Preparation method and application of lactoferrin-sodium alginate hydrogel system

Abstract

The invention discloses a preparation method and application of a lactoferrin-sodium alginate hydrogel system, and the preparation method of the lactoferrin-sodium alginate hydrogel system comprises the following steps: S1, preparing a lactoferrin subunit solution; s2, preparing a lactoferrin-active substance co-assembly; s3, purifying; s4, preparing a sodium alginate solution; and S5, preparing the lactoferrin-sodium alginate hydrogel loaded with the active substance. According to the method disclosed by the invention, small-molecule active substances are encapsulated on the basis that lactoferrin has a natural cage-shaped structure and a self-assembly characteristic, and lactoferrin is modified by utilizing good gel characteristic and gastrointestinal digestion resistance characteristic of sodium alginate, so that a lactoferrin-sodium alginate co-protected active substance hydrogel system is constructed; the constructed system shows obvious superiority in processing adaptability and bioaccessibility, and the problems of poor stability, low bioavailability and the like of natural small-molecule active substances in the food processing process are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and more specifically, to a preparation method and application of a lactoferrin-sodium alginate hydrogel system. Background Art

[0002] Utilizing the wide range of biological activities of natural active substances to achieve functional development of food is an important direction for the current development of the food industry. However, many natural active substances have problems such as poor stability, weak palatability, poor solubility, and low bioavailability in food. Finding appropriate strategies to improve the processing adaptability of natural bioactive substances has become a hot topic in the field of food research. Food proteins have natural amphiphilicity and self-assembly properties and have attracted widespread attention. However, the stabilized systems constructed by single proteins often have problems such as general stability effects and high preparation process costs.

[0003] Lactoferrin (LF) is an iron storage protein with a natural cage structure and self-assembly properties. It has 8 hydrophilic channels and 6 hydrophobic channels (width of about 0.3-0.4 nm), which provides an effective way for metal ions and other small molecule functional factors / drugs to enter the protein cage. It is a good carrier for transporting functional factors and drug ingredients. It has been studied in the development of active molecules such as carotenoids, curcumin and some anticancer drugs. However, LF is a heat-labile protein and may denature and aggregate during heat treatment, which in turn affects its biological activity; in the gastric fluid environment (low pH), iron is released from LF, which increases the sensitivity of LF to digestive enzymes and causes a large degree of degradation.

[0004] Modification of protein structure by food polysaccharides is an effective method to improve protein stability and functional properties, mainly including non-covalent complexes formed by physical mixing and covalent complexes formed by enzymatic glycosylation, chemical cross-linking and Maillard glycosylation (dry heat method, wet heat method). However, existing technologies often have problems such as high cost, complicated process or unclear effect.

[0005] Sodium alginate is an anionic linear polysaccharide with good water solubility and gelling properties. It can form a stable gel-like substance in the acidic environment of gastric juice and dissolve in the small intestine. It can inhibit digestive enzymes, reduce the absorption of cholesterol and glucose, and control the digestion of fat.

[0006] Hydrogel is a new delivery system for bioactive compounds with good biocompatibility and biodegradability. The formation of a gel-like network hinders the hydrolysis of proteins during digestion, thereby achieving sustained release of bioactive substances.

[0007] Based on this, this patent aims at improving the research frontier of stability and bioabsorption characteristics of active substances, focusing on improving the processing adaptability and absorption characteristics of active substances, and innovatively proposes to combine the good encapsulation ability of lactoferrin, the digestibility advantage of sodium alginate and the good stability characteristics of hydrogel to construct an active substance gel co-protected by sodium alginate and lactoferrin. Summary of the invention

[0008] The purpose of the present invention is to provide a preparation method and application of a lactoferrin-sodium alginate hydrogel system.

