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Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice
Food Research International 39 (2006) 755–760 www.elsevier.com/locate/foodres
Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice A. Lam Quoc a, M. Mondor b
b,*
, F. Lamarche b, D. Ippersiel b, L. Bazinet a, J. Makhlouf
a
a Department of Food Sciences and Nutrition, Laval University, Sainte-Foy, Que., Canada G1K 7P4 Food Research and Development Center, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Que., Canada J2S 8E3
Received 30 August 2005; accepted 8 November 2005
Abstract The purpose of this work was to develop a process enabling the quick inactivation of the polyphenol oxidase and pectin methylesterase enzymes, which are present in cloudy or unclarified apple juice; These enzymes are respectively responsible for enzymatic browning and opalescence instability. In order to fulfill this objective, acidification of the apple juice to pH 2.0 was conducted by electrodialysis (bipolar–anionic membranes) followed by mild heat treatment at temperature of 40, 45 and 50 °C for a duration of 0–60 min. Then, juice pH was readjusted to its initial value by electrodialysis with bipolar–anionic membranes. It was shown that a mild heat treatment at 45 °C for 5 min performed on the acidified juice represents an appropriate condition to quickly inactivate the enzymes. Furthermore, the organoleptic properties of the juice after treatment were found to be preserved and the adjusted juice (pH readjusted to its initial value) shows a better color than an untreated apple juice. Opalescence of the adjusted juice was also more stable than for an untreated cloudy apple juice, when stored at 4 °C for 3 months. Crown Copyright Ó 2006 Published by Elsevier Ltd. All rights reserved. Keywords: Browning; Cloudy apple juice; Electrodialysis; Mild heat treatment; Opalescence stability
1. Introduction Cloudy or unclarified apple juice contains significant quantities of suspended pulp and is perceived as a natural food product that supplies dietary fiber and important nutrients. However, it is very difficult to produce superior quality juice since cloudy apple juice is very sensitive to enzymatic browning because it contains considerable quantities of polyphenols and polyphenol oxidase (PPO) (Lea, 1990). PPO refers to a group of copper-containing enzymes that catalyze the oxidation of phenolic compounds to o-quinones, which then polymerize to form complex dark pigments, thereby changing the color and aroma of the juice (Macheix, Fleuriet, & Billot, 1990). In addition, it is *
Corresponding author. Tel.: +1 450 773 1105; fax: +1 450 773 8461. E-mail address: [email protected] (M. Mondor).
very difficult to produce a cloudy or unclarified apple juice with good opalescence stability due to the presence of pectin methylesterases (PME). The pectin molecules present in suspension are degraded by the PME enzymes resulting in a loss of opalescence stability (Beveridge, 1997). Ze´mel, Sims, Marshall, and Balaban (1990) showed that it is possible to irreversibly inhibit PPO by acidifying the cloudy apple juice to pH 2.0, using HCl, and readjusting the juice pH at its initial value (3.35), by addition of NaOH. The PME can also be inactivated at low pH (pH 2.0) as shown by Owusu-Yaw, Marshall, Koburger, and Wei (1988) for the case of orange juice. The drawbacks of this approach are the dilution associated with the addition of acid and base, and the salty aftertaste. Based on these observations, Tronc (1996) and Tronc, Lamarche and Makhlouf (1997 and 1998) have demonstrated the feasibility of acidifying cloudy apple juice using
0963-9969/$ - see front matter. Crown Copyright Ó 2006 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.11.002
756
A. Lam Quoc et al. / Food Research International 39 (2006) 755–760
electrodialysis (ED) with bipolar–cationic membranes. Readjustment of pH to its initial value was achieved by ED with bipolar–anionic (BP–A) membranes. However, the process was found too lengthy and required addition of exogenous KCl in the juice to reach pH 2.0. At this pH, the juice was kept one hour at room temperature to inhibit the enzymes. The adjusted juice (pH 3.35), shows a PPO reactivation corresponding to 25% of its initial value (Tronc, 1996). Finally, from an industrial point of view, the energy required was too high (197 kWh/m3 of juice) due to the large spacer (8 mm) used in the electrodialysis cell. However, they demonstrated that organoleptic properties of the juice were preserved. In a more recent work, Lam Quoc, Lamarche, and Makhlouf (2000), improved the process by performing both acidification and pH readjustment steps using a BP– A configuration. Furthermore, thinner spacers (0.75 mm) were used in the electrodialysis cell and HCl was used as the base solution instead of KCl (Tronc, 1996; Tronc, Lamarche, & Makhlouf, 1998). The use of this configuration resulted in acceleration of the acidification step by a factor of 3, increasing the yield from 3.3 to 10 l of juice/m2 membrane/min. Furthermore, the adjusted juice (pH 3.35), shows only a very small reactivation of PPO activity of 0.8%, which is lower than the 25% level reported by Tronc (1996). The energy required by the process was also reduced to only 4–5 kWh/m3 of juice, which is a very low energy consumption from an industrial standpoint. However, the main drawback of this approach remains the fact that the juice at pH 2.0 still had to be kept at room temperature for one hour to inhibit the enzymes. In this paper, bipolar membrane ED followed by a mild heat treatment at 40, 45 or 50 °C for duration of 0–60 min was investigated to increase the rate of inactivation of the PPO and PME present in the unclarified apple juice. The influence of this treatment on the color and on the opalescence stability of the juice during storage will be discussed.
