Ohmic Treatment of Pear Purées ( cv . ‘ Conference ’ ) in Terms of Some Quality Related Attributes

The effect of ohmic treatment on some quality related characteristics of pear purée (cv. ‘Conference’) such as color, reducing sugars, total phenols, rheological behavior and microbial counts, was analyzed. The inactivation kinetics of pectin methyl esterase (PME) in pear crude extract and purée were studied by conventional thermal and ohmic treatments. Thermal inactivation of PME in crude extract was described by a first-order kinetic model. The activation energy values suggested the presence of two isoenzymes with different thermostability. The ohmic heating reduced PME activity by 96% at 25 V·cm. Minimal changes induced by ohmic heating on above quality related aspects were observed. Supporting this statement, there were no significant changes in the nutritional and sensorial attributes. It was reported an increase of 3% of reducing sugar content for the ohmic heated samples. The phenolic content of the treated samples registered a reduction of 59% in comparison with fresh pear purée. The pear purée presented a non-Newtonian pseudoplastic behaviour. The Ostwald de Waele model was fitted to rheograms and the consistency coefficient (m) and flow behavior index (n) were determined. Results obtained for the microbial charge were higher in the control samples. Thus, microbial counts showed complete inactivation of yeast and mold at voltage gradient higher than 17.5 V·cm.


Introduction
During processing of plant-based food products the aim is to guarantee food safety and quality traits among which texture forms one of the most prominent quality attributes.Therefore, texture is an important quality factor in both fresh and processed fruits.During ripening, structural changes in the middle lamella and primary cell walls occur, leading to the cell separation and softening of tissue (Barrett et al., 1994).Several enzymes are responsible for these structural changes, such as polygalacturonase, pectin methyl esterase, β-galactosidase, cellulose etc.
Pectin methylesterase (EC 3.1.1.11)(PME) is an enzyme that acts mainly in the hydrolysis of methyl ester groups in pectin chains to form carboxylate groups, releasing methanol and H3O + (Jayani et al., 2005).Pectin greatly influences the textural properties of the plant cell wall and thus the textural properties of plant-based food products (Vandevenne, 2011).PME inactivation should be achieved without compromising other quality or safety aspects (Hendrix et al., 1995).Current inactivation of PME is accomplished by heat (Chen et al., 1993) however, conventional thermal treatments cause adverse effects such as color alterations, flavor changes, bioactive compounds loss and nutritional value reduction (Giner et al., 2001).Thus, inactivation of PME by alternative methods becomes a matter of interest, to avoid quality degradation of fruits or vegetables based products by thermal processing (Elez-Martínez et al., 2007).
Ohmic treatment of food products involves the passage of alternating current, thus generating internal heat as a result of electrical resistance (Reznick, 1996).The amount of heat generated inside of food is directly related to the current induced by the voltage gradient in the field and the electrical conductivity (Sastry et al., 1996).The main advantage of this technology consists in rapid and uniform heating, resulting in less thermal damage to the product (Sastry et al., 2000).In addition, the absence of a hot surface reduces fouling problems.Although the technology of ohmic heating appears to be promising and highly effective, there is little information concerning the effects of this technique on specific food products compared to conventional pasteurization.
The objectives of this study was to evaluate the influence of ohmic treatment on pear purée in relation with some quality characteristics such as color, reducing sugars, total phenols, rheological behavior and microbial counts.As the development of an adequate ohmic processing requires information about the behavior and kinetic data of enzymes in order to achieve enzymatically stable products without over processing, inactivation kinetics of PME in crude extract and pear purée were studied by conventional thermal and respectively ohmic treatments.

Materials
Pear fruit of Conference variety were purchased at commercial maturity during the year 2012 by a local producer (Galati, Romania).Apple pectin (75% degree of esterification) was obtained from Sigma-Aldrich Chemie GmbH Germany.All chemicals were of analytical grade.

