Deacidification of Cranberry Juice by Electrodialysis with Bipolar

Article pubs.acs.org/JAFC

Deacidification of Cranberry Juice by Electrodialysis with Bipolar Membranes Elodie Rozoy,† Leslie Boudesocque,§ and Laurent Bazinet*,† †

Institute of Nutrition and Functional Foods (INAF) and Department of Food Sciences, Pavillon Paul Comtois, Université Laval, Quebec, QC, Canada G1V 0A6 § UMR INRA 1282 Infectiology and Public Health, Faculty of Pharmacy Philippe Maupas, University François Rabelais, 31 avenue Monge, 37200 Tours, France ABSTRACT: Cranberry is recognized for its many benefits on human health; however, its high acidity may be a limiting factor for its consumption. This study aimed to investigate the deacidification of cranberry juice using a two simultaneous step electrodialysis with bipolar membranes (EDBM) process. In step 1 (deacidification), during the 6 h treatment, the pH of the juice increased from 2.47 to 2.71 and a deacidification rate of 22.84% was obtained, whereas in step 2 (pH lowering) the pH of juice 2 was almost stable. Citric, quinic, and malic acid were extracted with a maximum of 25% and were mainly transferred to the KCl 2 fraction. A significant loss of anthocyanins in juice 2 (step 2) was observed, due to their oxidation by oxygen incorporated by the centrifugal pump. This also affected its coloration. The first step of the EDBM process was successful for cranberry juice deacidification and could be improved by increasing the number of membranes stacked. KEYWORDS: cranberry juice, deacidification, electrodialysis, bipolar membranes

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INTRODUCTION Cranberry juice has a long history of scientific interest regarding its many positive benefits on human health. Particularly, it has a strong activity against Helicobacter pylori,1 reduces urinary tract infection,2 and can prevent gastrointestinal disorders.3 All of these health benefits should attract consumers, but the high content of organic acids and low pH of cranberry juice are inconveniences that limit its consumption. The main acids present in cranberry juice are citric, malic, and quinic acids, and there is also the presence of benzoic and glucuronic acids, in lower quantities.4 Those acids are responsible for the high titratable acidity of the juice and its astringency. Different acidic juices such as mandarin orange juice,5 pineapple juice,6 passion fruit juice,7 and tropical fruit juice8 have already been deacidified by electrodialysis. Electrodialysis is an electrochemical process based on the migration of electrically charged species through selective membranes, under the influence of an electrical field. This electrical field is created by the application of a potential difference between the two electrodes present in the cell. This process is affordable, environmentally friendly as it does not use solvents, and easy to use. More recently, bipolar membranes (BM), which are membranes dissociating water molecules in protons (on the cationic side of the BM) and hydroxyls (on the anionic side) under the effect of electric field, were stacked in electrodialysis cells. Hence, electrodialysis with bipolar membranes (EDBM) has found applications in food systems regarding production or separation of organic acids,9−14 production of hydroxide and hydrochloric acids,15,16 inhibition of juice enzymatic browning,17 and deacidification of fruit juices.5,18 A configuration already published for the deacidification of red wine19 was preliminarily tested in the present study on cranberry juice (results not shown), but the results were not satisfying due to the difference in initial pH between cranberry © 2014 American Chemical Society

juice and wine, influencing the migration of the acids according to their pKa. A system more complex and more eco-efficient was then proposed here for cranberry juice deacidification. The objectives of this work were (1) to use a two-step deacidification process to deacidify and then decrease the pH of the juice to its initial pH, which has never been performed to our knowledge; (2) to study the composition evolution of a cranberry juice deacidified during EDBM treatments; and (3) to study the EDBM parameters evolution and the potential fouling of the membranes during repetitive uses.

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MATERIALS AND METHODS

Materials. Food grade Neosepta AMX-SB anion-exchange membrane, CMX-SB cation-exchange membrane, and BP-1 bipolar membrane from Tokuyama Soda Ltd. (Tokyo, Japan) were used. Solutions of 20 g/L NaCl and 2 g/L KCl (Mat Laboratory, Québec, QC, Canada) were prepared. Treatments were carried out on a pasteurized and clarified cranberry juice produced by Fruit d’Or (Notre-Dame-de-Lourdes, QC, Canada). The juice was stored at −28 °C and thawed at 4 °C before each experiment. Electrodialysis Cell. The electrodialysis cell was an MP type cell with 100 cm2 of effective electrode surface (ElectroCell AB, Täby, Sweden) with two AMX-SB anionic membranes, two bipolar membranes, and two CMX-SB cationic membranes (Figure 1). Each membrane was placed behind two rubber gaskets, and there was one polypropylene spacer behind each membrane, representing the cell compartment. Food grade stainless steel cathode and a dimensionally stable anode (DSA-O2) were used. The KCl and NaCl solutions were connected to their own external tanks and circulated in the compartments using two centrifugal pumps (Iwaki Magnet Pump, Received: Revised: Accepted: Published: 642

September 3, 2014 December 16, 2014 December 22, 2014 December 22, 2014 DOI: 10.1021/jf502824f J. Agric. Food Chem. 2015, 63, 642−651

