Enzymatic inactivation of apple juice using HPCD High Pressure Carbon Dioxide technology and its effect on the quality parameters of the juice
The quality of cloudy apple juice seems to be better than the clarified juice due to the loss of phenolic compounds during processing. Enzymatic browning and cloud stability are the main problems in processing and storage of the juice. In this work, HPCD is proposed as an alternative to the traditional heat treatments that can degrade the quality of the juice. Polyphenol oxidase (PPO) and pectin methylesterase (PME) inactivation by HPCD were determined for both enzymes. The effect of stirring speed on PPO inactivation was studied; obtaining that at higher speed inactivation was also higher. Other quality parameters in the juice were also determined after HPCD treatment, such as particle size distribution, colour, turbidity and total phenolic compounds. INTRODUCTION Cloudy apple juice can be more beneficial to human health than the consumption of clear apple juice, due to its higher nutrient content, which is lost in juice processing [1] The main problem with cloudy apple juice during production and storage is the color and cloud stability. The discoloring of cloudy apple juice is a result of enzymatic browning, which involves the action of polyphenol oxidase (PPO) catalyzing oxidation of phenolic compounds. The mechanism of cloud stability in juices is not yet understood, but it is closely related to the pectin methylesterase (PME) activity. Among the different mechanisms, electrostatic repulsion by negative charges present in the partly demethylated pectin due to galacturonic residues could be responsible of cloud stability avoiding aggregation [2] This way, the activity of pectinmethylesterase (PME) causes pectin demethylation and formation of insoluble calcium pectate gels that could precipitate causing clarification and the loss of turbidity. Traditionally, thermal treatments are used to inactivate the enzymes but cause undesirable effects in the juice quality attributes such as flavor and loss of nutritional components [3] As an alternative, high pressure carbon dioxide (HPCD) has been proposed as a non thermal technique that can inactivate certain enzymes under mild operation conditions. This technology is growing attention as a cold pasteurization method through the effect of CO2 held under pressures below 50 MPa and temperatures lower than thermal treatments (below 50oC) The mechanism of pressurized CO2 on enzyme inactivation is not yet fully understood. In the literature different approaches can be found such as pH lowering due to dissolved CO2, conformational changes or formation of different complex with CO2 molecules. In this work, HPCD technology has been applied to cloudy apple juice. The influence of agitation on enzyme inactivation under high pressure environment is a key factor in the design of reactor volumes of industrial scale [4] Therefore, the effect of stirring speed on PPO inactivation was studied. Sampling procedure and the number of successive pressurization/depressurization cycles was also studied. Inactivation kinetics of PPO and PME in cloudy apple juice were determined in the pressure range from 100 to 200 bar and temperature range from 35 to 45 oC. Kinetic data have been successfully correlated by the Weibull model. Some quality parameters of cloudy apple juice, such as color, pH, oBrix, turbidity, antioxidant capacity and particle size distribution (PSD) were also determined right after HPCD treatment. MATERIALS AND METHODS Juice preparation: Golden deliciuos apples were peeled and cut in cubes and then were added to a 0.3 of L ascorbic acid solution to avoid enzymatic browning during processing. Apple cubes were wiped and immediately squeezed with a screw juice extractor and the liqueur was filtered with 2 layers of cheesecloth. The pH of the juice obtained was 3.89 0.01 and the soluble solid was 12.5 0.1 degBrix. The juice was stored frozen at 18 degC until further use. No significant change of PPO and PME activity was noticed during the storage of the frozen samples. HPCD equipment and processing: The HPCD cell was made of stainless steel (SS 116) and had an internal volume of 100 mL with a maximum operating pressure and temperature of 30 MPa and 80oC. For each experiment, approximately 40 mL of apple juice was charged into the high pressure cell and it was immersed in a thermostatic water bath at the operating temperature. Magnetic stirrer was connected. The system was then pressurized by using a syringe pump with a pressure controller (ISCO 260 D) up to the desired pressure. Samples were collected periodically at different treatment times up to 120 minutes to follow the inactivation kinetics of the enzyme. Experiments were carried out in a temperature (T) range from 35 to 45 oC, pressure (p) from 100 to 200 bar and stirrer speed from 200 to 600 rpm. The effect of sampling procedure and the number of depressurization cycles on enzyme activity was also analyzed. To compare