Peroxidase and polyphenol oxidase thermal inactivation by

ARTICLE IN PRESS

LWT 40 (2007) 852–859 www.elsevier.com/locate/lwt

Peroxidase and polyphenol oxidase thermal inactivation by microwaves in green coconut water simulated solutions K.N. Matsuia, L.M. Granadob, P.V. de Oliveirac, C.C. Tadinia, a

Department of Chemical Engineering, Escola Politećnica, Saõ Paulo University, P.O. Box 61548, Saõ Paulo, Zip code 05424-970, Brazil b Technology College Oswaldo Cruz, Rua Brig. Galvaõ, 540, Saõ Paulo, Zip code 01151-000, Brazil c Chemistry Institute, Saõ Paulo University, P.O. Box 26077, Saõ Paulo, Zip code 05513-970, Brazil Received 31 October 2005; received in revised form 20 March 2006; accepted 20 March 2006

Abstract Enzymes from coconut water such as peroxidase (POD) and polyphenol oxidase (PPO) when in contact with oxygen begin reactions causing nutritional and color losses. Solutions simulating the chemical constituents of coconut water were submitted to a batch process in a microwave oven. PPO and POD inactivation data could be characterized by: PPO/water D93 1C ¼ 16.5 s (z ¼ 35.5 1C); PPO/sugars D91 1C ¼ 18 s (z ¼ 331C); POD/water D91.5 1C ¼ 44 s (z ¼ 24 1C) and POD/sugars D92 1C ¼ 20.5 s (z ¼ 19.5 1C). The contact between salts and enzymes promoted a drastic reduction of the initial activity. After the incidence of microwave energy at temperatures above 90 1C, enzymes activity was not detected. These results can indicate an adequate choice of temperature conditions to inactivate coconut water enzymes. The knowledge of how green coconut water constituents influence POD and PPO activity will supply useful information about microwave processing of coconut water. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Microwaves; Peroxidase; Polyphenol oxidase; Thermal processing

1. Introduction Green coconut water can be considered a natural isotonic drink, due to its mineral and sugar content. It is a very popular drink in Brazil and can be found either in natura or processed. In order to avoid spoilage and enzymatic browning caused by peroxidase (POD) and polyphenol oxidase (PPO) when the product is exposed to air for a long time, coconut water can undergo different processes such as ultra-high temperature (UHT), conventional pasteurization, refrigeration and freezing (Abreu & Rosa, 2000; Campos, Souza, Coelho, & Glo´ria, 1996; Duarte, Coelho, & Leite, 2002). Microwave heating as an alternative method for liquid food pasteurization has gained acceptance because it offers several advantages over the conventional method. Microwaves are able to heat products internally, have greater penetration depth and faster heating rates that would Corresponding author. Tel.: +55 11 30912258; fax: +55 11 30912255.

E-mail address: [email protected] (C.C. Tadini).

potentially improve retention of thermolabile constituents in the food (Can˜umir, Celis, de Bruijn, & Vidal, 2002; Deng, Singh, & Lee, 2003; Gerard & Roberts, 2004; Heddleson & Doores, 1994; Nikdel, Chen, Parish, Mackellar, & Friedrich, 1993). Microwave energy induces thermal effects over microorganisms and enzymes similar to those of conventional heating mechanisms (Can˜umir et al., 2002). However, a problem that has often been encountered is the occurrence of temperature profiles within a product. The measurement of temperature profiles during microwave heating is conducted using fiber optic probes that could be easily incorporated into the process without disturbing it (Deng et al., 2003; Gerard & Roberts, 2004; Nott & Hall, 1999). Pasteurization involving enzyme inactivation by microwave energy has not been commonly studied and there are few reports on kinetic data for POD and PPO inactivation. (Soysal & Soylemez, 2005; Tajchakavit & Ramaswamy, 1997). POD and PPO are widely detected in many fruits and vegetables and are closely linked to enzymatic color

