Protein-polysaccharide-polyphenol ternary covalent complex, preparation thereof and functional fluid infusion containing protein-polysaccharide-polyphenol ternary covalent complex

Abstract

The invention relates to the technical field of nutritional supplements. The invention relates to a protein-polysaccharide-polyphenol ternary covalent complex, in particular to a protein-polysaccharide-polyphenol ternary covalent complex, preparation thereof and functional fluid infusion containing the protein-polysaccharide-polyphenol ternary covalent complex. The protein-polysaccharide-polyphenol ternary covalent complex stably exists in an acid environment with the pH smaller than or equal to 4, and the preparation method comprises the following steps that vitamin D3 and an oil carrying agent are fully mixed to be uniform and then added into a protein aqueous solution, homogenizing and emulsifying are conducted, and a vitamin D3 emulsion is obtained; adding a polysaccharide aqueous solution into the vitamin D3 emulsion, adjusting the pH value of the system to 8-10, and reacting at 85-95 DEG C for 2-3 hours; and cooling, continuously adding a polyphenol solution, adjusting the pH value to 9-10, reacting at 50-60 DEG C for 1-2 hours, and cooling to obtain the protein-polysaccharide-polyphenol ternary covalent complex. When the covalent complex is used for preparing functional rehydration rich in mineral substances and vitamin D3, the stability of the vitamin D3 in an acid environment and at a high temperature can be obviously improved.

Description

Technical Field

[0001] This invention relates to the field of nutritional supplement technology. More specifically, it relates to a protein-polysaccharide-polyphenol ternary covalent complex, its preparation, and a functional rehydration solution containing the same. Background Technology

[0002] Vitamin D3, also known as cholecalciferol, is called the "sunshine vitamin." It is an essential fat-soluble vitamin that plays a crucial role in maintaining the health of multiple systems throughout the body. It can significantly improve the absorption of calcium and phosphorus in the intestines, aid in bone mineralization, effectively prevent rickets in children and osteomalacia in adults, maintain bone density, reduce the risk of osteoporosis and fractures in middle-aged and elderly people, and accelerate the repair process after fractures.

[0003] Minerals are inorganic nutrients that maintain normal physiological functions in the human body. They are divided into two main categories: macrominerals (daily requirement greater than 100 mg) and microminerals (daily requirement less than 100 mg). Although they cannot provide energy for the human body, they participate in key processes such as growth and development, metabolic regulation, and organ function, and are an important cornerstone for ensuring good health.

[0004] Commercially available vitamin D3 and mineral supplements are mostly in solid dosage forms such as tablets and capsules. These products can reduce the direct reaction between vitamin D3 and minerals to some extent. However, solid dosage forms have problems such as slow onset of action, low bioavailability, and difficulty in swallowing, making them particularly unsuitable for infants, the elderly, and people with swallowing difficulties. Furthermore, vitamin D3 is extremely unstable in acidic, oxygen-rich, and mineral-rich liquid beverage systems, easily undergoing isomerization and oxidative degradation, leading to decreased product efficacy and flavor deterioration.

[0005] Existing technologies mainly employ the following methods for protection: ① Adding antioxidants: such as adding vitamin E and butylated hydroxytoluene. This method is prone to failure under high ionic strength, and the amount added is subject to regulatory limits; ② Using encapsulation technology: such as microcapsules and liposomes. This method is prone to damage to the inclusion structure in acidic beverage systems, and the inclusion structure is prone to rupture during high-temperature sterilization, leading to rapid release and degradation of vitamins.

[0006] Therefore, how to construct a structurally stable protection system that combines physical shielding and active antioxidant functions and is suitable for industrial production to significantly improve the stability of vitamin D3 in mineral drinks remains a technical problem that urgently needs to be solved. Summary of the Invention

[0007] Based on the above problems, the first objective of this invention is to provide a method for preparing a protein-polysaccharide-polyphenol ternary covalent complex. The raw materials (protein, polysaccharide, and polyphenol) used in this preparation method are all natural food-grade raw materials, without any chemically synthesized substances, meeting the modern food industry's requirements for green, healthy, and safe products. Furthermore, the composite network formed by the covalent cross-linking of protein, polysaccharide, and polyphenol effectively stabilizes vitamin D3 in acidic environments and minerals.