0.75 mm. The anode, a dimensionally stable electrode (DSA), and the cathode, a 316 stainless steel electrode, were supplied with the cell. The experiments were carried out using a BP–A configuration (Fig. 1) forming 10 compartments. A total of nine membranes were used: five bipolar membranes (Neosepta BP-1) and four anionic membranes (Neosepta AMX) from Tokuyama Soda Ltd. (Japan). This arrangement defines three closed loops containing the solution to alkalinize (0.1 N HCl), the cloudy apple juice to acidify and a 0.25 M K2SO4 solution used as rinsing solution for the electrodes. Each closed loop was connected to a separate external reservoir, allowing for continuous recycling. The electro-acidification was carried out with electrolytes volumes of 1 l for the solution to alkalinize (HCl), the apple juice to acidify and the K2SO4 solution. During treatment, juice temperature was maintained at 25 °C using a Haake G refrigerated bath (Haake, Berlin, Germany). The acidification of the juice by ED was conducted at a constant current density of 40 mA/cm2. The ED configuration for pH readjustment was the same as that shown in Fig. 1. However, the juice and HCl compartments were reversed. During the acidification and pH readjustment steps, conductivity and juice pH were measured at 1–2 min intervals until the end of the treatments, along with applied voltage. 2.3. Protocols 2.3.1. Electrodialysis combined with mild heat treatment The electro-acidified juices (pH 2.0) were subsequently heated at 40, 45 and 50 °C, as a function of time (0– 60 min) to inhibit the PPO and PME enzymes. The heat treatments were done on 50 ml samples placed in a water bath with a thermostat and under agitation. At the end of the heat treatments, the samples were immediately cooled by immersion in ice/cold water. Samples were analysed for PPO and PME activities, color and where compared with an acidified juice (pH 2.0) kept at room temperature for the corresponding time.
2. Materials and methods 2.1. Apple juice Juice was extracted from McIntosh apples that had been stored commercially under controlled atmosphere. The apples were crushed and pressed in a crusher-press model no. EG 260-X6 (Goodnature Products Inc., Buffalo, United States) under maximum pressure of 1500 psi. About 2 l of juice was extracted from 4 kg of apples for each experiment. The freshly extracted juice underwent bipolar membrane ED treatment immediately. 2.2. Electrodialysis cell configuration Electro-acidification was carried out using an ED-1-BP unit (100 cm2 of effective electrode surface) from Electrosynthesis Co. (Lancaster, NY, USA), with spacing of
Fig. 1. Electrodialysis experimental systems: bipolar–anionic membrane configuration. Bipolar membrane (BP) and anionic membrane (A). Acidification step: A = Juice and B = HCl. Alkalinization step: A = HCl and B = Juice.
A. Lam Quoc et al. / Food Research International 39 (2006) 755–760
2.4. Apple juice analysis PPO activity was assayed by the absorbance measurement of a mixture containing 0.1 ml of apple juice, 1.9 ml of phosphate buffer at pH 6.5 and 1 ml of 0.05 M catechol based on the method developed by TraversoRueda and Singleton (1973). PME activity was measured by the titration of carboxylic groups that are released at pH 7.0 (25 °C) based on the method of Owusu-Yaw et al. (1988). Enzymatic activities were expressed as a percentage of the control juice enzymatic activities immediately after the extraction. Color was determined with a Labscan Tristimulus colorimeter following the method reported by Lam Quoc et al. (2000). The results were reported as L (luminescence or lightness) and a (intensity of brown color varying from green to red values) values. Opalescence stability was determined by measuring the juice turbidity with a Hach turbidimeter (model 2100AN, Hach Company, Colorado, USA) before and after centrifugation at 320 g for 15 min (Versteeg, Rombouts, Spaansen, & Pilnik, 1980). The ratio between the turbidity before and after centrifugation gives an indication of the stability of the juice opalescence.
tively. However, at 50 °C, the juice shows a slight cooked fruit aftertaste. A similar behaviour than for the PPO was observed for the PME (Fig. 3). At ambient temperature, the PME are completely inactivated after 45 minutes, while at 40 °C the time required to inactivate the enzymes is reduced by half. At 45 and 50 °C, the inactivation time is 5 and 3 min, respectively. These results indicate that by warming acidified juice at pH 2.0 at temperatures between 45 and 50 °C it was possible to inactivate the PPO and the PME enzymes very quickly after the extraction step. Nicolas (1994) reported that a fast inactivation of PPO present in apple juice can be obtained only at temperature between 70 and 90 °C. In the same context, Demeaux and Bidan (1966) reported that heat treatment at 65 °C for a duration varying between 120 100
Activity (%)
2.3.2. Opalescence stability To complete the previous observations and, based on the optimal conditions determined, the acidified juice (pH 2.0) was heated to 45 °C for 5 min in a water bath with a thermostat, and then the pH was readjusted to its initial value (pH 3.35) by ED. A juice freshly pressed was prepared as the control juice. The juices were conditioned in 250 ml glass bottles. Sodium azide (0.1 g/l) was added to the juices to prevent any microbial growth. The bottles were stored at 4 °C for 3 months. During the storage, the opalescence stability was measured every month.
757
80 60 40 20 0 0
2
4
5
10
20
40
60
Time (min) 20˚C
40˚C
45˚C
50˚C
Fig. 2. Polyphenol oxidases activity of apple juice at pH 2.0 as a function of temperature and treatment duration.
2.5. Statistical analyses
3. Results 3.1. Effect of mild heat treatment on PPO and PME activity of apple juice acidified by ED The rate of decrease of PPO activity for an unclarified apple juice at pH 2.0 increases with an increase in temperature (Fig. 2). At ambient temperature, the time required to completely and irreversibly inactivate the PPO is 1 h. By heating the acidified juice at 40 °C, the PPO activity is reduced to 0% after 20 min. At 45 and 50 °C, the complete inactivation of PPO is obtained after 4 and 2 min respec-
120 100
Activity (%)
A completely randomized experimental design with three replicates was applied. An analysis of variance (ANOVA) was performed using SAS software to establish if combination of ED and mild heat treatment has an effect on the inactivation of PPO and PME enzymes, juice color and juice opalescence during storage.