Article

Journal of Agricultural and Food Chemistry

Juice from the first step was reused as juice 2 in the second step. The KCl 1 in the first step was also reused as KCl in the second step, but 250 mL of new solution (2 g/L KCl) was added to the previous one to reach a volume of 550 mL. A new juice called juice 1 was used (Figure 1b). The volumes for juice 1, juice 2, and KCl were 550 mL, and the volume of NaCl remained identical to that in the first step. All flow rates were identical to those of the first step, and the experiments were also performed at room temperature. Every electrodialytic parameter was recorded according to the first step, and samples were also taken to proceed to further analyses. At the end of the treatment, the electrodialysis system was cleaned as previously. Analyses. Conductivity. A YSI conductivity meter, model 3100, was used with a YSI immersion probe (model 3252, cell constant K = 1 cm−1; Yellow Springs Instrument, Yellow Springs, OH, USA) to measure the conductivity of all solutions. Global System Resistance. The global resistance of the system was calculated using Ohm’s law (U = RI). The values of voltage were read directly from the power supply indicators and the current passing through the electrodes from a multimeter (model 52-0060-2, Mastercraft, Toronto, Canada). Membrane Electrical Conductivity. The electrical conductivity of the membrane was measured with a specially designed cell (conductivity clip) from the Laboratoire des Matériaux Echangeurs d’Ions (Créteil, France). The membrane’s electrical conductivity κ (mS/cm) was calculated as

Figure 1. Configuration used for (a) the first and (b) second electrodialysis steps. AEM was an anion-exchange membrane, BP was a bipolar membrane, and CEM was a cation-exchange membrane.

κ= Iwaki Co. Ltd., Tokyo, Japan). The flow rates were controlled using flowmeters (Aalborg Instrument and Controls, Inc., Orangeburg, NY, USA), whereas cranberry juice was circulated using a peristaltic pump (Kate, Barnant Co., Barrington, IL, USA) to avoid oxygen incorporation. Protocol of Deacidification by ED. The deacidification was performed in two steps; the first was carried out to decrease the quantity of acids in the juice, as well as increase the pH of the juice. The second step was to reduce the pH of the juice previously deacidified to its initial value, in order to not affect its color, and to deacidify in the same time a new batch of juice to optimize the efficiency of the whole process. Step 1 (Figure 1a). The volume of cranberry juice used in the first step was 750 mL. In the electrode compartment, 900 mL of 20 g/L NaCl was used, and 650 mL of 2 g/L KCl was put in KCl 1 and KCl 2 compartments. The flow rates were 1000 mL/min for the electrolyte (NaCl), 600 mL/min for the juice and KCl 1 (the flow is divided in two circuits in the cell, and consequently the flow rate is equal to 300 mL/min), and 300 mL/min for KCl 2. This study was carried out at room temperature. The first step was performed during 6 h. During the electrodialysis treatment, the pH of the juice, KCl 1, and KCl 2 was recorded, as well as the conductivity of all those compartments. The current intensity, as well as the temperature of the compartment containing the juice, was also recorded. Samples (15 mL) were taken every hour to perform further analyses concerning the titratable acidity, total polyphenols, anthocyanins, organic acids, and degrees Brix. After the experiment, the juice that was deacidified and KCl 1 were stored at 4 °C. KCl 2 and NaCl solutions were discarded as they were only necessary for beginning the first step of the process. Then the electrodialysis system was cleaned as follows: 10 min of circulation of hot water, then 30 min of alkaline solution, rinsing with water at 55 °C and circulation for 10 min, then circulation of acidic solution for 20 min. Finally, every compartment was rinsed with water at 55 °C for 4− 5 min and then rinsed with distilled water. Step 2 (Figure 1b). The objective of the second step was to deacidify the cranberry juice (juice 1) as in the first step, as well as to decrease the pH of the juice deacidified in the first step under the action of the bipolar membranes, to restore the initial pH of this juice, with a diminished level of organic acids.

l R mA

where l is the membrane thickness (cm), Rm the transversal electric resistance of the membrane (Ω), and A the electrode area (1 cm2). Membrane Thickness. The membrane thickness was measured using a Mitutoyo Corp. IDC type digimatic indicator with absolute encoder (model ID-C112EB, Kanagawa-Ken, Japan) with a resolution of 1 μm and a range of 12.7−0.001 mm. The membrane thickness value was averaged from four measurements at different locations on the membrane. pH. The pH was measured using a VWR Symphony electrode equipped with an automatic temperature compensation device and connected to a VWR Symphony SP20 portable pH meter (Thermo Orion, West Chester, PA, USA). Titratable Acidity. The titratable acidity was evaluated according to AOAC method 942.15. Forty milliliters of degassed distilled water was added to 4 mL of juice, and then 0.1 M NaOH was added drop by drop until pH 8.2 was reached. The pH was measured with the same electrode and pH meter as for the pH measurements. The titratable acidity was expressed as milliliters of NaOH (0.1 M) added. Degrees Brix. The degrees Brix of cranberry juices was measured using a Digital Pocket Refractometer (model PAL-1, 0−53%, Atago, Tokyo, Japan). Total Polyphenol Content. Total polyphenol content measurements in raw juice, juice step 1 (S1), juice 1−step 2 (S2), and juice 2− step 2 (S2) were performed using the microscale protocol for Folin− Ciocalteu colorimetry of Waterhouse.20 The samples and the standards of gallic acid absorbances were measured at 765 nm using a UV− visible Agilent 8453 spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). Concentration in total polyphenol was expressed as gallic acid equivalent per liter of juice (mg/L). Anthocyanin Content. Total anthocyanin concentration was quantified by UV−visible spectroscopy following the method described by Giusti and Wrolstad.21 Each sample of cranberry juice was diluted 10 times in two distinct aqueous buffers: 0.025 M potassium chloride buffer, pH 1.0; and 0.4 M sodium acetate buffer, pH 4.5. Diluted solutions were equilibrated for 15 min at room temperature before analysis. The absorbance of each diluted sample was measured at 520 and 700 nm using a UV−visible Agilent 8453 spectrophotometer (Agilent technologies, Palo Alto, CA, USA). Absorbance (A) due to the contribution of total anthocyanins was calculated as follows: 643

DOI: 10.1021/jf502824f J. Agric. Food Chem. 2015, 63, 642−651

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Journal of Agricultural and Food Chemistry A = (A510nm − A 700nm )pH1.0 − (A510nm − A 700nm )pH4.5

red to green)m and b* (intensity of color varying from yellow to blue).24 Statistical Analyses. Data obtained during treatments for pH, conductivity, titratable acidity, and organic acids content were subjected to a repeated-measures analysis of variance using SigmaPlot software (version 11.0) or for anthocyanin and total polyphenol content to an analysis of variance. Tukey tests were also performed on data using SigmaPlot software (version 11.0) to allow the determination of treatments that were statistically different from the other at a probability level of P < 0.05.