[0008] A second objective of this invention is to provide a protein-polysaccharide-polyphenol ternary covalent complex prepared by the preparation method described above.

[0009] A third objective of this invention is to provide a functional rehydration solution rich in minerals and vitamin D3.

[0010] The fourth objective of this invention is to provide a method for preparing the functional rehydration solution rich in minerals and vitamin D3 as described above.

[0011] To achieve the first objective mentioned above, the present invention adopts the following technical solution: This invention discloses a method for preparing a protein-polysaccharide-polyphenol ternary covalent complex, wherein the protein-polysaccharide-polyphenol ternary covalent complex is stably present in an acidic environment with a pH less than or equal to 4, and its preparation includes the following steps: After thoroughly mixing vitamin D3 with the oil carrier, it is added to the protein aqueous solution and homogenized and emulsified to obtain vitamin D3 emulsion. Add polysaccharide aqueous solution to vitamin D3 emulsion, adjust the pH of the system to 8-10, react at 85-95℃ for 2-3 hours, and stir at 4000-6000 rpm / min to allow polysaccharide to covalently graft with proteins in the aqueous phase and at the interface to form a stable cross-linked structure. Cool down, continue adding polyphenol solution, adjust pH to 9-10, react at 50-60℃ for 1-2 hours, cool down, and obtain protein-polysaccharide-polyphenol ternary covalent complex.

[0012] In this invention, a protein-polysaccharide-polyphenol ternary covalent complex system is constructed. First, the Maillard reaction or amidation reaction of the protein and polysaccharide forms a covalent bond, constructing a stable cross-linked network. Then, polyphenols are introduced, and the phenolic hydroxyl groups of the polyphenols interact covalently or strongly non-covalently with the amino / thiol groups of the protein or the hydroxyl groups of the polysaccharide to achieve molecular self-assembly and further form a dense and stable three-dimensional network structure. Vitamin D3 is firmly embedded in this network structure, improving the stability of vitamin D3 in acidic environments and minerals. At the same time, the strong chemical antioxidant properties of polyphenols are utilized to achieve synergistic physical isolation and chemical scavenging, blocking the degradation of vitamins through multiple pathways.

[0013] Proteins undergo Maillard or amidation reactions with polysaccharides before cross-linking with polyphenols. This reaction process allows the product to have higher α-helices, β-sheets, and random coils, forming a more compact and stable complex structure. However, if the protein is first covalently bound to polyphenols and then grafted onto polysaccharides, the protein's native tertiary structure and conformation are more likely to unfold or denature, resulting in a reduction of α-helices and an increase in disordered structure.

[0014] Furthermore, the protein in the protein aqueous solution is selected from one or more of whey protein isolate, pumpkin seed protein, bone gelatin, sodium caseinate, and soy protein isolate.

[0015] Furthermore, the polysaccharide in the polysaccharide aqueous solution is selected from one or more of the following: locust bean gum, sodium alginate, pullulan, gellan gum, hyaluronic acid with a molecular weight of 50-100 kDa, and octenyl succinic acid starch (OSA starch).

[0016] Furthermore, the polyphenols in the polyphenol solution are selected from one or more of citrus polyphenols, grape seed extract, rosmarinic acid, and tea polyphenols.

[0017] Furthermore, the protein is selected from whey protein isolate and / or pumpkin seed protein, the polysaccharide is selected from one or more of locust bean gum, sodium alginate, and pullulan, and the polyphenol is selected from citrus polyphenols.

[0018] Furthermore, the pH values ​​of both the vitamin D3 emulsion and the polysaccharide aqueous solution are greater than 7.

[0019] Furthermore, sodium citrate was used to adjust the pH of the system.

