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Introduction
The applied thermal processes in fruits, such as the mango, have been to pasteurize them but also to inactivate enzymes (Lewis & Heppell, 2000; Diaz-Cruz et al., 2016).
The advantage of the enzymatic inactivation is based upon the damage, color change, viscosity changes natural processes or the separation of the solid-liquid phase. The pectin methylesterase (PME) enzyme has the capacity of separate the methoxyl C-6 of the galacturonic residues of pectin polymeric structures, causing changes in the turbidness and in the separation of phases of vegetable products (Carabalí-Miranda et al., 2009). It has been reported that the PME contains thermo-stable and thermo-capable fractions stability causing thermal stability variations in pitaya, orange, carrot and acerola juices (Castaldo et al., 1997). In this regard, it should be emphasized that the technological problems associated with PME have been related to thermostable pectin esterase of citric juices, being a determinant for the pasteurization (Versteeg et al., 1980).
Studies about the enzymatic inactivation of the PME in fruits such as Ataulfo mango are limited, and having different results about the activity of this enzyme, for example: (Ashraf et al.,1981), determined that within the senescent mango’s pulp the pectin esterase activity is high in ripeness stages and that this information allows to select the most convenient maturity stage, nonetheless Roe & Bruemmer (1981), reported that in the first ripening stage of the fruit, the enzyme increases its activity but afterwards it stabilizes and it even decreases in certain occasions, in the same manner, it has been demonstrated a major activity in the pericarp than in the mesocarp, with 39 (PME) units for each 100 g in the pericarp and 4.5 (PME) units for each 100 g in the mesorcarp (Labib et al., 1995). The studies carried out by Jamsazzadeh et al. (2015) indicate that there is an inhibition of the PME on behalf of the glycoproteins, which in this manner explains the variety of results in the literature as well as the low or null occasional activity detected of this enzyme in different fruits.
In general, for the preservation of the turbidness and texture of the mango pulps, it is necessary for the PME to be inactivated or inhibited. Several studies have been reported about the inactivation of the PME, as much in its traditional process (heat exchangers) as well as ohmic heating process.
Ohmic heating or heating by means of Joule effect, is an emerging technology in food processing, that takes advantage of the movement of ions and electrolyte to heat a food matrix when an electrical potential is applied (Castro et al., 2003). The ohmic heating has shown efficiency in different food preservation areas, from blanching processes and up to the more specific processes such as the inactivation of enzymes and microorganisms (Jakób et al., 2010).
One of the important parameters for the ohmic heating is the electric conductivity (σ [S/m]) of the food. The electric conductivity depends mainly on the temperature, voltage frequency applied, concentration of electrolyte, and solid particle size. The presence of ionic substances such as acids and salts within the food increases the electrical conductivity (Palaniappan & Sastry, 1991; Somavat et al., 2012).
In the studies reported about pasteurization and inactivation of enzymes by ohmic heating, the initial conductivity value is not taken into account, yet it is known that it is an important parameter because it determines the delivered initial power and therefore the heating rate. Hence, the objective of this research work is to determine the effect of ohmic heating parameters as initial electrical conductivity, electric potential applied and temperature in the PME thermal inactivation of Ataulfo mango.
Material and Methods
Extraction of the PME.
Pulp bagasse from ripe Ataulfo mango was obtained (17 oBrix, pH 4.1+0.2) using a juice commercial extractor (Jack Lalanne Power Juicer, USA). The pulp bagasse obtained was used for the extraction of PME. The utilized method for the extraction of the PME was reported by Vercet et al. (1999) and Labib et al. (1995) with certain modifications. The pulp bagasse of the mango was suspended in a mixture with a buffer solution of borum acetate (B-A) (0.45 mol/L boric acid, 0.1 mol/L sodium tetraborate, 0.3 mol/L sodium acetate) pH 8.3 proportion 1:3 containing 0.2 mol/L NaCI. The mixture was shaken for 4 h continuously followed by 12 hours of resting time at 4 °C.
The obtained extract was centrifuge at 3000 g and the supernatant was dyalized for 12 h against B-A diluted 1:1000, later the dialyzed product was subjected to a selective precipitation (salting out) with a saturated 30-80 % ammonia sulfate. The precipitated was re-dissolved in a 1:3 at 1:1000 B-A proportion for a later dialyzed against the solution of 1:1000 B-A. The dialyzed sample was considered as being a enzymatic raw extract containing PME, which was called E-PME.
Obtaining a thermostable fraction of PME from mango Ataulfo.
