Near field communication (NFC) cloudy apple juice stabilization method combining high-pressure micro-fluidic nano homogenization and medium-temperature sterilization

Abstract

The invention provides an NFC (Near Field Communication) cloudy apple juice stabilization method by combining high-pressure micro-fluidic nano homogenization and medium-temperature sterilization. The method comprises the following steps: S1, cleaning fresh apples, cutting into blocks, removing kernels, and soaking in a color protection solution; s2, the apple juice is put into a wall breaking machine to be squeezed, meanwhile, color protection liquid is added for color protection, filtering is conducted through filter cloth, and cloudy apple juice is obtained; and S3, homogenizing the cloudy apple juice by using an ultrahigh-pressure micro-fluidic nano homogenizer, and performing medium-temperature sterilization to obtain the stabilized NFC apple juice. According to the method disclosed by the invention, the cloudy degree stability (P is less than 0.05) of the cloudy apple juice can be remarkably improved, the turbidity and the centrifugal precipitation rate are respectively reduced by 38.6% and 34.1%, the particle size is reduced to (129.95 + / -0.39) nm from a contrast 1 (502.06 + / -9.79) nm, negative charges carried by a juice system are remarkably increased (P is less than 0.05), the pectin content and the apparent viscosity are remarkably increased, the dynamic instability is remarkably reduced, and the stability is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of functional food processing technology, specifically to a method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and medium-temperature sterilization. Background Technology

[0002] NFC (Not From Concentrate) apple cloudy juice is popular among consumers for its smooth taste and outstanding flavor. Because it is not subjected to high-temperature concentration and reduction processing, it retains more of the bioactive polyphenols, giving it higher nutritional value and further enhancing its flavor through interaction with components such as pectin and protein. However, the presence of cloud-like particles composed of polyphenols, pectin, and protein makes cloud stability a critical concern during apple cloudy juice processing. Cloud instability is mainly caused by the gravitational settling of cloud-like particles and the deesterification effect of pectin methyl esterase (PME). When cloudy juice cells rupture, PME and high molecular weight compounds are released and suspended during mechanical juicing. Methoxylated pectin undergoes deesterification through the activity of PME, calcium ions react with demethylated pectin to form insoluble pectin, and other larger and coarser particles also settle under gravity. Different sterilization methods have different effects on the pectin in apple cloudy juice, thus affecting the system viscosity and cloud stability. Heat sterilization often induces the hydrolysis of glycosidic bonds, and cell wall pectin polysaccharides are modified by high temperatures, participating in heat dissolution. β Polysaccharides undergoing elimination, depolymerization, and deesterification reactions lead to pectin degradation. Ensuring microbial safety during processing and sterilization of apple cloudy juice while simultaneously improving cloud particle stability is crucial for enhancing the quality of NFC apple cloudy juice.

[0003] High-pressure microfluidic nanoscale homogeneous combined with moderate temperature (HP-TR) sterilization utilizes the combined action of ultra-high pressure and moderate temperature to achieve the lethal effect on bacterial vegetative cells. However, different fruit juice systems have their own unique phenolic compounds and other nutrients. Therefore, selecting suitable high-pressure and moderate-temperature conditions to maximize their synergistic effect in cloudy apple juice systems—achieving sterilization while minimizing the degradation of phenolic compounds and flavor loss caused by traditional heat sterilization, and reducing nutrient loss from fresh fruit—is crucial for improving the nutritional quality of cloudy apple juice. Furthermore, moderate moderate-temperature heating can avoid the problem of insufficient enzyme inactivation caused by non-thermal sterilization, moderately promote pectin hydrolysis, reduce the size of cloud-like particles, increase their negative charge, thereby improving the stability of cloud-like particles in the juice system, enhancing the quality of NFC apple products, and promoting continuous industrial production of NFC apple juice. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention uses Fuji apples as raw material and aims to provide an NFC (Not From Concentrate) apple turbidity stabilization method that combines high-pressure microfluidic nano-homogenization and medium-temperature sterilization. This method can significantly improve the cloudiness stability of apple turbidity juice. P <0.05), turbidity and centrifugal sedimentation rate decreased by 38.6% and 34.1% respectively, particle size decreased from (502.06±9.79) nm in Comparative Example 1 to (129.95±0.39) nm, and the negative charge carried by the juice system increased significantly ( P <0.05), the kinetic instability TSI value is small, and the stability is greatly improved. At the same time, the atomic force microstructure of apple juice in Example 1 shows a decreasing trend in both width and height dimensions, the peak structure becomes shorter and flatter, and the large particles are reduced; scanning electron microscopy observation shows that the surface of Example 1 has a dense granular structure, which is small in size and uniformly distributed, and the overall structure is highly compact. Technical solution

