Thermal inactivation of peroxidase and polyphenoloxidase enzymes in mate leaves (Ilex paraguariensis) in a conveyor belt oven

Abstract

The inactivation of oxide reductases in mate leaves, a process known in Brazil as sapeco, is carried out at temperatures of 400 to 620°C. High temperatures can produce pyrolysis compounds, such as polycyclic aromatic hydrocarbons. In this study, the time/temperature binomial was determined for the inactivation of peroxidase (POD) in mate in a conveyor belt oven. POD showed greater thermal resistance than polyphenoloxidase. A 2² full factorial design (levels +1 and – 1), with two axial points (levels –α and +α) and three central points (level zero), was used. The biphasic and consecutive step models showed the best fit to the data obtained for the inactivation of POD (R² = 0.99). The best inactivation was obtained after treatment at 255°C for 20 to 24 s, achieving a green colour the same as that obtained through processing using the traditional procedure.

La inactivación térmica de las enzimas óxido reductasas en hojas yerba-mate mediante el procedimiento denominado en Brasil como ‘Sapeco’ ocurre a la temperatura de 400 hasta 620°C. Altas temperaturas pueden producir compuestos de pirólisis, tales como hidrocarburos aromáticos policíclicos (HAP). En el presente trabajo, el binomial tiempo/temperatura se determinó para la inactivación de peroxidasa (POD) más resistente, comparada a polifenoloxidasa (PPO). Un diseño factorial 2² (con nivel de +1 y – 1), con dos puntos axiales (niveles – α y + α) y tres puntos centrales (nivel cero) fueron utilizados. Los modelos bifásicos y de pasos consecutivos mostraron el mejor ajuste a los datos de la inactivación térmica de la peroxidasa (R² = 0,99). La mejor inactivación se obtuvo después del tratamiento a 255°C durante 20 a 24 segundos, atingiendo la misma color verde que uno procesado por lo método tradicional.

Keywords:

• mate
• peroxidase
• polyphenoloxidase
• inactivation

Palabras claves:

• yerba-mate
• peroxidasa
• polifenoloxidasa
• inactivación

Introduction

The oxide reductases polyphenoloxidases (PPOs) and peroxidases (PODs) are the main enzymes in mate. These enzymes are responsible for undesirable changes in the colour of mate, which is consumers’ first criterion for choice (Primo et al., 2007; Provesi, Nabechima, Vieira, & Amante, 2010). In Brazil, consumers prefer mate that has a bright green colour. However, in other South American countries, mate is stored for a longer period of time until it acquires an olive-green to golden-yellow colour in order to attend consumers’ preference (Morawicki, Schmalko, & Kanzig, 1999).

PPO is a group of enzymes which have copper in their structure. They catalyse the oxidation of monophenols into diphenols, and of diphenols into quinones, which can polymerize and produce melanins (Fatibello-Filho & Vieira, 2002; Goupy et al., 1995).

POD is a group of haemoproteins which use hydrogen peroxide to catalyse the oxidation of phenolic compounds (Fatibello-Filho & Vieira, 2002; Martínez-Parra & Muñoz, 2001). These enzymes can restore activity after thermal treatment. Besides that, they are considered to be the most thermally stable enzymes in vegetal systems and because of such characteristic they are used as a parameter for blanching efficiency (Agüero, Ansorena, Roura, & Del Valle, 2008; Gonçalves, Pinheiro, Abreu, Brandão, & Silva, 2007) and kinetic studies on mate leaves can thus be carried out considering the resistance of this enzyme. PODs are responsible for undesirable changes in flavour, colour, aroma, texture and chlorophyll degradation, and they also decrease the nutritional value of processed fruits and vegetables (Goupy et al., 1995; Prabha & Patwardhan, 1986).

In the process of bleaching mate leaves, known as mate sapeco, the leaves and stems of the plant are quickly passed through the heat of the flames generated by burning wood or propane in a rotating oven. The use of a conveyor oven is not common in this process.

