lg-MY prepared in the example.
[0040] Circular dichroism spectrum analysis: The secondary structure of the samples was determined by a Japanese JASCO J-1500 circular dichroism spectrum (CD) instrument, and the determination wavelength range was 190-260 nm. The products of the example and Comparative Examples 1-13 after lyophilization were dissolved in pH 7.0, 10 mmol / L PBS to ensure a final concentration of 0.2 mg / mL. The instrument parameters were set as a path length of 0.1 cm, a bandwidth of 1 nm, and a scanning speed of 50 nm / min. The secondary structure of the complexes was analyzed by a calculation program Dichroweb, and the results are shown in the following table.
[0041] Table 4 Secondary structure contents of β-Lg-flavonoid covalent complexes before and after ultrasonic treatment
[0042] From the above table, it can be seen that after ultrasonic treatment, the content of α-helix of β-Lg decreased, and the content of β-sheet increased. This phenomenon may be due to the mechanical action and cavitation effect of ultrasonic, which leads to the breakage of the secondary bonds inside β-Lg, and thus causes the unfolding of the structure of β-Lg. Compared with β-Lg, the content of α-helix in the covalent complex significantly decreased (p<0.05). This phenomenon may be attributed to the change in the conformation of β-Lg molecules in an alkaline environment, which causes the transition from an ordered structure to a disordered structure.
[0043] Antioxidant activity analysis, including DPPH, ABTS, FARP detection, the specific detection method is: (1) DPPH DPPH stock solution was prepared by dissolving 4 mg of DPPH powder in 80% ethanol solution and making up the volume to 100 mL. Then, 1 mg / mL of the finished product solutions of Examples and Comparative Examples 1-13 were mixed with the DPPH ethanol solution at a ratio of 1:1. After 30 min of reaction in the dark, the absorbance was measured at 517 nm. Using a Trolox standard solution, a standard curve was prepared by the same procedure, and the DPPH radical scavenging activity of each sample was expressed as Trolox equivalent.
[0044] (2) ABTS ABTS stock solution was prepared by mixing 7.0 mmol / L of ABTS with 2.45 mmol / L of potassium persulfate at a ratio of 1:1 and reacting for 12-16 h in the dark. Before the experiment, the ABTS stock solution was diluted with pH 7.4 10 mmol / L PBS so that the absorbance at 734 nm was 0.70±0.02. 100 μL of 0.5 mg / mL of the finished product solutions of Examples and Comparative Examples 1-13 was mixed with 3.9 mL of the ABTS solution, and then reacted for 10 min in a 37°C water bath. Finally, the absorbance at 734 nm was measured using a UV spectrophotometer, and the ABTS scavenging activity of the sample was expressed as Trolox equivalent.
[0045] (3) FARP TPZP working solution was prepared by first preparing a 10 mmol / L TPZP solution using 40 mmol / L HCl, and then preparing a pH 3.6 0.3 mol / L sodium acetate buffer and a 20 mmol / L ferric chloride solution (also dissolved in 40 mmol / L HCl). The acetate, TPZP and ferric chloride were mixed at a ratio of 10:1:1, and then reacted for 30 min in a 37°C water bath for use. 900 μL of the TPZP working solution was mixed with 100 μL of 1 mg / mL of the finished product solutions of Examples and Comparative Examples 1-13, and then reacted for 30 min in a 37°C water bath. After the mixture returned to room temperature, the absorbance at 597 nm was measured using an enzyme marker, and the FARP scavenging activity of the sample was expressed as Trolox equivalent. The results are shown in the following table.
[0046] Table 5 Antioxidant properties of β-Lg-flavonoid covalent complexes before and after ultrasonication
[0047] The antioxidant properties of the complexes were evaluated using three methods: DPPH, ABTS, and FARP. According to the experimental data in Tables 3-4, after ultrasonic treatment, the ability of β-Lg to scavenge DPPH free radicals increased from the previous 72.03 μmol Trolox / g sample to 75.79 μmol Trolox / g sample. This data change fully indicates that ultrasonic treatment can significantly enhance the antioxidant capacity of β-Lg, providing strong data support for subsequent exploration of the application of ultrasonic technology in improving the antioxidant properties of proteins. Compared with β-Lg, after adding API, LUT, MY, AGL, L7G, and MYR, the ability of β-Lg-API, β-Lg-LUT, and β-Lg-MY to scavenge DPPH free radicals increased to 85.75 μmol Trolox / g sample, 91.32 μmol Trolox / g sample, 113.71 μmol Trolox / g sample, 76.43 μmol Trolox / g sample, 81.25 μmol Trolox / g sample, and 88.84 μmol Trolox / g sample, respectively. This indicates that the covalent combination of β-Lg with flavonoids can significantly enhance the DPPH antioxidant activity.