PME extraction
The extraction of PME was performed according to the method of Ly-Nguyen et al. (2002).A portion of 1.0 kg of pear was homogenized with the addition of 500 mL of distilled water.The suspension was filtered using cheesecloth.The supernatant was discarded and the pellet was washed twice with 500 mL distillated water to remove other organic and color compounds.Then the pellet was mixed overnight in a 0.2 M Tris (hydroxylmethyl-amino-methane) buffer (Tris buffer) containing 1 M NaCl, pH 8.0 (1:1 w/v).After extraction, the suspension was filtered using cheesecloth and the pellet was discarded.As a result, the pear salt PME extract was obtained.Salt PME extracts were partially purified by ammonium sulphate precipitation at 30% saturation.After stirring for 30 min and centrifugation at 18,000 g for 15 min, the pellets were discarded.The supernatants were precipitated again by ammonium sulphate up to 80% saturation for 30 min.The precipitates containing PME were collected by centrifugation at 18,000 g for 15 min.These precipitates were dissolved in 20 mM citrate buffer (pH 7.0), using 5 mL of buffer per 100 g of fresh material, to obtain the crude extracts.

Pectin methylesterase activity
Pectin methylesterase (PME) activity was determined by a pH stat titration (Titrino, Metrohm, Switzerland) of the acid produced with a mixture consisting of 500 µL of crude enzyme extract in 20 mM citrate buffer for the thermal inactivation experiments and 5 g of un-treated samples for the ohmic treatment studies, 30 mL of 3.5 mg•mL -1 apple pectin solution containing 0.117 M NaCl at pH 7.0 and 25 °C.The PME activity unit is defined as the amount of enzyme required to release 1 µmol of acid per minute, under the above mentioned assay conditions.

Thermal inactivation kinetics
Kinetic parameter values for thermal inactivation of PME were determined on the basis of isothermal inactivation experiments and determination of the remaining activity of heattreated samples.The experiments of PME were conducted using the test tube method.Aliquots (0.200 mL) of enzyme solutions were placed in test tubes and immersed in a water bath (Digibath-2 BAD 4, RaypaTrade, Barcelona, Spain) at temperatures ranging between 50 °C and 80 °C for different holding times (0-35 min).After thermal treatment, the test tubes were immediately immersed in ice water to allow rapid cooling.

Purées preparation
The fruit were peeled and the seeds were removed.The purées (1.0 kg of fruit) were prepared by grinding the pulp with a blender.Fresh fruit purées were immediately analyzed.Three experimental samples were obtained for un-treated and ohmic treated purée samples.The pH was controlled with a Mettler Toledo pH meter.The physico-chemical values of fresh pear purée were pH 3.7, the titrable acidity at 9.0 g malic acid per 1,000 g and dry matter value 11.3%.

Ohmic treatment of fruit purées
Discontinuous ohmic treatment equipment, consisting of a power supply of 10 kW, an ohmic heating cell (a thermoresistant tank, two stainless steel electrodes of 5 mm diameter placed at an equal distance of 10 cm) was used.Temperatures were monitored using a thermocouple, placed at the geometrical centre of the cell.Voltage and current intensity were measured with a voltmeter, respectively an ampermeter.For PME inactivation kinetics, the samples were treated using an alternative current source of 50 Hz, with voltage gradients ranging from 15 V•cm -1 to 25 V•cm -1 .For each value of the voltage gradients, the temperature was maintained for 3 minutes by covering the sample with aluminum foil, while the temperature of the samples was measured with a thermocouple.The changes in quality related parameters were measured by using a voltage gradient of 25 V•cm -1 for 3 minutes.

Color analysis
Pear purée color was measured using a tristimulus colorimeter (Model AA200, Zetalab, Milano, Italy).Samples of 10 g were placed in a cell and measured for L, a, and b values.An increasing L value represented an increase in lightness (L = 0, dark; L = 100, light).An increase in a value indicated an increase in redness (-a = green, +a = red).Three measurements were taken on each sample and values were averaged.Browning index and color changes (∆E) were calculated as described by Hutchings (1994).

Reducing sugar analysis
The concentration of reducing sugar was determined by using 3, 5-dinitrosalycilic acid (DNS) reagent with small changes (Miller, 1959).The filtrate was centrifuged at 4,500 rpm for 10 minutes and the supernatant was collected.DNS reagent was prepared by mixing 40 mL water with 1 g of DNS and 2 mL of 50% NaOH.30 g Rochelle salt (potassium sodium tartrate) was added to the mixture.The reducing sugar was measured as follows: 3 mL of DNS reagent was added to 1 mL sample, the mixture was boiled for 5 min, cooled and the reduced sugar concentration was measured at 540 nm wavelength using a Lambda 25 UV/Vis Spectrometer (Perkin Elmer, Beaconsfield, UK) with 1 cm glass cells.Calibration standard solutions with concentrations in the range of 0.2 -1.2 mg mL -1 were prepared by diluting the stock solution of glucose with water.