0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.03.019

ARTICLE IN PRESS K.N. Matsui et al. / LWT 40 (2007) 852–859

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changes with consequent loss of sensorial properties and nutritional quality (Duarte et al., 2002; Robinson, 1991). Different names have been associated with PPO including tyrosinase, cresolase, cathecolase and phenolase and generally reflect the ability of this enzyme to utilize many different phenolic compounds as substrates. POD is a group of enzymes that catalyses oxidation reactions reducing hydrogen peroxide to water while oxidizing a variety of substrates (Robinson, 1991). PPO and POD are very resistant to heat and therefore are considered biological indicators of thermal processing (Robinson, 1991; Weng, Hendrickx, Maesmans, & Tobback, 1991). The aim of this work was to determine POD and PPO inactivation by microwave heating in coconut water simulated solutions and to verify possible influences of the major chemical constituents in coconut water on enzyme activities.

Samples of simulated solutions were individually submitted to a batch process in the microwave oven at different temperatures in the 60–100 1C. Real temperature–time profiles during batch processing were acquired using an optic fiber probe, calibrated with distilled water using a calibrated thermometer model TRB (Gavea Sensors, Rio de Janeiro, Brazil) inserted centrally inside the glass tube. Temperature readings were recorded using a continuous data acquisition system. After microwaves incidence, the glass tube was removed from the microwave oven and inserted into an ice bath to accelerate cooling. Subsequently 2 ml samples were collected to determine the enzymatic activity and were quickly cooled in liquid nitrogen and kept in a freezer at 80 1C, model MDF-U3086S (Sanyo Electric Co. Ltd., Japan).

2. Materials and methods

POD activity was monitored at 405 nm in a spectrophotometer UV–VIS, model 700 PLUS (Femto, Saõ Paulo, Brazil) according to the method described by Pu¨tter and Becker (1983). A test tube containing 7.0 ml of buffer (Na2HPO4 2H2O+KH2PO4) pH 6.0 and 0.8 ml of ABTS (2.2 azino-bis 3-ethylbenzthiazoline-6-sulfonic acid) solution (2 102 mole/l) and 0.8 ml of hydrogen peroxide solution (0.1% v/v), was immersed in a controlled temperature bath model U2C (Veb MLW, Saxony, Germany) at 25 1C, for 5 min for thermal stabilization. After that 1.0 ml aliquot of simulated solution was added to this solution. The reference value of POD (0.000 absorbance) was determined using a blank solution containing ABTS and hydrogen peroxide. PPO activity was monitored spectrophotometrically at 425 nm, according to the method described by Campos et al. (1996). A test tube containing 5.5 ml of 0.2 mole/l sodium phosphate buffer (pH 6.0) and 1.5 ml of 0.2 mole/l pyrocatechol solution (15890, Fluka) was immersed in a controlled temperature bath at 25 1C, for 5 min for thermal stabilization. After that 1.0 ml aliquot of simulated solution was added to this solution. The reference value of PPO (0.000 absorbance) was determined using a test tube containing 5.5 ml of 0.2 mole/l sodium phosphate buffer (pH 6.0) and 1.5 ml of 0.2 mole/l pyrocatechol solution. For both enzymes the absorbance was acquired every 10 s during 30 min. The data obtained was plotted against time and the PPO and POD activity was calculated from the slope of the initial linear portion of the curve. All enzyme activities were analysed in duplicate. In both cases, one unit of enzyme activity was defined as the quantity necessary to produce an increase in absorbance of 0.001 per ml of sample per second. The residual activity was determined as (A/A0) where: A ¼ mean enzyme activity (after microwave heating); A0 ¼ mean initial enzyme activity (before microwave heating).

