Changes in physicochemical profiles and quality of apple juice treated by ultrafiltration and during its storage

Abstract Effects of various factors, such as membrane materials, molecular weight cutoff, transmembrane pressure (TMP), and cross flow rate (CFR) on flux and physicochemical properties of apple juice during ultrafiltration and storage have been investigated. Clarity, color, total phenols, total proteins, total sugars, total soluble solids (TSS), pH, and some specific polyphenols of juices were evaluated. Results show that at conditions of PES‐10 kDa, CFR 30 L/hr, and TMP 0.75 MPa, a clarified juice obtained with color 0.15 A, clarity 96.94%T, TSS 9.55 °Brix, pH 4.2, and total phenols, total proteins, and total sugars were 64.12 and 13.20 μg/ml and 50.70 mg/ml, respectively. Chlorogenic acid, epicatechin, phloridzin, catechin, and caffeic acid decreased differently from 32.63, 17.33, 3.25, 7.58, and 0.75 μg/ml to 17.24, 12.38, 1.79, 5.27, and 0.25 μg/ml, respectively. Storage in refrigeration for 4 weeks, clarity, total sugars, and total phenols reduced by 2.5%, 6.4%, and 16.6%, respectively, while TSS increased by 3.1%.


| INTRODUC TI ON
As a health drink, apple juice stored nutrients, minerals, and micronutrients in apples and can be quickly absorbed by human body (Gerhauser, 2008). Clarified apple juice is popular among consumers because of its unique light transmittance, flavor, and taste. Some typical technologies, such as clarification agents, enzymatic methods, and membrane techniques have been widely used for clarification of apple juice.
Fining agents, such as gelatin, bentonite, silicasol, and diatomaceous earth, could create some problems of environmental impact due to their disposal. Addition of these clarifiers might affected some active ingredients loss and change the characteristics of juices (Vaillant et al., 1999). Enzyme treatment refers to the enzymatic hydrolysis of some components of juices with enzyme preparation.
It can not only improve the yield and taste of juice, but also reduce the viscosity and color. However, enzyme treatment was time consuming and the optimal treatment conditions were difficult to be controlled (Girard & Fukumoto, 2000).
In 1977, Heatherbell, Short, and Strubi (1977) successfully applied ultrafiltration (UF) technology to produce a stable clarified juice. Accordingly, membrane technology as a non-thermal technique has been emerged as a substitute to traditional juice clarification techniques because of low temperature, less operating cost, and less manpower. Additionally, it involves no phase change or chemical agents. UF is the most widely used membrane technology for clarification of fruit and vegetable juice in juice industry. Some studies found the application of UF to apple and lemon juices were successful, with reductions in color (99%) and viscosity (98%), subsequently achieving a high level of clarity (De-Bruijn et al., 2003;Maktouf et al., 2014;Mirsaeedghazi, Emam-Djomeh, Mousavi, Aroujalian, & Navidbakhsh, 2009;Toker, Karhan, Tetik, Turhan, & Oziyci, 2013;Warczok, Ferrando, Lopez, & Guell, 2004). Additionally, UF could be used to concentrate of phenolic compounds in juice, successfully in retaining a high percentage (85%) of polyphenols in its retentate (Conidi, Cassano, Caiazzo, & Drioli, 2017). But in most of these investigations, the changes of main ingredients during the processes have not been demonstrated clearly. Accordingly, the main components in juices, especially polyphenols can be affected by the membrane treatment. It is necessary to understand the changes of physicochemical profiles of juices by UF treatment, especially the phenolics. And the stability of ultrafiltrated juice during storage should be also demonstrated.
In this study, effects of various factors on apple juice during UF have been investigated. Changes of physicochemical properties and some specific phenolic compounds during the process and its storage have been demonstrated.

| Materials and reagents
Fresh "Fuji" apples were purchased from a local market (Zhejiang, China). The apples were washed, peeled, and the cores were also removed, after which the apple flesh was cut into slices.
Immediately, the slices were immersed into 0.6% ascorbic acid solution to avoid the enzymatic browning. Afterward, the slices were squeezed by a juice extractor (JYL-C022E, Joyoung). The juice was collected and filtered with a 100 mesh filter. After sterilization at 98°C for 30 s and filled in brown glass bottles, the juice was cooled to room temperature (25°C) for further UF immediately.
Coomassie brilliant blue was purchased from Shanghai Baoman Co. Ltd.

| UF membranes and system
Five membranes with different materials and molecular weight cutoff (MWCO), as shown in Table 1, were employed in this study.
The selection of MWCO was referred to the literatures (He, Ji, & Li, 2007;Onsekizoglu, Bahceci, & Acar, 2010) and our preliminary experiments. The schematic diagram of UF system is shown in Figure 1.
where J v is the permeate flux during UF process (L/(m 2 ·hr)), ΔV is the permeate volume (L) collected at the same interval t (hr) and A m is the active area of membrane (A m = 2.38 × 10 -3 m 2 ).
Effects of different membrane materials, MWCO, flow rates, and TMPs on the quality of juice were investigated.

| Physicochemical analysis
Color of fruit juice was measured by a spectrophotometer at 420 nm according to a published method (Rai et al., 2006).
According to percentage of transmittance (%T), clarity was measured by the method with some modification according to the following equation.
(1) where A is the optical absorbance at a wavelength of 660 nm.

