A pyrroloquinoline quinone disodium salt modified whey protein-hyaluronic acid complex nanogel embedded with ergothioneine and its preparation and food application

Abstract

The application discloses a pyrroloquinoline quinone disodium salt modified whey protein-hyaluronic acid composite nanogel embedded ergothioneine and preparation and food application thereof, and belongs to the field of functional food and targeted delivery technology. The application is aimed at the core problems of poor gastrointestinal stability, low intestinal absorption efficiency, insufficient intracellular mitochondrial targeting of ergothioneine in food application, and non-food grade raw materials for constructing a delivery system. Pyrroloquinoline quinone disodium salt modified whey protein-hyaluronic acid composite nanogel is prepared by using food grade raw materials as an embedding carrier, and high efficient embedding of ergothioneine is realized by a pH driven self-assembly method. The nanogel prepared by the application has an encapsulation efficiency of greater than or equal to 86%, a drug loading of greater than or equal to 12%, a retention rate in simulated gastric juice of greater than or equal to 91% in 2 hours, an intestinal absorption efficiency that is more than 8.5 times that of free ergothioneine, an intracellular mitochondrial enrichment rate of greater than or equal to 65%, and a mitochondrial ROS clearance efficiency that is 8.2 times that of free ergothioneine, and has the functions of synergistic intestinal absorption and antioxidant effect.

Description

Technical Field

[0001] This invention belongs to the field of functional food and targeted delivery technology, specifically relating to a whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt encapsulating ergothione, its preparation, and its food application. Background Technology

[0002] Ergothioneine (EGT) is a natural sulfur-containing imidazole amino acid widely found in edible fungi, grains, nuts, and other foods. As a potent natural antioxidant, ergothioneine can specifically accumulate in the mitochondrial matrix, directly scavenging reactive oxygen species (ROS) produced by mitochondria and protecting mitochondrial DNA and respiratory chain complexes from oxidative damage. Its antioxidant capacity is 14 times that of glutathione and 400 times that of vitamin C.

[0003] In recent years, the application of ergothioneine in the food industry has received widespread attention. Studies have shown that ergothioneine supplementation can improve post-exercise fatigue, enhance immunity, delay aging, and prevent chronic diseases. However, the application of ergothioneine in food still faces the following technical bottlenecks: 1) Poor gastrointestinal stability: Ergothioneine is easily degraded under the action of gastric acid (pH 1.2-3.0) and digestive enzymes, and the loss rate exceeds 60% when it passes through the gastrointestinal tract after oral administration.

[0004] 2) Low intestinal absorption efficiency: Ergothioneine is a water-soluble small molecule. Although it can be absorbed through the OCTN1 transporter protein of intestinal epithelial cells, its bioavailability is less than 10% due to its short residence time in the gastrointestinal tract.

[0005] 3) Insufficient intracellular mitochondrial targeting: Even if a small amount of ergothioneine is absorbed into the bloodstream, due to the lack of mitochondrial targeting mechanisms, less than 7% can reach the mitochondria to exert its effects, and most of it is consumed in the cytoplasm.

[0006] Existing ergothioneine encapsulation technologies are mainly concentrated in the cosmetics and pharmaceutical fields, using carriers such as liposomes and microspheres. However, these technologies suffer from high costs, poor stability, and the use of non-food-grade raw materials, making them difficult to apply to industrial food production. Therefore, developing a food-grade, highly stable, highly bioavailable ergothioneine encapsulation and delivery system with mitochondrial targeting capabilities is of great significance for expanding the application of ergothioneine in the food industry.

[0007] Pyrroloquinoline quinone disodium salt (PQQ) is a new food ingredient approved by the National Health Commission of China in 2022 (consumption amount ≤20mg / day, applicable scope does not include infant food). It has excellent mitochondrial targeting and mitochondrial protection functions. It can specifically bind to receptors on the mitochondrial membrane, promote mitochondrial biogenesis, scavenge mitochondrial reactive oxygen species, and has a synergistic antioxidant effect with ergothioneine. It is an ideal food-grade mitochondrial targeting ligand. Summary of the Invention

[0008] Technical challenges: Addressing the issues of poor gastrointestinal stability, low intestinal absorption efficiency, insufficient intracellular mitochondrial targeting, and the need for non-food-grade raw materials for the delivery system in ergothioneine applications in food.