2.1. Simulated solutions The solutions PPO/sugars, POD/sugars, PPO/salts, POD/salts, PPO/salts/sugars and POD/salts/sugars were prepared with sugars and salts concentrations similar to average contents of green coconut water reported in literature (Aleixo, No´brega, Santos Juńior, & Muller, 2000; Santoso, Kubo, Ota, Tadokoro, & Maekawa, 1996). Commercial horseradish POD (Sigma-P6140) and mushroom Tyrosinase (Sigma-T3824) were used to prepare the enzyme stock in distilled water: 1.7 104 g PPO/100 ml and 7.8 104 g POD/100 ml and maintained under freezing conditions at 80 1C. Simulated solutions were prepared as following: 1 ml of enzyme stock was diluted in 100 ml of distilled water to obtain PPO/water solution; 1 ml of enzyme stock was diluted in 100 ml of sugars solution containing 0.28 g of sucrose, 2.38 g of glucose and 2.40 g of fructose to obtain PPO/sugars solution; 1 ml of enzyme stock was diluted in 100 ml of salts solution containing 44 mg of KH2PO4, 336 mg of K2SO4, 31 mg of Na2SO4, 47 mg of CaCl2 and 20 mg of MgCl2 to obtain PPO/salts solution; 1 ml of enzyme stock was diluted in 100 ml of sugars and salts solution containing 0.28 g of sucrose, 2.38 g of glucose, 2.40 g of fructose, 44 mg of KH2PO4, 336 mg of K2SO4, 31 mg of Na2SO4, 47 mg of CaCl2 and 20 mg of MgCl2 to obtain PPO/sugars/salts solution. The same procedure was done to POD. 2.2. Microwave thermal treatment The microwave oven (model star system, CEM Corporation, Matthews, USA) at 2450 MHz contains two cavities where specific glass tubes (310 mm length and 41 mm diameter) were inserted and an automatic program that controls microwaves incidence. Maximum volume of 20 ml was used to obtain homogeneity of microwaves incidence.

2.3. Determination of enzymatic activity


854

2.4. Physical chemical analyses

90

Temperature (°C)

80

Total acidity and soluble solids were determined according to AOAC methods. Total acidity was expressed as malic acid percentage. Titration was carried out in the pH-Stat PHM-290 (Radiometer Analytical S.A., Lyon, France), until pH 8.2 was reached (Association of Official Analytical Chemists (AOAC), 1995). Soluble solids, expressed as 1Brix were determined for a portable refractometer and corrected by temperature (AOAC, 1995). The pH was directly measured using the pH-Stat PHM290 (Radiometer Analytical S.A., Lyon, France) (AOAC, 1995).

70 60 50 40 30 20 10 0

50

100

150

200

250

300

350

300

350

400

450

Time (s)

(a) 90

2.5. Kinetic data analysis: nonisothermal heating conditions

70

Temperature (°C)

In most thermal processing situations, food products are subjected to nonisothermal heating conditions. The accumulated lethality (L) is obtained by integration of the lethal effects of the temperature profile during the come-up, hold and cooling periods using the relationship: Z t L¼ 10ðTT ref Þ=z dt. (1)

80

50 40 30 20

0

Computation of the lethality requires data on the z value that can be obtained from a regression of log D value vs. temperature and Tref is the reference temperature. In batch microwave heating there is no isothermal hold period. First estimates of D values can be calculated based on the total residence time of the product in the microwave cavity, and from D values a first estimate of z value is obtained. Correction of heating times and calculation of D and z can then be repeated to get convergence of D and z values (Tajchakavit & Ramaswamy, 1997; Tajchakavit, Ramaswamy, & Fustier, 1998; Toledo, 1991). According to Tajchakavit and Ramaswamy (1997), since there is no specific isothermal hold period, any temperature within the range of study could be used as a reference temperature. The experiments were organized in groups according to maximum temperature (Tmax), and for each group the highest temperature was considered as reference temperature (Tref). This allowed the determination of the equivalent time (tequiv) and kinetic parameters (D and z). This procedure was applied for both enzymes (PPO and POD) for all simulated solutions. A z value of 15 1C was considered the initial value used to calculate the first equivalent time.