There is no consensus on the ideal temperature for the sapeco process, and the temperatures reported in literature are between 400 and 620°C (Peralta & Schmalko, 2007; Schmalko, Ramallo, Ferreira, & Berlingheri, 2002; Vieira et al., 2008). Mate leaves vary greatly in area and thickness; therefore, the heat transfer is uneven, and such fact hampers a quality control of the final product (Coelho, Mariath, & Schenkel, 2002).

Given this scenario, the mate sapeco has undergone very few modifications over the years in relation to the processing mechanisms and process control. The current process wastes 80% of the energy used (Vieira et al., 2008). Moreover, direct exposure of mate branches to the flames and smoke results in the incorporation and/or formation of pyrolysis compounds due to the high temperatures applied. These conditions are appropriate to form and incorporate of polycyclic aromatic hydrocarbons (PAHs), which are mutagenic and carcinogenic organic compounds (Camargo & Toledo, 2002; Usepa, 1998; Vieira et al., 2010; Zuin, Montero, Bauer, & Popp, 2005).

In South America, mate consumption is high; therefore, it justifies the need for improvements in the mate sapeco process to decrease incorporation of PAHs in order to obtain a safe and good-quality product and consequently favour traditional generation and consumption of products with higher added value. There are few studies on thermal inactivation of POD and PPO enzymes in mate leaves (Ceni et al., 2009; Provesi et al., 2010; Xander, Acosta, Scipioni, & Argüello, 2000) and none on the kinetics of the inactivation of these enzymes in the plant matrix of mate, as performed in this study. This raw material has irregularities in its dimensions which should be considered for future industrial projects. Therefore, when studying the kinetics of enzyme inactivation in mate leaves, these irregularities must also be considered. The objective of this work was to study the thermal inactivation of POD and PPO enzymes in mate leaves in a conveyor belt oven.

Results for the inactivation of isolated enzymes cannot be directly useful in the designing of new equipment where mild temperatures are used in the mate processing, according to Nabechima (2010) that studied several temperatures on mate processing. In this context, the aim of this work was to investigate low temperatures for the inactivation of POD and PPO in the matrix of mate leaves which can be applicable in new equipment design.

Experimental method

Materials

The leaves and stems used in the experiments were collected from three mate factories, from June to December 2009, in the municipal district of Catanduvas, located in mid-western Santa Catarina State, Brazil, latitude 27°04′14″ south, longitude 51°40′42″ east, and altitude of 945 m. For each experiment on the thermal inactivation of the enzymes, the samples were collected simulating the same conditions as those at the reception of mate leaves in the industrial sapeco process.

Samples were also collected from the industrial sapeco to establish a comparison with the sapeco in a conveyor oven. Therefore, the different samples of this experiment consisted of fresh leaves, leaves that were thermally treated under several different conditions in a conveyor oven, and leaves that were thermally treated through the traditional sapeco process.

All reagents used were of analytical grade and purchased from Vetec (Duque de Caxias, RJ, Brazil).

The conveyor oven used for the thermal treatment of mate has an electrically heated mat that was specifically designed for this experiment by the LABTUCAL Department of Mechanical Engineering at UFSC – Federal University of Santa Catarina, Brazil.

Determination of enzyme activity in mate leaves and production of the enzyme extract

The specific enzyme activity in mate leaves that were submitted to the industrial sapeco and in mate leaves treated in the conveyor oven was analysed. A crude enzyme extract was obtained based on the methodology proposed by Primo et al. (2007) and Ceni et al. (2008). Samples of 20 g of mate leaves were homogenized in 90 mL of natrium phosphate (Na₂HPO₄) buffer 0.05 M (pH 7.5) and polvinylpyrrolidone K30 3.0% (w/w) as protector agent. This homogenized mixture was filtered in 4-layer cotton gauze and centrifuged (Centrifuge 5840R, Eppendorf do Brasil, São Paulo, Brazil) at 11,000 g at 4°C for 30 min. The supernatant solution was used as crude enzyme extract and kept in ice until the analysis for PPO and POD activity. For the determination of their initial activities, an enzyme extract was made from samples of 20 g of fresh, untreated mate leaves.