[0005] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh apples, cut them into pieces, remove the cores, and soak them in color-protecting solution; S2. Put the juice into a blender and add color-protecting solution to protect the color. Filter the juice with a filter cloth to get cloudy apple juice. S3. After homogenizing the apple juice using an ultra-high pressure microfluidic nano-homogenizer, and then sterilizing it at medium temperature, stable NFC apple juice is obtained.

[0006] Furthermore, the color-protecting solution mentioned in S1 is a 0.9-1.1% D-isoascorbic acid sodium aqueous solution, and the soaking time is 10 min.

[0007] Furthermore, the amount of color-protecting liquid added in S2 is 0.1% of the apple's mass.

[0008] Furthermore, the filter cloth described in S2 is two layers of 300-mesh filter cloth.

[0009] Furthermore, the homogenization conditions described in S3 are 2-4 cycles at 120-180 MPa.

[0010] Furthermore, the medium-temperature sterilization conditions described in S3 are: 40-60℃ water bath, 10-50 min.

[0011] The present invention also provides stabilized NFC apple juice prepared by the above method.

[0012] Beneficial effects:

[0013] (1) Significantly improves the stability of NFC apple turbid juice and extends shelf life: This invention uses a combination of high-pressure microfluidic nano-homogenization and medium-temperature sterilization technology to improve the cloudiness stability of apple turbid juice to (17.16±0.03)%, which is about 5 times higher than the control group; the centrifugal sedimentation rate is reduced to (1.94±0.03)%, which is significantly lower than pasteurization (Comparative Example 3), microwave sterilization (Comparative Example 2) and the unsterilized control group (Comparative Example 1); the turbidity is reduced by 61.6%, 62.6% and 38.6% compared with pasteurization (Comparative Example 3), microwave sterilization (Comparative Example 2) and the unsterilized control group (Comparative Example 1), respectively. At the same time, this treatment reduces the particle size of turbid juice from (502±9.79) nm in the control group to (129.95±0.39) nm, significantly increases the absolute value of Zeta potential, and enhances the electrostatic repulsion between particles, effectively inhibiting the flocculation, sedimentation and stratification of cloud particles during storage. Multiple light scattering analysis showed that the system remained stable after standing at 25°C for 9 hours, with a significantly lower kinetic instability (TSI) value than other groups, thus greatly extending the product's shelf life. (2) Optimize the physicochemical quality of the product and retain its natural flavor and nutrition: The gentle shearing action of high-pressure microfluidic nano-homogenization combined with medium-temperature sterilization at 40-60℃ (below the pectin backbone breakage threshold) avoids the damage to the quality of the juice caused by high-temperature treatment. After treatment, the pH value (4.31±0.01) and total acid content (0.19±0.01%) of the juice were not significantly different from those of Comparative Example 1, and the solid-acid ratio (38.51±1.35) reached the optimal level, maximizing the preservation of the natural sweet and sour flavor of apples. Compared with the high-temperature degradation of pasteurization (Comparative Example 3) and the abnormal increase of soluble solids in microwave sterilization (Comparative Example 2), this invention can reduce the loss of nutrients such as organic acids and polyphenols, and ensure the nutritional characteristics of the product; (3) Improve the sensory and rheological properties of the product and enhance the consumer experience: The turbidity of the juice is significantly reduced and the clarity is significantly improved after processing. The appearance is uniform and clear without obvious layering and sedimentation. The viscosity and apparent viscosity are optimized, and the system exhibits uniform and stable pseudoplastic fluid characteristics with a smooth and delicate taste. Atomic force microscopy and scanning electron microscopy observation show that the turbid juice cloud particles have a dense and uniform micro-particle structure without obvious large particle agglomeration. This not only ensures the typical turbid juice morphology of NFC apple turbid juice, but also solves the problem of poor sensory quality of traditional products. (4) Highly efficient sterilization and reduced processing energy consumption, suitable for industrial production: The combined technology of this invention can completely kill the total number of colonies, molds and yeasts in fruit juice (all test results are ND), and the sterilization effect is comparable to pasteurization and microwave sterilization, ensuring the microbial safety of the product. Compared with the high temperature or high power consumption of pasteurization (85℃) and microwave sterilization, the medium temperature condition reduces energy consumption; the high-pressure microfluidic nano-homogenization process can realize continuous operation, the process parameters are stable and controllable, and the auxiliary steps such as filter cloth filtration and color protection are simple and easy to operate, which is suitable for the needs of large-scale industrial production and has significant economic and application value; Optimizing the system's microstructure and enhancing its inherent stability mechanism: High-pressure microfluidic shearing and extrusion depolymerizes pectin, improves the linearity of homogalacturonic acid polysaccharides, and reduces the proportion of neutral sugar branches; medium-temperature treatment prevents excessive pectin degradation, exposes more carboxyl groups in the pectin backbone, and enhances the system's negative charge density. The synergistic effect of these two methods ensures uniform dispersion of macromolecules such as proteins and pectin in the cloudy juice, forming a stable colloidal system. This addresses the core technical problem of poor stability of traditional NFC apple cloudy juice particles at the microstructural level. Attached Figure Description