60

10 0

(b)

50

100

150

200

250

400

450

Time (s)

Fig. 1. Temperature–time profiles for PPO simulated solutions (a) [(m) PPO/water; (’) PPO/salts; (&) PPO/sugars; (J) PPO/salts/sugars] and for POD simulated solutions (b) [(m) POD/water; (’) POD/salts; (&) POD/sugars; (J) POD/salts/sugars] submitted to microwave heating.

solutions at the same conditions of microwave incidence. The direct contact between the optic fiber probe and the simulated solutions allowed real acquisition of temperature data. It was observed that the temperature–time profiles of PPO and POD solutions have similar behaviors. Solutions with sugars and water reached about 75 1C while solutions containing salts (PPO/salts, PPO/salts/sugars, POD/salts and POD/salts/sugars) reached higher temperatures up to 85 1C. These results were expected since ionized components collide randomly with ionized and nonionized molecules when submitted to an electromagnetic field, causing heat generation by friction. Ionic movement is one of the most important mechanisms that contribute to convert electromagnetic energy into heat (Heddleson & Doores, 1994; Mudgett, 1986).

3. Results and discussion 3.2. Polyphenol oxidase microwave inactivation kinetics 3.1. Time–temperature profiles PPO and POD simulated solutions were tested at different temperature conditions. Fig. 1 shows typical temperature profiles obtained for PPO and POD simulated

Table 1 presents the maximum temperature (Tmax) of each condition, reference temperature (Tref), equivalent heating time (tequiv) calculated based on accumulated lethality (Eq. (1)) and D value for PPO/water, PPO/sugars,


855

Table 1 Maximum temperature (Tmax), reference temperature (Tref), equivalent heating time (tequiv) and D value for PPO simulated solutions submitted to microwaves PPO simulated solutions Watera

Sugarsb

Saltsc

Salts/sugarsd

Tmax (1C)

Tref (1C)

tequiv (s) D* (s)

Tmax (1C)

Tref (1C)

tequiv (s) D* (s)

Tmax (1C)

Tref (1C)

tequiv (s) Dl* (s)

Tmax (1C)

Tref (1C)

tequiv (s) Dl* (s)

74.1 76.5

76.5

78.7 74.4

48

74.4 75.6

75.6

68.3 86.1

51

73.4

70.1 72.9 75.7

75.7

16.9 30.3 42.9

18

82.1

70.0 81.7 85.4

33

78.3 79.8

79.8

76.1 83.0

41

11.3 19.0 33.2 44.4

10

81.0 82.0 82.1

64.3 69.1 71.7 73.4

26.7 41.8 50.8

20

26

81.9 83.4 85.7

85.7

63.5 75.1 67.5 70.4

14.2 22.6 32.7 38.3

14

84.3

80.8 84.1 87.1 89.6

89.6

82.6 83.0 83.1 84.3

36.2 39.1 19.7 26.1

—

18

96.2 96.5 97.2 98.7

98.7

50.4 46.6

46.0 55.7 22.8 29.9

—

91.1

97.4 97.5 98.5 99.2

99.2

90.0 91.1

84.6 85.3 86.8

86.8

66.6 71.0 72.6

27

90.9 91.7 92.9

92.9

44.6 44.9 45.8

17

2

2

1.5

1.5

Log (A/Ao x100)

Log (A/Ao x100)

*Determined according to Fig. 2.Dl ¼ thermo labile fraction. a pH 6.770.1 and (0.02670.005) % malic acid. b pH 6.570.2, (0.02770.007) % malic acid and (4.5370.15) 1Brix. c pH 5.070.0 and (0.06570.004) % malic acid d pH 5.070.1, (0.06870.005) % malic acid and (4.8670.38) 1Brix.

