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Thermosonication as a potential quality enhancement technique of apple juice
Ultrasonics Sonochemistry 21 (2014) 984–990
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Thermosonication as a potential quality enhancement technique of apple juice Muhammad Abid a,b, Saqib Jabbar a, Bing Hu a, Malik Muhammad Hashim a,c, Tao Wu a, Shicheng Lei a, Muhammad Ammar Khan a, Xiaoxiong Zeng a,⇑ a b c
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan Department of Food Science and Technology, Gomal University, Dera Ismail Khan, Pakistan
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 3 December 2013 Accepted 6 December 2013 Available online 13 December 2013 Keywords: Apple juice Thermosonication Quality Enzymes Microbes
a b s t r a c t Enzymatic browning and microbial growth lead to quality losses in apple products. In the present study, fresh apple juice was thermosonicated using ultrasound in-bath (25 kHz, 30 min, 0.06 W cm3) and ultrasound with-probe sonicator (20 kHz, 5 and 10 min, 0.30 W cm3) at 20, 40 and 60 °C for inactivation of enzymes (polyphenolase, peroxidase and pectinmethylesterase) and microflora (total plate count, yeast and mold). Additionally, ascorbic acid, total phenolics, flavonoids, flavonols, pH, titratable acidity, °Brix and color values influenced by thermosonication were investigated. The highest inactivation of enzymes was obtained in ultrasound with-probe at 60 °C for 10 min, and the microbial population was completely inactivated at 60 °C. The retention of ascorbic acid, total phenolics, flavonoids and flavonols were significantly higher in ultrasound with-probe than ultrasound in-bath at 60 °C. These results indicated the usefulness of thermosonication for apple juice processing at low temperature, for enhanced inactivation of enzymes and microorganisms. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Apple is one of the most valuable fruits with a global production estimate of about 75.64 million tons per annum [1]. Being an important part of a healthy human diet, it contributes substantial energy, antioxidants, vitamins, minerals and dietary fiber. In addition to fresh consumption, it is processed into a variety of products such as juice, jam and jelly. There has been growing interest of consuming apple juice owing to its specific pleasant taste and high nutritional value. However, quality defects arising during the processing and storage of apple juice, if not checked, lower its scope and market value. Discoloration of apple juice due to enzymatic browning is the major problem that deteriorates its quality. Polyphenoloxidase (PPO) and peroxidase (POD) are the major enzymes responsible for browning, especially, the former oxidizes phenolic compounds to o-quinones that further polymerize and ultimately produce undesirable brown color and off-flavored product [2–5]. Since quality is of utmost importance for the consumers, it is desirable to inactivate these deteriorative enzymes during processing and storage of apple juice.
⇑ Corresponding author. Fax: +86 25 84396791. E-mail address: [email protected] (X. Zeng). 1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2013.12.003
Processing methods affect the quality, safety and shelf life of food products. Thermal processing ensures the food safety and improves the shelf life but may also adversely affect the nutritional quality of the foods [6]. Alternatively, researchers are looking for the most suitable innovative techniques which could get results with the application of little or no heat [7]. The application of power ultrasound in the food industry has become an innovative and attractive tool [8]. Improved homogeneity, minimal loss of flavor and significant energy saving are the merits of sonoprocessing over thermal treatment [9]. It has been regarded as a potential technique to meet the criteria of the Food and Drug Administration, concerning a 5 log reduction of microbial cells in the fruit juices [10]. We have recently reported that the quality of apple juice treated with ultrasound at low temperature has been improved [10,11]. Thermosonication has been attempted as an alternative to thermal treatment for processing of fruit juices such as strawberry juice, blackberry juice and orange juice [12–14]. Improvement in the quality of tomato juice, watercress and yoghurt gels by thermosonication has been reported in the literature [8,15,16]. The inactivation of POD and retention of color and vitamin C were successfully achieved in watercress and guava using thermosonication [17–19]. However, the effects of thermosonication on PPO, POD, pectinmethylesterase (PME), microbial cells, total phenols, flavonoids, flavonols, ascorbic acid, pH, titratable acidity, Brix and color of apple juice have not been studied
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to date. Thus, the clear objective of the present study was to investigate thermosonication (ultrasound combined with mild heat treatment) as a potential quality enhancement technique of apple juice. 2. Materials and methods 2.1. Chemicals Gallic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin Ciocalteu reagent was purchased from Fluka (Buchs, Switzerland). Catechin was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Quercetin was purchased from Kayon Biological Technology Co., Ltd. (Shanghai, China). HPLC grade methanol was purchased from Hanbon Science and Technology (Nanjing, China). Sodium nitrite, sodium carbonate, sodium hydroxide, sodium chloride (NaCl), pectin, catechol, tartaric acid, molten agar and potato dextrose agar (PDA), aluminum trichloride (AlCl3), ascorbic acid, diabasic potassium phosphate (K2HPO4), monobasic potassium phosphate (KH2PO4), hydrogen peroxide (H2O2) and pyrogallol were obtained from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. 2.2. Apple juice preparation Fresh apples (Malus domestica) of variety Fuji were procured from a local fruit market of Nanjing, China. The apples were washed with tap water, dried with paper towels and cut into four pieces. The stems, seeds and overripe portions were discarded. Juice extractor of MJ-M176P (Panasonic Manufacturing Berhad, Malaysia) was used to extract the juice which was then filtered through sterilized double layered muslin cloth. The freshly extracted juice was vortex mixed and selected as the control. It was put in sterilized and air tight media bottle of 100 mL which was wrapped by aluminum foils and stored at 4 °C for 48 h until further analysis.
bath type sonicator was 0.06 W cm3. In case of ultrasound treatments with probe, apple juice sample was taken in a jacketed vessel through which water was circulated. Ultrasonic processor of 750 W (VC 750, Sonics and Materials Inc., Newtown, CT, USA), with 0.5 inch probe operating at 20 kHz frequency was used for sonication. The ultrasonic density of the probe type sonicator was 0.30 W cm3. The schematic diagram of the exposure system has been presented elsewhere [20]. Samples were treated at 20, 40 and 60 °C by radiating 70% of power (750 W) for 5 and 10 min (USP20-5, USP20-10, USP40-5, USP40-10, USP60-5 and USP60-10) keeping pulse durations of 5 s on and 5 s off. The depth of the probe was kept 25 mm in the juice samples. Overheating of the samples (temperature controlled by thermocouple ±0.5 °C) during the ultrasound treatment was prevented by circulating ice-water through the treatment chamber. Sonication of samples was started when the set temperature was reached. The observed rise in temperature of the samples due to sonication was 2–5 °C. All the treatments were carried out in triplicate. 2.4. Determination of POD residual activity POD activity was determined using pyrogallol as a substrate according to the method described by Kwak et al. [21]. The sample was centrifuged at 10,000g, 4 °C for 10 min with Avanti J-E Centrifuge (Beckman Coulter, Inc., USA). The total volume of the reaction mixture was 3.0 mL which contained 2.2 mL sample, 0.32 mL of 100 mM (w/v) potassium-phosphate buffer (pH 6), 0.32 mL of 5% (w/v) pyrogallol and 0.16 mL of 0.147 M (v/v) H2O2. The reaction was initiated by the addition of H2O2 and the increase in absorbance was recorded using a spectrophotometer (LabTech Bluestar-A UV spectrophotometer) at 420 nm in 3 min. Following equation was used to calculate the percent residual activity of POD:
Residual activityð%Þ ¼ 100 At =A0
ð1Þ
where At is the enzyme activity of treated sample and A0 is the enzyme activity of the control sample.
2.3. Ultrasound treatment 2.5. Determination of PPO residual activity Ultrasound treatments of apple juice were performed by putting 80 mL of sample in 100 mL jacketed vessel. As shown in Table 1, two types of ultrasonic treatments were carried out: in-bath (a) and with ultrasonic probe (b). In case of a, jacketed vessel containing apple juice sample was put in an ultrasonic bath cleaner of SB-500 DTY (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) working at 25 kHz frequency, radiating 70% of power (500 W) and the samples were ultrasound treated at 20, 40 and 60 °C for 30 min (USB20-30, USB40-30 and USB60-30). The ultrasonic density of the
PPO activity was assessed by the method reported by Augustin et al. [22]. The sample was centrifuged (10,000g) for 10 min at 4 °C. The total volume of the reaction mixture was 5.0 mL which contained 1.5 ml of sample, 0.5 mL of 0.05 M (w/v) catechol and 0.2 M (w/v) potassium phosphate buffer (pH 6.8). Increase in absorbance at 410 nm was recorded after every 60 s for 10 min, using a spectrophotometer. The percent residual activity of PPO was calculated by applying Eq.(1).
