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LWT 39 (2006) 492495 www.elsevier.com/locate/lwt

Thermal inactivation of polyphenoloxidase in pineapple puree

Benjar Chutintrasria, Athapol Noomhormb,
b

Food Technology Department, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand Food Engineering and Bioprocess Technology, School of Environment, Resources and Development, Asian Institute of Technology, Pathumthani, Klongluang 12120, Thailand Received 3 December 2004; received in revised form 31 March 2005; accepted 12 April 2005

Abstract Prevention of browning in pineapple puree by thermal inactivation of enzyme, Polyphenoloxisase (PPO), was examined between 40 and 90 1C and in relation to exposure time. The amount of inactivation was measured as a function of time and temperature under isothermal conditions. Reaction rate constant and activation energy (Ea) as well as Decimal reduction time (D) and z-value of thermal inactivation, were determined. The rate of inactivation varied with temperatures and follows a logarithmic law. Kinetic studies showed that the thermal inactivation (4090 1C) of the PPO followed rst-order kinetics. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Pineapple puree; Polyphenoloxidase; Enzymatic browning; Thermal inactivation; Kinetics

1. Introduction Pineapple is an important tropical fruit (Bartholomew, Paul, & Rohrbach, 2002), particularly in the form of processed products. Of these, pineapple puree, derived from crushing those portions of the fruit not otherwise used and thermally processing in cans or aseptic packs, is marketed as a high value-added product at a premium price. Important to the preparation of stable fruit purees is the inactivation of the enzyme, polyphenoloxidase (PPO), that catalyzes deterioration reactions after tissue is damaged in the size reduction process. Among these reactions is the formation of a brown pigment that is undesirable with respect to color, avor and market value. The pineapple PPO was found in three isoforms (Das, Boht, & Eowda, 1997), with the major isoform being a tetramer of identical subunits of 25 kDa and having optimum activity between pH 6 and 7. The PPO is stable to heat when extracted, but loses over 50% of its activity

Corresponding author. Tel.: +66 2 524 5476; fax: +66 2 524 6200.

E-mail address: athapol@ait.ac.th (A. Noomhorm).

following 20 min exposure to 60 1C in vivo (Teisson, 1977). Natural phenolic compounds in fruit and vegetables in the presence of PPO and oxygen are oxidized to oquinone that subsequently polymerizes nonenzymatically to brown pigments (Golan-Goldhirsh & Whitaker, 1984; Sapers, 1993). This browning process leads also to a change in avor and a reduction in nutritional quality, especially ascorbic acid. (Vamos-Vigyazo, 1981; GolanGoldhirsh & Whitaker, 1984). The most important factors that determine the rate of the enzymatic browning of fruit and vegetables are the concentrations of both active PPO and phenolic compounds present, the pH, the temperature and the oxygen availability of the tissue. pH and oxygen also inuence subsequent nonenzymatic browning (Martinez & Whitaker,1995). In general, exposure of PPO to temperatures of 7090 1C destroys their catalytic activity (Vamos-Vigyazo, 1981). Thermal inactivation proles of PPO in fruit and vegetable processing follow rst-order reaction kinetics with the time required varying with the product. Of the studies on heat inactivation of PPO only a few have included the calculations of Arrhenius and the kinetic parameters of heat inactivation of PPO from

0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2005.04.006

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various foods. These include apple (Stru bi, Escher, & Neukom, 1975), Sultana grapes (Aquilera, Oppermann, & Sanchez, 1987), apricot (Heil, McCarthy, & Merson, 1988), rice (Ansah, 1989) and mango (Askar, ElAshwah, Omran, & Labib, 1994). No information is available for pineapple puree on the quantitative effects of temperature and time on the inactivation of PPO which was the subject of this study.