The presence of thermolabile and thermostable fractions of mango PME was reported previously by Labib et al. (1995). In order to avoid the effects of the thermolabile fraction a procedure was applied to the mango E-PME consisting of a isothermal treatment at 70 °C by traditional heating (warm water bath) according to the report by Diaz-Cruz et al. (2016). Figure 1 shows the inactivation kinetic of E-PME where the inactivation of the thermolabile and thermostable fraction were measured. The thermostable fraction of the PME (PME-T) represented 15 % of the total activity of the E-PME mango extract. The thermostable extract of PME (PME-T) was lyophilized and stored at 4 ºC. The PME-T lyophilized was re-suspended in distilled water and again dialyzed against distilled water for the inactivation kinetic, in as much by traditional treatment as well as by ohmic heating.
Determination of the PME activity.
The PME activity was quantified by tritation of the carboxyl groups liberated from the pectin using an automatic pH-Stat (Metrohm, Herisau, Switzerland).
The trials were carried out using a 10 mL of apple pectin (Sigma Aldrich, 70-75 % esterification) at a 1 % substrate, containing 0.2 mol/L NaCI, adjusting the pH of the mixture at 8.5 with 0.1 N NaOH under auto-degradation consideration of the pectin describe by Renard & Tibault, (1996), And finally adjusting the pH at 7 with 10mM HCI, immediately after adding 1.6 mL of PME raw extract for the control of the pH during the reaction time. The pH was kept at 7 by means of adding 10 mmol NaOH for a 10-15 min period at 30° C. One unit of PME activity (UPME) was defined as the quantity of enzyme capable of releasing 1 mmol of carboxyl groups / min (Balaban et al., 1991) and calculated according to Equation 1.
Thermal treatments by the traditional process TP.
Samples of 1.6 mL of E-PME were processed at different times and different temperatures (68-78 °C) in 1.7 mL glass tubes, in triplicate and placed in a water bath at a selected temperature. After such treatments, the samples were immediately cooled in a cold water bath, and the residual activity of the PME was analyzed within the first 60 min after each treatment. The heating time was recorded beginning at the time when the samples reached the desired temperature.
Treatments by ohmic heating.
The treatments by ohmic heating were performed applying three different intensities of electric field (17, 20 y 23 V/cm) in a ohmic heating device built in the laboratory. A cubic heat cell was used with a magnetic stirrer feature, in order to guarantee a homogeneous temperature in the whole volume. The temperature was monitored at different points of the cell. Once the applied treatment, the samples were immediately cooled in a cold water bath and the PME residual activity was analyzed.
Ohmic heating treatment equipment.
The Ohmic Heating device used for experiments was built in CICATA-IPN Unidad Querétaro, Mexico and is shown in Figure 2. The set up consists of an AC Variable Transformer (Variac, 220 VAC); a temperature control with heating ramp and interface to PC type RS-232 (Watlow Series 981); 2 desktop multimeters (Model 34410A, Agilent) to acquire the values of voltage and current in a PC (not shown in Figure 2); an isolated T-type thermocouple (Physitemp Intruments, model IT-18), and a magnetic stirrer plate (CORNING, model PC-320).
The ohmic heating cells used for measuring PME activity were made of acrylic cubes of 1 cm x 1.7 cm x 1 cm with 2 titanium electrodes of 1 cm x 1.7 cm and a 4 mm magnetic stirrer was used for to shake the E-PME.
Determination of the KE and D values.
The residual activity obtained from the PME resulting from each ohmic heating and traditional treatment was calculated as a percentage of enzymatic activity of the extract without thermal treatment. A first order kinetic model was used to adjust the experimental data that corresponds to the residual activity. Equation 2.
Where a and b are respectively initial and remaining activities at time t [min], kE is the inactivation rate constant [1/min] and t is the time [min].
For a better representation of the data, the decimal reduction time or D value was used. The D value is defined as the necessary time, at a given temperature, for the enzymatic activity to be reduced in a logarithmic cycle, that is to say 90 %. For the calculation of the D value equation 3 was used as follows:
Where D is the decimal reduction time [min].
Statistical analysis
The obtained data was analyzed by means of ANOVA, and the comparison of the measurements was conducted with the Tukey a p≤0.05 test. The statistical analysis was performed using Minitab 16 (Minitab Inc, 2010). All measurements were carried out by triplicate. Data were expressed as the mean ± standard deviation (SD).
Results and Discussion
Electrical conductivity of mango’s pulp having three ripening stages.
The electrical conductivity was measured as per the Ataulfo type mango’s temperature with a ripening level of L3 (14.1º Brix), L4 (15.4º Brix) and L5 (17.0º Brix) according to the Kader (2015) classification. The electrical conductivity has a lineal dependency in regards to the temperature exhibited on Figure 3. It is appreciated that the 3 ripening stages did not show any significant difference (p≤0.05) but, indeed demonstrated a tendency where the L5 corresponding electrical conductivity is higher than the ripening conductivities L4 and L3. These electric conductivities are in accordance to the results reported by Sosa-Morales et al. (2009). The conductivity values obtained from Ataulfo mango having different ripening grades were taken as a reference in order to study the effect of the initial conductivity during the ohmic heating (Table 1).