Table 1 Processing conditions used during ultrasound (in-bath and with-probe) treatments. Conditions
Samples
Temperature (°C)
Time (min)
Power (W)
US density (W cm 3)a
Control
Fresh
–
–
–
–
US (in-bath)
USB20-30 USB40-30 USB60-30
20 40 60
30 30 30
500 500 500
0.06
US (with-probe)
USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
20 20 40 40 60 60
5 10 5 10 5 10
750 750 750 750 750 750
0.30
The treatments USB and USP represent ultrasound in-bath and ultrasound with-probe, respectively. a Determined according to Jambrak et al. [55].
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2.6. Determination of PME residual activity PME activity was determined by measuring free carboxyl groups produced as a result of action of enzyme on pectin. The reaction mixture contained 10.0 mL of centrifuged juice sample (10,000g for 10 min at 4 °C) and 40.0 mL of 1% (w/v) pectin solution in 0.15 M (w/v) NaCl solution. The pH of the mixture was maintained to 7.7 by adding 100 lL of 0.05 mol L1 (w/v) NaOH and the time required to reach pH 7.7 was recorded (50 ± 2 °C). The unit of PME is defined as the amount of enzyme that released 1 mmol of carboxyl groups in 1 min [23]. The percent residual activity of PME was calculated by using Eq.(1). 2.7. Microbiological analysis The FDA’s standard method of Bacteriological Analytical Manual [24] was used to determine the microbial population of juice samples. Pour plate method was used to determine the total plate counts. Proper serial dilutions were prepared by mixing sterilized distilled water followed by further decimal dilutions of samples (up to 105). These dilutions were poured into sterile petri dishes with the help of a pipette. Molten agar (15 mL) was added to each petri dish. Mixing of sample dilutions with agar medium was immediately done by moving each petri dish 5 times in vertical, clockwise and horizontal, anticlockwise directions. The petri dishes were held at room temperature (25 ± 1 °C) for 30 min followed by incubation at 37 °C for 48 h in an incubator (GSP-9080 MBE, Shanghai Boxun Industry & Commerce Co., Ltd., China). The bacterial colonies were counted by multiplying with reciprocal in the samples and the results were expressed as log colony-forming units (CFU)/ mL of juice. Yeast and mold counts were determined by pour plate method using PDA. A known amount of PDA powder (31.2 g) was dissolved in distilled water (800 mL) to prepare media. Tartaric acid (10%, w/v) was added in PDA media to protect it from the growth of microbes by cross contamination. All the media plates were put in an incubator at 32 ± 1 °C. After 48 h of incubation, yeast and mold were counted in each plate and the results were expressed as CFU/mL of juice. All the analysis was carried out in triplicate.
photometer at 760 nm. Standard gallic acid solutions were used to prepare a calibration curve and results of total phenols were shown as lg of gallic acid equivalents (GAE) per gram of sample. Total flavonoids content was measured by the method reported by Jia et al. [27] with some modification. Briefly, a known sample of 0.25 mL was mixed with 1.25 mL of de-ionized water and then a 75 lL solution of 5% (w/v) sodium nitrite was added and left it for 6 min. After that, 150 lL solution of a 10% (w/v) AlCl3 solution was added. After 5 min, 0.5 mL of 1 M NaOH was added. Then, distilled water was added to make the final volume of 2.5 mL. The absorbance at 510 nm was determined by a spectrophotometer. The results were shown as lg of catechin equivalents per gram of sample. Total flavonols content was measured using the method reported by Kumaran and Joel Karunakaran [28]. A known sample of 2.0 mL was mixed with 2.0 mL solution of AlCl3 solution (2%, w/v) and then 3.0 mL solution of sodium acetate (50 g/L) was added in it. The mixture was placed at 20 °C for 2.5 h. The same procedure was carried out with standard. Spectrophotometer was used to measure the absorbance at 440 nm and the results were expressed as lg of quercetin equivalents per gram of sample. 2.10. Determination of °Brix, pH and color attributes ° Brix of samples was determined by a hand refractometer (WYT-80, Quanzhou Wander Experimental Instrument Co., Ltd., China) at 20 ± 0.5 °C. Distilled water was used to wash the prism of the instrument after each sample. All the determinations were carried out in triplicate. The pH was analyzed by using a digital pH meter (Delta 320 pH meter, Metller Toledo Instruments (Shanghai) Co., Ltd., China). Before determination, buffer solutions of pH 7 and 4 were used to calibrate the pH meter. 10 mL sample was taken in a beaker and continuously stirred with a magnetic stirrer and pH was measured at 20 ± 0.5 °C. Color values, L⁄ (whiteness or brightness/darkness), a⁄ (redness/ greenness) and b⁄ (yellowness/blueness) of the samples were determined by a colorimeter (Chroma Meter CR-400 Konica Minolta, Sensing, Inc., Japan). A white reference tile was used to calibrate the instrument. All the measurements were carried out in triplicate.
2.8. Determination of ascorbic acid 2.11. Determination of titratable acidity The ascorbic acid contents were determined by the method reported by Lee and Coates [25] with slight modifications using Agilent 1100 series HPLC (Agilent Technologies, USA), which consisted of a model G1379A degasser, a model G1311A pump, a model G1316A column oven and model G1315B diode array detector (DAD). Before injection into column, 5.0 mL sample was filtered through syringe filters of 0.45 lm diameter. Sample volume of 20 lL with known concentration was injected into a Tskgel ODS100Z C18 column (4.6 mm 150 cm, 5 lm, Tosoh, Japan). Methanol 30% (v/v) was used as a mobile phase with a flow rate of 1.0 mL/min. Eluate was detected at 280 nm. Standard ascorbic acid solutions were used to prepare a calibration curve and the results were shown as mg/100 mL of apple juice.
Titratable acidity of the sample was determined by the standard method of AOAC [29]. 2.12. Statistical analysis All of the data obtained in the present study were presented as mean value ± standard deviation (SD). Analysis of variance (one way-ANOVA) was performed, and significant differences between mean values were determined by LSD pairwise comparison test at a significance level of P < 0.05. Statistical analyses were conducted using Statistix 9.0 software (Analytical Software, Tallahassee, FL, USA).
2.9. Determination of total phenolics, flavonoids and flavonols
3. Results and discussion
Total phenolic content was spectrophotometrically measured by Folin–Ciocalteu colorimetric method described by Singleton et al. [26] with slight modifications. 1 mL reagent of Folin–Ciocalteu (10%, v/v) was mixed with a 0.5 mL sample of known concentration and left this mixture for 6 min. Then 2.0 mL sodium carbonate (20%, w/v) was added to it and kept the mixture for 60 min at 30 °C. The absorbance was determined using a spectro-
3.1. Effects of thermosonication on PPO, POD and PME residual activities The results regarding the effects of ultrasound treatments on enzyme activities of PPO, POD and PME in apple juice are presented in Table 2. The inactivation of all the enzymes under study was more pronounced for apple juice treated with-probe
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M. Abid et al. / Ultrasonics Sonochemistry 21 (2014) 984–990 Table 2 Effects of thermosonication on PPO, POD and PME residual activity percentage in apple juice (n = 3). Samples
POD (residual activity%)
PPO (residual activity%)
PME (residual activity%)
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
100.00 ± 0.00a 99.97 ± 0.32a 96.15 ± 0.24d 70.00 ± 0.49e 99.00 ± 0.35b 98.12 ± 0.27c 68.00 ± 0.19f 57.00 ± 0.17g 28.00 ± 0.29h 9.00 ± 0.19i
100 ± 0.00a 99.10 ± 0.16b 90.00 ± 0.19e 63.00 ± 0.22f 98.16 ± 0.29c 97.04 ± 0.15d 61.00 ± 0.32g 53.00 ± 0.21h 21.15 ± 0.16i 6.15 ± 0.18j
100.00 ± 0.00a 99.12 ± 0.25b 91.10 ± 0.32e 62.00 ± 0.19f 98.00 ± 0.25c 97.00 ± 0.29d 60.00 ± 0.24g 52.00 ± 0.31h 23.70 ± 0.16i 7.10 ± 0.19j
Values with different letters in the same column (a–j) are significantly different (P < 0.05) from each other.