[0009] According to one aspect of the present invention, a method for preparing a lactoferrin-sodium alginate hydrogel system is provided, comprising the following steps:

[0010] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution, and the pH value of the lactoferrin solution was adjusted to 2.0 with HCl solution to obtain a lactoferrin subunit solution;

[0011] S2. The lactoferrin subunit solution is slowly added to the anhydrous ethanol solution of the active substance, and after ultrasonic treatment, the pH value is adjusted to 7.5 with a NaOH solution to obtain a lactoferrin complex solution encapsulating the active substance, ie, a lactoferrin-active substance co-assembly;

[0012] S3. The lactoferrin complex solution of the active substance embedded in step S2 is placed in a dialysis bag and dialyzed three times with Tris-HCl buffer, with the dialysate replaced every 6.0h to remove free active substances;

[0013] S4. Disperse sodium alginate in deionized water, stir until completely dissolved to obtain a sodium alginate solution, and leave overnight;

[0014] S5. The lactoferrin complex solution containing the active substance is mixed with the sodium alginate solution, and the mixture is stirred magnetically to obtain a lactoferrin-sodium alginate mixed solution containing the active substance. The lactoferrin-sodium alginate mixed solution containing the active substance is sucked with a sterile syringe and added dropwise to the CaCl2 solution. After solidification, gel beads are obtained. The gel beads are washed with ultrapure water, and the excess CaCl2 is removed. 2+ The lactoferrin-sodium alginate hydrogel loaded with active substances was obtained and stored at 4°C.

[0015] In some embodiments, the molar concentration of the lactoferrin solution in step S1 is 0.1 μM, and the molar concentration of the HCl solution is 1M.

[0016] In some embodiments, the molar concentration of the anhydrous ethanol solution of the active substance in step S2 is 0.5 mM, and the molar ratio of lactoferrin to the active substance in the lactoferrin-active substance co-assembly is 1:200-1:500.

[0017] In some embodiments, the active substance is any one of astaxanthin, curcumin, quercetin or resveratrol.

[0018] In some embodiments, the conditions for ultrasonic treatment in step S2 are: 35° C., 100 W for 30-60 min.

[0019] In some embodiments, the molar concentration of the Tris-HCl buffer is 50.0 mmol / L, the pH value thereof is 8.0, and the molecular weight retained by the dialysis bag in step S3 is 100 kDa.

[0020] In some embodiments, the concentration of the sodium alginate solution in step S4 is 0.1-1% (w / v).

[0021] In some embodiments, in step S5, the lactoferrin complex solution embedded with the active substance is mixed with the sodium alginate solution at a volume ratio of 8:2-7:3.

[0022] In some embodiments, the concentration of the CaCl2 solution in step S5 is 0.5-1% (w / v), and the solidification time in step S5 is 30-60 min.

[0023] According to another aspect of the present invention, there is provided a method for preparing a lactoferrin-sodium alginate hydrogel system for use in improving the loading amount of active substances and processing adaptability, wherein the active substance is any one of astaxanthin, curcumin, quercetin or resveratrol.

[0024] The beneficial effects of the present invention are as follows: (1) The method of the present invention utilizes the cage structure and self-assembly characteristics of lactoferrin to achieve the embedding and protection of active substances, and at the same time utilizes the good gel properties, thermal stability and gastric digestion resistance of sodium alginate to modify and protect the lactoferrin encapsulating the active substances, and realizes efficient loading and stabilization processing of the active substances through the co-stabilized hydrogel of the two;

[0025] (2) Lactoferrin and sodium alginate are both edible food raw materials. Lactoferrin has physiological functions such as promoting iron absorption, regulating immunity, antibacterial, antiviral, blocking oxygen free radicals and promoting the growth of bifidobacteria; while sodium alginate is mostly used as a food thickener and stability enhancer, and also has the effects of anticoagulation, lowering blood lipids and reducing blood viscosity. The present invention combines the structures and physicochemical properties of the two, designs a simple and easy-to-obtain active substance stabilization processing system, combines the functional advantages of proteins and polysaccharides, and provides dual protection for active substances, which not only effectively improves the physicochemical stability of active substances during processing, but also has good resistance to extreme environments and enzymolysis during gastrointestinal digestion, and has good universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a comparison chart of the astaxanthin embedding rate and loading rate of Example 4 of the present invention and Comparative Example 4.

[0027] Figure 2 It is a comparison chart of the astaxanthin embedding rate and loading rate of Example 5 of the present invention and Control Example 5.

[0028] Figure 3 This is a comparison diagram of the difference in bioaccessibility of astaxanthin between Example 6 of the present invention and Control Example 6.