80 60 40 20 0 0
3
5
10
20
45
Time (min) 20˚C
40˚C
45˚C
50˚C
Fig. 3. Pectin Methylesterases activity of apple juice at pH 2.0 as a function of temperature and treatment duration.
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5 and 20 min is required to inactivate the PPO contain in apple juices. The latter also reported that the lower the apple juice pH, the quicker the PPO inactivation. Usually, heat treatment at least at 75 °C is required if a fast inactivation of the PME present in the apple juice is desired (Macdonald & Evans, 1996). For example, for heat treatment at 90 °C, treatment duration of at least 1 min is needed to inactivate the PME (Castaldo, Quagliuolo, Servillo, Balestrieri, & Giovane, 1989). Since the fresh taste of the products is usually preserved at temperatures lower than 50 °C (Cheftel, Cheftel, & Besanc¸on, 1977) and since the juice treated by a combination of ED and heat treatment at 50 °C had a cooked apple aftertaste, the combination of ED (juice pH 2.0) and heat treatment at 45 °C is preferred. 3.2. Effect of mild heat treatment on the color of apple juice acidified by ED Immediately after the mild heat treatment, an effect on the color of the acidified juice was observed, as indicated by the parameters L and a (Table 1). The heat treatment resulted in high values of parameter L and low values of parameter a, when compared to an acidified juice without heat treatment. At a confidence level of 95%, the difference in parameter L and parameter a between both juices is significant. As a result, the color of acidified juice combining ED and mild heat treatment is lighter than the color of acidified juice treated by ED alone. 3.3. Opalescence stability of the juice during storage The opalescence of the adjusted juice (pH 3.35) remains more stable than the opalescence of the control juice, for which no treatment was done to inhibit the PME enzymes (Fig. 4). After one month of storage at 4 °C, the turbidity of the juice treated by ED is 56% as compared to 47% for the control juice. After 3 months, the turbidity of the juice treated by ED still represents 33% of its initial value, while the turbidity of the control juice is decreased to 24%. At a confidence level of 99.9%, the differences in relative turbidity between the adjusted juice (pH 3.35) treated by ED and the control juice is significant for all storage time.
Table 1 Effect of a mild heat treatment at pH 2.0 on the color of cloudy apple juice Color indice
L a a
Temperature (°C) 20a
45b
28.2 ± 0.9ac 0.9 ± 0.6a
32.1 ± 1.1b 2.2 ± 0.6b
Acidified juice to pH 2.0 without heat treatment. Acidified juice to pH 2.0 with heat treatment at 45 °C for 5 min. c Values with the same letter are not significantly different at a confidence level of 95%. b
Fig. 4. Opalescence stability of adjusted apple juice during storage at 4 °C. Values with the same letter are not significantly different at a confidence level of 99.9%.
4. Discussion Our study has demonstrated that a combination of ED, which enables the acidification of the cloudy apple juice to pH 2.0, and mild heat treatment at 45 °C has a positive effect on the inactivation of PPO and PME enzymes present in the apple juice. The combined treatment has reduced the time required to irreversibly inactivate the enzymes from 60 min without heat treatment to only 5 min. Our results also suggest an improvement of the juice stability during storage at 4 °C for a period of 3 months. The decrease in PPO activity obtained by combining ED and mild heat treatment may in part be due to the fact that PPO are metallo-enzymes having copper as co-factor. This copper prosthetic group of PPO must be present for the enzyme to be active and enzymatic browning reactions to occur. In the case of PPO, each of the two active site coppers is coordinated by three histidine residues. Since the link copper-enzyme is fragile, the acidification by ED may easily break this link which will release the copper and thus inactivate the PPO. This phenomenon explains the high sensitivity of the PPO at low pH (Vamos-Vigyazo, 1981). Concerning the PME, it is clear that electrostatic interactions between PME and pectin have a major impact, and these interactions are function of pH. As already reported in previous works on orange juice, as pH decreases, free carboxyl groups on PME become increasingly protonated as do similar groups on pectin molecules (Christensen, Nielsen, Kreiberg, Rasmussen, & Mikkelsen, 1998). This inhibits formation of the enzyme–substrate complex and may explain the weak activity of the enzyme at low pH (Versteeg, Rombouts, & Pilnik, 1978). The decrease in PPO and PME enzymes activity obtained by coupling ED and mild heat treatment also suggests that this treatment may be able to denature enzymes whose catalytic activity relies on the native configuration of their active site and the conformation of surrounding
A. Lam Quoc et al. / Food Research International 39 (2006) 755–760
proteins. The amino acid groups present in PPO and PME enzymes create highly asymmetric spatial distributions of charge that lead to strongly polar and charged regions in the molecular structure of enzymes. Because of a complex network of noncovalent (hydrogen bonding, hydrophobic and electrostatic bonds, van der Waals forces) and covalent (disulphide bonds) interactions, the structural stability and catalytic functions of enzymes are maintained (Wong, 1995). However, ionisable groups of the protein structure of enzymes are affected by the pH of the apple juice. These groups must be in the appropriate ionic form in order to maintain the conformation of the active site, bind substrates, or catalysis the enzymatic reaction. Although, changes in the ionization status of enzymes are generally reversible, irreversible denaturation can however occur under conditions of extreme pH. At extreme pH, strong electrostatic repulsions between the ionized groups arise in the enzymes, resulting in their unfolding and molecular rearrangement to forms with less functional or non-functional active sites (Schmid, 1992). This is believed to occur in the apple juice at pH 2.0. This would explain the negligible PPO reactivation observed in our