compared to those treated in-bath type sonicator. Very low reduction in enzyme activities of juice samples treated in-bath USB20-30, a bit higher reduction in USB40-30 and considerable reduction in USB60-30 were observed. The relative higher reduction in enzyme activities of PPO, POD and PME in USB60-30 that were 63%, 70% and 62%, respectively, might be due to higher treatment temperature. The less reduction in enzyme activities at lower temperature indicates that ultrasound treatment can only inactivate enzymes in combination with heat and/or pressure [30]. A considerable inactivation of PPO, POD and PME enzyme activities with treatment time was observed in ultrasound treated apple juice with-probe at a temperature of 40 °C that were 61%, 68% and 60%, respectively, in treatment USP40-5 and 53%, 57% and 52%, respectively, in treatment USP40-10. We found that the highest inactivation of enzymes in ultrasound treated apple juice with-probe at 60 °C showed minimum residual activity of PPO, POD and PME in USP60-10 that were 6.15%, 9.00% and 7.10%, respectively, compared to USP60-5 that were 21.15%, 28% and 23.70%, respectively. It has recently been reported that the inactivation of PPO and POD enzymes increase with increase in treatment time [31]. Similar results of higher POD and PME inactivation in carrots treated with-probe compared to bath have been reported [32]. Ultrasound treatments whether in-bath or with-probe resulted in the reduction of enzyme activities in a temperature- and timedependent manner. However, ultrasound with-probe showed significantly higher inactivation of enzymes at low temperature and short time than ultrasound in-bath treatments. The main benefit of thermosonication is that it allows enzyme inactivation at lower temperature and shorter time [33]. Thermosonicated samples at same temperature showed better results in terms of enzyme inactivation compared to thermal treatment [16,34]. Thermal effect and mechanical shock due to microstreaming might be the reasons of enzyme inactivation caused by thermosonication [33,35]. The protein structure of enzymes can be damaged by these factors, alone or in combination that results in the decrease of enzyme activity [36]. The mechanical force exerted by the collapse of bubbles and cavitations produced due to sonication with acoustic field can also cause enzyme inactivation [37]. Free radicals produced due to sonoprocessing have also been reported to cause inactivation of POD and catalase enzymes [38]. Furthermore, different intrinsic and extrinsic control parameters are responsible for the efficiency of ultrasound treatment [39]. Increase in localized temperature and pressure due to cavitations produced during sonication might be the other reasons of enzyme inactivation, but free radicals as described by Vercet et al. [40] do not play a role as their production decreases with increase in temperature whereas, the enzyme inactivation increases with an increase in temperature. Therefore, heat and mechanical damage are the leading factors of thermosonication to inactivate enzymes.
3.2. Effects of thermosonication on natural microflora of apple juice The results regarding total plate counts, yeast and mold in apple juice treated with thermosonication are illustrated in Table 3. We found that the total plate counts, yeast and mold were highly sensitive to temperature. Complete inactivation of microbes was achieved at 60 °C irrespective of type of sonication method. Previously, the highest inactivation of microbial cells at 60 °C in thermosonicated mango juice has been reported [41]. In our study, exposure at temperature of 20 °C, a non-significant reduction in total plate counts and significant reduction in yeast and mold were observed in-bath USB20-30 while, that of withprobe USP20-5, significant reductions were observed in total plate counts, yeast and mold and more reductions were observed in case of probe with USP20-10. Similar results of inactivation were observed for total plate counts, yeast and mold in the samples treated at 40 °C in both cases as treated in-bath USB40-30 and with-probe USP40-5. However, a clear difference in the inactivation of all these microbial cells was observed in case of with-probe USP40-10, which show the higher reduction efficiency of microbial cells in apple juice treated for extended time with-probe compared to inbath. A higher inactivation rate of natural microflora was found in samples treated with-probe even for a short time, at the same temperature as in-bath sonication. This might be attributed to severe physical and chemical conditions due to probe treatment. Generally, thermosonication of apple juice, whether in-bath or with-probe, showed better results of microbial inactivation. More reductions in microbial cells of the sample treated with ultrasound at higher temperatures revealed that synergism might exist between sonication and heat [42,43]. It has been reported that thermosonication cause the highest inactivation of pathogens as recorded in cranberry, pineapple and grapefruit juices [44]. Ultrasound has been proved to increase microbial sensitivity to heat, high osmotic pressure and low pH due to cavitations and other changes in the outer membrane of the cell structure [43,45]. In our previous study, we found microbial inactivation of apple juice treated with ultrasound but it could not achieve a complete decontamination of juice which might be due to low treatment temperature [10]. So, the results indicated that the increased level of microbial inactivation of apple juice could be obtained by coupling ultrasound with heat while reducing other adverse changes in the food. 3.3. Effects of thermosonication on ascorbic acid The amount of ascorbic acid in fresh apple juice was 5.27 mg/ 100 mL (Table 4). We observed an increase in ascorbic acid content of apple juice sonicated at 20 °C, whether in-bath (USB2030) or with-probe (USP20-5 and USP20-10). In our previous study, a similar increase in ascorbic acid content of apple juice treated with ultrasound at a temperature of 20 °C was observed
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Table 3 Effects of thermosonication treatment on the survival of microorganisms in apple juice (n = 3). Samples
Total plate counts (log CFU/mL)
Yeast & mold counts (log CFU/mL)
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
3.95 ± 0.12a 3.91 ± 0.25a 2.26 ± 0.13d ND 3.73 ± 0.21b 3.61 ± 0.19c 2.23 ± 0.12d 2.16 ± 0.13e ND ND
3.51 ± 0.26a 2.80 ± 0.20b 2.16 ± 0.15d ND 2.60 ± 0.19c 2.23 ± 0.24d 2.16 ± 0.18d 2.06 ± 0.14e ND ND
Values with different letters in the same column (a–e) are significantly different (P < 0.05) from each other.
Table 4 Effects of thermosonication on ascorbic acid, total phenolics, flavonoids and flavonols in apple juice (n = 3). Samples
Ascorbic acid (mg/100 mL)
Total phenolics (gallic acid equivalent lg/g)
Total flavonoids (catechin equivalent lg/g)
Total flavonols (quercetin equivalent lg/g)
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
5.27 ± 0.02d 5.36 ± 0.03c 5.05 ± 0.04ef 4.75 ± 0.05h 6.07 ± 0.04a 5.90 ± 0.04b 5.06 ± 0.07e 5.00 ± 0.04ef 4.97 ± 0.04fg 4.90 ± 0.07g
697.49 ± 3.24d 758.29 ± 4.10c 670.55 ± 3.12f 478.83 ± 2.72j 780.91 ± 2.49a 771.49 ± 2.96b 674.33 ± 2.36e 663.17 ± 2.34g 551.72 ± 1.95h 513.40 ± 1.83i
436.82 ± 2.24d 466.52 ± 2.54c 419.05 ± 1.59f 373.78 ± 1.39j 476.52 ± 1.12a 471.15 ± 2.10b 425.13 ± 2.72e 411.10 ± 1.65g 410.00 ± 1.31h 399.78 ± 1.19i
2.70 ± 0.11c 3.50 ± 0.08b 2.53 ± 0.08d 1.79 ± 0.07g 3.64 ± 0.05a 3.61 ± 0.06ab 2.57 ± 0.08d 2.49 ± 0.07d 2.13 ± 0.06e 2.00 ± 0.05f
Values with different letters in the same column (a–j) are significantly different (P < 0.05) from each other.