[0029] Figure 4 This is a comparison chart of the differences in bioaccessibility of the active substances in Examples 7-9 of the present invention and Control Examples 7-9. DETAILED DESCRIPTION

[0030] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0031] Example 1

[0032] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0033] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0034] S2. The lactoferrin subunit solution was slowly added to a 0.5 mM resveratrol anhydrous ethanol solution at a molar ratio of 1:200, and after ultrasonic treatment at 35°C and 100W for 60 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a resveratrol-encapsulated lactoferrin complex solution, i.e., a lactoferrin-resveratrol co-assembly;

[0035] S3. The resveratrol-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialysate being replaced every 6.0 h to remove free resveratrol;

[0036] S4. Disperse sodium alginate in deionized water, stir until completely dissolved, prepare a 0.1% (w / v) sodium alginate solution, and leave overnight;

[0037] S5. The lactoferrin complex solution embedded with resveratrol was mixed with the sodium alginate solution in a volume ratio of 8:2, and magnetically stirred at 37°C for 2 hours to obtain a lactoferrin-sodium alginate mixed solution encapsulating resveratrol. The lactoferrin-sodium alginate mixed solution encapsulating resveratrol was drawn with a sterile syringe and added dropwise to a 0.5% (w / v) CaCl2 solution. After curing for 30 minutes, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The resveratrol-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0038] Example 2

[0039] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0040] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0041] S2. The lactoferrin subunit solution was slowly added to a 0.5 mM quercetin anhydrous ethanol solution at a molar ratio of 1:500, and after ultrasonic treatment at 35°C, 100 W for 60 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a quercetin-encapsulated lactoferrin complex solution, i.e., a lactoferrin-quercetin co-assembly;

[0042] S3. The quercetin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, and the dialysate was replaced every 6.0 h to remove free quercetin;

[0043] S4. Disperse sodium alginate in deionized water, stir until completely dissolved, prepare a 1% (w / v) sodium alginate solution, and leave overnight;

[0044] S5. The quercetin-encapsulated lactoferrin complex solution and the sodium alginate solution were mixed in a volume ratio of 7:3, and magnetically stirred at 37°C for 2 h to obtain a quercetin-encapsulated lactoferrin-sodium alginate mixed solution. The quercetin-encapsulated lactoferrin-sodium alginate mixed solution was drawn with a sterile syringe and added dropwise to a 1% (w / v) CaCl2 solution. After curing for 60 min, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The quercetin-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0045] Example 3

[0046] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0047] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0048] S2. The lactoferrin subunit solution was slowly added to a 0.5 mM curcumin anhydrous ethanol solution at a molar ratio of 1:350, and after ultrasonic treatment at 35°C, 100 W for 45 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a curcumin-embedded lactoferrin complex solution, i.e., a lactoferrin-curcumin co-assembly;

[0049] S3. The curcumin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialysate replaced every 6.0 h to remove free curcumin;

[0050] S4. Disperse sodium alginate in deionized water, stir until completely dissolved, prepare a 0.55% (w / v) sodium alginate solution, and leave overnight;

[0051] S5. The curcumin-encapsulated lactoferrin complex solution and the sodium alginate solution were mixed in a volume ratio of 8:2, and magnetically stirred at 37°C for 2h to obtain a curcumin-encapsulated lactoferrin-sodium alginate mixed solution. The curcumin-encapsulated lactoferrin-sodium alginate mixed solution was drawn with a sterile syringe and added dropwise to a 0.75% (w / v) CaCl2 solution. After curing for 60min, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The curcumin-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0052] Example 4

[0053] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0054] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0055] S2. The lactoferrin subunit solution was slowly added to an anhydrous ethanol solution of astaxanthin with a molar concentration of 0.5 mM at a molar ratio of 1:200-1:500, and ultrasonically treated at 35°C and 100W for 30-60 min, and then the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a lactoferrin complex solution encapsulating astaxanthin, i.e., a lactoferrin-astaxanthin co-assembly;

[0056] S3. The astaxanthin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialyzate being replaced every 6.0 h to remove free astaxanthin;

[0057] Comparative Example 4

[0058] Control Example 4 was carried out in accordance with the method of Example 4, except that: in step S2, the pH value of the NaOH solution with a molar concentration of 1 M was adjusted to 5.5, 6.0, 6.5, 7.5, and 8.0, respectively.