1 0.5 0 -0.5

0

-1 0

10

20

30

40

50

60

70

80

90

Equivalent time (s)

(a)

0

10

20

1000

1000

100

100

10

1

30

40

50

60

70

80

90

Equivalent time (s)

(c)

D-value (s)

D-value (s)

0.5

-0.5

-1

10

1 60

(b)

1

70

80

90

Temperature (°C)

100

60

110

(d)

70

80

90

100

Temperature (°C)

Fig. 2. Residual PPO activity in aqueous solution (a) [(m) 76.5 1C; (S) 82.1 1C; (J) 86.8 1C;(E) 92.9 1C]; and in sugars solution (c) [( ) 75.6 1C; (’) 79.8 1C; (K) 84.3 1C; (B) 91.1 1C] according to equivalent time at different temperatures when submitted to heating by microwaves; Temperature sensitivity curves of PPO/water (b) [(n) z ¼ 35.5 1C and R2 ¼ 0.99] and PPO/sugars (d) [(&) z ¼ 33 1C and R2 ¼ 0.98].

ARTICLE IN PRESS K.N. Matsui et al. / LWT 40 (2007) 852–859 2

Log (A/Ao x100)

1.5 1 0.5 0 -0.5 0

10

(a)

20 30 Equivalent time (s)

40

50

2 1.5

Log (A/Ao x100)

PPO/salts and PPO/salts/sugars simulated solutions submitted to microwaves heating. Fig. 2 shows the residual PPO activity in aqueous and in sugar simulated solutions as a function of equivalent heating time at various temperatures under microwave heating conditions and the regression of residual activity vs. corrected equivalent time originated the D values by iterative method. The log-linear decrease of enzyme activity as a function of equivalent heating time showed that for temperatures close to 80 1C up to two log reductions occurred, while above this temperature almost three log reductions were reached. The z values for PPO/water and PPO/sugar solutions were similar D93 1C ¼ 16.5 s (z ¼ 35.5 1C) and D91 1C ¼ 18 s (z ¼ 33 1C), respectively, as presented in Fig. 2. Weemaes et al. (1997) determined the kinetic parameters (D and z values) for mushroom PPO when submitted to conventional thermal processing. The results show irreversible first-order inactivation. A batch with enzyme units similar of this work was tested and the D53 1C value obtained was 55 min (z ¼ 6.521C). Campos et al. (1996) determined PPO residual activity in coconut water heated in a water-bath. Inactivations above 90%were only encountered for temperatures above 90 1C held for more than 90 s. Fig. 3 shows the inactivation curves of PPO/salts and PPO/salts/sugars solutions as a function of equivalent heating time under microwave heating conditions. It can be observed in these cases, two first-order rate curves denoting a thermoresistant fraction in these temperatures. The kinetic parameter of the thermolabile fraction can be obtained from the slope of first linear section of the curve. After microwaves incidence, PPO residual activity of the solution with salts at Tref ¼ 99.2 1C and of the solution with salts/sugars at Tref ¼ 98.7 1C was not detected. Even though the same amount of enzymatic extract was added in all simulated solutions, results showed that initial PPO/ salts activity was lower than for the other solutions. Campos et al. (1996), Duangmal and Apenten (1999), Mendonc- a and Guerra (2003), Zawistowsky, Biliaderis, and Eskin (1991) observed that in presence of salts there was a significant decrease in enzyme thermostability; this is due to salt-induced changes in enzyme conformation and possibly the dissociation of thermostable aggregated molecules. Catechol solution, even without the presence of PPO, presented an increase in absorbance. Exposure to light for a certain time period causes substrate oxidation and consequent darkening; this fact could result in overestimation of the residual PPO/salts activity after microwave incidence and suggest a false interpretation of the results. To minimize the catechol oxidation interference in this study, higher PPO concentrations than those found in natural coconut water were tested, because an increase in PPO activity would probably reduce the effect of the salts

1 0.5 0 -0.5 0

10

(b)

20 30 40 Equivalent time (s)

50

60

Fig. 3. Residual PPO activity in salts solution (a) [(J) 73.4 1C; (’) 89.6 1C] and in salts/sugars solutions (b) [(E) 75.7 1C; (n) 85.7 1C] according to equivalent time at different temperatures when submitted to heating by microwaves.