[10]. This positive effect of ultrasound is presumed to be due to the effective removal of occluded oxygen from the juice [46], a critical parameter influencing the stability of ascorbic acid [47]. However, in the present study as the processing temperature increased to 40 °C, the degradation of ascorbic acid increased in both cases of bath and probe, which were recorded as 5.05, 5.06 and 5.00 mg/100 mL in USB40-30, USP40-5 and USP40-10, respectively. Degradation of ascorbic acid at higher treatment temperature might be attributed to severe physical conditions occurred as a result of cavitational collapse of bubbles [12,13]. When ultrasound processing of apple juice was carried out at a further high temperature (60 °C), loss of ascorbic acid was also further increased. But at 60 °C, larger differences in loss of ascorbic acid occurred in the samples treated in-bath and with-probe, which were 4.75 mg/100 mL in USB60-30, 4.97 mg/100 mL in USP60-5, and 4.90 mg/100 mL in USP60-10. We found more losses in samples treated in-bath at 60 °C, which might be due to the increased processing time that might induce chemical decomposition of ascorbic acid at higher rates. The increased loss of ascorbic acid by increasing processing time in the present study even at low treatment temperature is in agreement with the earlier findings [48]. Ascorbic acid is not a stable compound, as it decomposes under severe conditions; thus higher retention of ascorbic acid in juices is possible under mild processing conditions [49]. The highest retention of ascorbic acid in thermosonically treated apple juice at 60 °C was found in USP60-5 (4.97 mg/100 mL). Retention of ascorbic acid in fruit juices has been employed as an indicator of quality, shelf life ends when its amount decreases to 50% [50]. Hence, thermosonication at 60 °C was found to be effective in apple juice processing with improved inactivation of enzymes and natural microflora as well as relative higher retention of ascorbic acid content.
3.4. Effects of thermosonication on total polyphenols, flavonoids and flavonols Fresh apple juice had a total phenolic content of 697.49 lg GAE/g, total flavonoid content of 436.82 lg CE/g and total flavonol content of 2.70 lg QE/g (Table 4). All compounds increased significantly at lower ultrasound processing temperature of 20 °C in all the treatments of USB20-30, USP20-5 and USP20-10 compared with untreated control samples. But as the processing temperature increased from 40 to 60 °C, the degradation of all these bioactive compounds also increased, indicating that the temperature has a significant effect on levels of all these compounds. Furthermore, it was also observed that the effect was more pronounced in case of ultrasound in-bath compared to ultrasound with-probe at higher temperature of 60 °C. Our results regarding a decrease in total phenolic content with an increase in temperature are in conformity with the observation of Rawson et al. [49], who reported that the total phenolic content decreased with increase in processing temperature from 25 to 45 °C in thermosonically treated watermelon juice. Gardner et al. [51], Spanos and Wrolstad [52] have also reported the reduction in total phenolic content in apple juice treated at 80 °C for 15 min. Phenolic compounds are the secondary plant metabolites which play a considerable role in the development of characteristic color and flavor of fruit juices. In the present study, at processing temperature of 60 °C, the highest retention of all these bioactive compounds was observed in USP60-5 compared to USP60-10 and USB60-30. 3.5. Effects of thermosonication on °Brix, pH, titratable acidity and color attributes Results regarding the effects of thermosonication on °Brix, pH and titratable acidity of apple juice are presented in Table 5. No
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M. Abid et al. / Ultrasonics Sonochemistry 21 (2014) 984–990 Table 5 Effects of thermosonication on °Brix, pH, acidity and color attributes in apple juice (n = 3). Samples
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
°
Brix
12.00 ± 0.10a 12.00 ± 0.00a 12.05 ± 0.06a 12.05 ± 0.06a 12.05 ± 0.05a 12.05 ± 0.05a 12.05 ± 0.05a 12.05 ± 0.05a 12.05 ± 0.00a 12.05 ± 0.00a
pH
3.87 ± 0.04a 3.86 ± 0.03a 3.85 ± 0.04a 3.86 ± 0.01a 3.88 ± 0.04a 3.87 ± 0.07a 3.87 ± 0.02a 3.86 ± 0.04a 3.86 ± 0.05a 3.86 ± 0.03a
Titratable acidity (%)
0.23 ± 0.01a 0.23 ± 0.01a 0.22 ± 0.00a 0.22 ± 0.01a 0.23 ± 0.02a 0.23 ± 0.01a 0.23 ± 0.01a 0.22 ± 0.01a 0.22 ± 0.01a 0.22 ± 0.02a
Color attributes L⁄
a⁄
b⁄
21.25 ± 0.05h 21.43 ± 0.04f 22.62 ± 0.04e 23.98 ± 0.02d 21.27 ± 0.04h 21.35 ± 0.04g 23.96 ± 0.03d 24.06 ± 0.05c 24.29 ± 0.05b 24.68 ± 0.06a
7.87 ± 0.07f 7.60 ± 0.06h 8.36 ± 0.03de 8.52 ± 0.05c 7.80 ± 0.07fg 7.77 ± 0.04g 8.31 ± 0.05e 8.43 ± 0.04d 8.71 ± 0.07b 8.82 ± 0.07a
0.33 ± 0.03g 0.53 ± 0.04e 0.64 ± 0.03d 0.73 ± 0.04c 0.45 ± 0.03f 0.51 ± 0.07ef 0.64 ± 0.06d 0.74 ± 0.07c 0.85 ± 0.06b 0.97 ± 0.04a
Values with different letters in the same column (a–h) are significantly different (P < 0.05) from each other.
significant change was observed in °Brix, pH and titratable acidity of treated and untreated apple juice samples. These results are in line with the findings of Walkling-Ribeiro et al. [53] who reported non-significant effect of combined treatment of thermosonication and pulse electric field on °Brix and pH of orange juice. Similarly, some other studies have shown non-significant changes in °Brix, pH and titratable acidity of fruit juices treated with ultrasound [10,54]. Color is the key quality parameter of fruit juices that directly influences the consumer criteria for acceptance or rejection. The results regarding the effects of different ultrasound treatments on lightness (L⁄), redness (a⁄) and yellowness (b⁄) of apple juice are illustrated in Table 5. Color values of fresh untreated juice samples were 21.25, 7.87 and 0.33 for L⁄, a⁄ and b⁄, respectively. We observed a significant increase in L⁄ and b⁄ values and a decrease in a⁄ value of the juice sample treated at lower temperature of 20 °C. Similar results for L⁄, a⁄ and b⁄ values of color in apple juice have been reported in our previous study [10]. During thermosonication treatment of apple juice at higher temperature (40 and 60 °C), the L⁄, a⁄ and b⁄ values were significantly increased with the highest increase at 60 °C, especially in the samples treated with-probe for 10 min. Similar effects have been observed by Rawson et al. [49] who reported a maximum increase in L⁄ value for longer exposure time in thermosonically treated watermelon juice. Precipitation of unstable particles in the juice due to thermosonication treatment might be responsible for the increase in L⁄ value. But Tiwari et al. [12,13] reported that increase in L⁄ value might be due to increase in the cloud value of juice under the effect of sonication resulting in improved homogenization. The changes in color values of thermosonically treated apple juice might be due to alone or combined effects of extrinsic control treatment variables of temperature and time. In addition, although the most severe treatment significantly reduced the residual PPO and POD activity as shown in Table 2, these enzymes were clearly very active during the treatment, as the colour change indicates the most phenol condensation of all of the treatments. Further study on the process optimization is needed. The changes in color values may adversely affect the organoleptic properties and ultimately degrade the quality of juice, however, these changes could not be perceived by the naked eye in the present study.
4. Conclusion The study was conducted to evaluate the influence of different thermosonication treatments on enzyme, microbial and nutritional quality of apple juice. It may be inferred from the present study that ultrasound with-probe treatment seems convenient for the inactivation of enzymes and natural microflora of apple juice. Ultrasound with-probe treatment at temperature up to 60 °C
resulted in the highest inactivation of enzymes and microbial cells with lower losses of ascorbic acid, total phenolics, flavonoids and flavonols. Although, further research would be necessary to optimize the conditions by evaluating different extrinsic parameters but under these conditions the application of ultrasound withprobe at 60 °C was found to be an appropriate treatment. The data may reflect the application of ultrasound treatments to produce good quality apple juice with reduced enzymes and microbial activities and improved retention of other bioactive compounds.
Acknowledgements This work was supported by a grant-in-aid from a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Muhammad Abid and Saqib Jabbar would like to express their thanks to the Ministry of Education, China, for financial assistance through the Chinese Government Scholarship Program.