Results of the thermal experiments were processed into kinetic parameter values, decimal reduction times, D, inactivation rate constants, k, z-values and activation energy, Ea. Decimal reduction time was calculated from the equation (Stumbo, 1973): t DT , log Ai log Af where Ai is the initial activity, Af , the residual activity after treatment and t, time of thermal treatment in minutes. Z was derived from log DT values in relation to treatment temperature: Z T 1 T 2 , log D1 log D2

2. Materials and methods Pineapple puree samples (20 l) were obtained from a commercial plant in Rayong Province, Thailand. The puree samples were sampled from the same production lot after size reduction process and kept in industrial plastic bags with metalized plastic layer on the outside (Scholle # 800465) at 20 1C until used in experiments. The physicalchemical characteristics of the sample were: 1Brix (20 1C) 15.070.1; total acidity (g citric acid/ 100 g sample) 0.6370.07; pH 3.7270.04; % pulp 68.6772.31; moisture content (% wet basis) 83.770.2. PPO was assayed according to the procedure of Palou, Lopez-Malo, Barbosa-Canovas, Welti-Chanes, and Swanson (1999). An aliquot of puree (10 g) was mixed with McIlvaine citric-phosphate buffer (10 ml, pH 6.5) at 3000 rpm for 60 s. The homogenate was centrifuged (3960g, 4 1C, 30 min) and the supernatant was ltered (Whatman no.1). To the ltrate (0.5 ml) was added 1 ml of 0.175 mol/l catechol solution and 2 ml of McIlvaine buffer (pH 6.5) and optical density at 420 nm was measured every 15 s up to 1 and 2 min in fresh and treated purees using a 1-cm glass cuvette and a UV/VIS spectrophotometer as described by Pizzocaro, Torreggiani, and Gilardi (1993). This duration time was based on preliminary test results that no changes in the optical density were found after those specied time intervals. PPO activity was calculated on the basis of the slope from the linear portion of the curve of DA420 vs. time for fresh and treated puree, respectively. One unit of PPO activity was dened as 0.001 DA420 min1 (ml of extract)1. Residual PPO activity was expressed as the ratio between treated and fresh pineapple puree. Thermal inactivation experiments were conducted in a water bath at constant temperature between 40 and 90 1C. Samples were exposed to each temperature for 030 min (5 min interval). Puree samples (10 g) were lled in Pyrex screw-capped test tubes (15 1.6 cm, wall thickness 1.8 mm) held in the water bath for the assigned time after the geometric center of sample had reached the desired temperature measured by thermocouples (Type T). Immediately afterwards, samples were immersed in an ice bath and enzyme activity measured. At each combination of temperature and time, three samples were analysed.

where T 1 and T 2 represent the lower and higher temperatures, 1C or 1K, and D1 and D2 , D-values at the lower and higher temperatures in minutes. First-order reaction rates, k were obtained from the linear portion of the relation between ln activity retention and treatment time. The z and E a values were estimated from the linear regression of log D and temperature and ln k and 1=T , respectively.

3. Results and discussion The extent of PPO denaturation increased with temperature and treatment time (Table 1). PPO activities were reduced by approximately 60% after exposure to 4060 1C for times to 30 min. Denaturation increased rapidly above 75 1C. Thus, residual activity was only about 7% after 5 min at 85 1C and 1.2% after 5 min at 90 1C. The logarithmic linear relationship between PPO activity and treatment time for the temperature range of 4090 1C followed rst-order kinetics and was consistent with the relationships found in earlier studies on fruits and vegetables (Dimick, Ponting, & Makower, 1951; Demeaux & Biden, 1967; Chan & Yang, 1971; Benjamin & Montgomery, 1973; Halim & Montgomery, 1978; Lee, Smith, & Pennesi, 1983; McCord & Kilara, 1983). Rate of PPO inactivation (Table 2), after ln transformation, decreased linearly with the inverse of temperature. This relationship is described by the equation: ln k 9955:61=T a 24:49 n 5; R2 0:991; Pp0:05, where T a represents absolute temperature (K). Similarly, decimal reduction time, D (Table 3) after logarithmic transformation declined linearly with temperature increase:tvs log D 0:0466T c 5:2466 n 5; R2 0:996; Pp0:05,

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494 B. Chutintrasri, A. Noomhorm / LWT 39 (2006) 492495 Table 1 Effect of treatment temperature and time on the inactivation of polyphenoloxidase in pineapple puree Temp (1C) % Enzyme remaining at each treatment time (min) 5 40 45 50 55 60 65 70 75 80 85 90 99.771.6 85.571.8 81.471.6 74.670.3 68.271.3 62.671.2 59.170.6 59.071.3 31.071.8 7.070.3 1.270.3
a