(1)

Concentration in total anthocyanins was expressed as milligrams of cyanidin-3-glucoside equivalent per liter of juice (mg/L) by applying the equation

monomeric cyanidin‐3‐glucoside pigment (mg/L) = (A × MWCyd‐3‐glu × dilution factor × 1000)/(εCyd‐3‐glu × L)

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(2) −1

−1

RESULTS AND DISCUSSION Evolution of the Electrodialytic Parameters. Global System Resistance. According to the repeated measures analysis of variance, during the first step of electrodialysis the global system resistance remained almost constant (P = 0.412) around 44 Ω (Figure 2). During the second electrodialysis step,

−1

with MWCyd‑3‑glu = 449.2 g mol , ε = 26900 L mol cm , and L = 1 cm. Proanthocyanidins (PACs), Catechin, PAC-A 2, and PAC-B 1 Contents. The content of global PACs content of juices was determined by the BL-DMAC (4-dimethylaminocinamaldehyde) method, adapted from the method described from Prior et al.22 The quantitative analysis of the global content of PACS of cranberry juices was carried out in triplicate for all three repetitions in 96-well plates. Prior to analysis, juices were freeze-dried to obtain dry residues. Standard calibration of PAC-A2 controls was performed in methanol from 0.05 to 0.5 mg/mL. Seven microliters of control solution was added to 63 μL of methanol in each well. Dry residues obtained previously were solubilized in distilled water at a concentration of 15 mg/mL, then 20 μL of sample solution was added to 50 μL of methanol in each well. DMAC reagent was solubilized in acidified ethanol at 0.1%, and 210 μL of DMAC solution was added to each well before the reading. Plate readings were performed at 630 nm every 2 min for 20 min, on a Biohit Bp 800 microplate reader, and the DO values used were the ones obtained after stabilization around 16− 18 min. A standard curve of PAC-A2 was then drawn, and a polynomial regression was applied to provide an equation allowing the calculation of the PACs content of samples. The contents of catechin, PAC-A2, and PAC-B1 of juices have been analyzed by densitometric HPTLC using a plate scanner at a wavelength of 225 nm according to the method developed by Boudesocque et al.23 Dry residues of juices obtained prior to the PACs content analysis were solubilized in distilled water at 75 mg/mL. Stock solution of controls was prepared by solubilization of an appropriate amount of catechin, PAC-A2, and PAC-B1 to obtain the initial concentration of 1 mg/mL. Diluted solutions at 0.02, 0.035, 0.05, and 0.07 mg/mL were obtained from the stock solution. Ten microliters of each control and sample solution in triplicate was sprayed by a Linomat 5 Automatic TLC Sampler (CAMAG, Switzerland), at 30 nL/s under nitrogen flow. Plate migration was performed in an automatic ADC 2 tank (CAMAG), with dichloromethane/ethyl acetate/formic acid (6:10:1, v/v) eluent. Plates are then analyzed by a TLC Plate Scanner (CAMAG) at 200, 225, and 279 nm, and quantification was performed at 225 nm through the calibration curve for each standard. Organic Acid Contents. Prior to analysis, organic acids were extracted from 1/5 diluted cranberry juices with a C18-SPE cartridge (6 mL, 500 mg, Silicycle, Québec, QC, Canada). The cartridges were previously conditioned with elution of 5 mL of methanol, then 5 mL of distilled water, and then 10 mL of acetonitrile/water (50:50 v/v) solution. After drying of the cartridges, 10 mL of sample was dropped off and the last 5 mL was kept for analysis. The individual organic acid contents of cranberry juices and KCl fractions were determined by HPLC based on AOAC method 986.13. The system used was a Waters 600 HPLC system (Milford, MA, USA) equipped with a UV detector (Waters, model 966). Organic acids were analyzed with an Allure Organic Acids column (300 × 4.6 mm, Restek, USA). Standards for all organic acids tested were used with external calibration. A solution of 0.2 M (v/v) KH2PO4 (pH 2.4) was used as solvent for elution at a 0.5 mL/min flow rate. The detection wavelength was 226 nm. Cranberry Juice Color. The color parameters of the cranberry juice were determined with a chromameter (model Minolta meter CR-200, Konica Minolta Inc., Mississauga, ON, Canada). Results were reported as L* (luminescence or lightness), a* (intensity of color varying from

Figure 2. Results of the global system resistance (ohms) of the first and second electrodialysis steps.

the global system resistance increased from 45.60 to 58.00 Ω during the first 15 min and then decreased to 48.26 Ω after 150 min of treatment and reached a plateau until the end of the experiment (P < 0.001). The decrease could be due to water dissociation after 15 min of treatment at the surface of the monopolar ion-exchange membranes. Indeed, during water dissociation, the high conductivity of the H+ ions produced facilitates the transport of the current, decreasing then the resistance.25 Membrane Parameters. Table 1 shows the thickness and electrical conductivity values of each membrane used for the EDBM treatments of the cranberry juices. The thickness was not significantly different after ED treatment for CMX-SB-1 membrane (P = 0.184) and for the two bipolar membranes (P = 0.259 and P = 0.791). For the other membranes, the thickness after the treatment was significantly higher (P < 0.05). However, the electrical conductivity of every CMX-SB membrane was not significantly different (P > 0.05) after use, meaning that their integrities were preserved, although they were in contact with the juice during the second step of the treatment. The electrical conductivity of AMX-SB membranes significantly decreased after the treatment (P < 0.001), as well as the conductivity of bipolar membranes (P < 0.001). During the two-step process, AEMs were in contact with raw juice in step 1 (see Figure 1a) and juice 1 in step 2 (see Figure 1b): 644


