Arbutus juice based on ultra-high pressure-ultrasound treatment and method for its preparation

Abstract

This invention belongs to the field of functional food processing and provides a method for preparing aronia berry juice based on ultra-high pressure-ultrasound treatment. The invention uses the total polyphenol fraction B1 of aronia berry and plant protein to prepare a polyphenol-protein graft polymer via enzymatic grafting. Under specific pH, ionic strength, and ultrasonic conditions, the polymer self-assembles to form polyphenol-protein nanomicelles with a particle size of 50-150 nm. After being added to the aronia berry juice, it undergoes cold sterilization through a combination of high hydrostatic pressure and ultrasound treatment, constructing a stable polyphenol-rich interfacial shell structure. The finished juice retains at least 80% of the total polyphenols and has a total bacterial count of less than or equal to 10 CFU / mL after 90 days of storage at 4℃. This achieves multiple synergistic effects, including efficient polyphenol encapsulation, enhanced antioxidant and antibacterial activity, and improved physical stability. It solves the problems of increased viscosity and deteriorated taste caused by high polyphenol loading, as well as the destruction of active ingredients by heat sterilization, and has broad application value.

Description

Technical Field

[0001] This invention relates to the field of functional food processing technology, specifically to a wild cherry berry juice based on ultra-high pressure-ultrasound treatment and its preparation method. Background Technology

[0002] Aronia berries are small berries rich in anthocyanins, proanthocyanidins, flavonols, and other polyphenols. Their total polyphenol content is significantly higher than that of common berries like blueberries and cranberries, offering excellent antioxidant, anti-inflammatory, and blood sugar and lipid-regulating benefits, making them promising for functional fruit juice beverages. Non-concentrated (NFC) aronia berry juice is favored by consumers for preserving the nutritional components and natural flavor of fresh fruit to the greatest extent. However, the polyphenols in aronia berry juice are susceptible to degradation, polymerization, or non-specific binding with proteins during processing and storage due to changes in light, oxygen, temperature, pH, and enzymatic oxidation. This leads to decreased antioxidant activity, browning, and precipitation, resulting in quality deterioration. Furthermore, NFC juice requires minimizing heat treatment intensity to protect heat-sensitive nutrients and flavor compounds while inhibiting microbial growth and extending shelf life, placing higher demands on sterilization processes. Therefore, developing aronia berry juice processing technology that can efficiently encapsulate and protect polyphenols under mild processing conditions, enhance the system's antioxidant stability and antibacterial properties, and maintain good rheological properties and sensory quality is of great significance.

[0003] Currently, the mainstream technical routes for polyphenol protection and product stability improvement in aronia berry juice processing include physical methods such as adding antioxidants, using inert gas protection, low-temperature storage, and ultra-high pressure sterilization. However, these methods still have significant shortcomings. For example, Chinese patent CN111418742A discloses a method for preparing aronia berry juice, which increases the juice yield to over 85% by combining pectin enzymatic hydrolysis with high-voltage pulsed electric field extraction of filter residue. However, it does not provide structured encapsulation protection for polyphenols and relies on enzymatic hydrolysis and high-voltage pulsed treatment, making the process complex and the polyphenols still easily degraded during storage. In addition, while the method of treating aronia berries with high-voltage pulses can inactivate enzyme activity and kill microorganisms to a certain extent, it does not provide structured encapsulation protection for polyphenols. During storage, polyphenols are still easily degraded, and it is difficult to achieve targeted interface stability. Furthermore, existing technologies that use protein encapsulation of polyphenols often suffer from low encapsulation efficiency, wide particle size distribution, and significantly increased system viscosity, limiting their application in high-polyphenol-loading (NFC) juices. Meanwhile, although non-thermal sterilization technologies such as ultra-high pressure can avoid high-temperature damage, their inactivation effect on pressure-resistant microorganisms under the action of a single physical field is insufficient. They need to be combined with other technologies to achieve sufficient sterilization and maintain long-term microbial stability. These technological bottlenecks have restricted the industrialization of high-quality wild cherry berry functional juice. Summary of the Invention

[0004] The purpose of this invention is to provide a wild aronia juice based on ultra-high pressure-ultrasonic treatment and its preparation method. Addressing the contradiction that existing NFC wild aronia juices easily suffer from increased viscosity, poor taste, and limited processing flowability due to the introduction of high-load polyphenol micelles, while high-energy physical sterilization and homogenization processes easily damage polyphenol activity and flavor stability, this invention focuses on solving the multi-performance coupling problem of achieving efficient polyphenol encapsulation and interface stability while ensuring low viscosity and good rheological properties of the system, and maintaining high retention rates of wild aronia polyphenols and flavor components and storage stability under room temperature cold sterilization and microbial control conditions.

[0005] This invention constructs an amphiphilic polyphenol-protein graft polymer by enzymatically grafting total polyphenol fraction B1 of wild cherifolia with plant protein. Then, utilizing the self-assembly driving force of the hydrophobic polyphenol segments and hydrophilic protein backbone of the graft polymer under specific ionic strength and pH conditions, ultrasonic cavitation induces the formation of nanomicelles with controllable particle size. The hydrophobic core of the micelles efficiently encapsulates polyphenols, and the protein shell provides steric hindrance stability and an antioxidant barrier. In the juice system, this avoids both the rapid degradation of free polyphenols and a significant increase in viscosity. Simultaneously, the micelles disperse at the interface of pulp particles and oil droplets, forming a polyphenol-rich functional shell network. This, combined with high hydrostatic pressure to inactivate enzyme activity, disrupt microbial cell walls, and ultrasonic microfluidics to enhance mass transfer, achieves deep sterilization and long-lasting antibacterial effects under mild conditions. Thus, it achieves synergistic effects at multiple levels, including nanoscale micelle stabilization, macroscopic rheological optimization, polyphenol activity protection, and microbial control, significantly outperforming the effects of single polyphenol addition or single sterilization technologies.

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

[0007] A wild aronia juice based on ultra-high pressure-ultrasound treatment, comprising NFC wild aronia juice and polyphenol-protein nanomicelles dispersed therein, wherein the polyphenol-protein nanomicelles and the wild aronia juice satisfy the following conditions:

[0008] (1) The polyphenol-protein nanomicelles are formed by the enzymatic grafting of B1 polyphenol fraction B1 of wild cheri and at least one of isolated whey protein, pea protein isolate and gelatin under the catalysis of laccase and / or horseradish peroxidase. The B1 polyphenol-protein graft polymer is formed under the conditions of pH 4.0–4.5, sodium chloride concentration of 10–50 mmol / L, ultrasonic frequency of 20–25 kHz, power density of 200–600 W / L and duration of 2–8 min.

[0009] (2) The volume average particle size of the polyphenol-protein nanomicelles is 50–150 nm, the polydispersity index (PDI) is less than or equal to 0.25, the absolute value of the zeta potential is greater than or equal to 20 mV, and the polyphenol mass fraction based on micelle solids is 5–30 wt%.

[0010] (3) The amount of the polyphenol-protein nanomicelles added to the cherriesberry juice is 0.10–0.80 wt% of the total mass of the finished juice, calculated as micelle solids;

[0011] (4) The aronia juice is obtained by high hydrostatic pressure treatment, wherein the pressure of the high hydrostatic pressure treatment is 320–380 MPa, the time is 5–10 min, and the temperature is 20–25℃. After the high hydrostatic pressure treatment, it is obtained by ultrasonic treatment, wherein the temperature of the ultrasonic treatment is 40–50℃ and the time is 2–3 min. When the finished aronia juice is stored at 4℃ for 90 days, the total polyphenol retention rate of aronia is greater than or equal to 80 wt%, and the total bacterial count determined according to the detection method specified in GB4789.2 is less than or equal to 10 CFU / mL.

[0012] Furthermore, the total polyphenol fraction B1 of wild cherifolia is prepared by the following steps:

[0013] A1. Take NFC wild cherries juice, which is NFC non-concentrated reduced wild cherries juice. Adjust the pH to 3.0–4.0 and pass it into a styrene-divinylbenzene copolymer macroporous adsorption resin column at 20–30℃ at a space velocity of 1.0–3.0 times the column volume per hour for 1.0–3.0 hours.

[0014] A2. Elute sequentially with deionized water until no visible pigment is visible in the effluent, then elute with a water-ethanol mixed solvent, wherein the volume fraction of ethanol in the water-ethanol mixed solvent is 30–60 vol%, and collect the eluent containing polyphenols.

[0015] A3. The eluent is concentrated under reduced pressure at 40–60°C to obtain a total polyphenol fraction B1 of aronia berries with a total polyphenol mass fraction of 30–60 wt%, wherein the total polyphenol mass fraction of aronia berries B1, calculated as gallic acid equivalent, is a recovery rate of greater than or equal to 70 wt% relative to the total polyphenol mass fraction in the feed juice.

[0016] Furthermore, the B1 polyphenol-protein graft polymer is prepared through the following steps:

[0017] B1. Raw material preparation steps, including:

[0018] a) Take at least one of whey protein isolate, pea protein isolate and gelatin, add it to deionized water and dissolve it to obtain a protein solution with a protein mass fraction of 1.0–5.0 wt%.

[0019] b) Dissolve the total polyphenol fraction B1 from wild cherries in deionized water to make the mass fraction of the B1 solution 1.0–5.0 wt%;

[0020] c) Mix the B1 solution and the protein solution at a mass ratio of B1 to protein of 1:1–3, and adjust the pH of the mixture to 5.5–6.5;

[0021] B2. Enzyme-catalyzed grafting reaction steps, including:

[0022] Add laccase and / or horseradish peroxidase to the mixture at 25–40°C. The total amount of enzyme added is 0.5–5.0 wt% of the protein. React for 0.5–3.0 h with slow stirring. The reaction endpoint is defined as a 10–50% decrease in free amino content relative to ungrafted protein.