[0059] Example 5

[0060] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0061] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0062] S2. The lactoferrin subunit solution was slowly added to an anhydrous ethanol solution of astaxanthin with a molar concentration of 0.5 mM at a molar ratio of 1:300, and after ultrasonic treatment at 35°C and 100W for 30 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a lactoferrin complex solution encapsulating astaxanthin, i.e., a lactoferrin-astaxanthin co-assembly;

[0063] S3. The astaxanthin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialyzate being replaced every 6.0 h to remove free astaxanthin;

[0064] Comparative Example 5

[0065] Control Example 5 was carried out according to the method of Example 5, except that: in step S2, the lactoferrin subunit solution and the 0.5 mM astaxanthin anhydrous ethanol solution were mixed at a molar ratio of lactoferrin to astaxanthin of 1:200, 1:400, 1:500 or 1:600, respectively.

[0066] Example 6

[0067] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0068] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0069] S2. The lactoferrin subunit solution was slowly added to an anhydrous ethanol solution of astaxanthin with a molar concentration of 0.5 mM at a molar ratio of 1:300, and after ultrasonic treatment at 35°C and 100W for 30 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a lactoferrin complex solution encapsulating astaxanthin, i.e., a lactoferrin-astaxanthin co-assembly;

[0070] S3. The astaxanthin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialyzate being replaced every 6.0 h to remove free astaxanthin;

[0071] S4. Sodium alginate was dispersed in deionized water and magnetically stirred at 37°C until completely dissolved to obtain a 0.2% (w / v) sodium alginate solution, which was allowed to stand overnight;

[0072] S5. The lactoferrin complex solution embedded with astaxanthin was mixed with the sodium alginate solution in a volume ratio of 7:3, and magnetically stirred at 37°C for 2h to obtain a lactoferrin-sodium alginate mixed solution loaded with astaxanthin. The lactoferrin-sodium alginate mixed solution loaded with astaxanthin was drawn with a sterile syringe and added dropwise to a 0.5% (w / v) CaCl2 solution. After curing for 60min, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The astaxanthin-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0073] Comparative Example 6

[0074] Control Example 6 was carried out in accordance with the method of Example 6, except that in step S5, the lactoferrin complex solution embedded with astaxanthin and the sodium alginate solution were mixed in a volume ratio of 5:5, 6:4, 8:2 or 9:1.

[0075] Example 7

[0076] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0077] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0078] S2. The lactoferrin subunit solution was slowly added to an anhydrous ethanol solution of astaxanthin with a molar concentration of 0.5 mM at a molar ratio of 1:300, and after ultrasonic treatment at 35°C and 100W for 30 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a lactoferrin complex solution encapsulating astaxanthin, i.e., a lactoferrin-astaxanthin co-assembly;

[0079] S3. The astaxanthin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialyzate being replaced every 6.0 h to remove free astaxanthin;

[0080] S4. Sodium alginate was dispersed in deionized water and magnetically stirred at 37°C until completely dissolved to obtain a 0.2% (w / v) sodium alginate solution, which was allowed to stand overnight;

[0081] S5. The lactoferrin complex solution embedded with astaxanthin was mixed with the sodium alginate solution in a volume ratio of 8:2, and magnetically stirred at 37°C for 2h to obtain a lactoferrin-sodium alginate mixed solution loaded with astaxanthin. The lactoferrin-sodium alginate mixed solution loaded with astaxanthin was drawn with a sterile syringe and added dropwise to a 0.5% (w / v) CaCl2 solution. After curing for 30min, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The astaxanthin-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0082] Comparative Example 7

[0083] Control Example 7: The astaxanthin-lactoferrin embedding material was prepared by using steps S1-S3 in Example 7, with free astaxanthin as a blank control.