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Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Thermosonication as a potential quality enhancement technique of apple juice Muhammad Abid a,b, Saqib Jabbar a, Bing Hu a, Malik Muhammad Hashim a,c, Tao Wu a, Shicheng Lei a, Muhammad Ammar Khan a, Xiaoxiong Zeng a,⇑ a b c
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan Department of Food Science and Technology, Gomal University, Dera Ismail Khan, Pakistan
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 3 December 2013 Accepted 6 December 2013 Available online 13 December 2013 Keywords: Apple juice Thermosonication Quality Enzymes Microbes
a b s t r a c t Enzymatic browning and microbial growth lead to quality losses in apple products. In the present study, fresh apple juice was thermosonicated using ultrasound in-bath (25 kHz, 30 min, 0.06 W cm3) and ultrasound with-probe sonicator (20 kHz, 5 and 10 min, 0.30 W cm3) at 20, 40 and 60 °C for inactivation of enzymes (polyphenolase, peroxidase and pectinmethylesterase) and microflora (total plate count, yeast and mold). Additionally, ascorbic acid, total phenolics, flavonoids, flavonols, pH, titratable acidity, °Brix and color values influenced by thermosonication were investigated. The highest inactivation of enzymes was obtained in ultrasound with-probe at 60 °C for 10 min, and the microbial population was completely inactivated at 60 °C. The retention of ascorbic acid, total phenolics, flavonoids and flavonols were significantly higher in ultrasound with-probe than ultrasound in-bath at 60 °C. These results indicated the usefulness of thermosonication for apple juice processing at low temperature, for enhanced inactivation of enzymes and microorganisms. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Apple is one of the most valuable fruits with a global production estimate of about 75.64 million tons per annum [1]. Being an important part of a healthy human diet, it contributes substantial energy, antioxidants, vitamins, minerals and dietary fiber. In addition to fresh consumption, it is processed into a variety of products such as juice, jam and jelly. There has been growing interest of consuming apple juice owing to its specific pleasant taste and high nutritional value. However, quality defects arising during the processing and storage of apple juice, if not checked, lower its scope and market value. Discoloration of apple juice due to enzymatic browning is the major problem that deteriorates its quality. Polyphenoloxidase (PPO) and peroxidase (POD) are the major enzymes responsible for browning, especially, the former oxidizes phenolic compounds to o-quinones that further polymerize and ultimately produce undesirable brown color and off-flavored product [2–5]. Since quality is of utmost importance for the consumers, it is desirable to inactivate these deteriorative enzymes during processing and storage of apple juice.
⇑ Corresponding author. Fax: +86 25 84396791. E-mail address: [email protected] (X. Zeng). 1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2013.12.003
Processing methods affect the quality, safety and shelf life of food products. Thermal processing ensures the food safety and improves the shelf life but may also adversely affect the nutritional quality of the foods [6]. Alternatively, researchers are looking for the most suitable innovative techniques which could get results with the application of little or no heat [7]. The application of power ultrasound in the food industry has become an innovative and attractive tool [8]. Improved homogeneity, minimal loss of flavor and significant energy saving are the merits of sonoprocessing over thermal treatment [9]. It has been regarded as a potential technique to meet the criteria of the Food and Drug Administration, concerning a 5 log reduction of microbial cells in the fruit juices [10]. We have recently reported that the quality of apple juice treated with ultrasound at low temperature has been improved [10,11]. Thermosonication has been attempted as an alternative to thermal treatment for processing of fruit juices such as strawberry juice, blackberry juice and orange juice [12–14]. Improvement in the quality of tomato juice, watercress and yoghurt gels by thermosonication has been reported in the literature [8,15,16]. The inactivation of POD and retention of color and vitamin C were successfully achieved in watercress and guava using thermosonication [17–19]. However, the effects of thermosonication on PPO, POD, pectinmethylesterase (PME), microbial cells, total phenols, flavonoids, flavonols, ascorbic acid, pH, titratable acidity, Brix and color of apple juice have not been studied
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to date. Thus, the clear objective of the present study was to investigate thermosonication (ultrasound combined with mild heat treatment) as a potential quality enhancement technique of apple juice. 2. Materials and methods 2.1. Chemicals Gallic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin Ciocalteu reagent was purchased from Fluka (Buchs, Switzerland). Catechin was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Quercetin was purchased from Kayon Biological Technology Co., Ltd. (Shanghai, China). HPLC grade methanol was purchased from Hanbon Science and Technology (Nanjing, China). Sodium nitrite, sodium carbonate, sodium hydroxide, sodium chloride (NaCl), pectin, catechol, tartaric acid, molten agar and potato dextrose agar (PDA), aluminum trichloride (AlCl3), ascorbic acid, diabasic potassium phosphate (K2HPO4), monobasic potassium phosphate (KH2PO4), hydrogen peroxide (H2O2) and pyrogallol were obtained from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. 2.2. Apple juice preparation Fresh apples (Malus domestica) of variety Fuji were procured from a local fruit market of Nanjing, China. The apples were washed with tap water, dried with paper towels and cut into four pieces. The stems, seeds and overripe portions were discarded. Juice extractor of MJ-M176P (Panasonic Manufacturing Berhad, Malaysia) was used to extract the juice which was then filtered through sterilized double layered muslin cloth. The freshly extracted juice was vortex mixed and selected as the control. It was put in sterilized and air tight media bottle of 100 mL which was wrapped by aluminum foils and stored at 4 °C for 48 h until further analysis.
bath type sonicator was 0.06 W cm3. In case of ultrasound treatments with probe, apple juice sample was taken in a jacketed vessel through which water was circulated. Ultrasonic processor of 750 W (VC 750, Sonics and Materials Inc., Newtown, CT, USA), with 0.5 inch probe operating at 20 kHz frequency was used for sonication. The ultrasonic density of the probe type sonicator was 0.30 W cm3. The schematic diagram of the exposure system has been presented elsewhere [20]. Samples were treated at 20, 40 and 60 °C by radiating 70% of power (750 W) for 5 and 10 min (USP20-5, USP20-10, USP40-5, USP40-10, USP60-5 and USP60-10) keeping pulse durations of 5 s on and 5 s off. The depth of the probe was kept 25 mm in the juice samples. Overheating of the samples (temperature controlled by thermocouple ±0.5 °C) during the ultrasound treatment was prevented by circulating ice-water through the treatment chamber. Sonication of samples was started when the set temperature was reached. The observed rise in temperature of the samples due to sonication was 2–5 °C. All the treatments were carried out in triplicate. 2.4. Determination of POD residual activity POD activity was determined using pyrogallol as a substrate according to the method described by Kwak et al. [21]. The sample was centrifuged at 10,000g, 4 °C for 10 min with Avanti J-E Centrifuge (Beckman Coulter, Inc., USA). The total volume of the reaction mixture was 3.0 mL which contained 2.2 mL sample, 0.32 mL of 100 mM (w/v) potassium-phosphate buffer (pH 6), 0.32 mL of 5% (w/v) pyrogallol and 0.16 mL of 0.147 M (v/v) H2O2. The reaction was initiated by the addition of H2O2 and the increase in absorbance was recorded using a spectrophotometer (LabTech Bluestar-A UV spectrophotometer) at 420 nm in 3 min. Following equation was used to calculate the percent residual activity of POD:
Residual activityð%Þ ¼ 100 At =A0
ð1Þ
where At is the enzyme activity of treated sample and A0 is the enzyme activity of the control sample.
2.3. Ultrasound treatment 2.5. Determination of PPO residual activity Ultrasound treatments of apple juice were performed by putting 80 mL of sample in 100 mL jacketed vessel. As shown in Table 1, two types of ultrasonic treatments were carried out: in-bath (a) and with ultrasonic probe (b). In case of a, jacketed vessel containing apple juice sample was put in an ultrasonic bath cleaner of SB-500 DTY (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) working at 25 kHz frequency, radiating 70% of power (500 W) and the samples were ultrasound treated at 20, 40 and 60 °C for 30 min (USB20-30, USB40-30 and USB60-30). The ultrasonic density of the
PPO activity was assessed by the method reported by Augustin et al. [22]. The sample was centrifuged (10,000g) for 10 min at 4 °C. The total volume of the reaction mixture was 5.0 mL which contained 1.5 ml of sample, 0.5 mL of 0.05 M (w/v) catechol and 0.2 M (w/v) potassium phosphate buffer (pH 6.8). Increase in absorbance at 410 nm was recorded after every 60 s for 10 min, using a spectrophotometer. The percent residual activity of PPO was calculated by applying Eq.(1).