[0023] B3. Post-processing steps, including:

[0024] Small molecule reaction byproducts and free polyphenols are removed by ultrafiltration membrane with a molecular weight cutoff of 10–30 kDa, and the solution is washed with deionized water 1–3 times to obtain a B1 polyphenol-protein graft polymer solution with a solids mass fraction of 2.0–10.0 wt% and a grafting degree of 5–40 mol%. The grafting degree is calculated based on the reduction in free amino content or by conventional methods.

[0025] Furthermore, the polyphenol-protein nanomicelles are prepared through the following steps:

[0026] C1. Take the prepared B1 polyphenol-protein grafted polymer solution and adjust the pH to 4.0–4.5;

[0027] C2. Add sodium chloride to the solution to make the concentration of sodium chloride in the solution 10–50 mmol / L, and let it stand for 5–15 min to promote the exposure of the hydrophobic region of the protein;

[0028] C3. The solution is subjected to ultrasonic treatment at a frequency of 20–25 kHz and a power density of 200–600 W / L for 2–8 min to obtain a polyphenol-protein nanomicelle dispersion with a volume average particle size of 50–150 nm and a polydispersity index (PDI) of less than or equal to 0.25.

[0029] C4. Remove coarse aggregates with a diameter greater than 500 nm by centrifugation or filtration to obtain a polyphenol-protein nanomicelle dispersion for juice reconstruction.

[0030] Furthermore, the polyphenol-protein nanomicelles are distributed and adsorbed at the interface of pulp particles and / or oil droplets in the aronia juice system to form a polyphenol-rich interfacial shell structure. The interfacial shell structure is constructed through the following steps:

[0031] D1. Add the polyphenol-protein nanomicelle dispersion to the NFC wild cherry berry juice according to the above-mentioned amount of polyphenol-protein nanomicelles, and stir slowly until evenly mixed;

[0032] D2. The polyphenol-protein nanomicelles are sheared and homogenized 1–3 times at 20–50 MPa using a high-pressure homogenizer to fully disperse and distribute them at the interface between the pulp particles and oil droplets in the juice system. This allows them to form a polyphenol-rich interfacial shell structure together with the pulp particles and oil droplet interface in the NFC wild cherry berry juice. This interfacial shell structure helps to improve the antioxidant capacity and antibacterial effect of the juice system and improve the physical stability of the system.

[0033] As a concept of this invention, this invention employs a design that combines the total polyphenol fraction B1 from aronia berries with plant protease to construct polyphenol-protein nanomicelles and uses ultra-high pressure-ultrasound synergistic cold sterilization. This design primarily aims to enhance the encapsulation stability, antioxidant activity protection, and microbial control performance of polyphenols in aronia berry juice. First, the total polyphenol fraction B1 is enriched from aronia berry juice using selective adsorption-elution technology with macroporous adsorption resin. Compared to crude extract, this significantly improves polyphenol purity and removes most sugars, organic acids, and other impurities that affect the grafting reaction, providing high-quality raw materials for subsequent enzymatic grafting. Laccase or horseradish peroxidase catalyzes the oxidation of the phenolic hydroxyl groups in polyphenols to generate quinone intermediates, which then undergo Michael addition or Schiff base condensation reactions with nucleophilic groups such as amino and thiol groups on the protein side chains, forming a covalently linked B1 polyphenol-protein graft polymer. This graft polymer combines the hydrophobicity of polyphenols with the hydrophilicity of the protein backbone, exhibiting amphiphilic molecular structure characteristics. Within a suitable pH range, the charged protein backbone provides electrostatic stability. By adjusting the ionic strength, the charge is partially shielded, reducing electrostatic repulsion and promoting the inward aggregation of hydrophobic polyphenol segments and the outward extension of hydrophilic protein segments. Under the action of ultrasonic cavitation shear force, core-shell structured nanomicelles are rapidly formed. The hydrophobic core of the micelles encapsulates the polyphenol fraction B1 and provides dual protection through the steric hindrance effect and antioxidant activity of the protein shell, effectively blocking the oxidative degradation of polyphenols by oxygen, light, and metal ions. The nanomicelle particle size is controlled within the range of 50-150 nm, ensuring long-term dispersion stability in the juice system and facilitating the adsorption at the interface of pulp particles and oil droplets to form a stable polyphenol-rich functional shell. High hydrostatic pressure treatment destroys the microbial cell membrane structure and inactivates polyphenol oxidase and peroxidase activity through instantaneous high pressure, avoiding damage to polyphenols and flavor substances caused by heat sterilization. Subsequent ultrasonic treatment uses acoustic cavitation microjets and shock waves to further destroy the residual structure of pressure-resistant bacteria and promote the penetration of antibacterial active substances into the bacterial cells. The two work synergistically to achieve deep sterilization under mild conditions. Polyphenol-protein nanomicelles can significantly improve the total polyphenol retention rate and antioxidant activity in fruit juice systems with low addition amounts. At the same time, they improve emulsification stability and prevent sedimentation and stratification through interfacial adsorption, achieving synergistic enhancement of function and quality without causing a significant increase in viscosity or deterioration in taste.

[0034] This invention also discloses a method for preparing aronia berry juice based on ultra-high pressure-ultrasound treatment, comprising the following steps:

[0035] S1. Preparation steps of total polyphenol fraction B1 of wild cherries: Total polyphenol fraction B1 of wild cherries was prepared from NFC wild cherries juice by adsorption-elution using a styrene-divinylbenzene copolymer macroporous adsorption resin column.

[0036] Preparation steps of S2.B1 polyphenol-protein graft polymer: The total polyphenol fraction B1 of wild cherry berry is grafted with at least one of whey protein isolate, pea protein isolate and gelatin under the catalysis of laccase and / or horseradish peroxidase to obtain B1 polyphenol-protein graft polymer solution.

[0037] S3. Construction steps of polyphenol-protein nanomicelles: Under the conditions of pH 4.0–4.5, sodium chloride concentration 10–50 mmol / L, ultrasonic frequency 20–25 kHz, power density 200–600 W / L, and ultrasonic time 2–8 min, the B1 polyphenol-protein graft polymer is allowed to self-assemble to form a polyphenol-protein nanomicelle dispersion with a volume average particle size of 50–150 nm and a polydispersity index (PDI) less than or equal to 0.25. Large aggregates with a diameter greater than 500 nm are removed by centrifugation or filtration.

[0038] S4. Juice Reconstruction and Synergistic Cold Sterilization Step: The polyphenol-protein nanomicelle dispersion is added to NFC aronia berry juice, with the amount of polyphenol-protein nanomicelles added being 0.10–0.80 wt% of the total juice mass based on micelle solids. The mixture is homogenized under high pressure at 20–50 MPa 1–3 times to obtain a uniform dispersion system. Subsequently, it is subjected to high hydrostatic pressure treatment at 320–380 MPa for 5–10 min at 20–25℃. After the high hydrostatic pressure treatment, it is subjected to ultrasonic treatment at 40–45℃ for 2–3 min. After cooling, it is aseptically filled to obtain aronia berry juice.

[0039] Furthermore, the styrene-divinylbenzene copolymer macroporous adsorption resin used in step S1 has a specific surface area of ​​400–900 m². 2 / g, with a pore size of 20–100 nm.

[0040] Furthermore, the ultrasonic treatment in step S3 adopts an intermittent mode, with a single working time of 5–10 seconds and a single pause time of 5–10 seconds.

[0041] Furthermore, the absolute value of the Zeta potential of the polyphenol-protein nanomicelle dispersion obtained after step S3 is 25–40 mV.

[0042] Furthermore, during the high hydrostatic pressure treatment in step S4, the pressure increase rate and pressure decrease rate are controlled at 100–300 MPa / min, the pressure holding time is 5–8 min, and the total bacterial count of the wild cherry berry juice obtained by this preparation method is less than or equal to 10 CFU / mL when stored at 4℃ for 90 days, and yeast and mold are not detected.

[0043] As another aspect of this invention, a step-by-step, controllable process route is employed, involving polyphenol fraction enrichment, enzymatic grafting polymerization, nanomicelle self-assembly, and ultra-high pressure-ultrasound synergistic cold sterilization. This route primarily enhances the process controllability, polyphenol activity protection efficiency, and product shelf-life stability in the preparation of aronia berry juice. In the polyphenol fraction preparation stage, a styrene-divinylbenzene copolymer macroporous adsorption resin with specific surface area and pore size is selected. Selective adsorption is achieved through the π-π interactions and hydrogen bonding between the resin's hydrophobic framework and the aromatic rings of the polyphenols. Elution is then performed using a water-ethanol mixed solvent with an appropriate ethanol volume fraction. This process improves polyphenol purity and recovery while removing interfering substances that could negatively impact subsequent reactions, providing a pure and highly active polyphenol substrate for enzymatic grafting. The enzymatic grafting reaction is catalyzed by laccase or horseradish peroxidase, offering advantages over chemical cross-linking methods, including milder reaction conditions, higher product safety, and controllable grafting sites. By precisely controlling the enzyme dosage, reaction time, and pH, the grafting degree is precisely regulated, ensuring that the B1 polyphenol-protein graft polymer possesses both sufficient polyphenol loading and good water solubility. Intermittent ultrasonic treatment was employed during the construction of the nanomicelles to avoid damage to the protein structure and polyphenol activity caused by localized overheating from continuous ultrasonication. By adjusting the pH value to near the isoelectric point of the proteins and adding appropriate salt ions to reduce electrostatic repulsion, the mechanical shearing provided by ultrasonic cavitation and the localized high temperature and high pressure microenvironment promoted the rapid aggregation and nucleation of hydrophobic polyphenol segments and the formation of a stable protective layer on the micelle surface by hydrophilic protein segments, resulting in uniform nanomicelles with narrow particle size distribution and high surface charge. High hydrostatic pressure cold sterilization, through precise control of the pressure increase and decrease rate and holding time, increased the permeability of microbial cell membranes and inactivated key enzymes without causing significant temperature rise, thus preserving the fresh flavor and heat-sensitive nutrients of aronia berry juice. Short-term ultrasonic treatment after pressure treatment utilized sound wave transmission to enhance the penetration of antibacterial components into residual microorganisms and disrupt the repair mechanism of sublethal bacteria. The synergistic effect of these two processes ensured the long-term microbial stability of the finished juice under refrigeration. The entire preparation process ensured batch-to-batch stability through multi-level quality control nodes and online monitoring of key parameters, enabling the large-scale production of high-quality functional aronia berry juice.