Total phenolic content
The total phenolic content was measured using a modified colorimetric Folin-Ciocalteu method (Budini et al., 1980).Briefly, a volume of 2.5 mL of deionized water and 100 mL of a known dilution of the sample were added to a 10 mL volumetric flask.Folin-Ciocalteu reagent (0.5 mL) was added to the solution and allowed to react for 5 min.Then, 1.5 mL aliquots of 20% sodium carbonate solution were added and the mixture was made up to 10 mL with deionized water.The color developed over 120 min, and the absorbance was read at 765 nm using a Hewlett-Packard spectrophotometer HP 8452A (Cheadle Heath, Stockport Cheshire, UK).The measurements were compared to a standard curve of gallic acid and expressed as mg gallic acid equivalent 100 g -1 fresh matter (mg GAE 100 g -1 FM).

Rheological measurements
The rheological measurements were made with the rotational viscometer Brookfield DV-E equipped with a LV2 (Liquid viscosity) (Brookfield Viscometers Ltd, Harlow, UK).The apparent viscosity, η, is defined as the ratio of shear stress, τ, to the shear rate, γ, (η = τ / γ).The LV2 spindle characteristics were: diameter 18.72 mm and height 115 mm.
The shear rate (γ, s -1 ) was calculated using the eq.1: where: 0.212 -is a spindle constant and N is the rotor speed in rot•min -1 .
The temperature of this system was kept constant at 21 °C through a liquid thermal circulator.In order to investigate the reproducibility of the results, three experimental runs were accomplished for each product, and the resulting shear stress values were averaged.

Microbial counts
Microbial counts followed the standard ISO 7218-1996 "Microbiology of food and animal feeding stuffs general rules for microbiological examinations" methods.
Kinetic data analysis PME inactivation was described by the first-order kinetic model (Eq.2): where A 0 and A t are the initial activity and the remaining activity at time t, respectively.
The relation presented in eq. 2 is valid under isothermal condition, thereby the inactivation rate constant, k, can be determined from a linear regression of ln (At/A 0 ) versus heating time.
The temperature dependence of the inactivation rate constants was estimated using the Arrhenius model (Eq.3): where T is the absolute temperature (K), k 0 is the rate constant at reference temperature (T0), E a is the activation energy (J•mol -1 ) and R is the universal gas constant (8.314J•mol -1 •K -1 ).The activation energy and standard deviation values were estimated using linear regression on Eq. (3).

Statistical analysis
All experiments were performed in triplicates, and the results were expressed as the average of resulting data.Statistical and data analysis were conducted with data analysis tool pack of the Microsoft Excel software.