[0009] Technical solution: In view of the above problems, the present invention provides a method for preparing ergothionein encapsulated in whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt and its food application.

[0010] The present invention specifically provides the following technical solution: A method for preparing ergothionein encapsulated in a whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt includes the following steps: (1) Dissolve whey protein isolate in deionized water to prepare a protein solution, add ergothioneine and stir to dissolve, adjust the pH to alkaline, and react at room temperature for a period of time; (2) Adjust the pH of the reaction solution after the reaction in step (1) to weak acidity, add hyaluronic acid solution and stir for a period of time to obtain unmodified drug-loaded nanogel suspension; (3) Adjust the pH of the obtained unmodified drug-loaded nanogel suspension to weak alkalinity, then add pyrroloquinoline quinone disodium salt solution and react in the dark for a period of time. (4) After the reaction is completed, the resulting reaction system is purified by dialysis and freeze-dried in sequence to obtain the product of ergothionein encapsulated in whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt.

[0011] As one embodiment of the preparation method described in this invention, in step (1), the pH is adjusted to 11.0-12.0, and the reaction is stirred at room temperature for 30-60 min.

[0012] As one embodiment of the preparation method of the present invention, in step (1), the mass ratio of ergothioneine to whey protein isolate is 1:(5-10).

[0013] As one embodiment of the preparation method described in this invention, in step (1), the concentration of the protein solution is 10-20 mg / mL.

[0014] As one embodiment of the preparation method described in this invention, the pH is adjusted to 5.0-5.5 in step (2).

[0015] As one embodiment of the preparation method described in this invention, the reaction is stirred for 20-40 min in step (2).

[0016] As one embodiment of the preparation method described in this invention, the mass ratio of hyaluronic acid to whey protein isolate used in step (2) is (3-8):(15-25).

[0017] As one embodiment of the preparation method described in this invention, the concentration of the hyaluronic acid solution in step (2) is 5-10 mg / mL.

[0018] As one embodiment of the preparation method described in this invention, the concentration of the disodium pyrroloquinoline quinone solution in step (3) is 10-20 mg / mL, and the solvent is deionized water.

[0019] As one embodiment of the preparation method described in this invention, the mass ratio of pyrroloquinoline quinone disodium salt to whey protein isolate used in step (3) is (0.5-2):(15-25).

[0020] As one embodiment of the preparation method described in this invention, in step (3), the pH is adjusted to 8.0-9.0.

[0021] As one embodiment of the preparation method described in this invention, in step (3), the reaction is carried out in the dark for 5-10 hours.

[0022] As one embodiment of the preparation method described in this invention, in step (1) or step (2), pH adjustment is performed using 0.5 mol / L NaOH solution and 0.5 mol / L HCl solution.

[0023] As one embodiment of the preparation method described in this invention, the stirring speed involved in the method is 300-500 r / min.

[0024] As one embodiment of the preparation method described in this invention, in step (4), the dialysis purification uses a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and is dialyzed with deionized water for 24 hours, with the dialysis solution being replaced every 6 hours.

[0025] As one embodiment of the preparation method described in this invention, in step (4), the freeze-drying conditions are -50℃, 0.1mPa, and drying time is 24-36h.

[0026] Another objective of this invention is to prepare a product containing ergothionein encapsulated in a whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt based on the above method.

[0027] As one embodiment of the preparation method described in this invention, the average particle size of the ergothione product encapsulated in the pyrroloquinoline quinone disodium salt modified whey protein-hyaluronic acid composite nanogel is 110-170 nm, the polydispersity index (PDI) is ≤0.14, and the zeta potential is -26 mV to -36 mV.

[0028] Another objective of this invention is to provide an application of pyrroloquinoline quinone disodium salt-modified whey protein-hyaluronic acid composite nanogel-encapsulated ergothione product in the preparation of functional foods with antioxidant, fatigue-relieving, and immune-enhancing properties.

[0029] As one embodiment of the preparation method described in this invention, the functional food includes functional beverages, sports nutrition powders, meal replacement foods, and dietary supplements.