[0044] In this invention, the total polyphenol fraction B1 from aronia berries and plant proteins (whey protein isolate, pea protein isolate, or gelatin) each play their respective key functions and produce a significant synergistic effect. Polyphenol fraction B1 focuses on providing strong antioxidant activity, antibacterial activity, and biofunctionality. Its rich content of anthocyanins, proanthocyanidins, and other polyphenols has the ability to scavenge free radicals, chelate metal ions, inhibit lipid peroxidation, and disrupt the integrity of microbial cell membranes. However, free polyphenols are easily degraded by environmental factors and exhibit poor dispersion stability in fruit juice systems. Plant proteins focus on providing structural framework, interfacial activity, and colloidal stability. Their amphiphilic molecular structure allows them to adsorb at the oil-water interface, reducing interfacial tension. They stabilize the emulsion system through steric hindrance and electrostatic repulsion, while the proteins themselves also possess certain antioxidant and antibacterial capabilities. By enzymatically grafting polyphenols onto a protein backbone, the resulting B1 polyphenol-protein graft polymer integrates the advantages of both at the molecular level: the hydrophobicity of the polyphenol segments drives their aggregation towards the micelle core during self-assembly, forming a stable core through π-π stacking and hydrogen bonding interactions, achieving efficient encapsulation and environmental protection of the polyphenols; the protein backbone forms a hydrophilic shell on the micelle surface, providing electrostatic stability and preventing micelle aggregation through the charge of amino acid side chains. Simultaneously, the antioxidant activity of the protein shell itself synergistically provides a dual antioxidant barrier with the core polyphenols, significantly slowing down the polyphenol degradation rate. In the juice system, polyphenol-protein nanomicelles adsorb onto the interface between fruit pulp particles and oil droplets. The polyphenol component anchors to the interface through hydrophobic interactions, enhancing adsorption strength, while the protein component prevents particle aggregation and sedimentation through steric hindrance and charge repulsion. The interfacial shell network synergistically constructed by both improves the physical stability of the system and enhances overall antioxidant and antibacterial properties, achieving multifunctional integration and synergistic performance enhancement that are difficult to achieve with a single polyphenol or protein.

[0045] Beneficial technical effects

[0046] Significantly enhances polyphenol encapsulation stability and antioxidant activity protection: Amphiphilic graft polymers were constructed by grafting total polyphenol fraction B1 from wild aronia berries with plant protease. Under specific pH and ionic strength conditions, these polymers self-assembled to form polyphenol-protein nanomicelles with a particle size of 50-150 nm. Hydrophobic polyphenol segments aggregated in the micelle core for efficient encapsulation, while the hydrophilic protein shell provided steric hindrance stability and an antioxidant barrier, effectively isolating the polyphenols from oxidation and degradation by oxygen, light, and metal ions. This resulted in a total polyphenol retention rate of over 80% in the finished juice after 90 days of storage at 4℃, significantly better than the polyphenol stability of the unencapsulated system, thus fully preserving the health benefits and antioxidant potential of wild aronia berry juice.

[0047] Achieving mild cold sterilization and long-lasting microbial control: High hydrostatic pressure (320-380 MPa) and ultrasound are used in synergistic treatment. High hydrostatic pressure destroys the cell membrane structure of microorganisms and inactivates key enzymes through instantaneous pressure, avoiding the damage of heat-sensitive polyphenols and flavor substances to heat sterilization. Ultrasonic treatment uses acoustic cavitation microjets and shock waves to further destroy the residual structure of pressure-resistant bacteria and promote the penetration of antibacterial active substances. The synergistic effect of the two reduces the total number of colonies to below 10 CFU / mL and yeast and mold are not detected, achieving deep sterilization and long shelf-life stability while maintaining fresh flavor and nutrients.

[0048] Constructing a polyphenol-rich functional interface shell network and improving the physical stability of the system: Polyphenol-protein nanomicelles are dispersed and adsorbed at the interface between fruit pulp particles and oil droplets in the juice system. Through the hydrophobic interaction of polyphenols and the steric hindrance effect of proteins, a stable interface shell structure is formed, which not only enhances the overall antioxidant capacity and antibacterial effect of the system, but also improves the emulsification stability by reducing interfacial tension and preventing particle aggregation. It effectively prevents precipitation, stratification and browning. At a low addition amount (0.10-0.80wt%), the quality of the juice can be significantly improved without causing a significant increase in viscosity and deterioration in taste.

[0049] Multi-stage purification and enrichment ensures high purity and high activity of polyphenol raw materials: The total polyphenol fraction B1 is enriched from wild cherries juice using selective adsorption-elution technology with styrene-divinylbenzene copolymer macroporous adsorption resin. Compared with crude extract, this significantly improves polyphenol purity (total polyphenol mass fraction reaches 30-60 wt%) and removes impurities such as sugars and organic acids that affect the grafting reaction. The total polyphenol recovery rate is greater than 70%, providing high-quality raw materials for subsequent enzymatic grafting, improving grafting efficiency and product performance stability.

[0050] The process is mild and controllable, balancing large-scale production with product safety: It adopts mild and controllable process strategies such as enzymatic grafting instead of chemical cross-linking, intermittent ultrasound to avoid overheating from continuous ultrasound, and precise control of pressure rise and fall rates to prevent pressure surges. Under the premise of ensuring polyphenol activity and protein structural integrity, it achieves batch stability and product quality consistency of nanomicelles. No harmful chemical reagents are introduced in the entire preparation process, which meets the requirements of food safety regulations and is suitable for industrial production and market application of functional fruit juice beverages. Attached Figure Description

[0051] Figure 1 This is a bar chart showing the volume average particle size of nanomicelles.

[0052] Figure 2 The images show the superimposed FTIR spectra of B1 polyphenols, proteins, physical mixtures, and grafted polymers.

[0053] Figure 3 This is a bar chart showing the total bacterial count of each sample after storage at 4℃ for 90 days.

[0054] Figure 4 This is a viscosity-shear rate rheological curve.

[0055] Figure 5 The DLS particle size distribution diagram of the polyphenol-protein nanomicelles prepared in Example 1 is shown.

[0056] Figure 6 The Zeta potential distribution diagram is shown for the polyphenol-protein nanomicelles prepared in Example 1.

[0057] Figure 7 This is the HPLC polyphenol analysis chromatogram of fraction B1 of total polyphenols from wild cherry in Example 1. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0059] Example 1

[0060] Preparation of total polyphenol fraction B1 from wild cherifolia:

[0061] Fresh NFC arugula juice was taken, and the pH was adjusted to 3.5 with citric acid. The juice was then passed through a styrene-divinylbenzene copolymer macroporous adsorption resin column at 25°C and a space velocity of 2.0 column volumes per hour. The resin used in this example had a specific surface area of ​​650 m². 2 / g, pore size 60 nm, adsorption time 2.0 h. After adsorption, it was eluted with deionized water until no visible pigment was visible in the effluent, and then eluted with a 45 vol% water-ethanol mixed solvent, collecting the eluent containing polyphenols. The eluent was concentrated under reduced pressure at 50 °C to obtain aroniaberry total polyphenol fraction B1 with a total polyphenol mass fraction of 45 wt%. The recovery rate of total polyphenol mass fraction relative to the total polyphenol mass fraction in the feed juice, calculated as gallic acid equivalent, was 75 wt%.

[0062] Preparation of B1 polyphenol-protein graft polymer:

[0063] Dissolve isolated whey protein in deionized water to obtain a protein solution with a protein mass fraction of 3.0 wt%. Dissolve the prepared aronia berry total polyphenol fraction B1 in deionized water to obtain a B1 solution with a mass fraction of 3.0 wt%. Mix the B1 solution and protein solution at a mass ratio of 1:2, i.e., mix 100 g of B1 solution with 200 g of protein solution to make the system contain 3.0 g of B1 and 6.0 g of protein. Adjust the pH of the mixture to 6.0. Add laccase to the mixture at 32℃, with the enzyme addition amount being 2.5 wt% of the protein mass. React for 1.5 h with slow stirring. At the endpoint of the reaction, the free amino content is reduced by 30% relative to the ungrafted protein. Small molecule reaction byproducts and free polyphenols were removed by ultrafiltration membrane with a molecular weight cutoff of 20 kDa, and the solution was washed twice with deionized water to obtain a B1 polyphenol-protein graft polymer solution with a solids mass fraction of 6.0 wt% and a grafting degree of 24 mol%.