1.4 1.2

Abs at 425 nm

856

1 0.8 0.6 0.4 0.2 0 0

200

400

600 Time (s)

800

1000

1200

Fig. 4. Initial PPO activity for each simulated solution in comparison to catechol oxidation [(&) PPO/water; (K) PPO/salts; (m) PPO/sugars; (n) PPO/salts/sugars; (J) catechol oxidation].

on enzyme denaturation and the efficiency of thermal microwave processing could then be determined. Absorbance vs. time readings for solutions containing PPO demonstrate that the salts considerably reduced the enzymes thermostability. Comparing initial activity of the


enzymes it can be observed in Fig. 4, that even though the same amount of enzyme extract was added to all the solutions, PPO/salts and PPO/salts/sugars presented lower initial activities, particularly the PPO/salts solutions. This decrease in the enzyme’s initial activity renders determination of the residual activity after microwave incidence impossible, since the reaction rate is then so low that the change in absorbance falls within the precision range of the equipment, causing significant errors in the results. 3.3. Peroxidase microwave inactivation kinetics Table 2 presents the maximum temperature (Tmax) of each condition, reference temperature (Tref), equivalent heating time (tequiv) calculated based on accumulated lethality (Eq. (1)) and D value for POD/water, POD/ sugars, POD/salts and POD/salts/sugars simulated solutions submitted to microwave heating. POD/water solution presented a slight decrease in activity when submitted to microwaves heating at the studied temperatures (Fig. 5). At similar process conditions, POD/sugars solutions presented higher activity decrease. The sugars that were added to the solution contributed to decrease POD activity. Gibriel, El-Sahrigi, Kandil, and El-Mansy (1978) showed that the presence of sucrose in the reaction mixture inhibited POD activity in apricot. Chang, Park, and Lund (1988) showed by differential scanning calorimetry that in the presence of 10% sucrose POD thermal stability was reduced. For a range of sugars tested, fructose was the most effective in reducing the enzyme thermostability and it was suggested that this was due to the interaction of

857

fructose with the protein amino acids. Once more, the equipment did not allow data at different exposure times to be obtained, so the inactivation behavior of POD could not be determined. The D and z values encountered for POD/water and POD/sugars solutions were D91.5 1C ¼ 44 s (z ¼ 24 1C) and D92 1C ¼ 20.5 s (z ¼ 19.5 1C), respectively, similar to those reported in the literature. Weng et al. (1991) reported a z value of 26.371.8 1C for horseradish peroxidase (HRP) in aqueous solution and they found biphasic behavior of the heat inactivation when submitted to conventional heat treatment. Joffe and Ball (1962) obtained a z value of 27.7 1C for HRP in the temperature range 85–150 1C. Ling and Lund (1978) reported a D82 1C ¼ 1.2 min (z ¼ 17 1C) for heatlabile and D82 1C ¼ 42 min (z ¼ 27.3 1C) for heat-stable HRP. The carrot POD activity was determined by Soysal and Soylemez (2005) and its inactivation was studied by thermal and microwave heating. Biphasic behavior of enzyme inactivation was observed for the microwave treatment at low microwave power and monophasic behavior was observed at high microwave power. The POD/salts solution did not present significant activity decrease for Tref ¼ 62.7 and 85.9 1C. For Tmax ¼ 95.2 and 95.4 1C, no residual activity was detected. The POD in the salts/sugars solution presented for Tref ¼ 69.4 and 80.9 1C activity decrease less than one log cycle, while for Tref ¼ 90.7 1C there was a one log cycle decrease and for Tref ¼ 96.4 1C no residual activity was detected. For all studied solutions, POD was more resistant at temperatures below 90 1C than PPO.