[0020] Furthermore, the protein to polysaccharide mass ratio is 1-2.5:1; exemplaryly, the protein to polysaccharide mass ratio can be 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, etc.

[0021] Furthermore, the mass ratio of protein to polyphenol is 5-15:1; for example, the mass ratio of protein to polyphenol can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, etc.

[0022] Furthermore, the mass ratio of protein to vitamin D3 is 20-50:1; for example, the mass ratio of protein to vitamin D3 can be 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, etc.

[0023] Furthermore, the oil carrier is selected from one or more of medium-chain triglycerides, sunflower seed oil, and corn oil.

[0024] Furthermore, the mass ratio of vitamin D3 to oil carrier is 1:10-50; the mass ratio of vitamin D3 to oil carrier can be 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.

[0025] Furthermore, the rotation speed of the homogenization emulsification is 7000-10000 rpm / min, and the homogenization emulsification time is 5-10 min.

[0026] To achieve the second objective mentioned above, the present invention adopts the following technical solution: This invention discloses a protein-polysaccharide-polyphenol ternary covalent complex prepared by the preparation method described above.

[0027] To achieve the third objective mentioned above, the present invention adopts the following technical solution: This invention discloses a functional rehydration solution rich in minerals and vitamin D3, comprising the protein-polysaccharide-polyphenol ternary covalent complex as described above, and comprising the following raw material components by mass percentage: Xanthan gum 0.1-0.5 wt%; Sugars and / or sugar alcohols 10-30 wt%; Acidity regulator 0.5-1.5 wt%; Flavoring agent 0.05-1wt%; The protein-polysaccharide-polyphenol ternary covalent complex as described above is 5-65 wt%; Mineral supplements 0.2-40 wt%; The rest is water; The pH value of the functional replenishment solution is 3.5-4.0.

[0028] Furthermore, the mineral supplement includes one or more of the following: calcium supplement, zinc supplement, selenium supplement, and iron supplement.

[0029] In one specific embodiment, the calcium supplement is selected from at least one of food-grade calcium carbonate, calcium gluconate, calcium citrate, L-calcium lactate, calcium hydrogen phosphate, calcium chloride, tricalcium phosphate, glycerophosphate, calcium oxide, and calcium sulfate.

[0030] In one embodiment, the zinc supplement is selected from at least one of food-grade zinc gluconate, zinc oxide, zinc lactate, zinc citrate, zinc chloride, and zinc acetate.

[0031] In one specific embodiment, the selenium supplement is selected from at least one of sodium selenate and sodium selenite.

[0032] In one specific embodiment, the iron supplement is selected from at least one of ferrous sulfate, ferrous gluconate, ferric ammonium citrate, ferrous fumarate, ferric citrate, ferric pyrophosphate, and sodium ferric ethylenediaminetetraacetate.

[0033] Furthermore, the sugar is selected from one or more of the following: granulated sugar, edible glucose, crystalline fructose, maltose, fructooligosaccharides, xylooligosaccharides, maltodextrin, and polydextrose.

[0034] Furthermore, the sugar alcohol is selected from one or more of xylitol, erythritol, maltitol, sorbitol, and isomaltitol.

[0035] Furthermore, the acidity regulator is selected from one or more of citric acid, malic acid, lactic acid, sodium citrate, and sodium malate.

[0036] Furthermore, the flavoring agent is one or more of concentrated fruit juice (concentrated apple juice, concentrated orange juice, concentrated blueberry juice, concentrated strawberry juice, etc.) and food flavorings (apple flavoring, orange flavoring, blueberry flavoring, strawberry flavoring).

[0037] To achieve the fourth objective mentioned above, the present invention adopts the following technical solution: This invention discloses a method for preparing the functional rehydration solution as described above, comprising the following steps: Add sugar and / or sugar alcohol, mineral supplements, xanthan gum, and flavoring agents to the protein-polysaccharide-polyphenol ternary covalent complex according to the formula, mix, add an acidity regulator to adjust the pH to 3.5-4.0, shear at 7000-10000 rpm / min for 5-10 min, homogenize and filter to obtain the final product.