[0064] Preparation of polyphenol-protein nanomicelles:

[0065] The pH of the prepared B1 polyphenol-protein grafted polymer solution was adjusted to 4.25. Sodium chloride was added to the solution to a concentration of 30 mmol / L, and the solution was allowed to stand for 10 min to expose the hydrophobic regions of the protein. The solution was then subjected to ultrasonic treatment at a frequency of 22.5 kHz and a power density of 400 W / L in intermittent mode, with a single session lasting 7.5 s and a single pause lasting 7.5 s, for a total ultrasonic treatment time of 5 min. This yielded a polyphenol-protein nanomicelle dispersion with a volume average particle size of 100 nm, a polydispersity index (PDI) of 0.20, a zeta potential of 32 mV, and a polyphenol mass fraction of 17.5 wt% based on micelle solids. Large aggregates with a diameter greater than 500 nm were removed by centrifugation to obtain the polyphenol-protein nanomicelle dispersion for juice reconstruction.

[0066] Juice Reconstitution and Synergistic Cold Sterilization:

[0067] Add the polyphenol-protein nanomicelle dispersion to NFC aronia berry juice at an addition rate of 0.45 wt% (based on micelle solids) of the total weight of the finished juice, and stir slowly until homogeneous. The mixture is then homogenized twice at 35 MPa using a high-pressure homogenizer to ensure thorough dispersion of the polyphenol-protein nanomicelles within the juice system, distributing them at the interface between the pulp particles and oil droplets. This creates a polyphenol-rich interfacial shell structure with the pulp particles and oil droplets in the NFC aronia berry juice. This interfacial shell structure enhances the antioxidant capacity and antibacterial effect of the juice system, and improves its physical stability. Subsequently, the mixture undergoes high hydrostatic pressure treatment at 350 MPa and 22.5℃, with the pressurization and depressurization rates controlled at 200 MPa / min and the pressure held for 6.5 min. After the high hydrostatic pressure treatment, the mixture is ultrasonically treated at 45℃ for 2.5 min. After cooling, the mixture is aseptically filled to obtain the finished aronia berry juice. When the finished aronia berry juice was stored at 4℃ for 90 days, the total polyphenol retention rate of aronia berries was 85%, and the total bacterial count, determined according to the test method specified in GB 4789.2, was 5 CFU / mL. Yeast and mold were not detected.

[0068] Features of this embodiment: This embodiment employs a moderate parameter configuration, with each process parameter selected within the middle range, including a B1 polyphenol mass fraction of 45 wt%, a grafting degree of 24 mol%, a nanomicelle particle size of 100 nm, a polyphenol mass fraction of 17.5 wt%, a micelle addition amount of 0.45 wt%, and a high hydrostatic pressure of 350 MPa. This parameter combination ensures the stability and repeatability of the process, with each process step having a wide operating window, facilitating industrial production control. The finished juice exhibits good polyphenol retention and microbial control during storage, with overall stable and reliable quality. This embodiment is suitable for standardized commercial production of aronia berry juice, particularly suitable for large-scale production scenarios with high requirements for process stability and product quality consistency, and can meet the demand for functional juice products in the daily consumer market.

[0069] Example 2

[0070] Preparation of total polyphenol fraction B1 from wild cherifolia:

[0071] Fresh NFC arugula juice was taken, and the pH was adjusted to 3.2 with citric acid. The juice was then passed through a styrene-divinylbenzene copolymer macroporous adsorption resin column at 28°C and a space velocity of 1.5 column volumes per hour. The resin used in this embodiment had a specific surface area of ​​750 m². 2 / g, pore size 40 nm, adsorption time 2.5 h. After adsorption, it was eluted with deionized water until no visible pigment was visible in the effluent, and then eluted with a water-ethanol mixed solvent of 52 vol% (v / v), and the eluent containing polyphenols was collected. The eluent was concentrated under reduced pressure at 55 °C to obtain aroniaberry total polyphenol fraction B1 with a total polyphenol mass fraction of 52 wt%, and the recovery rate of total polyphenol mass fraction relative to the total polyphenol mass fraction in the feed juice was 78 wt% (calculated as gallic acid equivalent).

[0072] Preparation of B1 polyphenol-protein graft polymer:

[0073] Pea protein isolate was dissolved in deionized water to obtain a protein solution with a protein mass fraction of 2.2 wt%. The total polyphenol fraction B1 of aronia berries prepared above was dissolved in deionized water to make the B1 solution with a mass fraction of 4.0 wt%. The B1 solution and protein solution were mixed at a mass ratio of 1:1.5, i.e., 100 g of B1 solution and 272.7 g of protein solution were mixed to make the system contain 4.0 g of B1 and 6.0 g of protein. The pH of the mixture was adjusted to 5.8. Horseradish peroxidase was added to the mixture at 36℃, with the enzyme added at 3.8 wt% of the protein mass. The reaction was carried out for 2.3 h with slow stirring. At the endpoint of the reaction, the free amino content was reduced by 40% relative to the ungrafted protein. Small molecule reaction byproducts and free polyphenols were removed by using an ultrafiltration membrane with a molecular weight cutoff of 15 kDa, and the solution was washed twice with deionized water to obtain a B1 polyphenol-protein graft polymer solution with a solids mass fraction of 7.5 wt% and a grafting degree of 32 mol%.

[0074] Preparation of polyphenol-protein nanomicelles:

[0075] The pH of the prepared B1 polyphenol-protein grafted polymer solution was adjusted to 4.12. Sodium chloride was added to the solution to a concentration of 38 mmol / L, and the solution was allowed to stand for 12 min to expose the hydrophobic regions of the protein. The solution was then subjected to ultrasonic treatment at a frequency of 23.5 kHz and a power density of 500 W / L in intermittent mode, with a single session lasting 8 s and a single pause lasting 6 s, for a total ultrasonic treatment time of 6.5 min. This yielded a polyphenol-protein nanomicelle dispersion with a volume average particle size of 72 nm, a polydispersity index (PDI) of 0.17, a zeta potential of 36 mV, and a polyphenol mass fraction of 25 wt% (based on micelle solids). Large aggregates with a diameter greater than 500 nm were removed by centrifugation to obtain the polyphenol-protein nanomicelle dispersion for juice reconstruction.

[0076] Juice Reconstitution and Synergistic Cold Sterilization:

[0077] Add the polyphenol-protein nanomicelle dispersion to NFC aronia berry juice at an addition rate of 0.65 wt% of the total mass of the finished juice (based on micelle solids), and stir slowly until homogeneous. Homogenize three times at 42 MPa using a high-pressure homogenizer to ensure the polyphenol-protein nanomicelles are fully dispersed in the juice system and distributed at the interface between pulp particles and oil droplets. This forms a polyphenol-rich interfacial shell structure with the pulp particles and oil droplets in the NFC aronia berry juice. This interfacial shell structure enhances the antioxidant capacity and antibacterial effect of the juice system and improves its physical stability. Subsequently, high hydrostatic pressure treatment is performed at 368 MPa and 21°C, with the pressurization and depressurization rates controlled at 240 MPa / min and the pressure held for 7.0 min. After high hydrostatic pressure treatment, ultrasonic treatment is performed at 45°C for 2.8 min. The mixture is then cooled and aseptically filled to obtain the finished aronia berry juice. When the finished aronia berry juice was stored at 4℃ for 90 days, the total polyphenol retention rate of aronia berries was 89%, and the total bacterial count, determined according to the test method specified in GB 4789.2, was 3 CFU / mL. Yeast and mold were not detected.

[0078] Features of this embodiment: This embodiment employs a high-antioxidant performance optimized configuration. By increasing the B1 polyphenol mass fraction to 52 wt%, grafting degree to 32 mol%, nanomicelle polyphenol loading to 25 wt%, and micelle addition to 0.65 wt%, and using pea protein isolate as the carrier protein, along with a small nanomicelle particle size of 72 nm and a high ultrasonic power density of 500 W / L, efficient enrichment and stabilization of polyphenols are achieved. This parameter combination significantly enhances the antioxidant capacity of the finished juice, achieving a polyphenol retention rate of 89 wt% after 90 days, with excellent microbial control and a total bacterial count of only 3 CFU / mL. This embodiment is particularly suitable for the development of high-end functional juice products and is well-suited for the health food market with high requirements for antioxidant performance and nutrient retention, such as sports nutrition drinks and anti-aging functional beverages.

[0079] Example 3

[0080] Preparation of total polyphenol fraction B1 from wild cherifolia:

[0081] Fresh NFC arugula juice was taken, and the pH was adjusted to 3.8 with citric acid. The juice was then passed through a styrene-divinylbenzene copolymer macroporous adsorption resin column at 22°C and a space velocity of 2.6 times the column volume per hour. The resin used in this embodiment had a specific surface area of ​​520 m². 2 / g, pore size 78 nm, adsorption time 1.3 h. After adsorption, it was eluted with deionized water until no visible pigment was visible in the effluent, and then eluted with a 35 vol% water-ethanol mixed solvent, collecting the eluent containing polyphenols. The eluent was concentrated under reduced pressure at 43 °C to obtain aroniaberry total polyphenol fraction B1 with a total polyphenol mass fraction of 36 wt%, and the recovery rate of total polyphenol mass fraction relative to the total polyphenol mass fraction in the feed juice was 72 wt%, based on gallic acid equivalent.

[0082] Preparation of B1 polyphenol-protein graft polymer:

[0083] Gelatin was dissolved in deionized water to obtain a protein solution with a protein mass fraction of 4.2 wt%. The total polyphenol fraction B1 of aronia berries prepared above was dissolved in deionized water to make the B1 solution with a mass fraction of 1.8 wt%. The B1 solution and protein solution were mixed at a mass ratio of 1:2.6, i.e., 100 g of B1 solution and 110.7 g of protein solution were mixed to make the system contain 1.8 g of B1 and 4.65 g of protein. The pH of the mixture was adjusted to 6.3. A mixture of laccase and horseradish peroxidase was added to the mixture at 27℃, with a mass ratio of 1:1. The total amount of enzyme added was 1.5 wt% of the protein. The reaction was carried out with slow stirring for 0.8 h. At the endpoint of the reaction, the free amino content was reduced by 18% relative to the ungrafted protein. Small molecule reaction byproducts and free polyphenols were removed by ultrafiltration membrane with a molecular weight cutoff of 25 kDa, and the solution was washed once with deionized water to obtain a B1 polyphenol-protein graft polymer solution with a solids mass fraction of 3.8 wt% and a grafting degree of 15 mol%.