[0030] Beneficial effects of this invention: Compared with the prior art, the present invention has the following significant advantages: 1) Food-grade ingredients, safe and non-toxic: The whey protein isolate, hyaluronic acid, and disodium pyrroloquinoline quinone used are all nationally approved food ingredients or new food ingredients, with no toxic side effects, and are suitable for all types of people except infants and young children.

[0031] 2) High encapsulation efficiency and drug loading: Prepared using pH-driven self-assembly method, the encapsulation efficiency can reach 86%-93%, and the drug loading can reach 12%-18%, which is significantly higher than the existing food-grade encapsulation technology.

[0032] 3) Excellent gastrointestinal stability: Ergothioneine retention rate ≥91% in simulated gastric juice after 2 hours and cumulative release rate ≥86% in simulated intestinal juice after 4 hours, achieving targeted intestinal release of ergothioneine.

[0033] 4) Significantly improved intestinal absorption efficiency and synergistic effect: The small particle size of the nanogel enables it to be efficiently absorbed through endocytosis of intestinal epithelial cells, with oral bioavailability more than 8.5 times higher than that of free ergothioneine; In addition, the surface covalently bound PQQ and whey protein-hyaluronic acid composite nanogel encapsulating ergothioneine system synergistically promote cellular uptake, further improving delivery efficiency.

[0034] 5) Significant mitochondrial targeted enrichment effect and synergistic effect: For the first time, food-grade pyrroloquinoline quinone disodium salt was used as a mitochondrial targeted ligand, with an intracellular mitochondrial enrichment rate of ≥65%, which is 9.3 times that of free ergothioneine; PQQ and ergothioneine synergistically clear mitochondrial ROS, with a mitochondrial ROS clearance efficiency of 8.2 times that of free ergothioneine. At the same time, PQQ can promote mitochondrial biogenesis, further enhancing the mitochondrial protective effect.

[0035] 6) The preparation process is greener and safer, suitable for industrial production: The entire preparation process requires no organic solvents, the process parameters are controllable, the reproducibility is good, and the cost is low, making it suitable for large-scale industrial production. The product can be widely used in functional beverages, sports nutrition powders, meal replacement foods, and other fields, significantly improving the bioavailability and physiological efficacy of ergothioneine. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The cumulative release rates of ergothionein during simulated gastrointestinal digestion in Examples 1 and 2 and Comparative Examples 1 and 3 of the present invention are shown. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] Unless otherwise specified, all raw materials used in this invention are commercially available food-grade products in the field.

[0041] Example 1 A whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt encapsulated ergothionein has the following raw material ratio: whey protein isolate: 2.0g, hyaluronic acid: 0.5g, pyrroloquinoline quinone disodium salt: 0.15g, and ergothionein: 0.25g.

[0042] Specific preparation methods include: (1) Weigh 2.0g of whey protein isolate and dissolve it in 100mL of deionized water to prepare a protein solution of 20mg / mL; add 0.25g of ergothioneine and stir at 400r / min to dissolve; then adjust the pH to 11.5 with 0.5mol / L NaOH solution and stir at room temperature for 45min. (2) After the reaction is complete, adjust the pH to 5.2 with 0.5 mol / L HCl solution, slowly add 50 mL of 10 mg / mL hyaluronic acid solution, and stir the reaction at 400 r / min for 30 min; (3) Then add 0.5 mol / L NaOH solution dropwise to slowly adjust the pH to 8.5, then heat the reaction system to 35°C and adjust the stirring speed to 300 r / min; (4) Weigh 0.15 g of disodium pyrroloquinoline quinone and dissolve it in 15 mL of deionized water in a brown bottle protected from light. Then, slowly add 15 mL of 10 mg / mL disodium pyrroloquinoline quinone solution to the reaction system in three separate additions, 5 mL each time, with a 20 min interval between each addition. After the three additions are completed, react at 35 °C in the dark for 6 h. (5) After the reaction is completed, cool to room temperature, then put the reaction solution into a dialysis bag with a molecular weight cutoff of 10000 Da, dialyze with deionized water for 24 hours, and change the dialysate every 6 hours; freeze dry the dialysis suspension at -50℃ and 0.1mPa for 30 hours to obtain the final product.