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Journal of Agricultural and Food Chemistry Table 1. Thickness and Conductivity of Each Membrane Used before and after Electrodialysis Treatmenta thickness (cm)

a

membrane

before treatment

CMX-SB-1 CMX-SB-2 AMX-SB-1 AMX-SB-2 BP-1E-1 BP-1E-2

0.170 0.172 0.136 0.140 0.237 0.240

± ± ± ± ± ±

0.002a 0.001a 0.002a 0.000a 0.001a 0.001a

conductivity (mS/cm) after treatment 0.173 0.175 0.146 0.146 0.236 0.244

± ± ± ± ± ±

0.003a 0.001b 0.004b 0.002b 0.004a 0.006a

before treatment 10.31 10.67 7.62 7.46 6.31 6.27

± ± ± ± ± ±

after treatment

0.28a 0.12a 0.17b 0.51b 0.09b 0.26b

8.67 9.24 5.50 6.02 5.68 5.45

± ± ± ± ± ±

1.50a 1.71a 0.59a 0.08a 0.31a 0.06a

Data in the same row with different letters for the same parameter are significantly different at a probability level of 0.05.

Figure 3. Photographs of anionic (AMX-SB), cationic (CMX-SB), and bipolar (BP-1) membranes.

organic acids and negative ions passed through this membrane during all of the process, but other negative molecules could have built up a deposit on the surface during the treatment, decreasing the conductivity of these membranes. As can be seen in Figure 3, the color of the CEM, AEM, and bipolar membranes changed (active and nonactive surface). These membranes may have interacted with a positively charged compound such as proanthocyanidin, anthocyanin, or other polyphenolic products, as well as negatively charged molecules such as organic acids and other molecules with high molecular weight such as pectin. All of these interactions could explain the difference of color of the membranes and their possible responsibity in fouling. This result is consistent with the literature. Indeed, a study demonstrated that the fouling appears in the AEM when this membrane transports the organic anion and that this fouling could appear when the current density is close to the limiting current.8 It has been demonstrated in a previous study26 that organic fouling can decrease the electrical conductivity of the ion-exchange membrane surface. Vera et al.18 also observed a fouling on the anion-exchange membranes they used that were in contact with the treated passion fruit juice and attributed it to the colored compounds present in the passion fruit juice. Bipolar membranes were in contact with juice in step 1 and both juices in step 2, so that increased the risk of fouling at the surface of this membrane. Indeed, the fouling of membranes by charged compounds present in the juice is dependent on the position of the membranes in the configuration cell as well as the solution they are in contact with, as demonstrated before.27 Juice Parameter Evolution. pH. On the basis of the results obtained from the repeated measures analysis of variance, the first-step treatment had a significant effect on the pH (P < 0.001) of juice and both KCl fractions (Figure 4a). The pH of raw juice increased from 2.47 ± 0.02 to 2.71 ± 0.02 during the 6 h treatment (P < 0.001). The pH increase was higher in the first 75 min of treatment. At the initial pH of the juice, >60% of all acids were in the molecular and monoionized form, because the pH of the juice was below their first pKa.8

Figure 4. Evolution of the pH as a function of time for (a) the first electrodialysis step of juice 1, KCl 1, and KCl 2 and (b) the second electrodialysis step of juice 1, juice 2, and KCl.

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Journal of Agricultural and Food Chemistry The fact that a minority of the acids were charged did not help their migration through the anionic membranes. Concerning KCl 1, its pH decreased quickly from 2.85 ± 0.27 to reach a plateau around 2.26 ± 0.25 (P < 0.001) after only 55 min. The pH of KCl 2 decreased quickly in the first 75 min from 3.37 ± 0.16 to 2.51 ± 1.89 (P < 0.001), its final value. The pH decrease in the KCl 2 compartment followed closely the pH increase observed for juice (S1); the organic acids that left the juice were concentrated in the KCl 2 compartment, as expected with the cell configuration. In step 2 (Figure 4b), the pH of juice 1 followed the same behavior as juice in step 1, with an increase from pH 2.46 ± 0.02 to 2.68 ± 0.05 (P < 0.001); meanwhile, the pH of juice 2 was almost stable during the first 75 min of the treatment and then decreased slightly (P = 0.019). Surprisingly, the pH of KCl in step 2 increased quickly from its initial value of 2.26 ± 0.13 to 2.45 ± 0.03 in the first 75 min and then continued to increase, but more slowly (P = 0.033). With regard to the previously reported results obtained with passion fruit juices,18,28 the pH variation obtained in our study was lower (0.3 pH unit versus >1.0 pH unit), which was explained by a lower initial pH in our study, far below the pKa of the acids, corresponding to less charged species able to be extracted from the juice, so higher time or energy was required to achieve the transfer. Conductivity. According to the repeated-measures analysis of variance, the electrodialysis treatment in step 1 had a significant effect on the conductivity values of juice and KCl fractions (P < 0.001). During the first step (Figure 5a), the conductivity of juice remained almost constant around 3152 ± 65 μS/cm for the first 75 min and then decreased quickly to 2856 ± 21 μS/cm at the end of the treatment (P < 0.001). This is explained by a demineralization of the juice, as well as a neutralization of the protons and the loss of organic acids and other charged molecules, phenomena already reported in the deacidification of passion fruit juices.8 At the same time, the conductivity of KCl 1 followed a neperian logarithm increase: KCl 1 conductivity increased quickly from 3605 ± 73 μS/cm to reach a plateau at 4996 ± 219 μS/cm after 120 min of treatment (P < 0.001). The conductivity of KCl 2 fraction increased also from 3364 ± 22 to 4454 ± 110 μS/cm (P < 0.001), but the slope of the curve was slightly higher in the first 100 min compared to the last 300 min of the treatment. As expected, the conductivity of both KCl fractions increased due to the migration of organic acids as well as other charged molecules and ions from the juice into these compartments. To our knowledge, the conductivity of other KCl fractions during deacidification by electrodialysis has never been reported in the literature. During the second step of electrodialysis (Figure 5b), the conductivity of juice 1 remained almost constant around 3046 ± 51 μS/cm during the first 60 min (P > 0.05) and then slightly decreased to reach 2801 ± 106 μS/cm at the end of the 240 min treatment. This behavior was similar to that of juice in step 1, due to the same movement of molecules. The conductivity of juice 2 (the juice obtained in the first step of the treatment) was not significantly different (P > 0.05) for the first 30 min and then decreased to reach a plateau at around 2264 ± 66 μS/cm. The conductivity of KCl in step 2 slightly decreased from 3711 ± 240 to 3476 ± 296 μS/cm during the first 90 min and then increased to reach 3929 ± 166 μS/cm at the end of the treatment (P < 0.001). In the second step, the KCl fraction seems to compensate for the decrease in conductivity of juice 1