Table 2 Maximum temperature (Tmax), reference temperature (Tref), equivalent heating time (tequiv) and D value for POD simulated solutions submitted to microwaves POD simulated solutions Watera

Sugarsb

Saltsc

Salts/sugarsd

Tmax (1C)

Tref (1C)

tequiv (s) D* (s)

Tmax (1C)

Tref(1C) tequiv (s) D* (s)

Tmax (1C)

Tref (1C)

t equiv (s)

D* (s)

Tmax (1C)

Tref (1C)

tequiv (s) D* (s)

76.4 77.4

77.4

57.9 61.6

167

75.8

57.1 62.7

62.7

18.8 37.3

—

61.9 69.4

69.4

14.2 38.4

—

81.5 83.9

83.9

41.4 57.7

93

67.0 68.0 75.4 75.8

85.9

75.1 80.9

80.9

18.5 45.1

—

82.3

5.2 10.7 17.2 49.9

—

81.0 82.3

72.2 77.1 80.3 85.9

90.7

91.9

95.2 95.4

95.4

56.1 51.4

—

16.0 19.3 50.3

—

90.6 91.9

83.5 84.0 90.7 95.1 96.4

96.4

69.1 48.5

—

88.5 91.5

91.5

27.9 31.1

15.6 17.9 54.4 48.9

144

50.8 50.9

54

31.4 24.2

21

44

*Determined according to Fig. 5. a pH 6.270.1 and (0.02470.003) % malic acid. b pH 6.370.1, (0.02170.004) % malic acid and (4.5370.22) 1Brix. c pH 5.070.0 and (0.04870.002) % malic acid d pH 5.270.1, (0.06870.005) % malic acid and (4.9370.14) 1Brix.


858

2

Log (A/Ao x100)

Log (A/Ao x100)

2

1.5

1

0

10

20

30

40

50

60

0.5

70

Equivalent time (s)

(a)

0

10

20

1000

1000

100

100

10

1

30

40

50

60

Equivalent time (s)

(c)

D value (s)

D-value (s)

1

0

0.5

10

1 70

(b)

1.5

80

90

100

Temperature (°C)

60

70

(d)

80

90

100

Temperature (°C)

Fig. 5. Residual POD activity in aqueous solution (a) [(E) 77.4 1C; (J) 83.9 1C; (’) 91.51C]; and in sugars solution (c) [(m) 75.8 1C; (J) 82.3 1C; (E) 91.9 1C] according to equivalent time at different temperatures when submitted to heating by microwaves; temperature sensitivity curves of POD/water (b) [(K) z ¼ 24 1C and R2 ¼ 0.99] and POD/sugars (d) [(’) z ¼ 19.5 1C and R2 ¼ 0.99].

POD/salts and POD/salts/sugars solutions presented the same behavior observed for PPO solutions, presenting lower initial activities demonstrating that the salts also affect POD activity. The activity of POD/salts and POD/ salts/sugars solutions treated at temperatures above 90 1C was not detected, due to the combined effect produced by the salts and microwaves. Assays with ABTS were undertaken but no change in absorbance was detected.

Acknowledgements The authors wish to thank FAPESP (The State of Saõ Paulo Research Foundation) for the research grant and Gavea Sensors Measurement Solutions Ltd. for technical support. P.V. de Oliveira is also thankful to The National Council for Scientific and Technological Development (CNPq) by the research ship provided.

4. Conclusions References Focused-microwave oven showed to be a good alternative for enzyme inactivation studies using microwave heating. The optic fiber probe allowed reliable temperature–time profiles by microwave heat processing. Kinetic parameters (D and z) were estimated for PPO/ water and PPO/sugars and presented similar behavior. The activity of PPO in both solutions was considerably reduced when they were submitted to microwave heating. Sugars influenced POD more than PPO inactivation. Even so, POD was more resistant to microwave heating for all the green coconut water simulated solutions studied. The salts significantly affected PPO and POD stability. At temperatures above 90 1C, the combined effect of salts and microwave energy reduced PPO and POD enzymatic activity to undetectable levels. These results can indicate an adequate choice temperature conditions to inactivate green coconut water enzymes.

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