previous work, when pH of apple juice was readjusted to its initial pH (Lam Quoc et al., 2000). In addition, the effect of the heat treatment would be to disrupt hydrogen bonds and thereby favour enzymes unfolding, increasing the rate of enzymes inactivation, when compared to ED treatment alone (Hayakawa, Linko, & Linko, 1996). Thus, it seems that the pH has two distinct effects: one on the link copper-enzyme for the PPO, one on the enzyme-substrate complex for the PME and one effect on the enzymes denaturation. However, our results suggest that the stability of the juice during storage at 4 °C does not depend solely on PME enzyme activity. Although it is less significant than for the control juice, the turbidity of the juice treated by the combined treatments is decreasing with storage time. Other works on apple juice (Beveridge, 1997) and orange juice (Lacroix, Fliss, & Makhlouf, 2005) reported that opalescence stability also depends on the size of particles in suspension, their nature, the ionic environment and the particles hydration. To remain in suspension in the juice, the particles must be sufficiently small (0.5–10 lm for orange juice, Mizrahi & Berk, 1970), since large particles would tend to settle. The effect of mild heat treatments would be to cause the deaggregation of large particles and also enhance pectin solubility (Beveridge, 1997). These phenomena could explain the better opalescence stabilisation for the treated juice as compared to the control juice. However, experimental measurements including particle size distribution would be required to confirm this hypothesis. 5. Conclusion The results of this study have demonstrated that a combination of acidification (by ED with bipolar membranes) with a mild heat treatment increases the rate of inactivation
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of the PPO and PME. The best combination was a mild heat treatment at 45 °C for a juice acidified at pH 2.0, which reduced the time required to inactivate the enzymes to only 5 min, as compared to one hour without heat treatment, and did not induce cooked apple aftertaste. Furthermore, the color of the juice treated by combining ED and mild heat treatment was clearer, and thus more appealing to the eye, than the color of the juice treated by ED only; its opalescence was also more stable. The combination of ED with bipolar membranes and heat treatment can be considered as a simple and efficient stabilization method applicable to fruit juices and other biological liquids for which the enzymatic or microbiologic stabilization is a strong function of pH and temperature. However, to fully address the commercial viability of the ED process for stabilization of cloudy apple juice, combination of both the acidification and pH readjustment steps in one ED operation (in parallel) will have to be carried out and the results compared with the current process operated in series. This final part of the project is currently under investigation. Acknowledgements The financial support of the ‘‘Conseil des Recherches en Peˆche et en Agroalimentaire du Que´bec’’, A. Lassonde inc. and Agriculture and Agri-Food Canada is acknowledged. References Beveridge, T. (1997). Haze and cloud in apple juices. Critical Reviews in Food Science and Nutrition, 37, 75–91. Castaldo, D., Quagliuolo, L., Servillo, L., Balestrieri, C., & Giovane, A. (1989). Isolation and characterization of pectin methylesterase from apple fruit. Journal of Food Science, 54, 653–655. Cheftel, J. C., Cheftel, H., & Besanc¸on, P. (1977). Introduction a` la biochimie et a` la technologie des aliments. Paris: Techniques et Documentation. Christensen, T. M. I. E., Nielsen, J. E., Kreiberg, J. D., Rasmussen, P., & Mikkelsen, J. D. (1998). Pectin methylesterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry. Planta, 206, 493–503. ´ tude de lÕinactivation par la chaleur de Demeaux, M., & Bidan, P. (1966). E la polyphe´noloxydase du jus de pomme. Annales de Technologies Agricole, 15, 349–358. Hayakawa, I., Linko, Y. Y., & Linko, P. (1996). Mechanism of high pressure denaturation of proteins. Lebensmittel-Wissenschaft Technologie, 29, 756–762. Lacroix, N., Fliss, I., & Makhlouf, J. (2005). Inactivation of pectin methylesterase and stabilization of opalescence in orange juice by dynamic high pressure. Food Research International, 38, 569–576. Lam Quoc, A., Lamarche, F., & Makhlouf, J. (2000). Acceleration of pH variation in cloudy apple juice using electrodialysis with bipolar membranes. Journal of Agricultural and Food Chemistry, 48, 2160–2166. Lea, A. G. H. (1990). Apple juice. In D. Hicks (Ed.), Production and packaging of non-carbonated fruit juices and fruits beverages (pp. 182–225). New York: Van Nostrand Reinhold. Macdonald, H. M., & Evans, R. (1996). Purification and properties of apple pectinesterase. Journal of the Science of Food and Agriculture, 70, 321–326. Macheix, J. J., Fleuriet, A., & Billot, J. (1990). Fruits phenolics. Boca Raton, Fl: CRC Press.
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Mizrahi, S., & Berk, Z. (1970). Physico-chemical characteristics of orange juice cloud. Journal of the Science of Food and Agriculture, 21, 250–253. Nicolas, J. J. (1994). Enzymatic browning reactions in apple and apple products. Critical Reviews in Food Science and Nutrition, 34, 109–157. Owusu-Yaw, J., Marshall, M. R., Koburger, J. A., & Wei, C. I. (1988). Low pH inactivation of pectinesterase in single strength orange juice. Journal of Food Science, 53, 504–507. Schmid, F. X. (1992). Kinetics of unfolding and refolding of single-domain proteins. In T. E. Creighton (Ed.), Protein folding (pp. 197–241). New York: W.H. Freeman and Company. Traverso-Rueda, S., & Singleton, V. L. (1973). Cathecolase activity in grape juice and its implications in winemaking. American Journal of Enology and Viticulture, 24, 103–107. Tronc, J-S. (1996). Inhibition des re´actions de brunissement enzymatique dÕun jus de pomme frais non clarifie´ par e´lectrodialyse, Master Thesis, Universite´ Laval. Tronc, J-S., Lamarche, F., & Makhlouf, J. (1997). Enzymatic browning inhibition in cloudy apple juice by electrodialysis. Journal of Food Science, 62, 75–78.