Results and discussions
Thermal inactivation kinetics of crude pear PME extract Isothermal inactivation of crude PME extract dissolved in 20 mM citrate buffer (pH 7.0) could be accurately described by a first order model in a temperature range of 50 -80 °C (Fig. 1).
Inactivation rate constant (k) and decimal reduction time (D) values, estimated using linear regression analysis of ln (A/A0) versus t, were reported in Table 1.As expected, the inactivation rate constant increased and D-values decreased with increasing temperature.The k and D values at 65 °C were 0.027 ± 0.004 min -1 and 84.98 ± 3.30 min, whereas the corresponding values estimated at 80 °C were 0.28 ± 0.19 min -1 and 8.25 ± 1.09 min.The obtained data suggested that pear PME is significantly heat stable when compared with the corresponding enzyme from other fruits and vegetables.The inactivation rate constants for purified PME from Navel oranges at 65 °C were 0.889, 1.536, 0.288 and 0.234 min -1 in deionized water and citric buffer of pH 3.2, 3.7 and 4.2 (Van Den Broeck et al., 1999).Ly-Nguyen et al. (1999) suggested an inactivation rate constant value of 0.009  The temperature dependence of inactivation rate constants in the temperature range studied could be adequately described by the Arrhenius equation (Fig. 2).The activation energy was 79.93 ± 7.13 and 158.06 ± 14.99 kJ•mol -1 in the temperature range of 50-65 °C and respectively 65-80 °C, suggesting the presence of two isoenzymes with different thermostability.These values were significantly lower than values reported in the literature: 289.2 kJ•mol -1 for carrots PME, 257.9 kJ•mol -1 for papaya pulp PME (Massaguer et al., 1994), 379.4 kJ•mol -1 for banana PME (Ly-Nguyen et al., 2002), 301.4-350.5 kJ•mol -1 for commercial orange PME (Van Den Broeck et al., 1999).Moreover, Van Den Broeck et al. (1999) suggested E a values ranging from 404.9 to 292.6 kJ•mol -1 for orange PME at pH ranging from 3.2 to 4.2.The current results indicated that the pear PME is more temperature stable and the k values less sensitive to thermal treatment.
Ohmic inactivation kinetics of PME from pear purée PME inactivation by ohmic treatment was performed at different voltage intensity, ranging from 15 V•cm -1 to 25 V•cm -1 .The time required for pear purée samples to reach the desired temperature at different voltage intensities are shown in Table 2.It can be seen that the coming-up time increases with temperature and decreases with increasing intensity voltage.Therefore, the time required to heat the pear purée from 50 to 70 °C at 15 V•cm -1 was 3 and 5.5 times longer than at 22.5 and 25 V•cm -1 , respectively.The activity values were expressed as relative to the enzyme activity in the raw sample (A0).After a treatment at 15 V•cm -1 and 50 °C, approximately 84% of the initial activity was retained, whereas only 4.8% of the activity remained after applying a treatment at 25 V•cm -1 and 70 °C.Leizerson et al. (2005) reported also a reduction of 90-98% in pectin esterase activity in orange juices during ohmic treatment.The relative activity of PME after ohmic treatments was presented in Fig. 3.It can be seen that the impact of ohmic treatment on PME activity increased by applying higher temperatures.Giner et al. (2005) suggested that any increase of electric field would yield considerable reduction in pectin esterase activity.
The thermal stability of PME in pear purée showed differences to the heat treatment on crude extract, probably due to the different behavior at various pH and medium composition.Therefore, PME was more sensitive to ohmic treatment at acidic pH, achieving a maximum inactivation of approximately 96% at 25 V•cm -1 and 70 °C, whereas a heat treatment at 70 °C for 3 minutes at neutral pH caused a 27% decrease in enzyme activity (Fig. 4).
An increase in temperature due to the ohmic treatment caused a darkening of the pear purée, which was reflected in the degree of lightness.This means that fresh pear purée was more susceptible to enzymatic browning than ohmic treated samples.When ohmic treatment was applied, a* values decreased, whereas b* values increased, indicating a loss in "greenness" and an increase in "yellowness".The corresponding value of color difference (∆E) was calculated at 3.27 ± 0.25.(Moreno et al., 2013) and suggested also a small difference in ∆E for the fresh apple (cv.Granny Smith) during ohmic heating/osmotic dehydration treatments.
As expected, ohmic-treated pear purée browns during processing compared to fresh purée.The BI increased from 55.05% in fresh purée to 59.42% in the treated samples.The decrease in lightness of ohmic-heated purée correlated with the increase in their browning.Leizerson et al. (2005) also reported an increase in browning index and a decrease in lightness in orange juices during ohmic treatment.
Hexoses (fructose and glucose) are directly involved in browning reactions as well as sucrose, which can hydrolyze into glucose and fructose during thermal treatments.The ohmic treatment did not have a significant influence on the Table 2. Color measurements (CIELAB coordinates) of the pear purées before and after ohmic treatment concentration of reducing sugars and the variation among data was within the standard error (4.54 ± 1.36 % in fresh purée and 4.68 ± 0.32 % in ohmic treated samples).However, the steady concentration of sugars during thermal treatment showed that they did not react with amino-acids and consequently did not influence browning.The 3% increase in reducing sugar content may be due to water evaporation at high temperature.Damasceno et al. (2008) suggested a thermally-induced reduction less than 4% compared to the initial sugar concentration for cashew apple juice.

Total phenolic content
The initial phenolic content of fresh pear was 398 ± 28 mg GAE 100 g -1 FM.A reduction of 59% in comparison with fresh pear purée in the total phenolic content was observed in treated samples (233 ± 58 mg GAE 100 g -1 FM).Mrad et al. (2012) suggested a total phenolic content of 254.90 ± 9.10 mg GAE 100 g -1 DM in fresh pear, whereas a significant decrease was observed by these authors during drying at 70 °C.The observed variations are related to the chemical and enzymatic oxidation reactions which occur during purée preparation and heating.
In pear and other fruit, phenolics are relatively more concentrated in the skin compared to the flesh.Damages of the membrane ultrastructure caused by blending permit rapid oxidation of the phenolics compounds.
The content in anthocyanin and other polyphenolics compounds may vary due to many others factors than just heat treatment or polyphenoloxidase activity, including the presence of air.Also, it has been suggested that the decrease in phenolic content can also be attributed to the binding of polyphenols with other compounds (such as proteins) or to the alterations in the chemical structure of polyphenols (Martín-Cabrejas et al., 2009;Qu et al., 2010).