Figure 5. Evolution of the conductivity as a function of time for (a) the first electrodialysis step of juice 1, KCl 1, and KCl 2 and (b) the second electrodialysis step of juice 1, juice 2, and KCl.

according to step 1; moreover, juice 2 seems to be subjected to a demineralization because its conductivity decreased. Titratable Acidity. The titratable acidity of the juice in step 1 decreased during the 6 h of treatment from 15.90 ± 0.87 mL of 0.1 N NaOH to reach pH 8.2, to 12.27 ± 0.64 mL (Figure 6a), which corresponds to a 22.84 ± 0.22% deacidification. At the same time, the titratable acidity of KCl 1 fraction increased from 0.10 ± 0.00 mL to reach an almost stable value of 0.30 ± 0.00 mL of 0.1 N NaOH at 240 min. The titratable acidity of KCl 2 in the first step increased from 0.10 ± 0.00 mL to 2.85 ± 0.39 mL of 0.1 N NaOH: the KCl 2 compartment seemed to recover the acidity loss from the juice during the treatment. Vera et al.28 have reported a deacidification of 70% of different clarified tropical fruit juices including passion fruit juices when they reached pH 4, using two electrodialysis cells with bipolar membranes of 20 and 200 cm2 membrane areas, with various applied current densities between 50 and 400 A/m2, but the duration of the ED treatment was not known. All of the juices used in this previous study had initial pH values higher than those of the cranberry juices (near pH 3.0), so citric and malic acids, both major acids in those juices, were mainly in their molecular form,8,29 but at lower percentages than in cranberry juice, allowing a higher migration rate of these charged species. In the second step (Figure 6b), the titratable acidity of juice 1 decreased from 15.97 ± 1.42 mL to 12.33 ± 1.46 mL of 0.1 N NaOH, corresponding to a deacidification of 22.78 ± 5.89%, comparable to the first step, with 2 h less treatment. During the 646


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0.12 during the 6 h treatment (P < 0.001). During the 4 h treatment, the degrees Brix of juice 1−step 2 also decreased significantly (P < 0.001) following the same behavior as juice in step 1 (P > 0.05). Because the degrees Brix refers to the total soluble solids (primarily sugars and acids) and the titratable acidity decreased for the juice in step 1 and juice 1 in step 2, it was expected that the degrees Brix would decrease in these juices, as seen for the deacidification of tropical fruit juices.28 However, juice 2 had a constant degrees Brix around 6.23 ± 0.15 during the 4 h treatment of step 2, lower than that of juice in step 1 and juice 1 in step 2 (P < 0.001), which is consistent with the titratable acidity results, because it was stable during the treatment for this juice. Total Polyphenol Content. On the basis of the results obtained from the analysis of variance, every electrodialysistreated juice had a significantly lower total polyphenol content than the cranberry control juice (P < 0.001, Figure 8). Indeed,

Figure 6. Evolution of the titratable acidity as a function of time for (a) the first electrodialysis step of juice 1, KCl 1, and KCl 2 and (b) the second electrodialysis step of juice 1, juice 2, and KCl.

Figure 8. Results of total polyphenols content of the raw juice and the juices at the end of each treatment.

same time the titratable acidity of juice 2 remained stable (P > 0.5) at a lower acidity than juice 1, whereas KCl increased from 0.23 ± 0.08 to 1.80 ± 0.20 mL, possibly compensating for the juice 1 acidity decrease. Degrees Brix. The degrees Brix of juice in the first step (Figure 7) significantly decreased from 7.37 ± 0.06 to 6.87 ±