[0014] Figure 1 The cloudiness stability changes of apple turbidity juice in Comparative Examples 1-7 and Examples 1-7 are shown; Note: Lowercase letters in the figure indicate significant differences between different treatments for the same indicator. P <0.05), the same below; Figure 2 The changes in centrifugal sedimentation rate of apple turbid juice in Comparative Examples 1-7 and Examples 1-7; Figure 3 The turbidity changes of apple juice in Comparative Examples 1-7 and Examples 1-7; Figure 4 The apparent viscosity and kinetic instability changes of apple turbid juice in Comparative Examples 1, 2 and Examples 1 and 2; Figure 5 The particle size and potential changes of apple juice from Comparative Example 1, Comparative Example 2, and Examples 1 and 2 are shown. Figure 6 The changes in pectin content and pectin methyl esterase activity in the apple turbid juice of Comparative Example 1, Comparative Example 2, and Examples 1 and 2 are shown. Figure 7 The changes in backscattered light of apple turbid juice in Examples 1 and 2 and Comparative Examples 1 and 2 are shown. Figure 8 Images of apple turbid juice obtained by atomic force microscopy; where a is Comparative Example 1; b is Comparative Example 2; c is Example 1; d is Example 2; Figure 9The images are scanning electron microscope (SEM) images of apple turbid juice; where a is comparative example 1; b is comparative example 2; c is example 1; and d is example 2. Detailed Implementation

[0015] This invention proposes a method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. Example 1

[0016] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. After homogenizing 100 mL of apple juice twice at 160 MPa using an ultra-high pressure microfluidic nano-homogenizer, the mixture was sterilized in a 60℃ water bath for 10 min to obtain stabilized NFC apple juice. Example 2

[0017] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice three times at 150 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 60℃ water bath for 10 min to obtain stabilized NFC apple juice. Example 3

[0018] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice twice at 180 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 50℃ water bath for 10 min to obtain stabilized NFC apple juice. Example 4

[0019] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice twice at 160 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 50℃ water bath for 25 min to obtain stabilized NFC apple juice. Example 5

[0020] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice three times at 160 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 50℃ water bath for 20 min to obtain stabilized NFC apple juice. Example 6

[0021] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice twice at 160 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 50℃ water bath for 30 min to obtain stabilized NFC apple juice. Example 7

[0022] A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice twice at 160 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 60℃ water bath for 20 min to obtain stabilized NFC apple juice.