[0084] Preparation of polyphenol-protein nanomicelles:

[0085] The pH of the prepared B1 polyphenol-protein grafted polymer solution was adjusted to 4.38. Sodium chloride was added to the solution to a concentration of 18 mmol / L, and the solution was allowed to stand for 7 min to expose the hydrophobic regions of the protein. The solution was then subjected to ultrasonic treatment at a frequency of 20.5 kHz and a power density of 280 W / L in intermittent mode, with a single session lasting 5.5 s and a single pause lasting 8.5 s, for a total ultrasonic treatment time of 3.5 min. This yielded a polyphenol-protein nanomicelle dispersion with a volume average particle size of 130 nm, a polydispersity index (PDI) of 0.23, a zeta potential of 26 mV, and a polyphenol mass fraction of 10 wt% (based on micelle solids). Large aggregates with a diameter greater than 500 nm were removed by filtration to obtain the polyphenol-protein nanomicelle dispersion for juice reconstruction.

[0086] Juice Reconstitution and Synergistic Cold Sterilization:

[0087] Add the polyphenol-protein nanomicelle dispersion to NFC aronia berry juice at an addition rate of 0.22 wt% (based on micelle solids) of the total weight of the finished juice, and stir slowly until homogeneous. Homogenize once at 25 MPa using a high-pressure homogenizer to ensure the polyphenol-protein nanomicelles are fully dispersed in the juice system and distributed at the interface between pulp particles and oil droplets. This forms a polyphenol-rich interfacial shell structure with the pulp particles and oil droplets in the NFC aronia berry juice. This interfacial shell structure enhances the antioxidant capacity and antibacterial effect of the juice system and improves its physical stability. Subsequently, high hydrostatic pressure treatment is performed at 332 MPa and 23.5℃, with the pressurization and depressurization rates controlled at 160 MPa / min and the pressure held for 5.3 min. After high hydrostatic pressure treatment, ultrasonic treatment is performed at 41℃ for 2.1 min. The mixture is then cooled and aseptically filled to obtain the finished aronia berry juice. When the finished aronia berry juice was stored at 4℃ for 90 days, the total polyphenol retention rate of aronia berries was 82%, and the total bacterial count, determined according to the test method specified in GB 4789.2, was 8 CFU / mL. Yeast and mold were not detected.

[0088] Features of this embodiment: This embodiment adopts a cost-effective and stability-balanced configuration, using gelatin as the carrier protein to reduce raw material costs. It employs milder process parameters, including a B1 polyphenol mass fraction of 36 wt%, a grafting degree of 15 mol%, a low ultrasonic power density of 280 W / L, a relatively large nanomicelle particle size of 130 nm, and a low micelle addition amount of 0.22 wt%. This parameter combination significantly reduces production costs and energy consumption while ensuring the product's basic functionality. The process conditions are mild, equipment wear is minimal, and it is suitable for long-term continuous production. The finished juice meets standard requirements, with a polyphenol retention rate of 82 wt% and good microbial control. This embodiment is particularly suitable for economical functional juice products in the mass consumer market, catering to price-sensitive consumers who still prioritize health benefits, such as student drinks and daily health beverages, for large-scale production applications.

[0089] Example 4

[0090] Preparation of total polyphenol fraction B1 from wild cherifolia:

[0091] Fresh NFC arugula juice was taken, and the pH was adjusted to 3.1 with citric acid. The juice was then passed through a styrene-divinylbenzene copolymer macroporous adsorption resin column at 26°C and a space velocity of 2.8 times the column volume per hour. The resin used in this example had a specific surface area of ​​830 m². 2 / g, pore size 28 nm, adsorption time 1.2 h. After adsorption, it was eluted with deionized water until no visible pigment was visible in the effluent, and then eluted with a water-ethanol mixed solvent of 57 vol% (v / v), and the eluent containing polyphenols was collected. The eluent was concentrated under reduced pressure at 58 °C to obtain aroniaberry total polyphenol fraction B1 with a total polyphenol mass fraction of 57 wt%, and the recovery rate of total polyphenol mass fraction of aroniaberry relative to the total polyphenol mass fraction of the feed juice was 77 wt%, based on gallic acid equivalent.

[0092] Preparation of B1 polyphenol-protein graft polymer:

[0093] Dissolve whey protein isolate and pea protein isolate in deionized water at a 1:1 mass ratio to obtain a protein solution with a total protein mass fraction of 4.6 wt%. Dissolve the total polyphenol fraction B1 of aronia berries prepared above in deionized water to obtain a B1 solution with a mass fraction of 4.6 wt%. Mix the B1 solution and protein solution at a B1:1.1 mass ratio, i.e., mix 100 g of B1 solution with 100 g of protein solution to make the system contain 4.6 g of B1 and 4.6 g of protein, with an actual mass ratio of 1:1. The amount of protein solution needs to be adjusted to 110 g to make the B1 mass 4.6 g and the protein mass 5.06 g, with a mass ratio of approximately 1:1.1. Adjust the pH of the mixture to 6.45. Horseradish peroxidase was added to the mixture at 39°C at an amount equivalent to 4.6 wt% of the protein mass. The reaction was carried out under slow stirring for 2.8 h. At the reaction endpoint, the free amino content was reduced by 48% relative to the ungrafted protein. Small molecule reaction byproducts and free polyphenols were removed by ultrafiltration with a molecular weight cutoff of 28 kDa. The mixture was then washed three times with deionized water to obtain a B1 polyphenol-protein graft polymer solution with a solids mass fraction of 9.2 wt% and a grafting degree of 38 mol%.

[0094] Preparation of polyphenol-protein nanomicelles:

[0095] The pH of the prepared B1 polyphenol-protein grafted polymer solution was adjusted to 4.03. Sodium chloride was added to the solution to a concentration of 46 mmol / L, and the solution was allowed to stand for 14 min to expose the hydrophobic regions of the protein. The solution was then subjected to ultrasonic treatment at a frequency of 24.5 kHz and a power density of 560 W / L in intermittent mode, with a single session lasting 9.5 s and a single pause lasting 5.5 s, for a total ultrasonic treatment time of 7.5 min. This yielded a polyphenol-protein nanomicelle dispersion with a volume average particle size of 58 nm, a polydispersity index (PDI) of 0.16, a zeta potential of 39 mV, and a polyphenol mass fraction of 28 wt% based on micelle solids. Large aggregates with a diameter greater than 500 nm were removed by centrifugation to obtain the polyphenol-protein nanomicelle dispersion for juice reconstruction.

[0096] Juice Reconstitution and Synergistic Cold Sterilization:

[0097] Add the polyphenol-protein nanomicelle dispersion to NFC aronia berry juice at an addition rate of 0.13 wt% of the total mass of the finished juice (based on micelle solids), and stir slowly until homogeneous. Homogenize twice at 47 MPa using a high-pressure homogenizer to ensure the polyphenol-protein nanomicelles are fully dispersed in the juice system and distributed at the interface between pulp particles and oil droplets. This forms a polyphenol-rich interfacial shell structure with the pulp particles and oil droplets in the NFC aronia berry juice. This interfacial shell structure enhances the antioxidant capacity and antibacterial effect of the juice system and improves its physical stability. Subsequently, high hydrostatic pressure treatment is performed at 374 MPa and 20.5℃, with the pressurization and depressurization rates controlled at 280 MPa / min and the pressure held for 7.5 min. After high hydrostatic pressure treatment, ultrasonic treatment is performed at 43℃ for 2.9 min. The mixture is then cooled and aseptically filled to obtain the finished aronia berry juice. When the finished aronia berry juice was stored at 4℃ for 90 days, the total polyphenol retention rate of aronia berries was 87%, and the total bacterial count, determined according to the test method specified in GB 4789.2, was 2 CFU / mL. Yeast and mold were not detected.

[0098] Features of this embodiment: This embodiment uses parameter configuration close to the boundary of the technical solution, with several key parameters selected in the boundary region of the range, including a B1 polyphenol mass fraction of 57 wt% close to the upper limit, a pH value of 3.1 close to the lower limit, and a resin specific surface area of ​​830 m². 2The parameters, including a g / g ratio close to the upper limit, resin pore size of 28 nm close to the lower limit, grafting degree of 38 mol% close to the upper limit, nanomicelle particle size of 58 nm close to the lower limit, polyphenol mass fraction of 28 wt% close to the upper limit, ultrasonic power density of 560 W / L close to the upper limit, and high hydrostatic pressure of 374 MPa close to the upper limit, along with a composite protein system, demonstrate the wide applicability and process flexibility of the technical solution. Even under relatively extreme process conditions, excellent product performance can be achieved, with a polyphenol retention rate of 87 wt% and excellent microbial control, resulting in a total bacterial count of only 2 CFU / mL. This embodiment is particularly suitable for professional applications with extremely high product performance requirements, such as medical and health beverages, professional nutritional supplements for athletes, and antioxidant function verification studies—applications that require exploring the limits of product performance at the technological boundaries.

[0099] Comparative Example 1: It is basically the same as Example 1, except that the mass fraction of total polyphenols B1 from wild cherries is 18 wt%, while the amounts of other components and preparation conditions remain unchanged.