Determination of PPO enzyme activity

PPO activity was determined at 420 nm in a UV-VIS spectrophotometer (Hitachi, U-1800, Tokyo, Japan) according to the methodology proposed by Primo et al. (2007) and by Ceni et al. (2008) with some adaptations. A test tube containing 2.8 mL of 0.05 M natrium phosphate (Na₂HPO₄) buffer at pH 8.5 and 0.1 mL of 0.1M pyrocatecol prepared in a 0.1% Tween 80 was kept in thermostat bath (‘Personal Shaker’, Taiyo Kagaku Kogyo Co., Tokyo, Japan) until reaching 25°C and then added with 0.1 mL of enzyme extract solution, and the absorbance was monitored during the linear phase time. A unit of PPO activity was defined as the quantity of enzymes necessary to produce 1 μmol of o-quinone from pyrocatechol (C₆H₆O₂) per minute, under the established conditions. The molar absorptivity coefficient of o-quinone considered in order to transform the absorbance values into moles of o-quinone was 2800 M⁻¹ cm⁻¹ (Ceni, 2005). The values obtained were used to evaluate residual enzyme activity in the cases of the thermally treated mate leaves.

Determination of POD enzyme activity

POD activity was determined at 470 nm in a UV-VIS spectrophotometer according to the methodology proposed by Argüello, Spioni, and Martos (1999) and Ceni et al. (2008) with some adaptations. A test tube containing 2.725 mL of 0.05 M natrium phosphate (Na₂HPO₄) buffer at pH 4.7, 0.1 mL of 0.2 M hydrogen peroxide (H₂O₂) and 0.1 mL of 0.1 M guayacol solution prepared in a 0.1 % Tween 80 solution was kept in a thermostat bath (‘Personal Shaker’, Taiyo Kagaku Kogyo Co., Tokyo, Japan) until reaching 25°C and then added with 25 μL of enzyme extract, and absorbance was monitored during linear phase time. A unit of POD activity was defined as the quantity of enzymes necessary to produce 1 μmol of tetraguaiacol from guayacol per minute, under the established conditions. The molar absorptivity of tetraguaiacol used to transform the values of absorbance into moles of tetraguaiacol was 32,800 M⁻¹ cm⁻¹ (Ceni, 2005). The values obtained were used to evaluate the residual enzyme activity in the cases of thermally treated mate leaves.

Determination of total protein

The enzyme activity was determined based on the total protein content in the enzyme extracts of mate leaves. The quantification of proteins in the enzyme extracts was performed following to the Kjeldahl method (N × 5.25) (AOAC, 2005).

Determination of the time/temperature binomial through response surface methodology

A full factorial design 2² (with levels of +1 and –1), with two axial points (levels –α and +α) and three central points (level zero) was used. The independent variables are the time and the temperature of the thermal treatment. Table 1 shows the levels of these variables.

Table 1. Levels of the independent variables of treatments for the study on thermal inactivation of mate’ PPO and POD in a conveyor oven.

Tabla 1. Niveles de las variables independientes de tratamiento para el estudio sobre la inactivación térmica de PPO y POD de la yerba mate en un horno de cinta transportadora.

Variables	Levels
	−α	−1	0	+1	+α
Temperature (°C)	220	230	255	280	290
Time (s)	19	20	22.5	25	26

Dependent variables are the residual PPO and POD activity. The tests were performed at random. Table 2 shows the 11 assays of the experimental design and the real values codified to the time and temperature of the treatment. Due to the variability of mate leaves and the temperature of the treatment, the experiment was performed at random and in duplicate, and the best treatments were selected. The determination of the enzyme activity was performed in triplicate. The STATISTICA 7.0 (StatSoft, Inc. Tulsa, OK, USA) software was used for the statistical analysis of the data at a significance level of 5%.