[0048] Similar results were obtained in the experiment, in which the ABTS and FARP scavenging abilities of the β-Lg-flavonoid covalent complex were 2.93-3.89 times and 1.39-6.92 times that of β-Lg, respectively, indicating that the combination of β-Lg with flavonoids can stabilize the expression of antioxidant properties. Overall, the β-Lg-MY covalent complex performed best in terms of DPPH free radical scavenging, ABTS free radical scavenging, and iron reduction capacity. This is due to the fact that the polyphenol combination of β-Lg and MY is higher than that of other complexes, and the number of phenolic hydroxyl groups in MY is the highest, making its own antioxidant activity better than that of other flavonoids, so the antioxidant activity of the β-Lg-MY covalent complex is the strongest. In addition, after ultrasonic treatment, the antioxidant activity of the β-Lg-flavonoid complex was further enhanced.
[0049] Thermal stability analysis: 5-10 mg of the finished product of the examples and Comparative Examples 1-13 after lyophilization was weighed into an aluminum pan and sealed with an aluminum cover. The sealed empty aluminum pan was used as a blank control. Test conditions: sample temperature range: 30-180°C, temperature rise rate: 10°C / min, nitrogen flow rate: 30 mL / min. The instrument's built-in software was used to analyze and calculate the maximum denaturation temperature (Tmax) and enthalpy change (ΔH) of the sample, and the results are shown in the table below.
[0050] Table 6 Tmax and ΔH of β-Lg-flavonoid covalent complexes before and after ultrasonic treatment
[0051] From the above, DSC is used to characterize the thermal stability of β-Lg and β-Lg-flavonoid covalent complex, Tmax of β-Lg is 90.53℃, after ultrasonic, Tmax of β-Lg is 91.28℃, which shows that ultrasonic is conducive to improve the thermal stability of β-Lg. When β-Lg is combined with API, LUT, MY, AGL, LGL, MYR, Tmax is improved to 97.03℃, 97.54℃, 102.03℃, 95.14℃, 96.84℃ and 100.15℃, which shows that the covalent combination of β-Lg and flavonoid compounds can improve its thermal stability. In addition, compared with β-Lg, the ΔH value of β-Lg-flavonoid complex increases, which shows that the complex needs more energy in the unfolding process. This phenomenon indirectly reflects that the covalent complex has higher stability, and the thermal stability of the finished product U-β-Lg-MY prepared in the example is finally.
[0052] An application of a β-lactoglobulin-flavonoid covalent complex system, comprising the following steps: Q1. Dissolve the fat-soluble functional factor in anhydrous ethanol to make its concentration 0.5 mg / mL, and obtain D product; Q2. Dissolve the finished product U-β-Lg-MY prepared in the example in deionized water to make its concentration 0.5 mg / mL, and obtain E product; Q3. Mix D product and E product in a volume ratio of 1:3, and after magnetic stirring for 1 h, centrifuge the mixture with a centrifuge at a speed of 8000 r / min for 10 min, take the supernatant for vacuum freeze-drying treatment, and prepare a β-lactoglobulin-flavonoid covalent complex carrying a fat-soluble functional factor.
[0053] The fat-soluble functional factor is curcumin or lycopene.
[0054] In order to prove that the β-lactoglobulin-flavonoid covalent complex can improve the delivery effect of the fat-soluble functional factor, the following embedding rate detection and in vitro simulation digestion experiment are carried out, and the finished product prepared by Comparative Example 1, Comparative Example 2 and Comparative Example 8 is used as a control instead of U-β-Lg-MY in Q2.
[0055] Embedding rate experiment: Cur: The prepared product is prepared into 1 mg / mL, centrifuged at 8000 r / min for 10 min, and the supernatant is taken, and the absorbance value at 426 nm is measured by an enzyme marker;
[0056] Lyc: The prepared product is prepared into 1 mg / mL, centrifuged at 8000 r / min for 10 min, and the supernatant is taken, and the absorbance value at 472 nm is measured by an enzyme marker.
[0057]
[0058] Results as shown in the accompanying drawings Figure 1 As shown in the accompanying drawings, the embedding rates of U-β-Lg for Cur and Lyc were 57.34% and 41.79%, respectively, and the embedding rates of U-β-Lg-MY-Cur / Lyc were increased by 21.32% and 20.68%, respectively, compared with U-β-Lg. This may be due to the addition of MY, which changes the structure of β-Lg, reduces the surface hydrophobicity, and can better bind Cur and Lyc. In addition, MY contains phenolic hydroxyl groups and has strong antioxidant activity, which can protect Cur and Lyc from oxidative damage and also improve the stability of β-Lg. The above results show that β-Lg-MY can deliver Cur and Lyc.
[0059] In vitro digestion experiment: configure the in vitro digestion solution. First, configure 2 mol / L NaCl, 1 mol / L NaHCO3, 0.5 mol / L KCl, 0.5 mol / L KH2PO4, 0.5 mol / L (NH4)2CO3, 0.15 mol / L MgCl2(H2O)6, 0.3 mol / L CaCl2(H2O)2 solution, and prepare simulated oral fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) according to the proportions described in the table below.