Tronc, J-S., Lamarche, F., & Makhlouf, J. (1998). Effect of pH variation by electrodialysis on the inhibition of enzymatic browning in cloudy apple juice. Journal of Agricultural and Food Chemistry, 46, 829–833. Vamos-Vigyazo, L. (1981). Polyphenol oxidases and peroxidases in fruits and vegetables. Critical Reviews in Food Science and Nutrition, 15, 49–127. Versteeg, C., Rombouts, F. M., & Pilnik, W. (1978). Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensmittel-Wissenschaft Technologie, 11, 267–274. Versteeg, C., Rombouts, F. M., Spaansen, C. H., & Pilnik, W. (1980). Thermostability and orange juice destabilizing properties of multiple pectinases from orange. Journal of Food Science, 45, 969–971, 998. Wong, D. W. S. (1995). Tailoring enzyme: structures and functions. In D. W. S. Wong (Ed.), Food enzymes: structure and mechanism (pp. 17–36). New York: Chapman & Hall. Ze´mel, G. P., Sims, C. A., Marshall, M. R., & Balaban, M. (1990). Low pH inactivation of polyphenol oxidase in apple juice. Journal of Food Science, 55, 562–563.
Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice A. Lam Quoc a, M. Mondor b
b,*
, F. Lamarche b, D. Ippersiel b, L. Bazinet a, J. Makhlouf
a
a Department of Food Sciences and Nutrition, Laval University, Sainte-Foy, Que., Canada G1K 7P4 Food Research and Development Center, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Que., Canada J2S 8E3
Received 30 August 2005; accepted 8 November 2005
Abstract The purpose of this work was to develop a process enabling the quick inactivation of the polyphenol oxidase and pectin methylesterase enzymes, which are present in cloudy or unclarified apple juice; These enzymes are respectively responsible for enzymatic browning and opalescence instability. In order to fulfill this objective, acidification of the apple juice to pH 2.0 was conducted by electrodialysis (bipolar–anionic membranes) followed by mild heat treatment at temperature of 40, 45 and 50 °C for a duration of 0–60 min. Then, juice pH was readjusted to its initial value by electrodialysis with bipolar–anionic membranes. It was shown that a mild heat treatment at 45 °C for 5 min performed on the acidified juice represents an appropriate condition to quickly inactivate the enzymes. Furthermore, the organoleptic properties of the juice after treatment were found to be preserved and the adjusted juice (pH readjusted to its initial value) shows a better color than an untreated apple juice. Opalescence of the adjusted juice was also more stable than for an untreated cloudy apple juice, when stored at 4 °C for 3 months. Crown Copyright Ó 2006 Published by Elsevier Ltd. All rights reserved. Keywords: Browning; Cloudy apple juice; Electrodialysis; Mild heat treatment; Opalescence stability
1. Introduction Cloudy or unclarified apple juice contains significant quantities of suspended pulp and is perceived as a natural food product that supplies dietary fiber and important nutrients. However, it is very difficult to produce superior quality juice since cloudy apple juice is very sensitive to enzymatic browning because it contains considerable quantities of polyphenols and polyphenol oxidase (PPO) (Lea, 1990). PPO refers to a group of copper-containing enzymes that catalyze the oxidation of phenolic compounds to o-quinones, which then polymerize to form complex dark pigments, thereby changing the color and aroma of the juice (Macheix, Fleuriet, & Billot, 1990). In addition, it is *
Corresponding author. Tel.: +1 450 773 1105; fax: +1 450 773 8461. E-mail address: [email protected] (M. Mondor).
very difficult to produce a cloudy or unclarified apple juice with good opalescence stability due to the presence of pectin methylesterases (PME). The pectin molecules present in suspension are degraded by the PME enzymes resulting in a loss of opalescence stability (Beveridge, 1997). Ze´mel, Sims, Marshall, and Balaban (1990) showed that it is possible to irreversibly inhibit PPO by acidifying the cloudy apple juice to pH 2.0, using HCl, and readjusting the juice pH at its initial value (3.35), by addition of NaOH. The PME can also be inactivated at low pH (pH 2.0) as shown by Owusu-Yaw, Marshall, Koburger, and Wei (1988) for the case of orange juice. The drawbacks of this approach are the dilution associated with the addition of acid and base, and the salty aftertaste. Based on these observations, Tronc (1996) and Tronc, Lamarche and Makhlouf (1997 and 1998) have demonstrated the feasibility of acidifying cloudy apple juice using
0963-9969/$ - see front matter. Crown Copyright Ó 2006 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.11.002
756
A. Lam Quoc et al. / Food Research International 39 (2006) 755–760
electrodialysis (ED) with bipolar–cationic membranes. Readjustment of pH to its initial value was achieved by ED with bipolar–anionic (BP–A) membranes. However, the process was found too lengthy and required addition of exogenous KCl in the juice to reach pH 2.0. At this pH, the juice was kept one hour at room temperature to inhibit the enzymes. The adjusted juice (pH 3.35), shows a PPO reactivation corresponding to 25% of its initial value (Tronc, 1996). Finally, from an industrial point of view, the energy required was too high (197 kWh/m3 of juice) due to the large spacer (8 mm) used in the electrodialysis cell. However, they demonstrated that organoleptic properties of the juice were preserved. In a more recent work, Lam Quoc, Lamarche, and Makhlouf (2000), improved the process by performing both acidification and pH readjustment steps using a BP– A configuration. Furthermore, thinner spacers (0.75 mm) were used in the electrodialysis cell and HCl was used as the base solution instead of KCl (Tronc, 1996; Tronc, Lamarche, & Makhlouf, 1998). The use of this configuration resulted in acceleration of the acidification step by a factor of 3, increasing the yield from 3.3 to 10 l of juice/m2 membrane/min. Furthermore, the adjusted juice (pH 3.35), shows only a very small reactivation of PPO activity of 0.8%, which is lower than the 25% level reported by Tronc (1996). The energy required by the process was also reduced to only 4–5 kWh/m3 of juice, which is a very low energy consumption from an industrial standpoint. However, the main drawback of this approach remains the fact that the juice at pH 2.0 still had to be kept at room temperature for one hour to inhibit the enzymes. In this paper, bipolar membrane ED followed by a mild heat treatment at 40, 45 or 50 °C for duration of 0–60 min was investigated to increase the rate of inactivation of the PPO and PME present in the unclarified apple juice. The influence of this treatment on the color and on the opalescence stability of the juice during storage will be discussed.