10 88.171.8 83.771.2 80.670.9 74.071.6 67.871.1 60.970.3 56.271.6 56.171.3 26.571.1 6.170.2 1.170.2

15 81.871.9 81.570.3 73.771.9 72.370.6 67.571.3 59.871.8 51.570.9 51.571.1 23.470.9 4.070.2 1.170.3

20 80.970.9 73.370.9 71.972.6 71.571.8 66.970.9 59.671.8 51.071.9 51.070.9 22.770.6 3.870.2 0.770.1

25 78.870.9 70.971.3 70.871.8 68.971.6 66.171.8 59.271.6 50.470.9 50.370.8 21.270.3 3.570.2 0.870.2

30 69.670.6 65.670.8 62.670.6 61.570.9 56.770.6 47.870.6 47.070.7 47.070.5 18.370.3 2.070.1 0.370.1

The PPO activity in fresh pineapple puree was 0.4470.02 mUAbs min1. a Mean (n 3)7standard deviation. Table 2 Rate constants for heat inactivation of polyphenoloxidase in pineapple puree at 4090 1C Temp (1C) 40 45 50 55 60 65 70 75 80 85 90
a

k (min1) 0.004770.0005a 0.004870.0003 0.006270.0002 0.007470.0004 0.008770.0003 0.009870.0002 0.01170.007 0.01270.007 0.02470.005 0.04170.002 0.05270.003

Table 3 D, z- and Ea-values for thermal inactivation of polyphenol oxidase in pineapple puree at low-temperature range (4070 1C) and hightemperature range (7090 1C) D, z and E a Values 190.373.6a 164.373.2 146.872.7 142.472.5 121.972.2 97.472.3 93.772.1 104.270.7 23.771.7 91.371.9 40.770.6 17.870.5 11.470.2 21.570.0 82.872.7

R2 0.966 0.967 0.964 0.962 0.975 0.981 0.986 0.987 0.991 0.994 0.992

Low-temperature range (4070 1C) D40 (min) D45 (min) D50 (min) D55 (min) D60 (min) D65 (min) D70 (min) z (4070 1C) E a (kJ/mol) )(4070 1C) High-temperature range (7090 1C) D75 (min) D80 (min) D85 (min) D90 (min) z (7090 1C) E a (kJ/mol) )(7090 1C)
a

Mean (n 3)7standard deviation.

where T c is temperature as 1C. The E a value for thermal inactivation of pineapple puree PPO at 7090 1C( Table 3) was 82.8 kJ/ mol, much higher than that reported for rice, 23.3 kJ/mol (Aquilera et al., 1987) but lower than that for other fruit purees which have ranged from 276 kJ/mol for grape to 502 kJ/mol for peach (Chan & Yang, 1971). E a values reported for whole banana and cranberry, 413 and 116 kJ/mol, respectively, (Dimick et al., 1951; Lee et al., 1983) are within the range reported for purees. High activation energy reects a greater sensitivity of PPO to temperature change. Thus, the PPO of pineapple puree is more sensitive to heat than wild rice but less than the other fruit examined to date. The z-value for pineapple puree, 21.5 1C, at 7090 1C, was high relative to values reported for other fruits which have ranged from 8.5 to 10.1 1C (Stru bi et al., 1975; Vamos-Vigyazo, 1981). In general, low z- values are thought to indicate greater sensitivity to heat (Barrett, Gryison, & Lewis, 1999). Interestingly, z-values

Mean (n 3)7standard deviation.

may be inuenced by the degree of ripeness and preparation method. Thus, Heil et al. (1988) reported a wide variance in z-value of 16 and 61.5 1C for PPO of ripe and soft apricot prepared for canning. Differences in the kinetics of heat activation of PPO for different products may result from differences in their composition, reective of their variety or the agronomic and climatic conditions under which they were grown. In summary, the efcient and effective heat treatment to reduce PPO and enzymatic browning in pineapple puree can be derived from any of D, z, Ea or k values. However, to achieve the optimum heat treatment, further studies should be carried out to nd the loss of desirable characteristics from selected conditions and to

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investigate the effects of residual enzyme on the product stability during subsequent storage to validate the thermal inactivation process.

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