[0100] Comparative Example 2: Basically the same as Example 1, except that the mass fraction of total polyphenols B1 from wild cherries is 68 wt%, while the amounts of other components and preparation conditions remain unchanged.

[0101] Comparative Example 3: It is basically the same as Example 1, except that the mass ratio of B1 to protein is 1:0.6, and the amounts of other components and preparation conditions remain unchanged.

[0102] Comparative Example 4: It is basically the same as Example 1, except that laccase was not added for enzymatic grafting reaction. Instead, the total polyphenol fraction B1 of wild cherry was directly mixed with isolated whey protein and then self-assembled. The amount of other components and preparation conditions remained unchanged.

[0103] Comparative Example 5: It is basically the same as Example 1, except that the volume average particle size of the polyphenol-protein nanomicelles is 280 nm, which is achieved by adjusting the ultrasonic power density to 120 W / L and the ultrasonic time to 1.5 min. The amount of other components and preparation conditions remain unchanged.

[0104] Comparative Example 6: It is basically the same as Example 1, except that the amount of polyphenol-protein nanomicelles added to cherriesberry juice is 0.05 wt% of the total mass of the finished juice, based on the micelle solids. The amounts of other components and the preparation conditions remain unchanged.

[0105] Comparative Example 7: It is basically the same as Example 1, except that the amount of polyphenol-protein nanomicelles added to cherifolia juice is 1.10 wt% of the total mass of the finished juice, based on the micelle solids. The amounts of other components and the preparation conditions remain unchanged.

[0106] Comparative Example 8: It is basically the same as Example 1, except that the pressure of the high hydrostatic treatment is 280 MPa, while the amount of other components and the preparation conditions remain unchanged.

[0107] Comparative Example 9: Basically the same as Example 1, except that the high hydrostatic pressure treatment pressure is 420 MPa, while the dosage of other components and preparation conditions remain unchanged.

[0108] Comparative Example 10: Basically the same as Example 1, except that the sodium chloride concentration during self-assembly was 5 mmol / L, while the amounts of other components and preparation conditions remained unchanged.

[0109] Comparative Example 11: Basically the same as Example 1, except that the pH value during the self-assembly process was 3.2, while the amounts of other components and preparation conditions remained unchanged.

[0110] Comparative Example 12: Basically the same as Example 1, except that the high-pressure homogenization pressure is 8 MPa, while the amount of other components and preparation conditions remain unchanged.

[0111] Comparative Example 13: It is basically the same as Example 1, except that the high hydrostatic pressure treatment step is omitted and only conventional pasteurization is performed. The sterilization conditions are 85°C for 15 seconds followed by rapid cooling. The dosage of other components and preparation conditions remain unchanged.

[0112] Performance testing:

[0113] Test Subject: Samples of finished aronia berry juice stored at 4℃ for 90 days. Test Objective: To evaluate the retention rate of total polyphenols in aronia berries during storage and to verify the protective effect of the polyphenol-protein nanomicelle encapsulation system on polyphenols. Test Principle: The total polyphenol content was determined using the Folin-Ciocalteu colorimetric method. A standard curve was plotted using gallic acid as a standard, and the change in polyphenol content before and after storage was quantitatively determined by colorimetry. Experimental Method: 0.5 mL of sample was taken, 2.5 mL of Folin-Ciocalteu reagent was added, mixed well, and allowed to stand for 3 minutes. Then, 2.0 mL of 7.5% Na2CO3 solution was added, and the mixture was reacted at 25℃ in the dark for 2 hours. The absorbance was measured at a wavelength of 760 nm. The total polyphenol content was calculated according to the standard curve. Polyphenol retention rate = (total polyphenol content after 90 days of storage / initial total polyphenol content) × 100%. Key Parameters: Test temperature 25±2℃, color development time 2 hours, wavelength 760 nm, parallel determination n≥3. Data processing: Take the average of three parallel determinations, calculate the standard deviation, and retain the polyphenol retention rate to the nearest integer.

[0114] Test Subject: Samples of finished wild cherries fruit juice stored at 4℃ for 90 days. Test Objective: To evaluate the effectiveness of the synergistic cold sterilization process in controlling microorganisms and ensuring the product's microbiological safety. Test Principle: The total bacterial count was determined using the plate count method. Viable bacteria in the sample were allowed to form visible colonies on agar medium through a dilution and plating method. The colony count was then converted to the number of colonies per unit volume. Experimental Method: Under aseptic conditions, 1 mL of sample was taken and serially diluted 10-fold with sterile physiological saline to 10⁻⁶. -3 Take 0.1 mL of each dilution sample and spread it onto plate counting agar medium. Perform two replicates for each dilution. Incubate at 36±1℃ for 48 hours. Select plates with colony counts between 30-300 for counting. Key parameters: Incubation temperature 36±1℃, incubation time 48±2 hours, effective counting range 30-300 CFU / plate, parallel determination n≥2. Data processing: Total colony count (CFU / mL) = average colony count on plate × dilution factor × 10, round the result to the nearest integer.

[0115] Test Subjects: Polyphenol-protein nanomicelle dispersion and finished aronia berry juice. Test Objective: To determine the volume average particle size and polydispersity index (PDI) of the nanomicelles, and to evaluate the uniformity of the micelle size and its stability in the juice system. Test Principle: Based on the principle of dynamic light scattering, the Brownian motion of particles in solution causes the intensity of scattered light to fluctuate over time. The diffusion coefficient of the particles is calculated using the autocorrelation function, and the hydrodynamic diameter is calculated according to the Stokes-Einstein equation. Experimental Method: An appropriate amount of sample was diluted with deionized water to a suitable concentration to avoid multiple scattering effects. The sample was added to a cuvette and measured at 25℃ using a dynamic light scattering particle size analyzer with a scattering angle of 173° and a measurement time of 3 minutes. Each sample was measured three times. The instrument automatically calculated the volume average particle size and PDI value. Key Parameters: Test temperature 25℃, scattering angle 173°, sample concentration 0.01-0.1wt%, equilibration time 2 minutes, parallel measurements n≥3. Data Processing: The average value of the three measurements was taken, with the particle size retained to the nearest integer and the PDI retained to two decimal places.

[0116] Test Subject: Polyphenol-protein nanomicelle dispersion. Test Objective: To determine the Zeta potential of nanomicelles and evaluate the surface charge and electrostatic stability of the dispersion system. Test Principle: Based on the principle of electrophoretic light scattering, charged particles undergo directional movement in an applied electric field. The electrophoretic mobility of the particles is measured using laser Doppler velocimetry, and the Zeta potential is calculated according to the Smoluchowski equation. Experimental Method: An appropriate amount of sample was diluted with deionized water until the conductivity was less than 2 mS / cm, added to the Zeta potential test cell, and measured using a Zeta potential meter at 25℃. Each sample was measured in triplicate, and the instrument automatically calculated the average and standard deviation of the Zeta potential. Key Parameters: Test temperature 25℃, sample conductivity <2 mS / cm, pH value consistent with the original sample, equilibration time 2 minutes, parallel measurements n≥3. Data Processing: The average of the three measurements was taken, with the Zeta potential rounded to the nearest integer and the standard deviation rounded to one decimal place.

[0117] Test Subjects: Finished aronia berry juice and control samples. Test Objective: To evaluate the antioxidant activity of the juice and verify the enhancing effect of polyphenol-protein nanomicelles on the antioxidant capacity of the juice. Test Principle: Based on the DPPH free radical scavenging method, the antioxidants in the sample react with DPPH free radicals, causing the purple color to fade. The free radical scavenging rate is calculated by measuring the absorbance change at 517 nm. Experimental Method: Take 0.2 mL of appropriately diluted sample, add 3.8 mL of 0.1 mmol / L DPPH-ethanol solution, mix well, and react at 25℃ in the dark for 30 minutes. Measure the absorbance of sample A at 517 nm. Use anhydrous ethanol instead of the sample to measure the absorbance of blank A. DPPH scavenging rate = (1 - sample A / blank A) × 100%. Key Parameters: Reaction temperature 25℃, reaction time 30 minutes, DPPH concentration 0.1 mmol / L, wavelength 517 nm, parallel determination n≥3. Data Processing: Take the average of three parallel determinations, and retain the scavenging rate to one decimal place.

[0118] Test Subjects: Samples of finished aronia berry juice stored for different periods. Test Objective: To evaluate the rheological properties of the juice and verify the effect of polyphenol-protein nanomicelle addition on the system's viscosity and flowability. Test Principle: The apparent viscosity of the sample at different shear rates was measured using a rotational rheometer. The fluid type and flow characteristics were determined through rheological curves. Experimental Method: 10 mL of sample was added to the rheometer test cup. A cone-plate system was selected, the test temperature was set to 25℃, the equilibrium time to 5 minutes, and the shear rate range to 0.1-100 s. -1 Twenty test points were set up using a logarithmic increment method to measure the shear stress and apparent viscosity at various shear rates, and rheological curves were plotted. Key parameters: test temperature 25±0.1℃, shear rate 0.1-100 s⁻¹ -1The equilibrium time was 5 minutes, the cone-plate gap was 1 mm, and the parallel measurements were performed (n≥3). Data processing: The average of the three measurements was taken, and a shear rate-viscosity curve was plotted. The viscosity was retained to two significant figures.

[0119] Figure 1 The bar chart shows the volume-average particle size of the nanomicelles. Fixed parameters included the types of aronia berry polyphenols and proteins, the enzymatic grafting process, 90-day storage at 4°C, and the same DLS instrument and refractive index setting. Variations included the degree of enzymatic grafting, protein / polyphenol ratio, and process conditions in Examples 1 to 4 and Comparative Examples 1 to 13. Seventeen nanomicelles were prepared, and particle size and standard deviation were determined using dynamic light scattering. Results showed that the particle sizes in Examples 1 to 4 were concentrated between approximately 58 and 130 nm with small errors, while some comparative examples showed increased particle sizes to 180 to 320 nm with significant fluctuations. This indicates that under the same testing conditions, optimized polyphenol-protease grafting conditions can significantly reduce particle size and obtain a narrower particle size distribution, providing a foundation for subsequent stability and flavor control.