[0084] Example 8

[0085] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0086] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0087] S2. The lactoferrin subunit solution was slowly added to a 0.5 mM curcumin anhydrous ethanol solution at a molar ratio of 1:400, and ultrasonically treated at 35°C, 100 W for 30 min, and then the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a curcumin-embedded lactoferrin complex solution, i.e., a lactoferrin-curcumin co-assembly;

[0088] S3. The curcumin-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialysate replaced every 6.0 h to remove free curcumin;

[0089] S4. Sodium alginate was dispersed in deionized water and magnetically stirred at 37°C until completely dissolved to obtain a 0.2% (w / v) sodium alginate solution, which was allowed to stand overnight;

[0090] S5. The curcumin-encapsulated lactoferrin complex solution and the sodium alginate solution were mixed in a volume ratio of 8:2, and magnetically stirred at 37°C for 2h to obtain a curcumin-encapsulated lactoferrin-sodium alginate mixed solution. The curcumin-encapsulated lactoferrin-sodium alginate mixed solution was drawn with a sterile syringe and added dropwise to a 0.5% (w / v) CaCl2 solution. After curing for 30min, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The curcumin-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0091] Comparative Example 8

[0092] Control Example 8 The curcumin-lactoferrin embedding material was prepared by using steps S1-S3 in Example 8, and free curcumin was used as a blank control.

[0093] Example 9

[0094] A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that it comprises the following steps:

[0095] S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution having a molar concentration of 0.1 μM, and the pH value of the lactoferrin solution was adjusted to 2.0 with 1 M HCl solution to obtain a lactoferrin subunit solution;

[0096] S2. The lactoferrin subunit solution was slowly added to a 0.5 mM resveratrol anhydrous ethanol solution at a molar ratio of 1:300, and after ultrasonic treatment at 35°C and 100W for 30 min, the pH value was adjusted to 7.5 with a 1 M NaOH solution to obtain a resveratrol-encapsulated lactoferrin complex solution, i.e., a lactoferrin-resveratrol co-assembly;

[0097] S3. The resveratrol-encapsulated lactoferrin complex solution obtained in step S2 was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyzed three times with a Tris-HCl buffer having a molar concentration of 50.0 mmol / L and a pH value of 8.0, with the dialysate being replaced every 6.0 h to remove free resveratrol;

[0098] S4. Sodium alginate was dispersed in deionized water and magnetically stirred at 37°C until completely dissolved to obtain a 0.2% (w / v) sodium alginate solution, which was allowed to stand overnight;

[0099] S5. The lactoferrin complex solution embedded with resveratrol was mixed with the sodium alginate solution in a volume ratio of 8:2, and magnetically stirred at 37°C for 2 hours to obtain a lactoferrin-sodium alginate mixed solution encapsulating resveratrol. The lactoferrin-sodium alginate mixed solution encapsulating resveratrol was drawn with a sterile syringe and added dropwise to a 0.5% (w / v) CaCl2 solution. After curing for 30 minutes, gel beads were obtained. The gel beads were washed with ultrapure water for 3 times, and the excess CaCl2 was removed. 2+ The resveratrol-loaded lactoferrin-sodium alginate hydrogel was removed and stored at 4°C.

[0100] Comparative Example 9

[0101] Control Example 9: Resveratrol-lactoferrin embedding material was prepared by using steps S1-S3 in Example 9, and free resveratrol was used as a blank control.

[0102] The following is a comparison of the properties of the products obtained in the above Examples 4-9 and Comparative Examples 4-9 by testing the embedding rate, loading rate, photostability, thermal stability, storage stability and bioaccessibility of the active substances.

[0103] 1. Experimental Methods

[0104] 1. Determination of embedding rate and loading rate

[0105] The prepared embedding system is centrifuged, the content of free active substances is determined, and the content of loaded active substances is deduced, thereby calculating the embedding rate and loading rate of the active substances in the embedding material.

[0106] Embedding rate (%) = content of active substance after loading / content of total added active substance (%) Loading rate (%) = content of active substance after loading / content of total embedded material (%).

[0107] 2. Photostability

[0108] The effect of light on the stability of free active substances, lactoferrin encapsulating active substances, and lactoferrin-sodium alginate hydrogel encapsulating active substances was determined under ultraviolet light and natural light. The samples were placed under 254nm ultraviolet light or natural light for 6 hours, and the retention rate of the active substances was recorded.

[0109] 3. Thermal stability

[0110] In order to evaluate the thermal stability of free active substances, lactoferrin encapsulated active substances, and active substances in lactoferrin-sodium alginate hydrogels encapsulated active substances, experiments were conducted at a temperature range of 30°C to 80°C to simulate the storage process (30°C), processing process (60°C), and pasteurization process (80°C). The samples were heated in a water bath at 30°C, 60°C, and 80°C for 2 h, and the retention rate of the active substances was calculated.