[0043] Example 2 The difference between this embodiment and Embodiment 1 is that the raw material ratio is adjusted: Whey protein isolate: 1.5g, hyaluronic acid: 0.4g, disodium pyrroloquinoline quinone: 0.1g, ergothioneine: 0.3g.

[0044] The remaining steps and processes are the same as in Example 1.

[0045] Example 3 The difference between this embodiment and Embodiment 1 is that the raw material ratio is adjusted: Whey protein isolate: 2.5g, hyaluronic acid: 0.6g, disodium pyrroloquinoline quinone: 0.2g, ergothioneine: 0.2g.

[0046] The remaining steps and processes are the same as in Example 1.

[0047] Example 4 The difference between this embodiment and embodiment 1 is that the pH value is adjusted: in step (1), the pH is adjusted to 11.0, and in step (3), the pH is adjusted to 9.0. The remaining steps are the same as in embodiment 1.

[0048] Example 5 The difference between this embodiment and embodiment 1 is that the reaction time is adjusted: the reaction time in step (1) is 30 min, the reaction time in step (2) is 20 min, and the reaction time in step (4) is 10 h. The process of the remaining steps is the same as that in embodiment 1.

[0049] Comparative Example 1 This comparative example uses free ergothioneine powder.

[0050] Comparative Example 2 This comparative example is an unmodified whey protein-hyaluronic acid composite nanogel encapsulating ergothionein. The preparation method is basically the same as that in Example 1, except that step (4) is omitted and the pyrroloquinoline quinone disodium salt modification step is not performed.

[0051] Comparative Example 3 This comparative example is the traditional sodium alginate microspheres encapsulated with ergothioneine, prepared by extrusion method: 1.0 g of sodium alginate was dissolved in 100 mL of deionized water, 0.25 g of ergothioneine was added and stirred to dissolve, and then added dropwise to a 2% calcium chloride solution for crosslinking for 30 min. The product was obtained by filtration and freeze-drying.

[0052] Comparative Example 4 This comparative example is the soy protein isolate substitution group. Except for replacing whey protein isolate with food-grade soy protein isolate of equal mass, the other preparation steps and process parameters are the same as in Example 1.

[0053] Comparative Example 5 This comparative example is the sodium caseinate substitution group. Except for replacing whey protein isolate with food-grade sodium caseinate, the other preparation steps and process parameters are the same as in Example 1.

[0054] Comparative Example 6 This comparative example is the low hyaluronic acid ratio group. The mass ratio of whey protein isolate to hyaluronic acid was adjusted to 10:1. The total amount of other raw materials, preparation steps and process parameters are the same as in Example 1.

[0055] Comparative Example 7 This comparative example is a high hyaluronic acid ratio group. The mass ratio of whey protein isolate to hyaluronic acid was adjusted to 2:1. The total amount of other raw materials, preparation steps and process parameters are the same as in Example 1.

[0056] Comparative Example 8 This comparative example is the PQQ physical adsorption group. In the preparation process, step (3) of adjusting pH was omitted. The disodium pyrroloquinoline quinone solution was directly added to the nanogel suspension for reaction. The other raw material ratios, preparation steps and process parameters were the same as in Example 1.

[0057] Performance testing 1. Determination of basic physicochemical properties The average particle size, PDI, and Zeta potential of the nanogel were determined using a Malvern Zetasizer Nano ZS90 nanoparticle size potentiometer at 25℃. Each sample was measured in triplicate. Encapsulation efficiency and drug loading were determined by dialysis: 10 mg of freeze-dried nanogel was dissolved in 10 mL of deionized water, placed in a dialysis bag, and dialyzed for 24 h. The content of free ergothioneine in the dialysate was measured, and the encapsulation efficiency and drug loading were calculated. The results are shown in Table 1.

[0058] Table 1. Basic physicochemical properties of different samples

[0059] As shown in Table 1, the nanogels prepared in each embodiment of the present invention exhibit excellent colloidal properties, with an average particle size stable in the range of 110~170nm, a PDI of less than 0.14, and a highly uniform particle size distribution; the absolute value of the Zeta potential is higher than 26mV, indicating good colloidal stability of the system; the encapsulation efficiency can reach 86%~93%, and the drug loading is 12%~18%, with comprehensive performance significantly better than traditional microsphere carriers and other control schemes.