Table 1 Processing conditions used during ultrasound (in-bath and with-probe) treatments. Conditions
Samples
Temperature (°C)
Time (min)
Power (W)
US density (W cm 3)a
Control
Fresh
–
–
–
–
US (in-bath)
USB20-30 USB40-30 USB60-30
20 40 60
30 30 30
500 500 500
0.06
US (with-probe)
USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
20 20 40 40 60 60
5 10 5 10 5 10
750 750 750 750 750 750
0.30
The treatments USB and USP represent ultrasound in-bath and ultrasound with-probe, respectively. a Determined according to Jambrak et al. [55].
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2.6. Determination of PME residual activity PME activity was determined by measuring free carboxyl groups produced as a result of action of enzyme on pectin. The reaction mixture contained 10.0 mL of centrifuged juice sample (10,000g for 10 min at 4 °C) and 40.0 mL of 1% (w/v) pectin solution in 0.15 M (w/v) NaCl solution. The pH of the mixture was maintained to 7.7 by adding 100 lL of 0.05 mol L1 (w/v) NaOH and the time required to reach pH 7.7 was recorded (50 ± 2 °C). The unit of PME is defined as the amount of enzyme that released 1 mmol of carboxyl groups in 1 min [23]. The percent residual activity of PME was calculated by using Eq.(1). 2.7. Microbiological analysis The FDA’s standard method of Bacteriological Analytical Manual [24] was used to determine the microbial population of juice samples. Pour plate method was used to determine the total plate counts. Proper serial dilutions were prepared by mixing sterilized distilled water followed by further decimal dilutions of samples (up to 105). These dilutions were poured into sterile petri dishes with the help of a pipette. Molten agar (15 mL) was added to each petri dish. Mixing of sample dilutions with agar medium was immediately done by moving each petri dish 5 times in vertical, clockwise and horizontal, anticlockwise directions. The petri dishes were held at room temperature (25 ± 1 °C) for 30 min followed by incubation at 37 °C for 48 h in an incubator (GSP-9080 MBE, Shanghai Boxun Industry & Commerce Co., Ltd., China). The bacterial colonies were counted by multiplying with reciprocal in the samples and the results were expressed as log colony-forming units (CFU)/ mL of juice. Yeast and mold counts were determined by pour plate method using PDA. A known amount of PDA powder (31.2 g) was dissolved in distilled water (800 mL) to prepare media. Tartaric acid (10%, w/v) was added in PDA media to protect it from the growth of microbes by cross contamination. All the media plates were put in an incubator at 32 ± 1 °C. After 48 h of incubation, yeast and mold were counted in each plate and the results were expressed as CFU/mL of juice. All the analysis was carried out in triplicate.
photometer at 760 nm. Standard gallic acid solutions were used to prepare a calibration curve and results of total phenols were shown as lg of gallic acid equivalents (GAE) per gram of sample. Total flavonoids content was measured by the method reported by Jia et al. [27] with some modification. Briefly, a known sample of 0.25 mL was mixed with 1.25 mL of de-ionized water and then a 75 lL solution of 5% (w/v) sodium nitrite was added and left it for 6 min. After that, 150 lL solution of a 10% (w/v) AlCl3 solution was added. After 5 min, 0.5 mL of 1 M NaOH was added. Then, distilled water was added to make the final volume of 2.5 mL. The absorbance at 510 nm was determined by a spectrophotometer. The results were shown as lg of catechin equivalents per gram of sample. Total flavonols content was measured using the method reported by Kumaran and Joel Karunakaran [28]. A known sample of 2.0 mL was mixed with 2.0 mL solution of AlCl3 solution (2%, w/v) and then 3.0 mL solution of sodium acetate (50 g/L) was added in it. The mixture was placed at 20 °C for 2.5 h. The same procedure was carried out with standard. Spectrophotometer was used to measure the absorbance at 440 nm and the results were expressed as lg of quercetin equivalents per gram of sample. 2.10. Determination of °Brix, pH and color attributes ° Brix of samples was determined by a hand refractometer (WYT-80, Quanzhou Wander Experimental Instrument Co., Ltd., China) at 20 ± 0.5 °C. Distilled water was used to wash the prism of the instrument after each sample. All the determinations were carried out in triplicate. The pH was analyzed by using a digital pH meter (Delta 320 pH meter, Metller Toledo Instruments (Shanghai) Co., Ltd., China). Before determination, buffer solutions of pH 7 and 4 were used to calibrate the pH meter. 10 mL sample was taken in a beaker and continuously stirred with a magnetic stirrer and pH was measured at 20 ± 0.5 °C. Color values, L⁄ (whiteness or brightness/darkness), a⁄ (redness/ greenness) and b⁄ (yellowness/blueness) of the samples were determined by a colorimeter (Chroma Meter CR-400 Konica Minolta, Sensing, Inc., Japan). A white reference tile was used to calibrate the instrument. All the measurements were carried out in triplicate.
2.8. Determination of ascorbic acid 2.11. Determination of titratable acidity The ascorbic acid contents were determined by the method reported by Lee and Coates [25] with slight modifications using Agilent 1100 series HPLC (Agilent Technologies, USA), which consisted of a model G1379A degasser, a model G1311A pump, a model G1316A column oven and model G1315B diode array detector (DAD). Before injection into column, 5.0 mL sample was filtered through syringe filters of 0.45 lm diameter. Sample volume of 20 lL with known concentration was injected into a Tskgel ODS100Z C18 column (4.6 mm 150 cm, 5 lm, Tosoh, Japan). Methanol 30% (v/v) was used as a mobile phase with a flow rate of 1.0 mL/min. Eluate was detected at 280 nm. Standard ascorbic acid solutions were used to prepare a calibration curve and the results were shown as mg/100 mL of apple juice.
Titratable acidity of the sample was determined by the standard method of AOAC [29]. 2.12. Statistical analysis All of the data obtained in the present study were presented as mean value ± standard deviation (SD). Analysis of variance (one way-ANOVA) was performed, and significant differences between mean values were determined by LSD pairwise comparison test at a significance level of P < 0.05. Statistical analyses were conducted using Statistix 9.0 software (Analytical Software, Tallahassee, FL, USA).
2.9. Determination of total phenolics, flavonoids and flavonols
3. Results and discussion
Total phenolic content was spectrophotometrically measured by Folin–Ciocalteu colorimetric method described by Singleton et al. [26] with slight modifications. 1 mL reagent of Folin–Ciocalteu (10%, v/v) was mixed with a 0.5 mL sample of known concentration and left this mixture for 6 min. Then 2.0 mL sodium carbonate (20%, w/v) was added to it and kept the mixture for 60 min at 30 °C. The absorbance was determined using a spectro-
3.1. Effects of thermosonication on PPO, POD and PME residual activities The results regarding the effects of ultrasound treatments on enzyme activities of PPO, POD and PME in apple juice are presented in Table 2. The inactivation of all the enzymes under study was more pronounced for apple juice treated with-probe
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M. Abid et al. / Ultrasonics Sonochemistry 21 (2014) 984–990 Table 2 Effects of thermosonication on PPO, POD and PME residual activity percentage in apple juice (n = 3). Samples
POD (residual activity%)
PPO (residual activity%)
PME (residual activity%)
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
100.00 ± 0.00a 99.97 ± 0.32a 96.15 ± 0.24d 70.00 ± 0.49e 99.00 ± 0.35b 98.12 ± 0.27c 68.00 ± 0.19f 57.00 ± 0.17g 28.00 ± 0.29h 9.00 ± 0.19i
100 ± 0.00a 99.10 ± 0.16b 90.00 ± 0.19e 63.00 ± 0.22f 98.16 ± 0.29c 97.04 ± 0.15d 61.00 ± 0.32g 53.00 ± 0.21h 21.15 ± 0.16i 6.15 ± 0.18j
100.00 ± 0.00a 99.12 ± 0.25b 91.10 ± 0.32e 62.00 ± 0.19f 98.00 ± 0.25c 97.00 ± 0.29d 60.00 ± 0.24g 52.00 ± 0.31h 23.70 ± 0.16i 7.10 ± 0.19j
Values with different letters in the same column (a–j) are significantly different (P < 0.05) from each other.