[0111] 4. Storage stability

[0112] In order to evaluate the storage stability of free active substances, lactoferrin encapsulating active substances, and active substances in lactoferrin-sodium alginate hydrogel encapsulating active substances, the samples were stored in 20 mL serum bottles and placed in incubators at 4°C and 25°C in the dark for 21 days, respectively, and the retention rate of the active substances was calculated.

[0113] 5. Bioaccessibility

[0114] The method for determining bioaccessibility refers to the method in the following literature: Chen L, Yokoyama W, Liang R, et al. Enzymatic degradation and bioaccessibility of protein encapsulated β-carotene nano-emulsions during in vitro gastro-intestinal digestion[J]. Food Hydrocolloids, 2020, 100: 105177.

[0115] 2. Experimental Results

[0116] 1. The effect of the co-assembly endpoint pH value of Example 4 and Comparative Example 4 on the embedding efficiency and loading efficiency of astaxanthin is shown in the following table. Figure 1 As shown. Figure 1It can be seen that with the increase of pH value, the embedding rate and loading rate of astaxanthin in the lactoferrin-astaxanthin co-assembly both showed a trend of first increasing and then decreasing. When the end pH value reached 7.0 and 7.5, the embedding rate reached the maximum value, which was 92.82% and 93.69% respectively, and the loading rate of the latter was greater, which was 9.57% higher than the former. Therefore, pH 7.5 is considered to be the most favorable pH for the co-assembly of lactoferrin and active substances.

[0117] 2. The effects of the molar ratio of lactoferrin to astaxanthin in Example 5 and Comparative Example 5 on the embedding efficiency and loading efficiency of astaxanthin are shown in the following table: Figure 2 As shown. Figure 2 It can be seen that as the molar ratio of lactoferrin to astaxanthin increases, the embedding rate of astaxanthin in the lactoferrin-astaxanthin co-assembly shows a downward trend, while the loading rate of astaxanthin shows a slowly increasing trend. However, the greater the molar ratio of lactoferrin to astaxanthin, the greater the loss of astaxanthin. Taking all factors into consideration, with less astaxanthin being used to obtain the maximum utilization rate as the standard, 1:300 is considered to be the most favorable molar ratio for the co-assembly of lactoferrin and active substances.

[0118] 3. The results of the effect of the volume ratio of lactoferrin to sodium alginate solution in Example 6 and Comparative Example 5 on the bioaccessibility of astaxanthin are as follows: Figure 3 As shown. Figure 3 It can be seen that with the increase of sodium alginate addition, the astaxanthin encapsulation rate shows a trend of increasing first and then decreasing, which is due to the good gastric digestion stability of sodium alginate. However, if the amount of sodium alginate added is too high, the drug loading is too low, and the overall bioaccessibility will also be affected. In general, when the volume ratio of lactoferrin to sodium alginate solution is 8:2, the bioaccessibility of astaxanthin is the highest, reaching 42.18%.

[0119] 4. The processing adaptability differences of the active substances in Examples 7-9 and Comparative Examples 7-9 are shown in Table 1.

[0120] Table 1 Comparison of the processing adaptability of active substances in different embedding systems

[0121]

[0122] As shown in Table 1, the light stability, thermal stability and storage stability of natural active substances (astaxanthin, curcumin and resveratrol) are not ideal. They are more stable under low temperature conditions. When the temperature is appropriately increased, the storage time is extended or they are exposed to ultraviolet light, the loss of active substances is more obvious. The stability of these active substances can be effectively improved by encapsulating them with lactoferrin cage structures. Taking the treatment condition with the lowest stability (90°C, 2h) as an example, the retention rates of astaxanthin, curcumin and resveratrol are 18.21%, 15.38% and 16.12% respectively; after encapsulating them with lactoferrin, the retention rates of the three active substances are increased to 45.41%, 21.11% and 22.28% respectively. Sodium alginate is an anionic polysaccharide with good thermal stability after forming a gel. It is used to interact with lactoferrin to prepare an active substance-encapsulated gel, and its stability is more than doubled compared to that of the free active substances (the retention rates of astaxanthin, curcumin and resveratrol are 57.03%, 33.92% and 32.56%, respectively).