Table 1 Electrical conductivity (S*m-1) of three different ripening stages of Ataulfo mango’s pulp and electrical conductivity (S*m-1) of PME-T to different concentrations of NaCI.
| Temperature (°C) |
Electrical conductivity of different ripe states of Ataulfo mango's pulp (S*m-1) |
Electrical conductivity of PME-T with different NaCl concentration(S*m-1) |
||||||
|---|---|---|---|---|---|---|---|---|
| L3 | L4 | L5 | 0.021 | 0.028 | 0.031 | 0.035 | 0.043 | |
| 30 | 0.38 ± 0.10 | 0.37 ± 0.06 | 0.40 ± 0.08 | 0.29 ± 0.02 | 0.34 ± 0.02 | 0.37 ± 0.02 | 0.46 ± 0.03 | 0.48 ± 0.02 |
| 36 | 0.43 ± 0.11 | 0.42 ± 0.09 | 0.45 ± 0.09 | 0.32 ± 0.02 | 0.38 ± 0.02 | 0.45 ± 0.02 | 0.53 ± 0.02 | 0.57 ± 0.06 |
| 42 | 0.45 ± 0.12 | 0.47 ± 0.10 | 0.50 ± 0.10 | 0.36 ± 0.02 | 0.44 ± 0.02 | 0.52 ± 0.02 | 0.61 ± 0.02 | 0.66 ± 0.04 |
| 48 | 0.50 ± 0.12 | 0.52 ± 0.12 | 0.55 ± 0.12 | 0.40 ± 0.02 | 0.51 ± 0.02 | 0.59 ± 0.02 | 0.70 ± 0.02 | 0.75 ± 0.04 |
| 54 | 0.55 ± 0.14 | 0.57 ± 0.13 | 0.59 ± 0.12 | 0.45 ± 0.02 | 0.58 ± 0.02 | 0.67 ± 0.02 | 0.77 ± 0.02 | 0.83 ± 0.05 |
| 60 | 0.59 ± 0.14 | 0.62 ± 0.14 | 0.63 ± 0.13 | 0.49 ± 0.02 | 0.65 ± 0.02 | 0.75 ± 0.02 | 0.85 ± 0.02 | 0.94 ± 0.05 |
| 66 | 0.65 ± 0.15 | 0.67 ± 0.15 | 0.69 ± 0.15 | 0.53 ± 0.02 | 0.71 ± 0.02 | 0.84 ± 0.02 | 0.93 ± 0.02 | 1.06 ± 0.05 |
| 72 | 0.72 ± 0.15 | 0.72 ± 0.15 | 0.74 ± 0.15 | 0.56 ± 0.02 | 0.75 ± 0.02 | 0.90 ± 0.02 | 1.00 ± 0.02 | 1.12 ± 0.05 |
Electrical conductivity of the PME-T.
The initial electrical conductivity is a very important parameter when ohmic heating is used. For such reason we proceeded to investigate the effect of the initial electrical conductivity in the heating profile when a constant electric field is applied. The initial electrical conductivity of the PME-T was adjusted with NaCI with the purpose of evaluating the effect of the initial electrical conductivity in the heating profile. The initial values of the electrical conductivity are the ones shown on Table 1 with its corresponding concentration of NaCI (initial conductivity range 0.29 to 0.48 S/m).
Figure 4 shows temperature profiles being as per the time for each initial PME-T electric conductivity obtained applying a constant 20 V/cm electric field and up to 72 °C. As can be observe, the heating rate (slope) depends on the initial electrical conductivity. This means that the initial value of the electrical conductivity determine the heating rate of the sample and therefore the time to reach the temperature. As the electrical conductivity depend of the water content of soluble solids then the initial electrical conductivity is a function of ripening state as was mentioned before. Therefore, PME enzyme inactivation by ohmic heating will depend of the initial electrical conductivity. In ohmic heating the electrical power is calculated by the joule law that say that delivered power is a function of the voltage and of the conductivity (Sarang et al., 2008). So, according with the results the heating rate is influenced by the initial conductivity and therefore of the applied power. At high initial conductivity, a high power is applied and quickly is reached the temperature for processing. Now will be necessary to know the effect of the initial electrical conductivity in the residual activity.
Residual activity of the PME-T with different concentrations of the NaCI by ohmic heating and traditional process.