0.75 mm. The anode, a dimensionally stable electrode (DSA), and the cathode, a 316 stainless steel electrode, were supplied with the cell. The experiments were carried out using a BP–A configuration (Fig. 1) forming 10 compartments. A total of nine membranes were used: five bipolar membranes (Neosepta BP-1) and four anionic membranes (Neosepta AMX) from Tokuyama Soda Ltd. (Japan). This arrangement defines three closed loops containing the solution to alkalinize (0.1 N HCl), the cloudy apple juice to acidify and a 0.25 M K2SO4 solution used as rinsing solution for the electrodes. Each closed loop was connected to a separate external reservoir, allowing for continuous recycling. The electro-acidification was carried out with electrolytes volumes of 1 l for the solution to alkalinize (HCl), the apple juice to acidify and the K2SO4 solution. During treatment, juice temperature was maintained at 25 °C using a Haake G refrigerated bath (Haake, Berlin, Germany). The acidification of the juice by ED was conducted at a constant current density of 40 mA/cm2. The ED configuration for pH readjustment was the same as that shown in Fig. 1. However, the juice and HCl compartments were reversed. During the acidification and pH readjustment steps, conductivity and juice pH were measured at 1–2 min intervals until the end of the treatments, along with applied voltage. 2.3. Protocols 2.3.1. Electrodialysis combined with mild heat treatment The electro-acidified juices (pH 2.0) were subsequently heated at 40, 45 and 50 °C, as a function of time (0– 60 min) to inhibit the PPO and PME enzymes. The heat treatments were done on 50 ml samples placed in a water bath with a thermostat and under agitation. At the end of the heat treatments, the samples were immediately cooled by immersion in ice/cold water. Samples were analysed for PPO and PME activities, color and where compared with an acidified juice (pH 2.0) kept at room temperature for the corresponding time.
2. Materials and methods 2.1. Apple juice Juice was extracted from McIntosh apples that had been stored commercially under controlled atmosphere. The apples were crushed and pressed in a crusher-press model no. EG 260-X6 (Goodnature Products Inc., Buffalo, United States) under maximum pressure of 1500 psi. About 2 l of juice was extracted from 4 kg of apples for each experiment. The freshly extracted juice underwent bipolar membrane ED treatment immediately. 2.2. Electrodialysis cell configuration Electro-acidification was carried out using an ED-1-BP unit (100 cm2 of effective electrode surface) from Electrosynthesis Co. (Lancaster, NY, USA), with spacing of
Fig. 1. Electrodialysis experimental systems: bipolar–anionic membrane configuration. Bipolar membrane (BP) and anionic membrane (A). Acidification step: A = Juice and B = HCl. Alkalinization step: A = HCl and B = Juice.
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2.4. Apple juice analysis PPO activity was assayed by the absorbance measurement of a mixture containing 0.1 ml of apple juice, 1.9 ml of phosphate buffer at pH 6.5 and 1 ml of 0.05 M catechol based on the method developed by TraversoRueda and Singleton (1973). PME activity was measured by the titration of carboxylic groups that are released at pH 7.0 (25 °C) based on the method of Owusu-Yaw et al. (1988). Enzymatic activities were expressed as a percentage of the control juice enzymatic activities immediately after the extraction. Color was determined with a Labscan Tristimulus colorimeter following the method reported by Lam Quoc et al. (2000). The results were reported as L (luminescence or lightness) and a (intensity of brown color varying from green to red values) values. Opalescence stability was determined by measuring the juice turbidity with a Hach turbidimeter (model 2100AN, Hach Company, Colorado, USA) before and after centrifugation at 320 g for 15 min (Versteeg, Rombouts, Spaansen, & Pilnik, 1980). The ratio between the turbidity before and after centrifugation gives an indication of the stability of the juice opalescence.
tively. However, at 50 °C, the juice shows a slight cooked fruit aftertaste. A similar behaviour than for the PPO was observed for the PME (Fig. 3). At ambient temperature, the PME are completely inactivated after 45 minutes, while at 40 °C the time required to inactivate the enzymes is reduced by half. At 45 and 50 °C, the inactivation time is 5 and 3 min, respectively. These results indicate that by warming acidified juice at pH 2.0 at temperatures between 45 and 50 °C it was possible to inactivate the PPO and the PME enzymes very quickly after the extraction step. Nicolas (1994) reported that a fast inactivation of PPO present in apple juice can be obtained only at temperature between 70 and 90 °C. In the same context, Demeaux and Bidan (1966) reported that heat treatment at 65 °C for a duration varying between 120 100
Activity (%)
2.3.2. Opalescence stability To complete the previous observations and, based on the optimal conditions determined, the acidified juice (pH 2.0) was heated to 45 °C for 5 min in a water bath with a thermostat, and then the pH was readjusted to its initial value (pH 3.35) by ED. A juice freshly pressed was prepared as the control juice. The juices were conditioned in 250 ml glass bottles. Sodium azide (0.1 g/l) was added to the juices to prevent any microbial growth. The bottles were stored at 4 °C for 3 months. During the storage, the opalescence stability was measured every month.