Table 2. Experimental design and respective real and codified values of the independent variables for the study of inactivation of PPO and POD in mate thermally treated in a conveyor oven.

Tabla 2. Diseño experimental y respectivos valores reales y codificados de las variables independientes para el estudio de la inactivación de PPO y POD en yerba mate tratada térmicamente en un horno de cinta transportadora.

Assay	Codified levels		Real levels
	Temperature	Time	Temperature (°C)	Time (s)
1	−1	−1	230	20
2	+1	−1	280	20
3	−1	+1	230	25
4	+1	+1	280	25
5	0	0	255	22.5
6	0	0	255	22.5
7	0	0	255	22.5
8	−1.14	0	220	22.5
9	+1.14	0	290	22.5
10	0	−1.14	255	19
11	0	+1.14	255	26

Thermal inactivation assays

The leaves and stems were manually separated and were thermally treated at a proportion of 1.79 g of leaves/g of stems (which corresponds to the average proportion used in the traditional sapeco process in factories). The temperature in the conveyor oven showed an average standard deviation of 7%. Thermal treatments for each time and temperature were performed in four repetitions, with 5 g of leaves for each repetition, totalizing 20 g of leaves and 11.2 g of stems for each condition tested. Immediately after the thermal treatment, the samples were cooled to 25°C, and the stems were discarded, and thus the enzyme activity and other parameters were determined only with the leaves.s

Thermal inactivation of mate POD enzyme in a conveyor oven

The study of the kinetics of POD inactivation was performed at 255°C. The times of the thermal treatment investigated were 5, 10, 15, 20, 22 and 24 s.

Four enzyme inactivation models were used in this study: consecutive steps (Polakovic & Vrábel, 1996), biphasic (Bobrovnik, 2000), first-order (Toledo, 1999) and fractional conversion (Loey, Indrawati, Smout, & Hendrickx, 2003).

The measurements of PPO and POD activity were performed in triplicate, following the same procedures as those for the fresh leaves as raw material.

The model adjustments were made with MATLAB® R14 (MathWorks Inc., Natick, MA, USA). The evaluation of the adjustments was performed based on statistical criteria, determination coefficient (R²) and standard error.s

Mate PPO and POD enzyme activity in the industrial sapeco process

Samples of mate submitted to traditional industrial thermal treatment were analysed to determine PPO and POD activity in this product. Fresh leaves (20 g) and 11 g of thermal treated leaves were used in order to obtain the enzyme extract. The determination of the enzyme activity in the industrial sapeco process followed the same procedures as those for the samples of mate treated in a conveyor oven and of fresh mate, as reported in this work.

The determinations were performed in triplicate. The Statistica 7.0 software (StatSoft, Inc. Tulsa, OK, USA) was used for the analysis of variance at a significance level of 5%.

Results and discussion

Definition of the time/temperature binomial

The conditions of each test performed, with independent variables (time and temperature of treatment) and their responses (residual POD and PPO activity) are shown in Table 3. Tests on the leaves that suffered blight cannot be considered valid in spite of showing acceptable levels of enzyme inactivation. However, although the traditional process produces efficient enzyme inactivation, it may also give the final product an undesirable ‘burnt’ taste, and this product will probably contain pyrolysis compounds, including PAHs. Moreover, due to the heterogeneity of the dimension of the leaves, scorching occurred mostly in the smaller leaves. This behaviour, which occurs because the leaves are of very different dimensions, poses great difficulty to propose an appropriate thermal treatment in the sapeco step in mate processing.

Table 3. Residual POD and PPO activity in thermally treated mate leaves in the different assays performed.

Tabla 3. Actividad residual de las enzimas POD y PPO de la yerba mate tratada térmicamente en los diferentes ensayos realizados.