compared to those treated in-bath type sonicator. Very low reduction in enzyme activities of juice samples treated in-bath USB20-30, a bit higher reduction in USB40-30 and considerable reduction in USB60-30 were observed. The relative higher reduction in enzyme activities of PPO, POD and PME in USB60-30 that were 63%, 70% and 62%, respectively, might be due to higher treatment temperature. The less reduction in enzyme activities at lower temperature indicates that ultrasound treatment can only inactivate enzymes in combination with heat and/or pressure [30]. A considerable inactivation of PPO, POD and PME enzyme activities with treatment time was observed in ultrasound treated apple juice with-probe at a temperature of 40 °C that were 61%, 68% and 60%, respectively, in treatment USP40-5 and 53%, 57% and 52%, respectively, in treatment USP40-10. We found that the highest inactivation of enzymes in ultrasound treated apple juice with-probe at 60 °C showed minimum residual activity of PPO, POD and PME in USP60-10 that were 6.15%, 9.00% and 7.10%, respectively, compared to USP60-5 that were 21.15%, 28% and 23.70%, respectively. It has recently been reported that the inactivation of PPO and POD enzymes increase with increase in treatment time [31]. Similar results of higher POD and PME inactivation in carrots treated with-probe compared to bath have been reported [32]. Ultrasound treatments whether in-bath or with-probe resulted in the reduction of enzyme activities in a temperature- and timedependent manner. However, ultrasound with-probe showed significantly higher inactivation of enzymes at low temperature and short time than ultrasound in-bath treatments. The main benefit of thermosonication is that it allows enzyme inactivation at lower temperature and shorter time [33]. Thermosonicated samples at same temperature showed better results in terms of enzyme inactivation compared to thermal treatment [16,34]. Thermal effect and mechanical shock due to microstreaming might be the reasons of enzyme inactivation caused by thermosonication [33,35]. The protein structure of enzymes can be damaged by these factors, alone or in combination that results in the decrease of enzyme activity [36]. The mechanical force exerted by the collapse of bubbles and cavitations produced due to sonication with acoustic field can also cause enzyme inactivation [37]. Free radicals produced due to sonoprocessing have also been reported to cause inactivation of POD and catalase enzymes [38]. Furthermore, different intrinsic and extrinsic control parameters are responsible for the efficiency of ultrasound treatment [39]. Increase in localized temperature and pressure due to cavitations produced during sonication might be the other reasons of enzyme inactivation, but free radicals as described by Vercet et al. [40] do not play a role as their production decreases with increase in temperature whereas, the enzyme inactivation increases with an increase in temperature. Therefore, heat and mechanical damage are the leading factors of thermosonication to inactivate enzymes.
3.2. Effects of thermosonication on natural microflora of apple juice The results regarding total plate counts, yeast and mold in apple juice treated with thermosonication are illustrated in Table 3. We found that the total plate counts, yeast and mold were highly sensitive to temperature. Complete inactivation of microbes was achieved at 60 °C irrespective of type of sonication method. Previously, the highest inactivation of microbial cells at 60 °C in thermosonicated mango juice has been reported [41]. In our study, exposure at temperature of 20 °C, a non-significant reduction in total plate counts and significant reduction in yeast and mold were observed in-bath USB20-30 while, that of withprobe USP20-5, significant reductions were observed in total plate counts, yeast and mold and more reductions were observed in case of probe with USP20-10. Similar results of inactivation were observed for total plate counts, yeast and mold in the samples treated at 40 °C in both cases as treated in-bath USB40-30 and with-probe USP40-5. However, a clear difference in the inactivation of all these microbial cells was observed in case of with-probe USP40-10, which show the higher reduction efficiency of microbial cells in apple juice treated for extended time with-probe compared to inbath. A higher inactivation rate of natural microflora was found in samples treated with-probe even for a short time, at the same temperature as in-bath sonication. This might be attributed to severe physical and chemical conditions due to probe treatment. Generally, thermosonication of apple juice, whether in-bath or with-probe, showed better results of microbial inactivation. More reductions in microbial cells of the sample treated with ultrasound at higher temperatures revealed that synergism might exist between sonication and heat [42,43]. It has been reported that thermosonication cause the highest inactivation of pathogens as recorded in cranberry, pineapple and grapefruit juices [44]. Ultrasound has been proved to increase microbial sensitivity to heat, high osmotic pressure and low pH due to cavitations and other changes in the outer membrane of the cell structure [43,45]. In our previous study, we found microbial inactivation of apple juice treated with ultrasound but it could not achieve a complete decontamination of juice which might be due to low treatment temperature [10]. So, the results indicated that the increased level of microbial inactivation of apple juice could be obtained by coupling ultrasound with heat while reducing other adverse changes in the food. 3.3. Effects of thermosonication on ascorbic acid The amount of ascorbic acid in fresh apple juice was 5.27 mg/ 100 mL (Table 4). We observed an increase in ascorbic acid content of apple juice sonicated at 20 °C, whether in-bath (USB2030) or with-probe (USP20-5 and USP20-10). In our previous study, a similar increase in ascorbic acid content of apple juice treated with ultrasound at a temperature of 20 °C was observed
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Table 3 Effects of thermosonication treatment on the survival of microorganisms in apple juice (n = 3). Samples
Total plate counts (log CFU/mL)
Yeast & mold counts (log CFU/mL)
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
3.95 ± 0.12a 3.91 ± 0.25a 2.26 ± 0.13d ND 3.73 ± 0.21b 3.61 ± 0.19c 2.23 ± 0.12d 2.16 ± 0.13e ND ND
3.51 ± 0.26a 2.80 ± 0.20b 2.16 ± 0.15d ND 2.60 ± 0.19c 2.23 ± 0.24d 2.16 ± 0.18d 2.06 ± 0.14e ND ND
Values with different letters in the same column (a–e) are significantly different (P < 0.05) from each other.
Table 4 Effects of thermosonication on ascorbic acid, total phenolics, flavonoids and flavonols in apple juice (n = 3). Samples
Ascorbic acid (mg/100 mL)
Total phenolics (gallic acid equivalent lg/g)
Total flavonoids (catechin equivalent lg/g)
Total flavonols (quercetin equivalent lg/g)
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
5.27 ± 0.02d 5.36 ± 0.03c 5.05 ± 0.04ef 4.75 ± 0.05h 6.07 ± 0.04a 5.90 ± 0.04b 5.06 ± 0.07e 5.00 ± 0.04ef 4.97 ± 0.04fg 4.90 ± 0.07g
697.49 ± 3.24d 758.29 ± 4.10c 670.55 ± 3.12f 478.83 ± 2.72j 780.91 ± 2.49a 771.49 ± 2.96b 674.33 ± 2.36e 663.17 ± 2.34g 551.72 ± 1.95h 513.40 ± 1.83i
436.82 ± 2.24d 466.52 ± 2.54c 419.05 ± 1.59f 373.78 ± 1.39j 476.52 ± 1.12a 471.15 ± 2.10b 425.13 ± 2.72e 411.10 ± 1.65g 410.00 ± 1.31h 399.78 ± 1.19i
2.70 ± 0.11c 3.50 ± 0.08b 2.53 ± 0.08d 1.79 ± 0.07g 3.64 ± 0.05a 3.61 ± 0.06ab 2.57 ± 0.08d 2.49 ± 0.07d 2.13 ± 0.06e 2.00 ± 0.05f
Values with different letters in the same column (a–j) are significantly different (P < 0.05) from each other.