[0038] Furthermore, the homogenization filtration is performed using a 5μm titanium rod in conjunction with a 0.22μm pleated membrane.

[0039] The beneficial effects of this invention are as follows: 1. Significantly improves the stability of vitamin D3: ① By cross-linking proteins and polysaccharides through Maillard / amidation reactions, a stable and dense network structure is constructed. Then, by utilizing the covalent or strong non-covalent interactions between the phenolic hydroxyl groups of polyphenols and the amino / thiol groups of proteins or the hydroxyl groups of polysaccharides, a dense three-dimensional network structure is further formed through molecular assembly, thereby embedding vitamin D3 into the network structure. Vitamin D3 is effectively physically encapsulated, reducing its degradation in acidic, mineral-rich environments.

[0040] ② By utilizing the powerful antioxidant properties of polyphenols, a synergistic effect of physical isolation and chemical removal is achieved, blocking the degradation of vitamins through multiple pathways.

[0041] 2. Green, natural, and safe: The raw materials used in this invention (protein, polysaccharide, polyphenol) are all natural food-grade raw materials, without any chemical synthesis, which meets the requirements of the modern food industry for green, healthy and safe products. Attached Figure Description

[0042] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0043] Figure 1 The images show the measured values ​​of the functional replenishment solutions prepared in different embodiments and comparative examples in stability test 2. Detailed Implementation

[0044] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 20g of whey protein isolate). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0046] 2. Add 500g of polysaccharide aqueous solution (pH > 7.0, containing 10g of locust bean gum) to the above vitamin D3 emulsion. Adjust the pH of the solution to 8.0 with sodium citrate. Heat the solution to 90℃ and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo Maillard reaction and complete the grafting. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 20g of polyphenol aqueous solution (containing 2g of citrus polyphenols), adjust the pH to 9.0, stir at 55℃ for 1-2h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a whey protein isolate-locus bean gum-citrus polyphenol ternary covalent complex solution.

[0047] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0048] Example 2 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of whey protein isolate). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0049] 2. Add 500g of polysaccharide aqueous solution (pH > 7.0, containing 6g sodium alginate) to the above vitamin D3 emulsion. Adjust the pH of the solution to 8.0 with sodium citrate. Heat the solution to 90℃ and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo Maillard reaction and complete the grafting. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 20g of polyphenol aqueous solution (containing 1.2g citrus polyphenols), adjust the pH to 9.0, stir at 55℃ for 1-2h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a whey protein isolate-sodium alginate-citrus polyphenol ternary covalent complex.

[0050] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0051] Example 3 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of whey protein isolate). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0052] 2. Add 500g of polysaccharide aqueous solution (pH > 7.0, containing 6g pullulan) to the above vitamin D3 emulsion. Adjust the pH of the solution to 8.0 with sodium citrate. Heat the solution to 90℃ and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo Maillard reaction and complete the grafting. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 20g of polyphenol aqueous solution (containing 1.2g of citrus polyphenols), adjust the pH to 9.0, stir at 55℃ for 1-2h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a whey protein isolate-pullulan-citrus polyphenol ternary covalent complex.

[0053] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0054] Example 4 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 20g of pumpkin seed protein). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0055] 2. Add 500g of polysaccharide aqueous solution (pH > 7.0, containing 10g of locust bean gum) to the above vitamin D3 emulsion. Adjust the pH of the solution to 8.0 with sodium citrate. Heat the solution to 90℃ and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo the Maillard reaction and complete the grafting. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 20g of polyphenol aqueous solution (containing 2g of citrus polyphenols), adjust the pH to 9.0, stir at 55℃ for 1-2h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a pumpkin seed protein-locust bean gum-citrus polyphenol ternary covalent complex.