control juice had total polyphenol content of 799.15 ± 46.13 mg/L gallic acid equivalent, compared to around 703.10 ± 23.04 mg/L for juice−step 1 and juice 1−step 2 and 633.80 ± 44.01 mg/L for juice 2−step 2. Those results can be correlated to the fouling of membranes, because the coloration of the different membranes suggested a polyphenol/membrane interaction and a possible anthocyanins/membrane interaction. Anthocyanin Content. Because the content of total polyphenol decreased for each juice treatment, the anthocyanin content was investigated. It appeared that this content was not significantly different (P > 0.05) for juice−step 1 and juice 1− step 2 at around 84.40 ± 1.14 mg/L cyanidin-3-glucoside equivalent before or after electrodialysis treatment, so those molecules did not migrate during the ED treatment. Otherwise, juice 2−step 2 had significantly lower anthocyanin content that other juices at the beginning of the experiment (P < 0.001) and even lower content at the end of the treatment (P < 0.001) (Figure 9). These results may suggest that there was a loss in anthocyanin between each step, due to storage (even for 0.05) in the citric acid content as a function of time, there was a tendency to decrease, due to the migration of citrate ions through the AEMs membranes to produce citric acid in the KCl compartment. Indeed, as expected because of the cell configuration, the content of citric acid increased in the KCl compartment to reach 301.84 ± 39.55 mg/100 mL. Juice 2−step 2 remained stable for its content in citric acid during the 240 min of treatment (P > 0.05). Furthermore, the citric acid content of juice 2−step 2 was significantly lower than that of juice−step 1 and juice 1− step 2 from the beginning of the treatment until 120 min of electrodialysis, because of the migration of citrate ions during the first step of treatment. These trends were similar for quinic acid (Figure 10b), except that juice 1−step 2 had a higher content of quinic acid at the beginning of the electrodialysis than at the end at 240 min (P = 0.022) and that the content of quinic acid of juice 2 in the second step was lower than for juice−step 1 and juice 1−step 2 during the 240 min of treatment. In that case, the quinic acid also concentrated in KCl 2−step 1 and the KCl−step 2, due to their same position in the cell. It has to be noted that the concentrations of quinic acid at times 0, 60, and 240 min were higher for KCl−step 2 compared to KCl 2−step 1. This can be due to the accumulation of quinic acid during the first passage in the electrodialysis, where the final concentration of KCl 1 was 17.43 ± 1.48 mg/100 mL. The content of malic acid (Figure 10c) significantly decreased from 846.52 ± 54.13 to 635.13 ± 30.97 mg/100 mL during the treatment of juice−step 1 (P < 0.001), which corresponds to an extraction of 211.38 ± 54.65 mg/100 mL or 25% of this acid, whereas the KCl 2 fraction was enriched in this acid to reach a final content of 127.45 ± 22.98 mg/100 mL. A very little content of malic acid passed in the KCl 1 fraction to reach a final value of 1.24 ± 0.30 mg/100 mL. In the second step of the electrodialysis treatment, juice 1 and the KCl fractions had the same behavior as in the first step (P < 0.001), whereas juice 2 was not affected by the second electrodialysis treatment (P > 0.05), as for the other acids. In their study, Bazinet et al.27 also found a migration of citric and malic acid through an ultrafiltration membrane, when enriching a cranberry juice in antioxidants, confirming the migration ability of those acids during electrodialysis treatment. The most abundant organic acid found in the KCl 2−step 1 and KCl−step 2 recovery compartment was the citric acid. Among the acids present in the juice, citric acid was the most available to migrate at the pH of the juice, due to its high concentration (in comparison with other acids), its low molecular weight (192.12 Da), and its first pKa (3.14), which was the lowest of all the acids.8

Figure 9. Results of total anthocyanins content at the end of each electrodialysis treatment.

Indeed, the decrease in total polyphenols for juice−step 1 and juice 1−step 2 was not due to anthocyanin degradation or fouling on membranes, contrary to what was suggested. PACs, Catechin, PAC-A2, and PAC-B1 Contents. According to the analysis of variance, raw juice, juice−step 1, juice 1−step 2, and juice 2−step 2 were not significantly different (P > 0.05) with regard to their global PACs content, with values between 26.53 ± 3.11 and 28.60 ± 0.70 μg/mL, so these flavonoids were not degraded by the treatment (Table 2). However, this level of PACs was 16 times lower than what was already reported in the literature.22,31 No catechin and PAC-B1 have been observed by HTPLC. PAC-A2 has been detected, but with values below the lower point of the standard curve applied, so its quantity in the different juices was below 0.02 mg/mL of PAC-A2 standard. Those compounds were also not detected in cranberry juices by HPLC in another study.32 Organic Acid Contents. The major organic acids found in cranberry juices determined by HPLC analysis were citric acid (molecular weight (MW) = 192.12 g/mol), malic acid (MW = 134.09 g/mol), and quinic acid (MW = 192.17 g/mol). The analysis of variance showed that the citric acid content of juice−step 1 (Figure 10a) was not significantly different (P > 0.05) throughout the treatment, but there was a tendency to decrease. Indeed, its concentration decreased from 3501.47 ± 215.75 mg/100 mL at 30 min to 2989.38 ± 355.41 mg/100 mL at the end of the treatment. On the other hand, the KCl 2 fraction had a content of citric acid that increased during the 6 h electrodialysis (P < 0.001) to reach 401.15 ± 36.58 mg/100 mL, compensating for the slight loss of citric acid in the juice, as expected with the cell configuration. Citric acid was produced in the KCl 2 fraction from citrate ion extracted from the juice and protons provided by BP-1 that went through CEM-1, as demonstrated in another study.8 It is not visible in Figure 10a, but the content of citric acid in KCl 1 fraction increased slightly

Table 2. Global Proanthocyanidins (PACs), Catechin, PAC-A2, and PAC-B1 Contents (μg/mL) in Cranberry Juicesa PACs catechin PAC-A2 PAC-B1

raw juice

juice (S1)

juice 1 (S2)

juice 2 (S2)

26.77 ± 3.57a ndb bdlc nd

26.53 ± 3.11a nd bdl nd

28.60 ± 0.70a nd bdl nd

28.00 ± 1.11a nd bdl nd

a

Data in the same row with different letters for the same parameter are significantly different at a probability level of 0.05. bnd, not determined (no peak corresponded to the peaks of the standards tested). cbdl, below detection level (below the lower point of the standard curve). 648


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Figure 10. Evolution of (a, top) citric acid, (b, middle) quinic acid, and (c, bottom) malic acid contents of juice 1, KCl 1, and KCl 2 in the first electrodialysis step (6 h), as well as of juice 1, juice 2, and KCl in the second step (4 h). Data with different letters for the same parameter are significantly different at a probability level of 0.05.