As follows: it was proceeded to observe the residual activity of the PME-T during the thermal treatment by ohmic heating (OH) and by the traditional process (TP) to determine the effect of the initial electric conductivity in the inactivation of the PME. A 20 V/cm electric field was applied. The results are shown on Figure 5. The treatments with ohmic heating presented a lower residual activity value of the PME than the treatments by traditional process, there being an inversely proportional relation between the initial electric conductivity and the residual activity. At a major initial electrical conductivity there were a lower residual activity. It is observed, in Figure 5, that in the traditional process (TP) samples with NaCl there were not a significant effect in the residual activity. It also can be observed that there were not an effect of the concentration of the NaCl in the inactivation of the enzyme since the residual activity was maintained constant. Therefore, we can affirm that the initial electric conductivity is an important parameter that has a significance effect in the residual activity of PME enzyme when ohmic heating is applied. This is an important result of the present investigation.
Inactivation kinetic of the PME during ohmic heating.
Once the effect of the initial electrical conductivity in the inactivation of the PME-T was determined, it is proceeded to carry out experiments to determine the effect of the electric field at a constant conductivity. The value of the initial electrical conductivity selected was 0.037 S/m at 30° C corresponding to 0.031 mM of NaCl. The inactivation kinetic of the PME-T extract were carried out for a traditional process. Figure 6 shows the PME-T inactivation kinetic by ohmic heating with a 20 V/cm electric field. The results show that there is an electric field effect on the inactivation of the PME-T extract. With a higher electric field, there is less residual activity at the same temperature. According to some authors the electric field effect in the inactivation of the PME enzyme it is due to the polarization of the molecules of the enzyme having an electrolysis effect; or the electric current through the food produces electro-pores in the cellular membrane inhibiting the enzymatic action (Camargo et al., 2010). This is an important result because show that ohmic heating process inactive PME enzyme at low temperature allowing that mango preserve its textural characteristics because had a low thermal treatment. This is very significant in a pasteurization process.
Table 2 shows the D and kE values obtained from the inactivation kinetic of the PME-T in as much as per ohmic heating as well as the traditional process. It is observed that the kE values increase according to the temperature but also according to the applied potential. In regards to the traditional process the D value at 78 °C is 20.95, but for the ohmic treatment at the same temperature the D values vary according to the applied voltage: for an electric field measuring 17 V/cm the D value at 78° was 13.34; for a 20 V/cm electric field value the D value at 78 °C was 5.73; and for a 23 V/cm field the D value at 78 °C was 3.99. As D value is the time that enzymatic activity is reduced in a logarithmic cycle, these results indicate that the enzyme inactivation by ohmic heating is carried on faster than the traditional process at the same temperature ie applying 20 V/cm it reduces almost a logarithmic cycle comparing with traditional process. It is also observed that it is possible to obtain the same D value at lower temperatures when different electric fields are applied. Such result indicates that the ohmic heating can achieve the same enzymatic inactivation as compared to a traditional process but at a lower temperature, having as an advantage a lower thermal treatment, keeping functional and textural properties (Jakób et al., 2010).
Table 2 D and kE values of the inactivation kinetic of PME.
| Treatment / Electric Field strength | Temperature (ºC.) | D Value (min) |
Constant of inactivation (ke [sec-1]) |
|---|---|---|---|
| 72 | 82.67 | 0.03 | |
| Traditional | 74 | 56.22 | 0.04 |
| 76 | 36.24 | 0.06 | |
| Process | 78 | 20.95 | 0.11 |
| 72 | 45.40 | 0.05 | |
| 74 | 31.08 | 0.07 | |
| 17 V/cm | 76 | 21.80 | 0.11 |
| 78 | 13.34 | 0.17 | |
| 72 | 38.97 | 0.06 | |
| 74 | 18.67 | 0.12 | |
| 20 V/cm | 76 | 12.29 | 0.19 |
| 78 | 5.73 | 0.40 | |
| 72 | 41.85 | 0.06 | |
| 74 | 30.36 | 0.18 | |
| 23 V/cm | 76 | 5.98 | 0.38 |
| 78 | 3.99 | 0.58 |
Because the discussion about the causes of the inactivation of the PME by ohmic heating are a subject of debate, this work contribute with two important facts: 1) the initial electrical conductivity has an effect in the inactivation of the PME-T at a constant electric field, y 2) the electric field has an inactivation effect on the PME-T at a constant conductivity. This means that the increase of charge carriers by addition of the NaCI (high initial electrical conductivity) benefits the inactivation of the PME-T by ohmic heating.
Conclusions
The present work shows that the initial electrical conductivity in a mango PME extract has an effect on the residual activity of the PME during ohmic heating. At a higher initial conductivity, there is a minor residual activity of the PME. It also demonstrates that the effect of the electric field during ohmic heating is directly proportional to the enzymatic inactivity of the extract of mango PME. At a higher electric field, the enzymatic inactivity of the PME of mango extract is higher as well. This demonstrates the synergic effect of the ohmic heating between the electrical conductivity and the electric field.
This study exhibits that the ohmic heating can achieve the same enzymatic inactivity of the PME of mango, during a traditional process, but at a lower temperature, having the advantage of a lower degradation of the texture and functional properties.