TA B L E 1 Properties of UF membranes
Total soluble solid (°Brix) was measured using Abbe refractometer as described by Ranganna (2005).
pH value of juice was measured by a multi-parameter pocket tester (Allometrics, Inc.).

| Determination of total phenolic
Total phenolic compounds in apple juice were determined by the Folin-Ciocalteu colorimetric method (Vasco, Ruales, & Eldin, 2008) with some modifications. 0.2 ml sample aliquot was mixed with 1 ml of a 10 fold diluted Folin-Ciocalteu reagent and 0.8 ml 7.5% sodium carbonate. The mixture was allowed to stand for 30 min at room temperature, measured at 760 nm by a UV-visible spectrophotometer (V-1800PC). Gallic acid solutions with concentrations ranging from 10 to 100 mg/L were used for calibration, and results were expressed as mg/L gallic acid equivalent (GAE).

. Principle of the Bradford Protein
Assay is based on an absorbance maximum at 595 nm for Coomassie brilliant blue G-250 (CBBG) when binding to protein occurs. The bovine serum albumin (BSA) as standard protein (10 mg) was dissolved in 10 ml 0.2 M phosphate buffered saline (PBS, pH 7.4) to be a concentration of 1 mg/ml as stock. 1, 2, 3, 4, and 5 μg/ml protein standards were prepared from the stock solution for the standard assay. One hundred milligram CBBG was dissolved in 50 ml 95% ethanol. One hundred milliliter phosphoric acid (85% w/v) was added, and the solution was diluted to be 1 L with deionized water and filtered twice immediately.
One milliliter protein standards were mixed with 5 ml CBBG dye. After being incubated for 5 min, the absorbance at 595 nm was measured.

| Determination of total sugars
Total sugars were analyzed by phenol-sulfuric acid method (Masuko et al., 2005) with some modifications. 1.0 ml samples diluted with 1.0 ml distilled water were placed in a tube. One milliliter phenol solution was added, shaken, following added 5 ml concentrated sulfuric acid, rapidly. The mixture heated for 5 min at 90°C in a static water bath. After cooling to a room temperature for 20 min, the absorbance was measured at 490 nm.

| Storage study
Ultrafilted apple juice was transferred into a sterile brown glass bottle with a sterile measuring cylinder and stored in a refrigerator at 4°C for 4 weeks in the dark. Changes of physicochemical properties, total proteins, polyphenols total sugars were determined weekly.

| Statistical analysis
Each experiment was conducted in triplicate. The data were processed and analyzed by using OriginPro 8, and the data were expressed by mean standard deviation. (2) %T = 100 × 10 −A Cross flow rate can also play an important role in flux, especially at a lower TMP. The flux can be improved when the   membrane surface differences, which can also affect the characteristics of apple juices. total phenols, total proteins, total sugars, and total soluble solids in clarified apple juice increased with the same membrane types.

| Effects of various conditions on quality of clarified apple juice
Total sugars in the filtrate treated by M3 were about 2.78 times of that by M5. Because the differences of a cake layer formed by some macromolecules such as proteins, polysaccharides, and aggregates on membrane surface. This cake layer will intercept some small molecular chemicals, such as phenols and monosaccharide.
As Huang et al. (2013) reported that with the increase of MWCO, when solutes with larger size that have been trapped on membrane surface to form a cake layer, the layer composed of polymer solutes has more holes and higher permeability. Concentration of protein can also affect the shelf life of fruit juice. Low protein content of apple juice treated with M4 was beneficial for storage, and the other components were higher. Meanwhile, the high permeate flux made the process more effective. Therefore, M4 was selected as the best membrane for clarification. Table 3, profiles of apple juice among nine groups at different conditions were almost the same. When the flow rate and pressure increasing, total phenols, total sugars, and total soluble solids all increased except the total proteins. Clarification degree decreased as the pressure increasing. Consequently, the optimal conditions for apple juice clarification should be at 30 L/hr and 0.75 MPa.

| Effects of UF on polyphenol profiles of apple juice
Polyphenols in apple juice might be combined with proteins, or co-colored with other compounds in the system, or oxidative condensation of polyphenols themselves. Other components in apple juice may also be directly or indirectly affected with polyphenols.
As shown in Figure 3, Table 4 shows the changes of quality properties of clarified apple juice in 4 weeks storage. It indicates that the properties changed slightly.

| Properties change of clarified juice during storage
Clarity, total sugars, and total phenols reduced by 2.5%, 6.4%, and 16.6%, respectively, while TSS increased by 3.1%. Concentration of phenolics decreased gradually, this degradation of polyphenols was accordance with the reported study (Knebel, Braun, & Dietrich, 2018).
Consequently, ultrafiltrated juice can be preserved at 4°C without significant quality deterioration for 4 weeks.

| CON CLUS IONS
Clarified apple juice is popular for consumers because of its unique light transmittance, flavor, and taste. An optimal membrane and operated conditions carried out could promote the quality of clarified juice. PES-10 kDa membrane, CFR 30 L/hr, and TMP 0.75 MPa were found to be the most suitable conditions for clarification of apple juice. The clarified apple juice with a color 0.15 A 420 , clarity 96.94%T, TSS 9.55 °Brix, pH value 4.2, and total phenols, total proteins, and total sugars were 64.12 and 13.20 μg/ml and 50.70 mg/ ml, respectively. Ultrafiltrated juice can be preserved at 4°C without significant quality deterioration for 4 weeks. However, there are still some components loss during the clarification process. It is necessary to find a way to improve the membrane technology for juice treatment.
F I G U R E 3 Changes in polyphenols before (a) and after (b) ultrafiltration.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation of China (No. 31401506).

CO N FLI C T O F I NTE R E S T
The authors declared that we had no any conflict of interest.

E TH I C A L A PPROVA L
The study did not include any animal or human tests.