[0023] Comparative Example 1 A method for processing unpasteurized fruit juice includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% D-isoascorbic acid sodium salt for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain NFC apple cloudy juice.

[0024] Comparative Example 2 A method for microwaving fruit juice includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Place 100 mL of Fuji apple juice in a microwave oven, set the microwave power to 700 W, and the sterilization time to 60 s to obtain microwave sterilized juice.

[0025] Comparative Example 3 A method for pasteurizing fruit juice includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Place 100 mL of Fuji apple juice in a water bath and sterilize at 85°C for 10 min to obtain pasteurized juice.

[0026] Comparative Example 4 A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. After homogenizing 100 mL of apple juice four times at 120 MPa using an ultra-high pressure microfluidic nano-homogenizer, the mixture was sterilized in a 60℃ water bath for 15 min to obtain stabilized NFC apple juice.

[0027] Comparative Example 5 A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. After homogenizing 100 mL of apple juice twice at 170 MPa using an ultra-high pressure microfluidic nano-homogenizer, the mixture was sterilized in a 60℃ water bath for 10 min to obtain stabilized NFC apple juice.

[0028] Comparative Example 6 A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. After homogenizing 100 mL of apple juice twice at 160 MPa using an ultra-high pressure microfluidic nano-homogenizer, the mixture was sterilized in a 40℃ water bath for 50 min to obtain stabilized NFC apple juice.

[0029] Comparative Example 7 A method for stabilizing NFC apple turbid juice using a combination of high-pressure microfluidic nano-homogenization and mesophilic sterilization includes the following steps: S1. Wash fresh Fuji apples, cut them into pieces, remove the cores, and soak them in a 1% D-isoascorbic acid sodium solution for 10 min; S2. Put the juice into a blender and add 0.1% sodium D-isoascorbate for color protection. Filter the juice using two layers of 300-mesh filter cloth to obtain cloudy apple juice. S3. Homogenize 100 mL of apple juice three times at 150 MPa using an ultra-high pressure microfluidic nano-homogenizer, and then sterilize it in a 50℃ water bath for 10 min to obtain stabilized NFC apple juice.

[0030] I. Testing Method: 1. Determination of sugar content and acidity The titration method using acid-base indicators in GB / T 12456-2021 "Determination of Total Acidity in Food" was modified appropriately. Sodium hydroxide solution was standardized with potassium dihydrogen phthalate, and the total acid (TA) content was calculated using citric acid as a conversion factor. pH was measured using a pH meter; total soluble solids (TSS) were directly measured using a saccharimeter, and the results are expressed as °Brix. The solid-acid ratio represents the ratio of soluble solids to total acid content.

[0031] 2. Cloudiness stability and centrifugal sedimentation rate The juice sample was centrifuged at 4200×g for 15 min, and the absorbance was measured at 625 nm. A 0 and A 1. The magnitude (%) of cloud stability is expressed as: A 1 / A 0×100%. Take 20 mL of juice, weigh it, and record the weight as 0. M 0; Weigh 4200×g, centrifuge for 15 min, and record the precipitate as . M 1. The centrifugal sedimentation rate (%) is expressed as follows: M 1 / M 0×100%.

[0032] 3. Turbidity and apparent viscosity The turbidity of the fruit juice was measured using a turbidimeter, with NTU representing the turbidity unit. The sample was added to a turbidity bottle, shaken thoroughly, and the surface was wiped with lens paper. The bottle was then placed in the test area of ​​the turbidimeter, zeroed with deionized water, and the turbidity was recorded after the reading stabilized. Apparent viscosity was measured using a HAAKE rheometer. The measurement temperature was set to 25℃, the sample-probe gap to be 0.1 mm, the rotor model to be P35 / Ti-01221945, and the continuous scanning time to be 0.01000 1 / s–100.0 1 / s. Each sample was measured three times.