[0056] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0057] Example 5 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of pumpkin seed protein). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0058] 2. Add 500g of polysaccharide aqueous solution (pH > 7.0, containing 6g sodium alginate) to the above vitamin D3 emulsion. Adjust the pH of the solution to 8.0 with sodium citrate. Heat the solution to 90℃ and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo the Maillard reaction and complete the grafting. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 20g of polyphenol aqueous solution (containing 1.2g citrus polyphenols), adjust the pH to 9.0, stir at 55℃ for 1-2h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a pumpkin seed protein-sodium alginate-citrus polyphenol ternary covalent complex.

[0059] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0060] Example 6 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of pumpkin seed protein). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0061] 2. Add 500g of polysaccharide aqueous solution (pH > 7.0, containing 6g pullulan) to the above vitamin D3 emulsion. Adjust the pH of the solution to 8.0 with sodium citrate. Heat the solution to 90℃ and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo Maillard reaction and complete the grafting. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 20g of polyphenol aqueous solution (containing 1.2g of citrus polyphenols), adjust the pH to 9.0, stir at 55℃ for 1-2h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a pumpkin seed protein-pullulan-citrus polyphenol ternary covalent complex.

[0062] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0063] Comparative Example 1 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of whey protein isolate). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0064] 2. Add 20g of polyphenol aqueous solution (pH > 7.0, containing 1.2g of citrus polyphenols) to the above vitamin D3 emulsion. Adjust the pH of the solution to 9.0 with sodium citrate. Heat the solution to 55℃ and react at 6000rpm / min for 1-2h to allow the protein and polyphenols to crosslink. Then, rapidly lower the solution temperature to 30℃ to terminate the reaction. Subsequently, add 500g of polysaccharide aqueous solution (containing 6g of pullulan), adjust the pH to 8.0, stir at 90℃ for 2-3h, and then rapidly lower the solution temperature to 30℃ to terminate the reaction, obtaining a whey protein isolate-citrus polyphenol-pullulan ternary covalent complex.

[0065] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0066] Comparative Example 2 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of whey protein isolate). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0067] 2. Mix 500g of polysaccharide aqueous solution (containing 6g pullulan polysaccharide) and 20g of polyphenol aqueous solution (containing 1.2g of citrus polyphenols), adjust the pH of the solution to 9.0 with sodium citrate, heat the solution to 55℃, and react at 6000rpm / min for 1-2h to allow the polysaccharide and polyphenols to crosslink.

[0068] 3. Add the vitamin D3 emulsion prepared in step one to the above solution, adjust the pH of the entire mixture to 8.0 with sodium citrate, then immediately heat the solution to 90°C and react at 6000 rpm / min for 2-3 hours. After that, quickly lower the solution temperature to 30°C to terminate the reaction, and obtain pullulan polysaccharide-citrus polyphenol-whey protein isolate ternary covalent complex.

[0069] 4. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0070] Comparative Example 3 This example provides a method for preparing a functional rehydration solution rich in minerals and vitamin D3, including the following steps: 1. Mix 0.4g of vitamin D3 and 12g of medium-chain triglycerides thoroughly and add them to 100g of protein aqueous solution (containing 12g of whey protein isolate). Homogenize and emulsify at 10000rpm / min for 5-10min to obtain vitamin D3 emulsion, and adjust the pH of the emulsion to >7.0.

[0071] 2. Add 500g of polysaccharide aqueous solution (pH>7.0, containing 6g pullulan polysaccharide) to the above vitamin D3 emulsion, adjust the pH of the solution to 8.0 with sodium citrate, heat the solution to 90℃, and react at 6000rpm / min for 2-3h to allow the protein and polysaccharide to undergo Maillard reaction to complete the grafting, and then quickly lower the solution temperature to 30℃ to terminate the reaction.

[0072] 3. Add 123g calcium citrate, 2.7g ferrous gluconate, 200g glucose, 2g xanthan gum, 3g sweetener and 2g flavoring to the above solution and mix. Adjust the pH to 3.5-4.0 with 10g acidity adjuster (citric acid and / or malic acid). Add purified water to make up to 1kg of total system weight. Then shear at 7000-10000rpm / min for 5-10min. Homogenize the solution using a 5μm titanium rod and a 0.22μm pleated membrane to obtain a mineral-rich vitamin D3 solution.