			Residual activity
Test	Temperature (°C)	Time (s)	POD (%)	PPO (%)
1	230	20	20	4
2*	280	20	14	3
3	230	25	17	3
4*	280	25	1	2
5	255	22.5	5	4
6	255	22.5	7	3
7	255	22.5	8	4
8	220	22.5	29	3
9*	290	22.5	0	3
10	255	19	15	4
11*	255	26	4	3

Note: *Test in which leaf blight occurred.

The treatment at 280°C for 20 s showed residual enzyme activity above 10%, indicating that besides scorching some smaller leaves, such treatment also produced unsatisfactory inactivation in the larger leaves (Table 3).

There was no significant difference in the residual PPO activity among the several assays performed, which was impeditive to obtaining a statistical model for the kinetics of PPO inactivation. The level of PPO inactivation was low for all the assays, with values lower than 5%, including assay 8, which represents the thermal treatment at 220°C and where residual POD activity was 29%. These results with temperatures lower than those in the traditional industrial processing show that the inactivation of PPO enzymes does not justify the use of excessive heat, commonly adopted in mate factories.

Provesi et al. (2010) evaluated the thermal treatment of mate leaves in an oven and reported that, after thermal treatment, PPO enzymes showed higher residual activity than POD enzymes. However, only POD showed recovery of enzyme activity after seven days of storage (Whitaker, Voragen, & Wong, 2003).

Ceni et al. (2009) studied mate POD and PPO thermal inactivation in a microwave oven and reported that POD showed greater thermal resistance because inactivation of POD occurred after 220 s whereas inactivation of PPO occurred after 30 s.

In the statistical model for residual POD activity, the parameters that were statistically significant were the linear and quadratic temperature and the linear time, as can be seen in the Pareto chart (Figure 1).

Figure 1. Pareto chart of standardized effects of the residual mate tea POD activity in function of temperature and time of thermal treatment in a conveyor belt oven.

Figura 1. Diagrama de Pareto de los efectos estandarizados de la actividad de POD residual de la yerba mate en función de la temperatura y el tiempo de tratamiento térmico en un horno de cinta transportadora.

The regression coefficients of significant (p < 0.05) parameters of the statistical model are shown in Table 4. The negative signs of the regression coefficients of linear temperature and time show a decrease in residual POD activity with the increase in these variables, and temperature showed greater influence on residual POD activity. The positive sign of the quadratic temperature indicates that the model has a minimum point.

Table 4. Regression coefficients for response of the residual POD activity in mate thermally treated in a conveyor oven.

Tabla 4. Los coeficientes de regresión para la respuesta de la actividad POD residual en yerba mate tratada térmicamente en un horno de cinta transportadora.

Effects	Regression coefficient	Standard error	t(7)	p
Average	8.21	1.54	5.33	0.00
Temperature (L)	−7.90	2.60	−6.09	0.00
Temperature (Q)	3.75	2.99	2.51	0.04
Time (L)	−3.96	2.60	−3.05	0.02

Note: R² = 0.88.

The coefficient of determination for the model was 0.88, i.e. it indicates that the model explains 88% of the variation of the experimental data.

The following formula shows the statistical model for residual POD activity in mate in function of temperature and time.

where T (°C) and t (s) are the temperature and the time of exposure to the thermal treatment, respectively.

To validate the statistical model, it is necessary to evaluate the analysis of variance of the process besides the coefficient of determination (Table 5). The statistical model obtained was significant (F calculated >3 F tabulated) and the lack of adjustment was not significant (F calculated < F tabulated) (Barros Neto, Scarminio, & Bruns, 1996). The value of F validates the significance of the model, which must be used only within the range studied. Although the methodology used allows extrapolation, this procedure is not adequate for studies with foods because the variables do not generally show linear behaviour.

Table 5. Analysis of variance for the quadratic model of POD inactivation in mate through thermal treatment in a conveyor oven.

Tabla 5. Análisis de la varianza para el modelo cuadrático de la inactivatión de la POD de la yerba mate submetida al tratamiento térmico en un horno de cinta transportadora.