[0033] 4. Particle size and zeta potential determination The particle size distribution of the juice from different treatment groups was determined using a 90Plus Zeta particle size and Zeta potential analyzer, with each sample measured three times. The Zeta potential of the juice from different treatment groups was determined using a Zetasizer Lab laser particle size analyzer, with the program set to scan five times and the measurement temperature at 25℃.

[0034] 5. Pectin content and pectin methylesterase activity Pectin was extracted from cloudy apple juice using the citric acid method. Four types of treated apple juice were taken, and the pH was adjusted to 2±0.1 with 1 M citric acid solution. The juices were then transferred to a 90℃ water bath with magnetic stirring and incubated for 2 h. After removal, the juices were centrifuged at 5000 rpm for 15 min. The supernatant was collected and precipitated with 4 volumes of 95% anhydrous ethanol for 12 h. The precipitate was obtained by centrifugation at 5000 rpm for 20 min and washed twice with anhydrous ethanol. For pectin content determination, 6.0 mL of concentrated sulfuric acid was added to 1 mL of pectin solution, and the mixture was heated in a boiling water bath for 20 min. After cooling to room temperature, 0.2 mL of 1.5 g / L carbazole-ethanol solution was added to each solution. The mixture was shaken well, treated in the dark for 30 min, and the absorbance was measured colorimetrically at 530 nm. A standard curve was plotted with galacturonic acid concentration on the x-axis and absorbance on the y-axis, and the linear regression equation was obtained as y = 0.006x + 0.0077, R² = 0.996.

[0035] Enzyme solution preparation: Apple turbid juice was mixed with 0.2 mol / L Tris-HCl buffer (pH=7.5, containing 0.1 mol / L HCl) at a volume ratio of 1:3, and allowed to stand at 4℃ for 12 h. The mixture was then centrifuged at 10000 r / min at 4℃ for 10 min, and the supernatant was used as the PME enzyme extract. Enzyme activity assay: 5 mL of the enzyme solution was mixed with 20 mL of 1% pectin solution (containing 0.1 mol NaCl) and the pH was adjusted to 7.5. 0.5 mL of 0.05 mol / L NaOH was added, and the time t required for the pH meter reading to return to 7.5 was recorded. According to the formula: PME (U / mL) = 0.5 mL × 0.05 mol / L / (5 mL × t).

[0036] 6. Determination of kinetic instability The Lab Expert multiple light scattering instrument was used to scan the sample from bottom to top using light emitted from the light source. The scanning temperature was set to 25°C, the scanning time to 9 hours, and the scanning frequency to 10 minutes per scan. Each sample was measured three times.

[0037] 7. Observation using atomic force microscopy and scanning electron microscopy The fruit juice sample was prepared to a concentration of 0.5 μL / mL using ultrapure water and sonicated for 5 min. 10 μL of the sample was added to a mica sheet, dried for 24 h, and then placed on a sample stage for image acquisition using an AFM probe. The lyophilized apple juice sample was then sputter-coated with gold and its microstructure was observed under SEM with an accelerating voltage of 5 kV.

[0038] II. Data Results: (1) Effect on the sugar and acidity of apple juice Table 1. Effects of Examples 1-7 and Comparative Examples 1-7 on pH, total acid, soluble solids, and solid-acid ratio of apple turbid juice.

[0039] Note: All experiments were repeated three times, and results are expressed as mean ± standard deviation; different lowercase letters in the table indicate significant differences between different treatment groups for the same indicator. P <0.05).

[0040] Sugar content and acidity are important indicators for evaluating the taste and flavor of fruit juice. Hydrolysis of polysaccharides and denaturation of proteins in fruit juice after sterilization can both cause changes in sugar and acidity. Table 1 shows that there were significant differences in TSS and glutamate ratio among the different treatment groups of cloudy apple juice. P <0.05). The pH and TA of Example 1 were not significantly different from those of Comparative Example 1 ( P >0.05), which can maintain the stability of apple juice acidity. It is possible that the medium temperature conditions in Example 1 are mild and will not destroy water-soluble nutrients such as organic acids in apple juice. High-pressure homogenization itself can reduce the impact on the quality of juice.