[0060] Compared with traditional sodium alginate microspheres (Comparative Example 3), the nanogel of the present invention has a particle size of only about 1 / 6 of that of sodium alginate microspheres, a distribution uniformity that is nearly 3 times higher, and an encapsulation efficiency that is 34.5 percentage points higher, which fully demonstrates the technical advantages of the pH-driven self-assembly system in terms of encapsulation efficiency and system uniformity.

[0061] Further comparison of the preparation effects under different process parameters reveals that the type of protein carrier, the ratio of raw materials, and the PQQ binding method all significantly affect gel performance. Replacing the protein with other commonly used food proteins or adjusting the amount of hyaluronic acid beyond the scope of this invention resulted in increased particle size, wider distribution, and decreased encapsulation efficiency in the gels. The physical adsorption group (omitting the activation step) showed no significant difference from the unmodified gel, indicating that covalent binding is a necessary condition for stable PQQ loading. These results collectively validate the rationality and necessity of the process parameters in this invention.

[0062] 2. In vitro simulated gastrointestinal release test Ergothioneine release was investigated using a continuous in vitro gastrointestinal digestion model: simulated gastric juice (SGF, pH 1.2) and simulated intestinal juice (SIF, pH 6.8) were prepared. 10 mg of lyophilized nanogel was reconstituted and placed in a dialysis bag. The bag was first immersed in 100 mL of SGF and incubated at 37°C and 100 r / min for 2 h (gastric digestion stage, sampling time points: 0.5, 1, and 2 h). After 2 h of incubation, the dialysis bag was transferred to 100 mL of SIF and incubated for a total duration of 12 h (intestinal digestion stage, sampling time points: 4, 6, 8, and 12 h). At each time point, 1 mL of sample was taken and an equal volume of fresh medium was added. The ergothioneine content was measured, and the cumulative release rate was calculated. The release curves are shown in [Figure number missing]. Figure 1 .

[0063] 0~2 h simulated gastric juice stage: The cumulative release rate of the nanogel in Example 1 was only 8.7% after 2 h, showing excellent gastric acid stability; the release loss of free ergothioneine (Comparative Example 1) reached 65.2% after 2 h, indicating that it is easily degraded by gastric acid.

[0064] 2-12 h simulated intestinal stage: After the sample was transferred to the intestinal fluid, the drug was released rapidly. In Example 1, the cumulative release rate reached 86.3% after 4 h (total time) of incubation, and the total release rate was 93.5% after 12 h, achieving targeted release in the intestinal tract. In contrast, the traditional sodium alginate microspheres in Comparative Example 3 leaked a large amount of gastric fluid during the gastric fluid stage, resulting in poor controlled release.

[0065] 3. Intestinal cell absorption test Intestinal absorption efficiency was determined using a Caco-2 cell monolayer model. Caco-2 cells were seeded in Transwell chambers and cultured for 21 days to form a dense monolayer. 1 mL of sample solution containing 50 μg / mL ergothioneine was added to the upper chamber, and 1.5 mL of HBSS buffer was added to the lower chamber. The chambers were incubated at 37°C. The lower chamber solution was collected at 1, 2, and 4 hours to determine the ergothioneine content and calculate the apparent permeability coefficient (Papp). The results are shown in Table 2.

[0066] Table 2 Apparent permeability coefficients of Caco-2 cell monolayer membranes from different samples

[0067] The results showed that the nanogel of the present invention can significantly improve the intestinal absorption efficiency of ergothioneine, with an apparent permeability coefficient that is 8.1 to 8.9 times that of free ergothioneine, effectively solving the problems of short intestinal residence time and low bioavailability of water-soluble small molecules.