Source of variation	Quadratic sum	Degrees of freedom	Mean square	Fcalculated	Ftabulated
Regression	703.48	3	234.49	17.57	4.35
Residues	93.43	7	13.35
F adjustment	88.76	5	17.75	7.61	19.30
Pure error	4.67	2	2.33
Total	796.91	10

Note: F 0.95, 3, 7 = 4.35 ; f 0.95, 5, 2 = 19.30. ^aF-reason (Regression/Residues). ^bF-reason (Lack of adjustment/Pure error).

Another aspect to be considered for the validation of a model is the distribution of errors; the errors should follow a normal distribution with their mean closest to zero. The errors from the model adjustment (Equation (1)) follow a normal distribution, with greater concentration of errors in the range between 0 and 2.

By analysing the contour surface (Figure 2), it was noted that within the ranges studied, the residual POD activity below 10% was obtained at temperatures above 240°C and for times longer than around 19 s.

Figure 2. Contour surface for mate POD inactivation in function of temperature and time of thermal treatment in a conveyor belt oven.

Figura 2. Superficie de contorno para la inactivación de la POD de la yerba mate en función de la temperatura y del tiempo de tratamiento térmico en un horno de cinta transportadora.

When considering the best thermal treatment, i.e. the time/temperature binomial to be used, one should take into consideration the changes in mate besides its residual POD activity.

In this study, the thermal treatment performed at 255°C for 22.5 s showed residual POD enzyme activity below 10% and no scorching of the mate leaves. The temperature of 255°C was chosen to study the behaviour of the POD inactivation and to identify the treatment time in which >90% enzyme inactivation occurs. Only the kinetics of POD was investigated because of the higher thermal stability.

Thermal inactivation of mate POD enzyme

The kinetic models that best describe the inactivation of POD were the biphasic inactivation and the consecutive steps (Table 6).

Table 6. Coefficient of determination and standard error of the models adjusted for inactivation of mate POD enzyme through thermal treatment in conveyor oven.

Tabla 6. Coeficiente de determinación y el error estándar de los modelos ajustados para la inactivación de la enzima POD de la yerba mate submetida a lo tratamiento térmico en horno con cinta transportadora.

Model	R^2	Standard error
First order	0.96	6.82
Fractional conversion	0.98	4.47
Biphasic inactivation	0.99	3.55
Inactivation by consecutive steps	0.99	3.55

Both models (biphasic and consecutive steps) showed a good coefficient of determination; however, they show high values of standard error. Such result occurs because of the great heterogeneity of mate leaves in thickness and dimensions, which consequently affects the efficiency of the thermal treatment. When plotting both models, an overlap in the same range studied is noted, thus indicating no difference between the values predicted by these models (Figure 3).

Figure 3. Adjustment of the biphasic inactivation and consecutive steps to the data of thermal inactivation of mate POD enzymes in a conveyor belt oven.

Figura 3. Ajuste de la inactivación bifásica y pasos consecutivos a los datos de la inactivación térmica de las enzimas POD de la yerba mate en un horno de cinta transportadora.

The parameters, a_l, a_s, k_l and k_s, in the biphasic inactivation model, which are the most usual values, were 83.12 and 16.88 (%) and 6.67 and 0.056 (s⁻¹), respectively. Because the samples were not submitted to a constant temperature during the assays, these parameters have no significant theoretical value; even so, they can be used to predict the behaviour of mate POD inactivation in the condition studied.

There are no published studies on the kinetics of the inactivation of oxide reductase enzymes in mate leaves in their plant matrix or in enzyme extract for comparison. However, studies on the kinetics of POD inactivation at constant temperature performed with enzyme extracts of plants showed biphasic behaviour during inactivation. Soysal and Söylemez (2005) studied carrot POD inactivation using a microwave oven. Thermal treatments were performed at 35 to 75°C for 0.5 to 1.8 min. The kinetics of inactivation showed biphasic behaviour, except at 75°C, where the behaviour was of first order. Połata, Wilińska, Bryjak, and Polakovič (2009) studied the kinetics of broccoli POD inactivation at temperatures from 58 to 74°C, while Terefe, Yang, Knoerzer, Buckow, and Versteeg (2010) performed the same study with strawberry puree to 45 and at 55°C. The kinetics of enzyme inactivation was described through a biphasic model in both studies.