Cranberry Juice Color. The cranberry juice color was studied on raw juice, final juice 1−step 2, and juice 2−step 2 because the volume required (30 mL) to do the analysis was not available for juice−step 1, because it was always used for the second step of electrodialysis (Figure 11). Considering that there were no significant differences (P > 0.05) for each L*a*b parameters between the control juice and juice 1−step 2, there should not be significant differences for juice−step 1. However, juice 2−step 2 was significantly different from other juices

regarding component a*. Indeed, this juice had a lower intensity of red and was visibly affected by the second electrodialysis treatment. Due to the presence of anthocyanins in cranberry juices, particularly cyanidin galactoside, peonidin galactoside, cyanidin arabinoside, and peonidin arabinoside, responsible for the red color of the juice,33,34 and the variation of the intensity of ,a* parameter, already demonstrated to be correlated with anthocyanin content variation,25 we can conclude that the significant degradation of anthocyanin in 649



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ABBREVIATIONS USED

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REFERENCES

EDBM, electrodialysis with bipolar membrane; S1, step 1; S2, step 2; PACs, proanthocyanidins; DMAC, dimethylaminocinamaldehyde; CEM, cation-exchange membrane; AEM, anionexchange membrane; HPLC, high-performance liquid chromatography; MW, molecular weight

(1) Burger, O.; Weiss, E.; Sharon, N.; Tabak, M.; Neeman, I.; Ofek, I. Inhibition of Helicobacter pylori adhesion to human gastric mucus by a high-molecular-weight constituent of cranberry juice. Crit. Rev. Food Sci. Nutr. 2002, 42, 279−284. (2) Blatherwick, N. R.; Long, M. L. Studies of urinary acidity. II. The increased acidity produces by eating prunes and cranberries. J. Biol. Chem. 1923, 57, 815−818. (3) Vattem, D. A.; Ghaedian, R.; Shetty, K. Enhancing health benefits of berries through phenolic antioxidant enrichment: focus on cranberry. Asia Pac. J. Clin. Nutr. 2005, 14, 120−130. (4) Raz, R.; Chazan, B.; Dan, M. Cranberry juice and urinary tract infection. Clin. Infect. Dis. 2004, 38, 1413−1419. (5) Kang, J. J.; Rhee, K. C. Deacidification of mandarin orange juice by electrodialysis combined with ultrafiltration. Nutraceuticals Food 2002, 4, 411−416. (6) Sairi, M.; Jeng Yih, L.; Roji Sarmidi, M. Chemical composition and sensory analysis of fresh pineapple and deacidified pineapple juice using electrodialysis. In Regional Symposium on Membrane Science and Technology; uteri Pan Pacific Hotel, Johor Bahru, Johor, Malaysia, 2004; pp 1−9. (7) Vera Calle, E.; Ruales, J.; Dornier, M.; Sandeaux, J.; Sandeaux, R.; Pourcelly, G. Deacidification of the clarified passion fruit juice (P. edulis f. f lavicarpa). Desalination 2002, 149, 357−361. (8) Vera, E.; Sandeaux, J.; Persin, F.; Pourcelly, G.; Dornier, M.; Ruales, J. Deacidification of clarified tropical fruit juices by electrodialysis. Part I. Influence of operating conditions on the process performances. J. Food Eng. 2007, 78, 1427−1438. (9) Boyaval, P.; Seta, J.; Gavach, C. Concentrated propionic acid production by electrodialysis. Enzyme Microb. Technol. 1993, 15, 683− 686. (10) Tongwen, X.; Weihua, Y. Citric acid production by electrodialysis with bipolar membranes. Chem. Eng. Process.: Process Intensification 2002, 41, 519−524. (11) Siebold, M.; Frieling, P. V.; Joppien, R.; Rindfleisch, D.; Schügerl, K.; Röper, H. Comparison of the production of lactic acid by three different Lactobacilli and its recovery by extraction and electrodialysis. Process Biochem. 1995, 30, 81−95. (12) Trivedi, G. S.; Shah, B. G.; Adhikary, S. K.; Indusekhar, V. K.; Rangarajan, R. Studies on bipolar membranes. Part II  Conversion of sodium acetate to acetic acid and sodium hydroxide. React. Funct. Polym. 1997, 32, 209−215. (13) Wang, Y.; Zhang, X.; Xu, T. Integration of conventional electrodialysis and electrodialysis with bipolar membranes for production of organic acids. J. Membr. Sci. 2010, 365, 294−301. (14) Voss, H. Deacidification of citric acid solutions by electrodialysis. J. Membr. Sci. 1986, 27, 165−171. (15) Mazrou, S.; Kerdjoudj, H.; Che’Rif, A. T.; Mole’Nat, J. Sodium hydroxide and hydrochloric acid generation from sodium chloride and rock salt by electro-electrodialysis. J. Appl. Electrochem. 1997, 27, 558− 567. (16) Gineste, J. L.; Pourcelly, G.; Lorrain, Y.; Persin, F.; Gavach, C. Analysis of factors limiting the use of bipolar membranes: a simplified model to determine trends. J. Membr. Sci. 1996, 112, 199−208. (17) Quoc, A. L.; Mondor, M.; Lamarche, F.; Ippersiel, D.; Bazinet, L.; Makhlouf, J. Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice. Food Res. Int. 2006, 39, 755−760. (18) Vera, E.; Sandeaux, J.; Persin, F.; Pourcelly, G.; Dornier, M.; Ruales, J. Deacidification of passion fruit juice by electrodialysis with