[0060] Table 7 Proportion of in vitro simulated digestion solution
[0061] Oral cavity: The sample was prepared into 1 mg / mL with deionized water, 1 mL of the finished product solution, 0.7 mL of SSF, 0.1 mL of 4.0 mg / mL α-amylase, 5 μL of 0.3 mol / L CaCl2, and the pH of the solution was adjusted to 7.0. The solution was mixed thoroughly with a magnetic stirrer, placed in a 37℃ water bath oven in the dark for 1 min, and the oral cavity digestion solution was collected.
[0062] Stomach: 1.5 mL of SGF, 0.32 mL of 3 mg / mL pepsin, and 1 μL of 0.3 mol / L CaCl2 were added to the above oral cavity digestion system. The pH was adjusted to 3.0 with 1 mol / L HCl, and the system was placed in a 37℃ water bath oven in the dark for 1 h, and the gastric digestion solution was collected.
[0063] Intestine: continue to add 2.2 mL SIF, 1 mL 4 mg / mL trypsin, 0.5 mL 160 mmol / L porcine bile salt, 8 μL 0.3 mol / L CaCl2 in the system digested by gastric juice above, adjust pH to 7.0 with 1 mol / L NaOH, place in a 37℃ water bath oven in the dark for 2 h, collect intestinal juice. The test results are shown in the drawings Figure 2
[0064] The release rate of free Cur in the oral digestion stage is only 5.68%, and the release rate of Cur increases when the finished product enters the gastric digestion stage: in the U-β-Lg system, the release rate reaches 7.23%; in the U-β-Lg-MY system, the release rate is 6.6%, which shows a small growth trend compared with the oral stage. The trend of Lyc in the simulated oral, gastric and intestinal stages is similar to that of Cur, both of which have the lowest release rate in the oral digestion stage and the highest release rate in the intestinal digestion stage. The embodiment shows good sustained-release effect in in vitro simulated digestion when used to carry fat-soluble functional factors.
Claims
1. A method for preparing a β-lactoglobulin-flavonoid covalent complex system, characterized by: The method comprises the following steps: S1. 1 g of β-Lg is dissolved in 50 mL of deionized water, the pH value of the solution is adjusted to 9.0 by using 0.1 mol / L NaOH, and ultrasonic treatment is performed at room temperature to obtain product A; S2. The flavonoid compound is dissolved in an appropriate amount of ethanol, then diluted with deionized water to 50 mL to obtain a final concentration of 0.35 mmol / L, and the pH value is adjusted to 9.0 to obtain product B; S3. Product A and product B are mixed and stirred for 12 h, and reacted at room temperature for 24 h to obtain product C; S4. Product C is continuously reacted in a 3500 Da dialysis bag for 48 h, finally, ethanol in the solution is removed by using a rotary evaporator, and then freeze-drying treatment is performed to obtain the finished product.
2. The method for preparing a β-lactoglobulin-flavonoid covalent complex system according to claim 1, characterized in that, The ultrasonic treatment in S1 is performed at a power of 400 W for 15 min.
3. The method for preparing a β-lactoglobulin-flavonoid covalent complex system according to claim 1, characterized in that, The flavonoid compound in S2 can be apigenin, luteolin, myricetin, apigenin-7-O-glucoside, luteolin-7-O-glucoside, or myricitrin.
4. The method for preparing a β-lactoglobulin-flavonoid covalent complex system according to claim 3, characterized in that, The flavonoid compound in S2 is myricetin.
5. The method for preparing a β-lactoglobulin-flavonoid covalent complex system according to claim 1, characterized in that, The volume ratio of product A to product B in S3 is 1:
1.
6. Use of a β-lactoglobulin-flavonoid covalent complex system, characterized in that, The method comprises the following steps: Q1. The liposoluble functional factor is dissolved in anhydrous ethanol to obtain product D with a concentration of 0.5 mg / mL; Q2. The finished product prepared according to any one of claims 1-5 is dissolved in deionized water to obtain product E with a concentration of 0.5 mg / mL; Q3. Product D and product E are mixed at a volume ratio of 1:3, the mixture is subjected to magnetic stirring for 1 h, then centrifuged by using a centrifuge, the supernatant is subjected to vacuum freeze-drying treatment, and a β-lactoglobulin-flavonoid covalent complex carrying a liposoluble functional factor is prepared.
7. The use of a beta-lactoglobulin-flavonoid covalent complex system according to claim 6, characterized in that, The centrifuge in Q3 is set at a speed of 8000 r / min for 10 min.
8. The use of a beta-lactoglobulin-flavonoid covalent complex system according to claim 6, characterized in that, The liposoluble functional factor is curcumin or lycopene.
9. The use of a beta-lactoglobulin-flavonoid covalent complex system according to claim 6, characterized in that, The finished product is the product prepared by using myricetin as the flavonoid compound in S2, and then by using S3 and S4.