757
80 60 40 20 0 0
2
4
5
10
20
40
60
Time (min) 20˚C
40˚C
45˚C
50˚C
Fig. 2. Polyphenol oxidases activity of apple juice at pH 2.0 as a function of temperature and treatment duration.
2.5. Statistical analyses
3. Results 3.1. Effect of mild heat treatment on PPO and PME activity of apple juice acidified by ED The rate of decrease of PPO activity for an unclarified apple juice at pH 2.0 increases with an increase in temperature (Fig. 2). At ambient temperature, the time required to completely and irreversibly inactivate the PPO is 1 h. By heating the acidified juice at 40 °C, the PPO activity is reduced to 0% after 20 min. At 45 and 50 °C, the complete inactivation of PPO is obtained after 4 and 2 min respec-
120 100
Activity (%)
A completely randomized experimental design with three replicates was applied. An analysis of variance (ANOVA) was performed using SAS software to establish if combination of ED and mild heat treatment has an effect on the inactivation of PPO and PME enzymes, juice color and juice opalescence during storage.
80 60 40 20 0 0
3
5
10
20
45
Time (min) 20˚C
40˚C
45˚C
50˚C
Fig. 3. Pectin Methylesterases activity of apple juice at pH 2.0 as a function of temperature and treatment duration.
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5 and 20 min is required to inactivate the PPO contain in apple juices. The latter also reported that the lower the apple juice pH, the quicker the PPO inactivation. Usually, heat treatment at least at 75 °C is required if a fast inactivation of the PME present in the apple juice is desired (Macdonald & Evans, 1996). For example, for heat treatment at 90 °C, treatment duration of at least 1 min is needed to inactivate the PME (Castaldo, Quagliuolo, Servillo, Balestrieri, & Giovane, 1989). Since the fresh taste of the products is usually preserved at temperatures lower than 50 °C (Cheftel, Cheftel, & Besanc¸on, 1977) and since the juice treated by a combination of ED and heat treatment at 50 °C had a cooked apple aftertaste, the combination of ED (juice pH 2.0) and heat treatment at 45 °C is preferred. 3.2. Effect of mild heat treatment on the color of apple juice acidified by ED Immediately after the mild heat treatment, an effect on the color of the acidified juice was observed, as indicated by the parameters L and a (Table 1). The heat treatment resulted in high values of parameter L and low values of parameter a, when compared to an acidified juice without heat treatment. At a confidence level of 95%, the difference in parameter L and parameter a between both juices is significant. As a result, the color of acidified juice combining ED and mild heat treatment is lighter than the color of acidified juice treated by ED alone. 3.3. Opalescence stability of the juice during storage The opalescence of the adjusted juice (pH 3.35) remains more stable than the opalescence of the control juice, for which no treatment was done to inhibit the PME enzymes (Fig. 4). After one month of storage at 4 °C, the turbidity of the juice treated by ED is 56% as compared to 47% for the control juice. After 3 months, the turbidity of the juice treated by ED still represents 33% of its initial value, while the turbidity of the control juice is decreased to 24%. At a confidence level of 99.9%, the differences in relative turbidity between the adjusted juice (pH 3.35) treated by ED and the control juice is significant for all storage time.
Table 1 Effect of a mild heat treatment at pH 2.0 on the color of cloudy apple juice Color indice
L a a
Temperature (°C) 20a
45b
28.2 ± 0.9ac 0.9 ± 0.6a
32.1 ± 1.1b 2.2 ± 0.6b
Acidified juice to pH 2.0 without heat treatment. Acidified juice to pH 2.0 with heat treatment at 45 °C for 5 min. c Values with the same letter are not significantly different at a confidence level of 95%. b
Fig. 4. Opalescence stability of adjusted apple juice during storage at 4 °C. Values with the same letter are not significantly different at a confidence level of 99.9%.