[0123] 5. The results of the comparison of the bioaccessibility of the active substances in Examples 7-9 and Comparative Examples 7-9 are as follows: Figure 4 shown.

[0124] from Figure 4 The results show that the bioaccessibility of natural active substances (astaxanthin, curcumin, and resveratrol) after simulated in vitro digestion is at a low level, among which natural astaxanthin has the lowest bioaccessibility, only 8.27%. After encapsulation with lactoferrin, the gastrointestinal digestion stability is significantly improved, and the bioaccessibility can be increased to 26.67%. After further protection of the system with sodium alginate, the bioaccessibility is increased to 42.19%, which is 4.2 times higher than that of natural astaxanthin.

[0125] Similar to the changes, the bioaccessibility of curcumin and resveratrol also increased from 10.27% and 24% to 53.28% and 57.76%, respectively. This is due to the cage structure, self-assembly characteristics and acid stability of lactoferrin, which can effectively embed and protect the active substances and avoid the extreme gastrointestinal environment and enzymatic hydrolysis; at the same time, sodium alginate has good gel properties, thermal stability and gastric digestion resistance, and the modification and protection of lactoferrin that encapsulates the active substances can further improve the bioabsorption characteristics of the active substances.

[0126] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for preparing a lactoferrin-sodium alginate hydrogel system, characterized in that: The following steps are involved: S1. The lactoferrin was separated in deionized water to obtain a lactoferrin solution, and the pH value of the lactoferrin solution was adjusted to 2.0 with HCl solution to obtain a lactoferrin subunit solution; S2. The lactoferrin subunit solution is slowly added to the anhydrous ethanol solution of the active substance, and after ultrasonic treatment, the pH value is adjusted to 7.5 with a NaOH solution to obtain a lactoferrin complex solution encapsulating the active substance, ie, a lactoferrin-active substance co-assembly; S3. The lactoferrin complex solution of the active substance embedded in step S2 is placed in a dialysis bag and dialyzed three times with Tris-HCl buffer, with the dialysate replaced every 6.0h to remove free active substances; S4. Disperse sodium alginate in deionized water, stir until completely dissolved to obtain a sodium alginate solution, and leave overnight; S5. The lactoferrin complex solution embedded with the active substance is mixed with the sodium alginate solution, and magnetic stirring is performed to obtain a lactoferrin-sodium alginate mixed solution encapsulating the active substance, and the lactoferrin-sodium alginate mixed solution encapsulating the active substance is sucked with a sterile syringe, and added dropwise to the CaCl2 solution, and gel beads are obtained after solidification, and the gel beads are washed with ultrapure water, and the excess CaCl2 is removed. 2+ The lactoferrin-sodium alginate hydrogel loaded with active substances was obtained and stored at 4°C.

2. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The molar concentration of the lactoferrin solution in step S1 is 0.1 μM, and the molar concentration of the HCl solution is 1M.

3. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The molar concentration of the anhydrous ethanol solution of the active substance in step S2 is 0.5 mM, and the molar ratio of lactoferrin to the active substance in the lactoferrin-active substance co-assembly is 1:200-1:

500.

4. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The active substance is any one of astaxanthin, curcumin, quercetin or resveratrol.

5. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The conditions of the ultrasonic treatment in step S2 are: 35° C., 100 W for 30-60 min.

6. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The molar concentration of the Tris-HCl buffer is 50.0 mmol / L, and the pH value thereof is 8.

0. The molecular weight retained by the dialysis bag in step S3 is 100 kDa.

7. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The concentration of the sodium alginate solution in step S4 is 0.1-1% (w / v).

8. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: In the step S5, the lactoferrin complex solution embedded with the active substance is mixed with the sodium alginate solution at a volume ratio of 8:2-7:

3.

9. The method for preparing a lactoferrin-sodium alginate hydrogel system according to claim 1, characterized in that: The concentration of the CaCl2 solution in step S5 is 0.5-1% (w / v), and the curing time in step S5 is 30-60 minutes.

10. Use of the preparation method of a lactoferrin-sodium alginate hydrogel system according to any one of claims 1 to 9 in improving the loading amount of active substances and processing adaptability, wherein the active substance is any one of astaxanthin, curcumin, quercetin or resveratrol.

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