[0041] (2) Effect on the stability of apple juice Stability is a crucial indicator for evaluating fruit juice products. During storage, suspended particles in fruit juice can flocculate and settle due to gravity and other factors, affecting the product's appearance. Cloudiness stability, as one of the main parameters for the stability of fruit juice suspensions, is measured under conditions equivalent to one year of storage. Figure 1 It can be seen that the cloudiness stability of apple juice after Example 1 was (17.16±0.03)%, which was about 5 times higher than that of other treatment groups. Figure 2 It can be seen that the centrifugal sedimentation rate of apple juice in Example 1 was (1.94±0.03)%, significantly lower than that of other treatment groups. The cloudiness stability was significantly increased, and the centrifugal sedimentation rate was significantly reduced. This may be because high-pressure homogenization can break down fruit pulp particles to a certain extent, thus maintaining them stably in suspension, reducing sedimentation, and improving the overall uniformity and stability of the cloudy apple juice. Turbidity reflects the binding state of the cloud-like particle system in the juice; particle aggregation increases turbidity, and vice versa. Figure 3It can be seen that the turbidity of Comparative Examples 1, 2, 3, and 4 is significantly higher than that of the Example.

[0042] (3) Effects on apparent viscosity and kinetic instability Due to Brownian motion, suspended particles and macromolecules in cloudy juice are randomly distributed. This results in significant flow resistance at low shear rates, leading to a high viscosity in the apple juice system. For example... Figure 4 As shown, Example 1 exhibited the highest initial apparent viscosity. Combined with particle size analysis, this is likely due to the high-pressure homogenization process reducing particle size, resulting in a larger interfacial area and a smaller average distance between particles. This leads to stronger interparticle interactions, and the smaller particles disperse better in the turbid juice, thus causing a higher viscosity. With increasing shear rate, the externally applied motion rate gradually increases, exceeding the rate of new entanglement formation between hydrated macromolecules. The cross-linked network formed by pectin interactions in the juice is disrupted, leading to a decrease in viscosity. Ultimately, the apparent viscosity of the juice from different treatments tends to converge. The Turbiscan stability index (TSI) measurement utilizes the principle of multiple light scattering, providing a more intuitive and rapid reflection of the system's changing trends without damaging the original sample structure. A smaller TSI value indicates a more stable sample system, and vice versa. The TSI values ​​of the juices in Comparative Examples 1 and 2 showed an increasing trend over time, indicating that their stability decreased with prolonged time, and stratification occurred within 9 hours of standing. The TSI value of the juice in Example 2 showed a slow increasing trend over time, starting to increase at 5.5 hours, indicating that stratification occurred in the PA group juice with increasing standing time. Example 1 showed higher stability, with the apple juice system remaining relatively stable at 9 hours. This suggests that Example 1 can stably suspend apple juice particles. Considering the changes in particle size and potential, it is speculated that Example 1 reduces the size of cloud-like particles in the apple juice system, increases the specific surface area, increases the negative charge, and increases the electrostatic repulsion between particles. Since the cloud-like particles maintain a dynamic equilibrium within the apple juice system, this also indicates that reducing the particle size promotes improved physical stability.

[0043] (4) Effects on particle size and potential The stability of turbid fruit juice is controlled by the Stokes equation, in which particle size and apparent viscosity are the main factors affecting the sedimentation rate. Figure 5It is known that sterilization treatment significantly reduces the particle size of apple juice. The smallest particle size was observed in Example 1. This is likely because the shearing and extrusion action of high-pressure homogenization caused the cloud-like particles to collide with each other, breaking large particles into smaller ones. After subsequent medium-temperature (60°C) treatment, the temperature is below the pectin backbone breaking threshold (≈70°C), resulting in limited backbone degradation. This reduces the exposure of protein and other groups encapsulated in pectin, thereby decreasing the aggregation of protein and other groups with other small molecules and improving the stability of the system.