The rheological behavior of pear purées
The importance of rheological properties of fruits and vegetables are given by the human perception of product quality, the causes and extent of damage during harvesting, transport and storage and the physiological changes, being important during growth, maturation, ripening and storage after harvest (Rao et al., 1992).
In order to evaluate the rheological behavior of pear purées, the shear stress (τ) and apparent viscosity (η) against shear rate (γ) were depicted as seen in Fig. 5. From these figures it can be evidenced that the variation was exponential and therefore the purée were non-Newtonian pseudo-plastic fluids.The shear stress increased with the shear rate (Fig. 5).This behavior could be explained by the structural breakdown of the molecules due to the hydrodynamic forces generated and the increased alignment of the constituent molecules (Izidoro et al., 2008).
The minimum values for the shear stress (0.73 Pa for the raw pear purée and 0.89 Pa for the ohmic heated one) were corresponding to the shear rate values of 0.035 s -1 .When shear rate values increased from 0.035 to 0.353 s -1 , the dynamic viscosities decreased from 21.1 to 7.9 Pa•s for the fresh purée and from 25.5 Pa•s to 3.68 Pa•s for the ohmic treated one.These differences in dynamic viscosities values were probably due to the water evaporation during thermal treatment.
Fitting the Power law model to the flow curves resulted in n values less than one (Fig. 5) which indicated pseudo-plastic flow behavior.Considering the n values (0.53 for fresh purée and 0.17 for the treated one), it seemed that ohmic treatment significantly altered the size of particles.Many solutions exhibit the value of n in the range of 0.3-0.7 depending upon the concentration and molecular weight of the polymers.Smaller values of n (0.1-0.15) are characteristic for finer particle suspensions (Chhabra et al., 2008).It has been reported that fruit purées behave as non-Newtonian fluid as a result of complex interactions among soluble sugars, pectic substances and suspended solids (Ahmed et al., 2004).The current results were in good agreement with data from literature, which enclose the fruit purées in pseudo-plastic fluids category (Balestra et al., 2011;Steffe, 1996).

Microflora
Table 3 summarizes the molds and yeasts content in control and treated pears purée after processing and during one week of storage at 4 °C.It can be seen that the amount of CFUs in both cases was higher in the control, whereas the ohmic treatment at voltage intensities higher than 17.5 V•cm -1 caused a complete inactivation of microorganisms.In the case of storage, the amount of CFUs decreased in positive samples with 1 log during storage.
236  Changes in pectin methylesterase activity during conventional thermal treatment of crude extract were described by first order kinetics, with activation energy of 79.93 ± 7.13 and 158.06 ± 14.99 kJ•mol -1 in the temperature range of 50-65 °C and respectively 65-80 °C.In pear purée, PME was more sensitive to ohmic treatment, achieving a maximum inactivation of approximately 96% at 25 V•cm -1 and 70 °C.No significant changes were observed in nutritional (total polyphenols, reducing sugars), sensorial (color) and rheological attributes.The rheological experiments evidenced that pear purées had a complex non-Newtonian pseudo-plastic behavior.Furthermore, the microbial counts was zero in ohmic treated purées at voltage intensities higher that 17.5 V•cm -1 , whereas microbial counts decreased with 1 log during one week of storage at 4 °C for the positive samples.Based on the results obtained, ohmic treatment can be used in food processing and bioengineering for the inactivation of microorganisms and quality-degrading enzymes, as well as the retention of health-related compounds and the extension of shelflife.

Fig. 3 .Fig. 4 .
Fig. 3.The relative activity of PME crude extract after ohmic treatment at different temperatures and voltage gradient

Fig. 5 .
Fig. 5. Variations of the rheological parameters for the fresh pear purée (black) and ohmic treated (grey) pear purée at 25 V/cm; a) Shear stress dependence on shear rate; b) Dynamic viscosity dependence on shear rate

Table 1 .
Estimated kinetic parameters (D-values and z, rate constant k and activation energy Ea) of PME inactivation in 0.01 Tris buffer at pH 7.0

Table 3 .
Number of yeasts and molds per gram sample pear purée