[0073] Performance evaluation methods Stability test Determination of Vitamin D3 content: The determination method in GB 5009.296-2023 shall be followed.

[0074] Stability Test 1 The samples of each embodiment and comparative example were placed in a sterilizer and sterilized for 30 min at high temperatures of 85±2℃, 95±2℃, 105±2℃ and 115±2℃, respectively, to investigate the detection rate of vitamin D3.

[0075] Stability Test 2 According to the accelerated stability test conditions, the samples of each embodiment and comparative example were placed in an environment with a temperature of 40±2℃ and a relative humidity of 75%±5% for 3 months to examine their stability.

[0076] The results of stability test 1 are shown in Table 1. All embodiments of the present invention exhibit a certain protective effect against vitamin D3, especially Example 3, which demonstrates excellent heat resistance. Under high-temperature sterilization conditions (95℃, 105℃, and 115℃), the detection rate of vitamin D3 in Example 3 remained the highest, and the decrease with increasing temperature was the smallest. This proves that the "whey protein isolate-pullulan-citrus polyphenol" ternary covalent network constructed therefrom has the optimal thermal barrier effect and can effectively pass through relatively strict sterilization processes. Examples 2 and 6 also showed good thermal stability, with significantly higher vitamin D3 detection rates than the comparative examples at the test temperatures, indicating that they also have strong thermal stability and can meet the requirements of heat processing conditions. However, the detection rate of vitamin D3 in Example 4 decreased significantly at 115℃, indicating that its covalent network structure lacked integrity under extreme thermal stress, and therefore it is not recommended for such high-temperature sterilization scenarios.

[0077] Table 1

[0078] The results of stability test 2 are shown in Tables 2 and 3. Figure 1 Accelerated stability test data further confirmed the above conclusions. Throughout the accelerated testing period, Example 3 consistently maintained the highest detection rate, proving that its constructed protection system not only remained stable at high temperatures but also effectively delayed chemical degradation during long-term storage, demonstrating a significant shelf-life advantage. Examples 2 and 5 also exhibited significantly better stability than traditional protection methods, with lower degradation rates. Conversely, traditional protection methods showed poor protective effects. Comparative Examples 1 and 2 showed a significant decrease in vitamin D3 detection rates after heat sterilization and accelerated storage, with degradation rates far exceeding those of the embodiments of this invention. While Comparative Example 3 showed some protective effect, its stability was still significantly inferior to the complete ternary system examples containing polyphenol crosslinking. This clearly demonstrates that simple physical mixing or traditional methods relying on a single protection mechanism cannot provide effective and durable stabilization protection for vitamin D3 in complex and demanding processing and long-term storage environments.

[0079] Table 3 and Figure 1 The results showed that the sensory characteristics, such as color and state, of all the samples in the examples did not change significantly during storage. However, Comparative Examples 1 and 2 showed slight stratification. This may be because the polyphenols first undergo strong and random cross-linking with the protein, easily causing protein denaturation and hydrophobic aggregation, forming a rigid "precipitate core." The polysaccharides, unable to effectively penetrate and bind later, can only loosely adhere, leading to slight stratification in the later products. This indicates that the method of the present invention not only effectively protects the stability of vitamin D3 but also has a positive impact on the stability of liquid beverages.

[0080] Table 2

[0081] Table 3

[0082] In summary, the method for protecting vitamin D3 using a protein-polysaccharide-polyphenol ternary covalent complex provided by this invention achieves significant technical advantages. The complex, through Maillard / amidation and secondary cross-linking with polyphenols, forms a dense network possessing both physical barrier and chemical antioxidant functions. Experiments have demonstrated that this system significantly improves the retention rate of vitamin D3 under stringent sterilization conditions and exhibits a lower degradation rate in accelerated stability tests. Compared with traditional techniques, this method overcomes the shortcomings of insufficient stability under high temperatures and long-term storage, making it particularly suitable for the development and production of acidic liquid foods and beverages with stringent requirements for stability and shelf life.