[10]. This positive effect of ultrasound is presumed to be due to the effective removal of occluded oxygen from the juice [46], a critical parameter influencing the stability of ascorbic acid [47]. However, in the present study as the processing temperature increased to 40 °C, the degradation of ascorbic acid increased in both cases of bath and probe, which were recorded as 5.05, 5.06 and 5.00 mg/100 mL in USB40-30, USP40-5 and USP40-10, respectively. Degradation of ascorbic acid at higher treatment temperature might be attributed to severe physical conditions occurred as a result of cavitational collapse of bubbles [12,13]. When ultrasound processing of apple juice was carried out at a further high temperature (60 °C), loss of ascorbic acid was also further increased. But at 60 °C, larger differences in loss of ascorbic acid occurred in the samples treated in-bath and with-probe, which were 4.75 mg/100 mL in USB60-30, 4.97 mg/100 mL in USP60-5, and 4.90 mg/100 mL in USP60-10. We found more losses in samples treated in-bath at 60 °C, which might be due to the increased processing time that might induce chemical decomposition of ascorbic acid at higher rates. The increased loss of ascorbic acid by increasing processing time in the present study even at low treatment temperature is in agreement with the earlier findings [48]. Ascorbic acid is not a stable compound, as it decomposes under severe conditions; thus higher retention of ascorbic acid in juices is possible under mild processing conditions [49]. The highest retention of ascorbic acid in thermosonically treated apple juice at 60 °C was found in USP60-5 (4.97 mg/100 mL). Retention of ascorbic acid in fruit juices has been employed as an indicator of quality, shelf life ends when its amount decreases to 50% [50]. Hence, thermosonication at 60 °C was found to be effective in apple juice processing with improved inactivation of enzymes and natural microflora as well as relative higher retention of ascorbic acid content.
3.4. Effects of thermosonication on total polyphenols, flavonoids and flavonols Fresh apple juice had a total phenolic content of 697.49 lg GAE/g, total flavonoid content of 436.82 lg CE/g and total flavonol content of 2.70 lg QE/g (Table 4). All compounds increased significantly at lower ultrasound processing temperature of 20 °C in all the treatments of USB20-30, USP20-5 and USP20-10 compared with untreated control samples. But as the processing temperature increased from 40 to 60 °C, the degradation of all these bioactive compounds also increased, indicating that the temperature has a significant effect on levels of all these compounds. Furthermore, it was also observed that the effect was more pronounced in case of ultrasound in-bath compared to ultrasound with-probe at higher temperature of 60 °C. Our results regarding a decrease in total phenolic content with an increase in temperature are in conformity with the observation of Rawson et al. [49], who reported that the total phenolic content decreased with increase in processing temperature from 25 to 45 °C in thermosonically treated watermelon juice. Gardner et al. [51], Spanos and Wrolstad [52] have also reported the reduction in total phenolic content in apple juice treated at 80 °C for 15 min. Phenolic compounds are the secondary plant metabolites which play a considerable role in the development of characteristic color and flavor of fruit juices. In the present study, at processing temperature of 60 °C, the highest retention of all these bioactive compounds was observed in USP60-5 compared to USP60-10 and USB60-30. 3.5. Effects of thermosonication on °Brix, pH, titratable acidity and color attributes Results regarding the effects of thermosonication on °Brix, pH and titratable acidity of apple juice are presented in Table 5. No
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M. Abid et al. / Ultrasonics Sonochemistry 21 (2014) 984–990 Table 5 Effects of thermosonication on °Brix, pH, acidity and color attributes in apple juice (n = 3). Samples
Control USB20-30 USB40-30 USB60-30 USP20-5 USP20-10 USP40-5 USP40-10 USP60-5 USP60-10
°
Brix
12.00 ± 0.10a 12.00 ± 0.00a 12.05 ± 0.06a 12.05 ± 0.06a 12.05 ± 0.05a 12.05 ± 0.05a 12.05 ± 0.05a 12.05 ± 0.05a 12.05 ± 0.00a 12.05 ± 0.00a
pH
3.87 ± 0.04a 3.86 ± 0.03a 3.85 ± 0.04a 3.86 ± 0.01a 3.88 ± 0.04a 3.87 ± 0.07a 3.87 ± 0.02a 3.86 ± 0.04a 3.86 ± 0.05a 3.86 ± 0.03a
Titratable acidity (%)
0.23 ± 0.01a 0.23 ± 0.01a 0.22 ± 0.00a 0.22 ± 0.01a 0.23 ± 0.02a 0.23 ± 0.01a 0.23 ± 0.01a 0.22 ± 0.01a 0.22 ± 0.01a 0.22 ± 0.02a
Color attributes L⁄
a⁄
b⁄
21.25 ± 0.05h 21.43 ± 0.04f 22.62 ± 0.04e 23.98 ± 0.02d 21.27 ± 0.04h 21.35 ± 0.04g 23.96 ± 0.03d 24.06 ± 0.05c 24.29 ± 0.05b 24.68 ± 0.06a
7.87 ± 0.07f 7.60 ± 0.06h 8.36 ± 0.03de 8.52 ± 0.05c 7.80 ± 0.07fg 7.77 ± 0.04g 8.31 ± 0.05e 8.43 ± 0.04d 8.71 ± 0.07b 8.82 ± 0.07a
0.33 ± 0.03g 0.53 ± 0.04e 0.64 ± 0.03d 0.73 ± 0.04c 0.45 ± 0.03f 0.51 ± 0.07ef 0.64 ± 0.06d 0.74 ± 0.07c 0.85 ± 0.06b 0.97 ± 0.04a
Values with different letters in the same column (a–h) are significantly different (P < 0.05) from each other.
significant change was observed in °Brix, pH and titratable acidity of treated and untreated apple juice samples. These results are in line with the findings of Walkling-Ribeiro et al. [53] who reported non-significant effect of combined treatment of thermosonication and pulse electric field on °Brix and pH of orange juice. Similarly, some other studies have shown non-significant changes in °Brix, pH and titratable acidity of fruit juices treated with ultrasound [10,54]. Color is the key quality parameter of fruit juices that directly influences the consumer criteria for acceptance or rejection. The results regarding the effects of different ultrasound treatments on lightness (L⁄), redness (a⁄) and yellowness (b⁄) of apple juice are illustrated in Table 5. Color values of fresh untreated juice samples were 21.25, 7.87 and 0.33 for L⁄, a⁄ and b⁄, respectively. We observed a significant increase in L⁄ and b⁄ values and a decrease in a⁄ value of the juice sample treated at lower temperature of 20 °C. Similar results for L⁄, a⁄ and b⁄ values of color in apple juice have been reported in our previous study [10]. During thermosonication treatment of apple juice at higher temperature (40 and 60 °C), the L⁄, a⁄ and b⁄ values were significantly increased with the highest increase at 60 °C, especially in the samples treated with-probe for 10 min. Similar effects have been observed by Rawson et al. [49] who reported a maximum increase in L⁄ value for longer exposure time in thermosonically treated watermelon juice. Precipitation of unstable particles in the juice due to thermosonication treatment might be responsible for the increase in L⁄ value. But Tiwari et al. [12,13] reported that increase in L⁄ value might be due to increase in the cloud value of juice under the effect of sonication resulting in improved homogenization. The changes in color values of thermosonically treated apple juice might be due to alone or combined effects of extrinsic control treatment variables of temperature and time. In addition, although the most severe treatment significantly reduced the residual PPO and POD activity as shown in Table 2, these enzymes were clearly very active during the treatment, as the colour change indicates the most phenol condensation of all of the treatments. Further study on the process optimization is needed. The changes in color values may adversely affect the organoleptic properties and ultimately degrade the quality of juice, however, these changes could not be perceived by the naked eye in the present study.
4. Conclusion The study was conducted to evaluate the influence of different thermosonication treatments on enzyme, microbial and nutritional quality of apple juice. It may be inferred from the present study that ultrasound with-probe treatment seems convenient for the inactivation of enzymes and natural microflora of apple juice. Ultrasound with-probe treatment at temperature up to 60 °C
resulted in the highest inactivation of enzymes and microbial cells with lower losses of ascorbic acid, total phenolics, flavonoids and flavonols. Although, further research would be necessary to optimize the conditions by evaluating different extrinsic parameters but under these conditions the application of ultrasound withprobe at 60 °C was found to be an appropriate treatment. The data may reflect the application of ultrasound treatments to produce good quality apple juice with reduced enzymes and microbial activities and improved retention of other bioactive compounds.
Acknowledgements This work was supported by a grant-in-aid from a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Muhammad Abid and Saqib Jabbar would like to express their thanks to the Ministry of Education, China, for financial assistance through the Chinese Government Scholarship Program.
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