the effect of HPCD treatment on enzyme activity under mild heating treatment, cloudy apple juice was subjected in the same temperature range (from 35 to 45oC) but at atmospheric pressure. Determination of PPO activity: Activity of PPO was determined spectrophotometrically by using a 0.05 M catechol solution prepared in a 0.1 M phosphate buffer (pH 6,5) as substrate. Oxidation of catechol was determined by the increase in absorbance at 420 nm at 30 oC during 3 minutes of reaction after mixing 100 mL of juice with 2900 mL of the catechol solution. A Jasco V 750 Spectrophotometer was used. PPO activity was taken as the very first linear part of the reaction curve. Determination of PME activity: PME activity was determined by using an automatic titrator system (Metrohm Titrando) by using a 1 of pectin solution (Alfa Aesar pectin citrus) prepared in NaCl 0.3 M as substrate. 50 mL of pectin solution was mixed with 1 mL of cloudy apple juice and pH was adjusted to 7.5 with NaOH 0.02 N. During pectin hydrolysis at 30oC, pH was maintained at 7.5 by adding NaOH 0.02 N. The amount of NaOH added for 15 minutes was recorded. One PME activity unit (UPE) is defined as the micromoles of carboxylic groups produced per minute and mL of juice at pH 7.5 and 30 oC [3] Physico chemical analysis: Some other important quality attributes of cloudy apple juice were also determined before and after HPCD treatment. Particle size distribution was determined by laser diffraction with a Mastersizer 2000 (Malvern(r) Inst. MA) Total phenolic compounds were determined by using the Folin Ciocalteau reagent, and antioxidant capacity was measured by the ABTS method. Turbidity was measured with a turbidimeter (Eutech Instruments, TN 100) and expressed as nephelometric turbidity units (NTU) pH of apple juice was determined with a pH meter (Crison pH Ion Meter GLP 22) Finally, color was measured using a colorimeter an L* a* and b* values were obtained representing brightness, red to green color and yellow to blue color, respectively. Kynetic data analysis: In this work, the inactivation kinetic data at different operating temperatures and pressures were correlated by the Weibull model. llllll AA AAoo bbttnn [1] where b and n are the scale and shape parameters, respectively. ZT or ZP (temperature or pressure increase needed for a 90% reduction of the D value was evaluated as the negative reciprocal slope of the regression of log D as function of T or p respectively: llllll DD1 DD2 TT2 TT1 ZZTT [2] llll DD lnDDaaaaaa 1 ZZpp [pp ppaaaaaa] [3] Statistical analysis: Analyses were conducted using software Statgraphics X64. The results are presented as a mean standard deviation of at least three replicates. The significance of the differences was determined based on an analysis of the variance with the Fisher's least significant (LSD) method at p value 0.05. RESULTS Stirring speed: The effect of agitation on the inactivation of PPO from cloudy apple juice was evaluated at 20 MPa at three different stirring speeds, 200, 400 and 600 rpm (Figure 1) When working at higher stirrer speeds, the magnetic stirrer did not work properly in our experimental equipment; therefore stirrer speeds higher than 600 rpm are not included. At any of the stirring speeds, the inactivation degree increased with increasing time. From Figure 1, it can be also observed that an increase in the stirrer speed led to a higher inactivation degree. Figure 1. Effect of stirrer speed on PPO inactivation from cloudy apple juice by HPCD at 20 MPa and 45oC 200 rpm, D 400 rpm, #600 rpm) Sampling procedure: PPO activity after HPCD treatment was compared when sample was obtained after system depressurization or removed while the system was under pressure for one single cycle pressurization. HPCD treatment was carried out at 45oC and 20 MPa at two different treatment times, 15 and 30 min. No significant differences have been determined among sample means of different sampling procedure when applying the Fisher's least significant (LSD) method at p value 0.05. The effect of a single cycle has been compared with a triple cycle for 15 and 30 minutes of total treatment time, that is 5 and 10 min of pressure treatment each cycle, respectively. For PPO inactivation same effectiveness for a single pressurization cycle as for a three cycle pressurization can be determined when applying the Fisher's least significant (LSD) method at p value 0.05. Inactivation kinetics for PPO and PME Inactivation kinetics for PPO of cloudy apple juice have been determined at 20 MPa in the temperature range from 35 to 45oC. By increasing temperature, enzyme inactivation rate also increased (Figure 2a) Figure 2b shows the inactivation kinetic at 45 oC in the pressure range from 10 MPa to 20 MPa. Higher operating pressure led to faster inactivation rates, but the fraction of the resistant form of the enzyme is nearly independent of the operating pressure, being around 6 Inactivation kinetic data by HPCD were correlated by the Weibull model. D values were calculated and