[0068] This effect is mainly attributed to the uniform nanoparticle size and excellent colloidal stability, enabling the gel particles to be efficiently taken up by the endocytosis pathway of intestinal epithelial cells. Traditional microspheres, due to their large particle size, have a cellular uptake efficiency only about 1 / 4 that of this invention. When the protein carrier is replaced or the ratio deviates from the optimal range, changes in particle size and aggregation state lead to varying degrees of decreased absorption efficiency. Furthermore, although the particle size of the product with simple physical adsorption of PQQ in Comparative Example 8 is also small, the cellular uptake effect of the product with covalently bound PQQ in this embodiment is still significantly better than that of the product with simple physical adsorption of PQQ in Comparative Example 8. This indicates that surface-stable PQQ not only targets mitochondria but also promotes cellular uptake, further enhancing delivery efficiency in conjunction with the whey protein-hyaluronic acid composite nanogel encapsulating ergothioneine system.

[0069] 4. Intracellular mitochondrial colocalization assay Colocalization assays were performed using human hepatocellular carcinoma HepG2 cells. Cells were seeded in confocal culture dishes and cultured for 24 h. FITC-labeled samples were then added to achieve a final ergothioneine concentration of 50 μg / mL, and the cells were incubated for another 4 h. Mitochondrial colocalization coefficients were calculated using the mitochondrial red fluorescent probe MitoTrackerRed and the nuclei were stained with DAPI. The results are shown in Table 3.

[0070] Table 3. Intracellular mitochondrial colocalization coefficients of different samples

[0071] The PQQ-modified nanogel prepared by this invention has excellent mitochondrial targeting enrichment ability, with an intracellular mitochondrial colocalization coefficient of over 0.65 and a relative enrichment rate more than 9 times that of free ergothionein, enabling precise delivery of the core material to the target site.

[0072] Free ergothionein, traditional microspheres, and unmodified nanogels all showed no significant mitochondrial targeting effect, indicating that the core material cannot effectively accumulate in mitochondria without specific ligand mediation. Only by covalently modifying PQQ to stably bind it to the gel surface can it exert a targeting-mediated effect throughout the entire cell internalization process; physically adsorbed PQQ is easily detached during delivery, offering almost no improvement in targeting efficiency. The ratio of protein carrier to raw materials also affects the targeting effect; when the parameters deviate from the optimal range, the amount and exposure of PQQ decrease, and the enrichment efficiency decreases accordingly.

[0073] 5. Mitochondrial ROS scavenging assay HepG2 cells were seeded in 6-well plates and cultured for 24 h. Different samples were then added to achieve a final ergothioneine concentration of 50 μg / mL, followed by incubation for 2 h. Cells were then treated with 200 μM H₂O₂ for 4 h to induce mitochondrial ROS production. Mitosolic ROS were stained with the mitochondrial-specific ROS probe MitoSOXRed, and fluorescence intensity was measured by flow cytometry to calculate ROS scavenging efficiency. The results are shown in Table 4.

[0074] Table 4. Scavenging efficiency of mitochondrial ROS by different samples

[0075] The product of this invention has a highly efficient ability to scavenge mitochondrial reactive oxygen species, with a scavenging efficiency of over 86%, and a relative scavenging rate that is more than 8 times that of free ergothioneine, thus fully realizing the synergistic antioxidant effect of PQQ and ergothioneine.

[0076] Free ergothioneine and untargeted carrier systems exhibit very limited clearance effects due to extremely low effective drug concentrations reaching the mitochondria. This invention, through precise mitochondrial-targeted delivery, allows both active ingredients to co-enrich at the site of action. This synergistic effect—directly scavenging ROS with ergothioneine and promoting mitochondrial biogenesis with PQQ—results in a significant increase in antioxidant capacity. Other control groups, due to insufficient targeting efficiency, failed to achieve the same level of synergistic protective effect.

[0077] Food Application Example: Preparation of Functional Beverages Formula (per 1000mL): Ergothioneine nanogel prepared in Example 1 of this invention: 1.0g (containing approximately 146mg of ergothioneine and approximately 14mg of PQQ). White sugar: 50g Citric acid: 1.5g Sodium citrate: 0.5g Vitamin C: 0.2g Edible flavoring: 0.1g Deionized water: Add to 1000 mL.

[0078] Preparation method: Dissolve white sugar, citric acid, sodium citrate and vitamin C in 800mL of deionized water, add ergothioneine nanogel and stir to dissolve, add edible flavoring, make up to 1000mL, pasteurize (85℃, 15min), cool and fill to obtain functional beverage.