[0083] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a protein-polysaccharide-polyphenol ternary covalent complex, characterized in that, The protein-polysaccharide-polyphenol ternary covalent complex is stable in an acidic environment with a pH less than or equal to 4, and its preparation includes the following steps: After thoroughly mixing vitamin D3 with the oil carrier, it is added to the protein aqueous solution and homogenized and emulsified to obtain vitamin D3 emulsion. Add polysaccharide aqueous solution to vitamin D3 emulsion, adjust the pH of the system to 8-10, and react at 85-95℃ for 2-3 hours; Cool down, continue adding polyphenol solution, adjust pH to 9-10, react at 50-60℃ for 1-2 hours, cool down, and obtain protein-polysaccharide-polyphenol ternary covalent complex.

2. The preparation method according to claim 1, characterized in that, The protein in the protein aqueous solution is selected from one or more of whey protein isolate, pumpkin seed protein, bone gelatin, sodium caseinate, and soy protein isolate. The polysaccharide in the polysaccharide aqueous solution is selected from one or more of the following: locust bean gum, sodium alginate, pullulan, gellan gum, hyaluronic acid with a molecular weight of 50-100 kDa, and octenyl succinic acid starch. The polyphenols in the polyphenol solution are selected from one or more of citrus polyphenols, grape seed extract, rosmarinic acid, and tea polyphenols.

3. The preparation method according to claim 2, characterized in that, The protein to polysaccharide mass ratio is 1-2.5:1; The mass ratio of the protein to the polyphenol is 5-15:1; The mass ratio of the protein to vitamin D3 is 20-50:

1.

4. The preparation method according to claim 1, characterized in that, The oil carrier is selected from one or more of medium-chain triglycerides, sunflower seed oil, and corn oil; Preferably, the mass ratio of vitamin D3 to oil carrier is 1:10-50.

5. The preparation method according to claim 1, characterized in that, The homogenization emulsification speed is 7000-10000 rpm / min, and the homogenization emulsification time is 5-10 min.

6. A protein-polysaccharide-polyphenol ternary covalent complex, characterized in that, It is prepared by the preparation method according to any one of claims 1-5.

7. A functional rehydration solution rich in minerals and vitamin D3, characterized in that, By mass percentage, including: Xanthan gum 0.1-0.5 wt%; Sugars and / or sugar alcohols 10-30 wt%; Acidity regulator 0.5-1.5 wt%; Flavoring agent 0.05-1wt%; 5-65 wt% of the protein-polysaccharide-polyphenol ternary covalent complex according to claim 6; Mineral supplements 0.2-40 wt%; The rest is water; The pH value of the functional replenishment solution is 3.5-4.

0.

8. The functional fluid replacement according to claim 7, characterized in that, The mineral supplements include one or more of the following: calcium supplements, zinc supplements, selenium supplements, and iron supplements.

9. The functional fluid replacement according to claim 7, characterized in that, The sugar is selected from one or more of the following: white granulated sugar, edible glucose, crystalline fructose, maltose, fructooligosaccharides, xylooligosaccharides, maltodextrin, and polydextrose. The sugar alcohol is selected from one or more of xylitol, erythritol, maltitol, sorbitol, and isomaltitol; The acidity regulator is selected from one or more of citric acid, malic acid, lactic acid, sodium citrate, and sodium malate. The flavoring agent is one or more of concentrated fruit juice and food flavoring.

10. A method for preparing a functional rehydration solution as described in any one of claims 7-9, characterized in that, Includes the following steps: Add sugar and / or sugar alcohol, mineral supplements, xanthan gum, and flavoring agents to the protein-polysaccharide-polyphenol ternary covalent complex according to the formula, mix, add an acidity regulator to adjust the pH to 3.5-4.0, shear at 7000-10000 rpm / min for 5-10 min, homogenize and filter to obtain the final product.

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