[0120] Figure 2 This is a superimposed FTIR spectrum of B1 polyphenols, proteins, physical mixtures, and grafted polymers, with fixed parameters including FTIR instrument model and scan wavenumber range of 800 to 1800 cm⁻¹. -1 The resolution and sample preparation conditions were consistent, with the parameters varying between B1 polyphenol alone, protein alone, the physical mixture prepared in Comparative Example 4, and the polyphenol-protein graft polymer prepared in Example 1. Results showed that B1 polyphenol was at 1715 cm⁻¹. -1 and 1230 cm -1 Characteristic peaks exist at 1650 and 1540 cm⁻¹, with proteins showing peaks at these values. -1 The absorption of amide I and amide II was characterized at the 1715 cm⁻¹, and the physical mixture essentially superimposed the absorption. -1 The original peak position was maintained, while the carbonyl peak in the grafted polymer changed from 1715 cm⁻¹. -1 Redshifted to 1698 cm -1 And it weakened significantly, while 1540 cm -1 Peak enhancement at 1280 cm⁻¹ -1 The appearance of a new absorption band indicates that the shift in peak position and the generation of new peaks suggest that a new covalent bond is formed between the polyphenol and the protein, rather than a simple physical adsorption, which supports the existence of an enzyme-catalyzed graft structure at the molecular level.

[0121] Figure 3A bar chart is presented to show the total bacterial count of each sample after 90 days of storage at 4℃. The fixed parameters were the same batch of wild cherifolia polyphenol matrix, the same ultra-high pressure-ultrasonic synergistic cold sterilization process, and the 4℃ storage time. The varying parameters were the differences in formulation and process combination between Examples 1 to 4 and Comparative Examples 1 to 13. The total bacterial count was determined using the plate count method, and the mean and standard deviation were plotted. The results showed that the total bacterial count of samples from Examples 1 to 4 was controlled below 10 CFU / mL, while the total bacterial count of some comparative examples, especially Comparative Example 8 and Comparative Example 13, increased to the hundreds or even higher, with significant fluctuations. This indicates that under the same storage conditions, the optimized nanomicelle structure and process combination can balance sterilization and system environment regulation, effectively suppressing microbial growth to a low level.

[0122] Figure 4 The viscosity-shear rate rheological curves are shown. The fixed parameters were measurement temperature, rheometer rotor specifications, and the samples were aronia berry beverage systems stored at 4℃ for 90 days. The varying parameters were the differences in nanomicelle structure, protein content, and formulation ratios between Examples 1 to 4 and Comparative Examples 6 and 7, ranging from 0.1 to 100 s⁻¹. -1 Viscosities were tested within the shear rate range and plotted on logarithmic coordinates. The results showed that all samples exhibited shear-thinning properties, but Examples 1 to 4 showed a significant decrease in viscosity at 10 s⁻¹. -1 The viscosity was approximately 12 to 22 mPa·s, and decreased smoothly with increasing shear, falling within the range suitable for beverage flow and mouthfeel. In contrast, Comparative Example 7 had an initial viscosity exceeding 50 mPa·s, exhibiting excessive thickening, while Comparative Example 6 had lower viscosity and poorer stability. This indicates that by using the polyphenol-protein shell to regulate the microstructure, the viscosity of the system can be controlled within a more suitable range while ensuring suspension stability.

[0123] Figure 5 The image shows the DLS particle size distribution of the polyphenol-protein nanomicelles prepared in Example 1. The fixed parameters were: total polyphenol fraction B1 from Aronia arguta (45 wt%), B1 to isolated whey protein ratio 1:2, grafting degree 24 mol%, enzyme addition 2.5 wt% of protein, reaction temperature 32°C, reaction time 1.5 h, ultrafiltration molecular weight cutoff 20 kDa, pH for nanomicelle preparation 4.25, and sodium chloride concentration 30 mmol·L⁻¹. -1 Ultrasonic frequency 22.5 kHz, power density 400 W·L -1The total sonication time was 5 min, and centrifugation removed coarse aggregates with a diameter greater than 500 nm. The parameters for variation were: the particle size distribution of the self-assembled polyphenol-protein graft polymer ranged from approximately 20 to 300 nm, with a volume average particle size concentrated at 100 ± 5 nm; and the polydispersity index (PDI) was 0.20 ± 0.02. The particle size distribution exhibited a narrow, unimodal distribution, with the main peak located around 100 nm. The proportion of particles smaller than 50 nm and larger than 200 nm was low, indicating that under grafting conditions of 24 mol% and pH 4.25, uniformly sized and well-dispersed polyphenol-protein nanomicelles were formed, providing a particle size basis for the subsequent construction of a stable interfacial shell in the juice system.

[0124] Figure 6 The image shows the Zeta potential distribution of the polyphenol-protein nanomicelles prepared in Example 1. The fixed parameters were: total polyphenol fraction B1 from aronia berries 45 wt%, B1 to isolated whey protein ratio 1:2, grafting degree 24 mol%, enzyme addition 2.5 wt% of protein, reaction temperature 32°C, reaction time 1.5 h, ultrafiltration molecular weight cutoff 20 kDa, pH for nanomicelle preparation 4.25, and sodium chloride concentration 30 mmol·L⁻¹. -1 Ultrasonic frequency 22.5 kHz, power density 400 W·L -1 The total sonication time was 5 min. The variation parameters were that the zeta potential distribution range of the nanomicelles was approximately -60 to 0 mV, with a peak potential of -32 ± 2 mV. The zeta potential distribution showed a single-peak shape, with a high proportion of particles having an absolute value higher than 30 mV. This indicates that the polyphenol-protein graft polymer has a high density of charge on its surface and maintains strong electrostatic repulsion under pH 4.25 conditions, thereby inhibiting the aggregation and sedimentation between nanomicelles and enabling the system to obtain good colloidal stability. This provides an interfacial electrical basis for maintaining the clarity of the juice and preventing stratification during storage.

[0125] Figure 7The HPLC chromatogram of total polyphenol fraction B1 from Aronia chinensis in Example 1 is shown. The fixed parameters were: NFC Aronia chinensis juice adjusted to pH 3.5 and adsorbed onto a styrene-divinylbenzene copolymer macroporous adsorption resin at 25°C, 2.0 column volumes, and a space velocity of 2.0 h. The adsorption was carried out sequentially with deionized water until no visible pigment was found, followed by elution with a 45 vol% water-ethanol mixed solvent. The fraction B1 with a total polyphenol content of 45 wt% was obtained by concentration under reduced pressure at 50°C. The total polyphenol recovery rate was 75 wt% based on gallic acid equivalent. The variable parameters were the peak area and relative peak height of each polyphenol component within the retention time range of 0 to 30 min. The HPLC polyphenol analysis chromatogram showed multiple well-separated peaks within the range of 6 to 22 min. The main peak area accounted for a high proportion of the total peak area, and the baseline was stable without obvious tailing. This indicates that the process of elution with 45 vol% water-ethanol and concentration under reduced pressure at 50°C can efficiently enrich and maintain the integrity of aronia berry polyphenol components. This is consistent with the fact that the total polyphenol retention rate of the finished aronia berry juice was 85±3 wt% and the DPPH scavenging rate was 78±3 wt% after storage at 4°C for 90 days. This proves that the preparation of B1 and the nanomicelle encapsulation together achieved efficient enrichment and stable delivery of polyphenols.

[0126] As can be seen from the performance of the examples and comparative examples in Table 1, Examples 1-4, by using the enzymatic grafting of total polyphenol fraction B1 from wild cherifolia with protein to form polyphenol-protein nanomicelles and combining it with ultra-high pressure-ultrasound synergistic cold sterilization technology, are significantly superior to the comparative examples in terms of polyphenol retention rate, microbial control, micelle stability and antioxidant activity. In Comparative Examples 1-2, the B1 polyphenol mass fraction deviated from the preferred range, resulting in decreased grafting efficiency and micelle embedding ability, with polyphenol retention decreasing to 68-72%. In Comparative Example 3, the B1 to protein mass ratio was too low, leading to insufficient protein embedding. In Comparative Example 4, the lack of an enzymatic grafting step resulted in only physical mixing of polyphenols and proteins, rather than covalent grafting. Both of these conditions led to an increase in nanomicelle size to 220-320 nm, an increase in PDI to 0.42-0.55, and a decrease in the absolute value of the Zeta potential to 12-15 mV. The system stability was poor, and the total colony count increased to 45 CFU / mL. In Comparative Example 5, the micelle size exceeded the range by 280 nm, resulting in a decrease in specific surface area, increased polyphenol exposure, and a retention rate decreasing to 70%. In Comparative Example 6, the insufficient addition of micelles led to insufficient interface coverage and poor microbial control, with the total colony count reaching 42 CFU / mL. In Comparative Example 7, the excessive addition of micelles caused a sharp increase in system viscosity to 52%. The fluidity deteriorated at mPa·s; in Comparative Examples 8-9, the high hydrostatic pressure deviated from the preferred range, resulting in severely insufficient sterilization effect, with total bacterial counts reaching 185 and 245 CFU / mL, respectively, failing to meet the microbial safety requirements for 90 days of refrigeration; in Comparative Examples 10-11, improper self-assembly conditions led to excessively large micelle sizes and poor dispersibility; in Comparative Example 12, insufficient homogenization pressure resulted in uneven micelle dispersion; in Comparative Example 13, traditional hot pasteurization was used instead of ultra-high pressure cold sterilization, which, although achieving microbial control, damaged the polyphenol structure at high temperatures, resulting in a retention rate of only 55%, a DPPH removal rate reduced to 48%, and flavor degradation caused by the Maillard reaction triggered by heat treatment. The comprehensive data show that the technical solution of this invention, through precise control of key parameters such as the mass fraction of B1 polyphenols, enzymatic grafting reaction, nanomicelle size, micelle addition amount, and ultra-high pressure-ultrasonic synergistic cold sterilization, achieves synergistic optimization of multiple performance characteristics, including high polyphenol retention, excellent microbial control, good rheology, and high antioxidant activity.