Table 7 shows data on the kinetics of POD enzyme inactivation at 255°C for mate leaves obtained in the present study.

Table 7. Residual POD activity of mate in function of the time of the treatment at 255°C in a conveyor oven.

Tabla 7. Actividad residual de la enzima POD de la yerba mate en función del tiempo de tratamiento a 255°C en un horno de cinta transportadora.

Time (s)	Residual activity POD ±(%)
0	100 ± 1^c
5	10 ± 4ª^,b
10	13 ± 3^b
15	7 ± 6^a,b
20	6 ± 4^a,b
22	3 ± 3^a
24	3 ± 2^a

Note: *Numbers followed by the same letter between lines are not statistically different at a significance level of 5%.

All the thermal treatments showed low values for residual POD activity. Only the treatment for 5 s showed a significant difference. However, for a deeper discussion, it is necessary to investigate the behaviour of POD enzymes during storage because they can restore their activity.

The criterion for the selection of the treatment was based on the treatments that showed residual enzyme activity below 10%, which was the value established as the most adequate in order to assure thermal inactivation of the enzymes (Provesi et al., 2010).

Evaluation of mate PPO and POD enzyme inactivation though industrial process

In this present work, the efficiency of enzyme inactivation through the traditional industrial sapeco process was evaluated. Mate leaves submitted to the sapeco process in all the factories studied showed signs of blight, which results from excessive thermal treatment.

There was no significant difference between the levels of residual activity of POD enzymes in mate after the sapeco process in all the factories studied (Table 8). The extreme levels of residual activity ranged from 6 to 13% for factories D and A, respectively. Only one mate factory showed leaves with residual enzyme activity level above 10%.

Table 8. Residual POD and PPO activity in mate leaves thermally treated through the traditional industrial sapeco process.

Tabla 8. Actividad residual de las enzimas POD y PPO en hojas de la yerba mate submetidas a lo procesado industrial del sapeco.

Industry	POD residual activity (%)	PPO residual activity (%)
A	13 ± 7^a	8 ± 4^a
B	9 ± 4^a	4 ± 1^a
C	7 ± 1^a	8 ± 2^a
D	6 ± 1^a	4 ± 1^a

Note: *Numbers followed by the same letter between lines are not statistically different at a significance level of 5%. A, B, C, D: industries of mate.

The residual PPO activity in mate leaves after thermal treatment in the traditional industrial sapeco process, where values ranged from 4 to 8%, did not show any significant differences among the factories studied this present work.

The high temperatures predominantly used in the industrial sapeco process, led residual PPO activity to range between 4 and 8%. This traditional process could be replaced by milder processes, such as one with the conditions studied in this present work.

Conclusion

The sapeco process carried out at 255°C for 20 to 24 s was sufficient to inactivate the POD enzymes in the mate to a satisfactory degree, indicating that it is possible to lower the temperatures traditionally used.

The sapeco process performed in the processing plants investigated in this study produced efficient enzyme inactivation. However, the scorching of some leaves indicates the need to modify this process, aiming to reduce the energy consumption and to avoid the production of pyrolysis compounds.

Future studies to adequate industrial equipment to mate tea processing at mild temperature using the time and temperature established in this work are necessary. Additionally, checking further effects of this mild processing on the sensorial properties and PHA quantification can offer a new opportunity to produce mate tea at safer conditions.

Acknowledgements

We thank the FINEP/SEBRAE and CNPq for their financial support and also thank Ms. Jozeane Caldartt for her contribution by providing the mate samples.