[0044] The cloud-like particles of apple juice are coated with a layer of negatively charged pectin, resulting in an overall negatively charged surface. Figure 5 The image shows the charge status of the apple juice after sterilization. In Example 1, the apple pectin underwent appropriate removal of some methyl ester groups, resulting in more carboxyl groups being exposed in the pectin backbone. This led to the stretching of the molecular chains, a slight increase in the degree of esterification, and enhanced carboxyl group vibration. After treatment at 60°C, the stretching of the pectin chains and the increase in charge resulted in an increase in the negative charge of the pectin, enhanced electrostatic repulsion, and increased interparticle repulsion. Consequently, the stability of the apple juice system in Example 1 was significantly higher than that of other treatment groups.

[0045] (5) Effects on apple juice pectin and pectin methylesterase activity Pectin-like substances in apple juice can be released or degraded depending on the sterilization conditions. Figure 6 The changes in pectin content in apple turbid juice under different treatments are shown. Example 1 showed a significantly higher pectin content than the other treatment groups, increasing the pectin content extracted using the weak acid method. This is likely because high-pressure homogenization alters the strength of chemical bonds between pectin molecules and between pectin molecules and the cell wall, promoting pectin release from the cell wall. The weak acid effect makes high-pressure homogenization more likely to disrupt and interfere with the cell wall network, thereby promoting pectin release and extraction by weakening cell interactions and forming a more open cell wall structure. Comparative Examples 2 and 3 typically only extracted weakly bound pectin, through divalent cation interactions.

[0046] Residual pectin methylesterase (PME) can lead to pectin demethylation and main chain breakage, which is detrimental to maintaining the integrity of pectin molecules and thus to the stability of the juice system. Figure 6The changes in pectin methyl esterase (PME) activity in apple cloudy juice under different treatments are shown. The PME activity in Example 1 was significantly lower than that in Comparative Examples 1 and 2. Comparative Examples 1 and 2 showed higher PME activity in their apple cloudy juice, which disrupted the ester bonds connecting the methyl ester group and the carboxyl group, leading to pectin hydrolysis and a decrease in viscosity. This explains why their apparent viscosity was lower than that of Example 1. The lower PME activity in Example 1 reduced the hydrolysis of the pectin methyl ester group, potentially increasing the degree of pectin esterification, which may explain the better stability of the apple cloudy juice system. It is possible that high-pressure homogenization caused the α-helix structure in PME to transform into a β-sheet structure. The reduction in α-helix content not only affects the catalytic active site of the enzyme, leading to decreased enzyme activity, but also disrupts hydrogen bonds within the molecular chain, resulting in more hydrogen bonds between molecular chains, thus improving its density and stability.

[0047] (6) Effect on juice kinetic instability Real-time monitoring of particle size and position changes caused by aggregation, flocculation, or agglomeration in samples is used to characterize sample stability. Based on the principle of multiple light scattering, changes in particle size and concentration, particle sedimentation, or flotation in the dispersion system can be determined by the light intensity deviation obtained from multiple scans. A smaller ΔBS indicates a more stable dispersion system, and vice versa. Figure 7 As shown, after apple juice underwent different treatments and was left to stand at 25°C for 9 hours, the backscattered light intensity at different heights showed significant changes over time. Specifically, the backscattered light intensity at the bottom (0–10 mm) of Comparative Example 1 and the bottom (0–5 mm) of Example 2 showed an increasing trend over time; however, the backscattered light intensity at the bottom (0–5 mm) of Comparative Example 2 showed an initial increase followed by a decrease. This indicates that significant particle settling occurred, leading to an increase in particle concentration at the bottom and a decrease in concentration at the top, resulting in stratification. The backscattered light intensity of the Example 1 sample did not change significantly over time, maintaining a relatively stable trend, indicating minimal change in particle concentration and good stability.