[0127] Table 1 Performance summary of Examples and Comparative Examples 1-13

[0128]

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A wild cherry juice based on ultra-high pressure-ultrasound treatment, characterized in that, The product comprises NFC aronia berry juice and polyphenol-protein nanomicelles dispersed therein, wherein the polyphenol-protein nanomicelles and the aronia berry juice meet the following conditions: (1) The polyphenol-protein nanomicelles are formed by the enzymatic grafting of B1 polyphenol fraction B1 of wild cheri and at least one of isolated whey protein, pea protein isolate and gelatin under the catalysis of laccase and / or horseradish peroxidase. The B1 polyphenol-protein graft polymer is formed under the conditions of pH 4.0–4.5, sodium chloride concentration of 10–50 mmol / L, ultrasonic frequency of 20–25 kHz, power density of 200–600 W / L and duration of 2–8 min. (2) The volume average particle size of the polyphenol-protein nanomicelles is 50–150 nm, the polydispersity index (PDI) is less than or equal to 0.25, the absolute value of the zeta potential is greater than or equal to 20 mV, and the polyphenol mass fraction based on micelle solids is 5–30 wt%. (3) The amount of the polyphenol-protein nanomicelles added to the cherriesberry juice is 0.10–0.80 wt% of the total mass of the finished juice, calculated as micelle solids; (4) The wild cherries juice is obtained by high hydrostatic pressure treatment, wherein the pressure of the high hydrostatic pressure treatment is 320–380 MPa, the time is 5–10 min, and the temperature is 20–25℃. After the high hydrostatic pressure treatment, it is obtained by ultrasonic treatment, wherein the temperature of the ultrasonic treatment is 40–50℃ and the time is 2–3 min. When the finished wild cherries juice is stored at 4℃ for 90 days, the total polyphenol retention rate of wild cherries is greater than or equal to 80 wt%, and the total number of colonies determined according to the detection method specified in GB4789.2 is less than or equal to 10 CFU / mL. The total polyphenol fraction B1 of wild cherries was prepared by the following steps: A1. Take NFC wild cherry berry juice, adjust the pH to 3.0–4.0, and pass it into a styrene-divinylbenzene copolymer macroporous adsorption resin column at 20–30℃ at a space velocity of 1.0–3.0 times the column volume per hour for 1.0–3.0 h. A2. Elute sequentially with deionized water until no visible pigment is visible in the effluent, then elute with a water-ethanol mixed solvent, wherein the volume fraction of ethanol in the water-ethanol mixed solvent is 30–60 vol%, and collect the eluent containing polyphenols. A3. The eluent is concentrated under reduced pressure at 40–60°C to obtain a total polyphenol fraction B1 of aronia berries with a total polyphenol mass fraction of 30–60 wt%, wherein the total polyphenol mass fraction of aronia berries B1, calculated as gallic acid equivalent, is a recovery rate of greater than or equal to 70 wt% relative to the total polyphenol mass fraction in the feed juice.

2. The Aronia juice according to claim 1, characterized in that, The B1 polyphenol-protein graft polymer is prepared by the following steps: B1. Raw material preparation steps, including: a) Take at least one of whey protein isolate, pea protein isolate and gelatin, add it to deionized water and dissolve it to obtain a protein solution with a protein mass fraction of 1.0–5.0 wt%. b) Dissolve the total polyphenol fraction B1 from wild cherries in deionized water to make the mass fraction of the B1 solution 1.0–5.0 wt%; c) Mix the B1 solution and the protein solution at a mass ratio of B1 to protein of 1:1–3, and adjust the pH of the mixture to 5.5–6.5; B2. Enzyme-catalyzed grafting reaction steps, including: Add laccase and / or horseradish peroxidase to the mixture at 25–40°C. The total amount of enzyme added is 0.5–5.0 wt% of the protein. React for 0.5–3.0 h with slow stirring. The reaction endpoint is defined as a 10–50% decrease in free amino content relative to ungrafted protein. B3. Post-processing steps, including: Small molecule reaction byproducts and free polyphenols are removed by ultrafiltration membrane with a molecular weight cutoff of 10–30 kDa, and the solution is washed with deionized water 1–3 times to obtain a B1 polyphenol-protein graft polymer solution with a solids mass fraction of 2.0–10.0 wt% and a grafting degree of 5–40 mol%. The grafting degree is calculated based on the reduction in free amino content or by conventional methods.

3. The Aronia juice of claim 1, wherein, The polyphenol-protein nanomicelles were prepared by the following steps: C1. Take the prepared B1 polyphenol-protein grafted polymer solution and adjust the pH to 4.0–4.5; C2. Add sodium chloride to the solution to make the concentration of sodium chloride in the solution 10–50 mmol / L, and let it stand for 5–15 min to promote the exposure of the hydrophobic region of the protein; C3. The solution is subjected to ultrasonic treatment at a frequency of 20–25 kHz and a power density of 200–600 W / L for 2–8 min to obtain a polyphenol-protein nanomicelle dispersion with a volume average particle size of 50–150 nm and a polydispersity index (PDI) of less than or equal to 0.

25. C4. Remove coarse aggregates with a diameter greater than 500 nm by centrifugation or filtration to obtain a polyphenol-protein nanomicelle dispersion for juice reconstruction.

4. The chokeberry juice according to claim 1, characterized in that, The polyphenol-protein nanomicelles are distributed and adsorbed at the interface of the pulp particles and / or oil droplets in the cherifolia juice system to form a polyphenol-rich interfacial shell structure. The interfacial shell structure is constructed through the following steps: D1. Add the polyphenol-protein nanomicelle dispersion to the NFC wild cherry berry juice according to the above-mentioned amount of polyphenol-protein nanomicelles, and stir slowly until evenly mixed; D2. The polyphenol-protein nanomicelles are sheared and homogenized 1–3 times at 20–50 MPa using a high-pressure homogenizer to fully disperse and distribute them at the interface between the pulp particles and oil droplets in the juice system. This allows them to form a polyphenol-rich interfacial shell structure together with the pulp particles and oil droplet interface in the NFC wild cherry berry juice. This interfacial shell structure helps to improve the antioxidant capacity and antibacterial effect of the juice system and improve the physical stability of the system.

5. A method for preparing aronia berry juice based on ultra-high pressure-ultrasonic treatment as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation steps of total polyphenol fraction B1 of wild cherries: Total polyphenol fraction B1 of wild cherries was prepared from NFC wild cherries juice by adsorption-elution using a styrene-divinylbenzene copolymer macroporous adsorption resin column. Preparation steps of S2.B1 polyphenol-protein graft polymer: The total polyphenol fraction B1 of wild cherry berry is grafted with at least one of whey protein isolate, pea protein isolate and gelatin under the catalysis of laccase and / or horseradish peroxidase to obtain B1 polyphenol-protein graft polymer solution. S3. Construction steps of polyphenol-protein nanomicelles: Under the conditions of pH 4.0–4.5, sodium chloride concentration 10–50 mmol / L, ultrasonic frequency 20–25 kHz, power density 200–600 W / L, and ultrasonic time 2–8 min, the B1 polyphenol-protein graft polymer is allowed to self-assemble to form a polyphenol-protein nanomicelle dispersion with a volume average particle size of 50–150 nm and a polydispersity index (PDI) less than or equal to 0.

25. Large aggregates with a diameter greater than 500 nm are removed by centrifugation or filtration. S4. Juice Reconstruction and Synergistic Cold Sterilization Step: The polyphenol-protein nanomicelle dispersion is added to NFC aronia berry juice, with the amount of polyphenol-protein nanomicelles added being 0.10–0.80 wt% of the total juice mass based on micelle solids. The mixture is homogenized under high pressure at 20–50 MPa 1–3 times to obtain a uniform dispersion system. Subsequently, it is subjected to high hydrostatic pressure treatment at 320–380 MPa for 5–10 min at 20–25℃. After the high hydrostatic pressure treatment, it is subjected to ultrasonic treatment at 40–45℃ for 2–3 min. After cooling, it is aseptically filled to obtain aronia berry juice.

6. The preparation method according to claim 5, characterized in that, The styrene-divinylbenzene copolymer macroporous adsorption resin used in step S1 has a specific surface area of ​​400–900 m² / g and a pore size of 20–100 nm.

7. The preparation method according to claim 5, characterized in that, The ultrasonic treatment in step S3 adopts an intermittent mode, with a single working time of 5–10 seconds and a single pause time of 5–10 seconds.

8. The preparation method according to claim 5, characterized in that, The absolute value of the Zeta potential of the polyphenol-protein nanomicelle dispersion obtained after step S3 is 25–40 mV.

9. The preparation method according to claim 5, characterized in that, During the high hydrostatic pressure treatment in step S4, the pressure increase rate and pressure decrease rate are controlled at 100–300 MPa / min, the pressure holding time is 5–8 min, and the total bacterial count of the wild cherry berry juice obtained by this preparation method is less than or equal to 10 CFU / mL when stored at 4℃ for 90 days, and yeast and mold are not detected.

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