4. Discussion Our study has demonstrated that a combination of ED, which enables the acidification of the cloudy apple juice to pH 2.0, and mild heat treatment at 45 °C has a positive effect on the inactivation of PPO and PME enzymes present in the apple juice. The combined treatment has reduced the time required to irreversibly inactivate the enzymes from 60 min without heat treatment to only 5 min. Our results also suggest an improvement of the juice stability during storage at 4 °C for a period of 3 months. The decrease in PPO activity obtained by combining ED and mild heat treatment may in part be due to the fact that PPO are metallo-enzymes having copper as co-factor. This copper prosthetic group of PPO must be present for the enzyme to be active and enzymatic browning reactions to occur. In the case of PPO, each of the two active site coppers is coordinated by three histidine residues. Since the link copper-enzyme is fragile, the acidification by ED may easily break this link which will release the copper and thus inactivate the PPO. This phenomenon explains the high sensitivity of the PPO at low pH (Vamos-Vigyazo, 1981). Concerning the PME, it is clear that electrostatic interactions between PME and pectin have a major impact, and these interactions are function of pH. As already reported in previous works on orange juice, as pH decreases, free carboxyl groups on PME become increasingly protonated as do similar groups on pectin molecules (Christensen, Nielsen, Kreiberg, Rasmussen, & Mikkelsen, 1998). This inhibits formation of the enzyme–substrate complex and may explain the weak activity of the enzyme at low pH (Versteeg, Rombouts, & Pilnik, 1978). The decrease in PPO and PME enzymes activity obtained by coupling ED and mild heat treatment also suggests that this treatment may be able to denature enzymes whose catalytic activity relies on the native configuration of their active site and the conformation of surrounding
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proteins. The amino acid groups present in PPO and PME enzymes create highly asymmetric spatial distributions of charge that lead to strongly polar and charged regions in the molecular structure of enzymes. Because of a complex network of noncovalent (hydrogen bonding, hydrophobic and electrostatic bonds, van der Waals forces) and covalent (disulphide bonds) interactions, the structural stability and catalytic functions of enzymes are maintained (Wong, 1995). However, ionisable groups of the protein structure of enzymes are affected by the pH of the apple juice. These groups must be in the appropriate ionic form in order to maintain the conformation of the active site, bind substrates, or catalysis the enzymatic reaction. Although, changes in the ionization status of enzymes are generally reversible, irreversible denaturation can however occur under conditions of extreme pH. At extreme pH, strong electrostatic repulsions between the ionized groups arise in the enzymes, resulting in their unfolding and molecular rearrangement to forms with less functional or non-functional active sites (Schmid, 1992). This is believed to occur in the apple juice at pH 2.0. This would explain the negligible PPO reactivation observed in our previous work, when pH of apple juice was readjusted to its initial pH (Lam Quoc et al., 2000). In addition, the effect of the heat treatment would be to disrupt hydrogen bonds and thereby favour enzymes unfolding, increasing the rate of enzymes inactivation, when compared to ED treatment alone (Hayakawa, Linko, & Linko, 1996). Thus, it seems that the pH has two distinct effects: one on the link copper-enzyme for the PPO, one on the enzyme-substrate complex for the PME and one effect on the enzymes denaturation. However, our results suggest that the stability of the juice during storage at 4 °C does not depend solely on PME enzyme activity. Although it is less significant than for the control juice, the turbidity of the juice treated by the combined treatments is decreasing with storage time. Other works on apple juice (Beveridge, 1997) and orange juice (Lacroix, Fliss, & Makhlouf, 2005) reported that opalescence stability also depends on the size of particles in suspension, their nature, the ionic environment and the particles hydration. To remain in suspension in the juice, the particles must be sufficiently small (0.5–10 lm for orange juice, Mizrahi & Berk, 1970), since large particles would tend to settle. The effect of mild heat treatments would be to cause the deaggregation of large particles and also enhance pectin solubility (Beveridge, 1997). These phenomena could explain the better opalescence stabilisation for the treated juice as compared to the control juice. However, experimental measurements including particle size distribution would be required to confirm this hypothesis. 5. Conclusion The results of this study have demonstrated that a combination of acidification (by ED with bipolar membranes) with a mild heat treatment increases the rate of inactivation
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of the PPO and PME. The best combination was a mild heat treatment at 45 °C for a juice acidified at pH 2.0, which reduced the time required to inactivate the enzymes to only 5 min, as compared to one hour without heat treatment, and did not induce cooked apple aftertaste. Furthermore, the color of the juice treated by combining ED and mild heat treatment was clearer, and thus more appealing to the eye, than the color of the juice treated by ED only; its opalescence was also more stable. The combination of ED with bipolar membranes and heat treatment can be considered as a simple and efficient stabilization method applicable to fruit juices and other biological liquids for which the enzymatic or microbiologic stabilization is a strong function of pH and temperature. However, to fully address the commercial viability of the ED process for stabilization of cloudy apple juice, combination of both the acidification and pH readjustment steps in one ED operation (in parallel) will have to be carried out and the results compared with the current process operated in series. This final part of the project is currently under investigation. Acknowledgements The financial support of the ‘‘Conseil des Recherches en Peˆche et en Agroalimentaire du Que´bec’’, A. Lassonde inc. and Agriculture and Agri-Food Canada is acknowledged. References Beveridge, T. (1997). Haze and cloud in apple juices. Critical Reviews in Food Science and Nutrition, 37, 75–91. Castaldo, D., Quagliuolo, L., Servillo, L., Balestrieri, C., & Giovane, A. (1989). Isolation and characterization of pectin methylesterase from apple fruit. Journal of Food Science, 54, 653–655. Cheftel, J. C., Cheftel, H., & Besanc¸on, P. (1977). Introduction a` la biochimie et a` la technologie des aliments. Paris: Techniques et Documentation. Christensen, T. M. I. E., Nielsen, J. E., Kreiberg, J. D., Rasmussen, P., & Mikkelsen, J. D. (1998). Pectin methylesterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry. Planta, 206, 493–503. ´ tude de lÕinactivation par la chaleur de Demeaux, M., & Bidan, P. (1966). E la polyphe´noloxydase du jus de pomme. Annales de Technologies Agricole, 15, 349–358. Hayakawa, I., Linko, Y. Y., & Linko, P. (1996). Mechanism of high pressure denaturation of proteins. Lebensmittel-Wissenschaft Technologie, 29, 756–762. Lacroix, N., Fliss, I., & Makhlouf, J. (2005). Inactivation of pectin methylesterase and stabilization of opalescence in orange juice by dynamic high pressure. Food Research International, 38, 569–576. Lam Quoc, A., Lamarche, F., & Makhlouf, J. (2000). Acceleration of pH variation in cloudy apple juice using electrodialysis with bipolar membranes. Journal of Agricultural and Food Chemistry, 48, 2160–2166. Lea, A. G. H. (1990). Apple juice. In D. Hicks (Ed.), Production and packaging of non-carbonated fruit juices and fruits beverages (pp. 182–225). New York: Van Nostrand Reinhold. Macdonald, H. M., & Evans, R. (1996). Purification and properties of apple pectinesterase. Journal of the Science of Food and Agriculture, 70, 321–326. Macheix, J. J., Fleuriet, A., & Billot, J. (1990). Fruits phenolics. Boca Raton, Fl: CRC Press.
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