[0048] (7) Effect on the microstructure of fruit juice cloud particles Sterilization treatment not only affects the size of apple juice cloud particles but also alters their morphology. Atomic force microscopy (AFM) images of apple juice treated with different sterilization methods are shown below. Figure 8As can be seen from the figure, Comparative Example 1 apple juice exhibits a spike-like granular structure with pointed peaks. The particle width ranges from approximately 1.204 to 1.526 μm, and the particle height ranges from 34.85 to 58.72 nm. Comparative Example 2 apple juice has more large particles adhering to it, with a particle width increased compared to Comparative Example 1, ranging from 1.511 to 2.804 μm, and a height ranging from 19.02 to 28.5 nm. Example 2 shows a significant reduction in large particles, but the uniformity of the system particles deteriorates. The particle length ranges from 1.064 to 1.443 μm, and the height ranges from 18.96 to 40.86 nm. Compared to Comparative Example 1, Example 1 shows a reduction in both width and height, a decrease in large particles, and a significantly shorter and flatter surface "peak-like structure," with a length ranging from 1.021 to 1.249 μm and a height ranging from 15.53 to 17.61 nm. Example 1 disrupted the aggregated structure of macromolecules such as proteins and pectin in the cloudy juice particle system, resulting in a more uniform dispersion of macromolecules within the system and thus reducing surface peak undulations. Combined with AFM microstructure observation and stability index analysis, Example 1 showed more uniform particle length and height, and a smaller range of particle size variation, indicating that Example 1 significantly improved the stability of the apple cloudy juice system.

[0049] Scanning electron microscopy (SEM) image of apple turbid juice after sterilization. Figure 9 Comparative Example 1 exhibits an irregular blocky agglomerate structure with some stacking between the layers; Comparative Example 2 shows particle aggregation, with irregularly shaped granules and fine agglomerates on the surface compared to Comparative Example 1; Example 2 exhibits a network micelle structure with some pores and gaps; Example 1 has a dense granular structure on the surface, with small and relatively uniform particle size, no obvious large particle flocculation, and high overall structural density. Under high pressure, Example 1 formed even smaller particles, pores, and aggregates, which also affected the size and morphology of the particles.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A NFC apple cloudy juice stabilization method combined with high-pressure microfluidic nanohomogenization-moderate-temperature sterilization, characterized by: The method comprises the following steps: S1. Fresh apples are cut into pieces and cored after washing, and then soaked in a color protection solution; S2. The apples are put into a wall-breaking machine to extract juice, and a color protection solution is added for color protection. A filter cloth is used for filtration to obtain apple cloudy juice; S3. The apple cloudy juice is homogenized by using an ultrahigh-pressure microfluidic nanohomogenizer, and then combined with medium-temperature sterilization to obtain stable NFC apple juice.

2. The NFC apple cloudy juice stabilization method combined with high-pressure microfluidic nanohomogenization and moderate-temperature sterilization according to claim 1, characterized in that: The color protection solution in S1 is a 0.9-1.1% D-sodium erythorbate aqueous solution, and the soaking time is 10 min.

3. The NFC apple juice stabilization method using high-pressure microfluidic nano-homogenization-intermediate sterilization combined with the method described in claim 1, characterized in that: The addition amount of the color protection solution in S2 is 0.1% of the mass of the apples.

4. The NFC apple cloudy juice stabilization method of claim 1, wherein the NFC apple cloudy juice stabilization method is a high-pressure microfluidic nanohomogenization-moderate-temperature sterilization combined method. The filter cloth in S2 is two layers of 300-mesh filter cloth.

5. The NFC apple juice stabilization method using high-pressure microfluidic nano-homogenization-intermediate sterilization combined with the method described in claim 1, characterized in that: The homogenization conditions in S3 are 120-180 MPa for 2-4 cycles.

6. The NFC apple cloudy juice stabilization method of claim 1, wherein the NFC apple cloudy juice stabilization method is a high-pressure microfluidic nanohomogenization-moderate-temperature sterilization combined method. The medium-temperature sterilization conditions in S3 are 40-60°C water bath for 10-50 min.

7. Stable NFC apple juice prepared by the method according to any one of claims 1-6.

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