[0079] Stability test: The functional beverage was stored at room temperature in the dark for 6 months, and the contents of ergothioneine and PQQ were measured periodically. The results showed that after 6 months of storage, the retention rate of ergothioneine was 92.5% and the retention rate of PQQ was 90.8%, with no obvious precipitation or discoloration, demonstrating excellent stability.

[0080] In summary, this invention provides a method for preparing ergothioneine encapsulated in a whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt and its food applications. Using food-grade raw materials, the method employs a pH-driven self-assembly approach, resulting in a simple, solvent-free, low-cost process suitable for industrial production. The nanogel prepared by this invention exhibits high encapsulation efficiency, high drug loading capacity, excellent gastrointestinal stability, and high intestinal absorption efficiency. For the first time, this invention utilizes pyrroloquinoline quinone disodium salt, a nationally approved new food ingredient, to achieve mitochondrial-targeted delivery of ergothioneine. Furthermore, PQQ and ergothioneine exhibit synergistic antioxidant and mitochondrial protective effects, significantly enhancing the bioavailability and physiological efficacy of ergothioneine. This invention has broad applicability in functional beverages, sports nutrition powders, meal replacement foods, and other fields, possessing significant market value and application prospects.

[0081] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing ergothionein encapsulated in a whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt, characterized in that: include, (1) Dissolve whey protein isolate in deionized water to prepare a protein solution, add ergothioneine and stir to dissolve, adjust the pH to alkaline, and react at room temperature for a period of time; (2) Adjust the pH of the reaction solution after the reaction in step (1) to weak acidity, add hyaluronic acid solution and stir for a period of time to obtain unmodified drug-loaded nanogel suspension; (3) Adjust the pH of the obtained unmodified drug-loaded nanogel suspension to weak alkalinity, then add pyrroloquinoline quinone disodium salt solution and react in the dark for a period of time. (4) After the reaction is completed, the resulting reaction system is purified by dialysis and freeze-dried in sequence to obtain the product of ergothionein encapsulated in whey protein-hyaluronic acid composite nanogel modified with pyrroloquinoline quinone disodium salt.

2. The preparation method according to claim 1, characterized in that: In step (1), the pH is adjusted to 11.0-12.0, and the reaction is stirred at room temperature for 30-60 min; the mass ratio of ergothioneine to whey protein isolate is 1:(5-10); the concentration of the protein solution is 10-20 mg / mL.

3. The preparation method according to claim 1, characterized in that: In step (2), the pH is adjusted to 5.0-5.5; the reaction is stirred for 20-40 min; the mass ratio of hyaluronic acid to whey protein isolate is (3-8):(15-25); and the concentration of the hyaluronic acid solution is 5-10 mg / mL.

4. The preparation method according to claim 1, characterized in that: The concentration of the disodium pyrroloquinoline quinone solution in step (3) is 10-20 mg / mL, and the solvent is deionized water; the mass ratio of the disodium pyrroloquinoline quinone to the whey protein isolate is (0.5-2):(15-25).

5. The preparation method according to claim 1, characterized in that: The pH is adjusted to 8.0-9.0 as described in step (3).

6. The preparation method according to claim 1, characterized in that: In step (4), the dialysis purification uses a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and is dialyzed with deionized water for 24 hours, with the dialysate being replaced every 6 hours; the freeze-drying conditions are -50℃, 0.1mPa, and drying time is 24-36 hours.

7. The pyrroloquinoline quinone disodium salt modified whey protein-hyaluronic acid composite nanogel encapsulated with ergothioneine product prepared by any of the preparation methods described in claims 1-6.

8. The product as described in claim 7, characterized in that: The average particle size of the ergothione product encapsulated in the pyrroloquinoline quinone disodium salt modified whey protein-hyaluronic acid composite nanogel is 110-170 nm, the polydispersity index (PDI) is ≤0.14, and the zeta potential is -26 mV to -36 mV.

9. The use of the product according to claim 7 or 8 in the preparation of functional foods with antioxidant, fatigue-relieving, and immune-enhancing properties.

10. The application as described in claim 9, characterized in that: The functional foods include functional beverages, sports nutrition powders, meal replacement foods, and dietary supplements.

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