Figure 11. Results for the L*a*b* color parameters of the raw juice, juice 1, and juice 2 in the second electrodialysis step. Data with different letters for the same parameter are significantly different at a probability level of 0.05.

juice 2−step 2 during the electrodialysis treatment influenced its color and confirmed the decrease observed previously for the anthocyanins content. This study demonstrated that it is possible to increase significantly the pH of a cranberry juice, as well as decrease its titratable acidity and organic acids contents by electrodialysis with bipolar membranes (EDBM). The first step of the treatment allowed better results than the second step concerning the polyphenols and anthocyanins content, as well as the color characteristics. The second step did not allow a pH decrease of juice 2 to recover its original characteristics; on the contrary, it seemed to deteriorate the cranberry juice. With regard to the electrodialytic parameters, the global system resistance decreased after each repetition, supported by the coloration of the different membranes and the diminution of the conductivity of the anionic and bipolar membranes due to the negatively charged molecules that passed through membranes or made a fouling at their surface. To improve the results obtained in this study, the use of a configuration considering only the first step but with a higher surface of membrane obtained by increasing the number of membranes stacked would increase the rate of juice deacidification. Finally, this study proves that it is possible to remove organic acids with electrodialysis, a green technique using no solvent, even when the pH of the juice is far lower than the pKa of the acids targeted.

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AUTHOR INFORMATION

Corresponding Author

*(L.B.) Phone: (418) 656-2131, ext. 7445. Fax: (418) 6563353. E-mail: [email protected]. Funding

We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Ministère de l’agriculture, Pêcheries et Alimentation du Québec (MAPAQ) for their financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Pascal Dubé and Alain Brousseau for their analytical help. 650


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Journal of Agricultural and Food Chemistry bipolar membrane after different pretreatments. J. Food Eng. 2009, 90, 67−73. (19) Rozoy, E.; Bazinet, L.; Gagné, F.; Bouissou, D.; Escudier, J.-L.; le Gratiet, Y.; Lutin, F.; d’Hauteville, J.; Bes, M. Développement d’un procédé de désacidification des vins par électrodialyse à membrane bipolaire: étude de faisabilité à l’échelle laboratoire. OIV 2013, 86, 187−208. (20) Waterhouse, A. L. Determination of total phenolics. In Current Protocols in Food Analytical Chemistry; Wiley: Hoboken, NJ, USA, 2002; pp I1.1.1−I1.1.8. (21) Giusti, M. M.; Wrolstad, R. E. Characterization and measurement of anthocyanins by UV-visible spectroscopy. In Current Protocols in Food Analytical Chemistry; Wiley: Hoboken, NJ, USA, 2001; pp F1.2.1−F1.2.13. (22) Prior, R. L.; Fan, E.; Ji, H.; Howell, A.; Nio, C.; Payne, M. J.; Reed, J. Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders. J. Sci. Food Agric. 2010, 90, 1473−1478. (23) Boudesocque, L.; Dorat, J.; Pothier, J.; Gueiffier, A.; EnguehardGueiffier, C. High performance thin layer chromatography− densitometry: a step further for quality control of cranberry extracts. Food Chem. 2013, 139, 866−871. (24) CIE. Recommendations on Uniform Color Spaces, Color-difference Equations, Psychometric Color Terms; CIE Publication 15 (E-1.3.1) 1971, Suppl. 2. Bureau Central de la CIE: Vienna, Austria, 1978. (25) Bazinet, L.; Cossec, C.; Gaudreau, H.; Desjardins, Y. Production of a phenolic antioxidant enriched cranberry juice by electrodialysis with filtration membrane. J. Agric. Food Chem. 2009, 57, 10245− 10251. (26) Grebenyuk, V. D.; Chebotareva, R. D.; Peters, S.; Linkov, V. Surface modification of anion-exchange electrodialysis membranes to enhance anti-fouling characteristics. Desalination 1998, 115, 313−329. (27) Bazinet, L.; Brianceau, S.; Dubé, P.; Desjardins, Y. Evolution of cranberry juice physico-chemical parameters during phenolic antioxidant enrichment by electrodialysis with filtration membrane. Sep. Purif. Technol. 2012, 87, 31−39. (28) Vera, E.; Sandeaux, J.; Persin, F.; Pourcelly, G.; Dornier, M.; Piombo, G.; Ruales, J. Deacidification of clarified tropical fruit juices by electrodialysis. Part II. Characteristics of the deacidified juices. J. Food Eng. 2007, 78, 1439−1445. (29) Novalic, S.; Jagschits, F.; Okwor, J.; Kulbe, K. D. Behaviour of citric acid during electrodialysis. J. Membr. Sci. 1995, 108, 201−205. (30) Markakis, P.; Jurd, L. Anthocyanins and their stability in foods. CRC Crit. Rev. Food Technol. 1974, 4, 437−456. (31) Cunningham, D. G.; Vannozzi, S.; O’Shea, E.; Turk, R. Analysis and standardization of cranberry products. In Quality Management of Nutraceuticals; American Chemical Society: Washington, DC, USA, 2001; Vol. 803, pp 151−166. (32) Biswas, N.; Balac, P.; Narlakanti, S. K.; Enamul Haque, M. D.; Mehedi Hassan, M. D. Identification of phenolic compounds in processed cranberries by HPLC method. J. Nutr. Food Sci. 2013, 3, 1− 5. (33) Neto, C. C.; Vinson, J. A. 6. Cranberry. In Herbal Medicine: Biomolecular and Clinical Aspects, 2nd ed.; Wachtel-Galor, S., Ed.; CRC Press: Boca Raton, FL, USA, 2011; pp 107−130. (34) Gu, L.; Kelm, M. A.; Hammerstone, J. F.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S.; Prior, R. L. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J. Nutr. 2004, 134, 613−617.

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Deacidification of Cranberry Juice by Electrodialysis with Bipolar

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