2014 Volume 20 Issue 6 Pages 1153-1164
“Sanhua” plums of 4 cultivars were processed into juice. Pretreatment including heat blanching (HB), microwave blanching (MB) and SO2 were studied on the inactivation of polyphenoloxidase (PPO), peroxidase (POD) and nutrition qualities. Enzymatic clarification, pasteurization and UHT were also investigated on nutrition qualities. Results showed that PPO and POD could be almost inactivated by HB (95°C − 5 min) and MB (700 W − 150 s). Pretreatments extracted more phytochemicals and antioxidant activity (AA) and MB produced higher effect than other two means. The high content of phytochemicals after pretreatment could correlate with the inactivation of PPO and POD. Clarification resulted in a better extraction of nutritional compounds. Pasteurization only slightly increased and UHT remained unchanged or slightly degraded the nutrition qualities. Significant loss of nutritional compounds was found during juicing process. Our findings are useful when evaluating health benefits of plum juice.
Plums are in the genus Prunus of the Rosaceae family and contain several unique health-promoting phytochemicals (Kim et al., 2003). According to prior studies, an intake of plums may be effective to eliminate an initiation or progress of various human diseases due to high content of secondary metabolites such as polyphenolics (Mubarak et al., 2012).
“Sanhua” plum (Prunus salicina Lindl.), which has a purple peel and flesh, is the most important plum cultivar in China and has been greatly planted in Southern China especially in Guangdong province (He et al., 2008). The planting area of “Sanhua” plums is the largest in all of the plum cultivar in China (He et al., 2008). “Sanhua” plums are appreciated for their taste, flavor, yield, fruit weight and percentage of stone but they are very perishable and seasonal, therefore requiring additional industrial procedure to process the seasonal excesses of “Sanhua” plums into new products including juice, wine and prunes for long-term storage.
To process plums into juice, pretreatment, clarification and thermal treatments are necessary steps. Previous studies have found phenolics, flavonoids, anthocyanins and other nutrients are significantly degraded during fruit processing into juice (Kader et al., 1997). The degradation was mainly caused by native oxidative enzymes which could be prevented by pretreatment such as HB or SO2 (Rossi et al., 2003, Lee et al., 2002). To improve the juice yield, enzymatic clarification is thus a requiring step which applies pectinase to lower the viscosity, turbidity and water binding capacity of pectin for easier extraction of components (Tu et al., 2013). Phytochemicals have been studied during juice clarification (Spanos et al., 1992; Skrede et al., 2000, Rinaldi et al., 2013), but few investigations on plum juice (Chang et al., 1994).
To prevent enzymatic changes or inactivate spoilage microorganisms or human pathogens, thermal treatment such as pasteurization or UHT is applied in juice production. However, fruit juice has been considered to have a lower nutritional value than their fresh fruits because thermal treatment may cause undesirable degradation of ascorbic acid, anthocyanins and phenolics. Despite of the disadvantages of thermal treatment, this treatment is still the most widely used and most effective technique to inactivate oxidative enzymes or destroy microorganisms or human pathogen, therefore prolonging the shelf-life of the product.
As we know, AA and other nutrition quality of fruits could be also influenced by genetics. Accordingly the objective of this study was conducted to compare the effect of cultivars (“Dami”, “Xiaomi”, “Jima” and “Zaoshi”), pretreatment (HB, MB and SO2), thermal treatments (pasteurization and UHT) and enzymatic clarification on plum juice in terms of total phenolics (TP), total flavonoids (TF), total ascorbic acid (TAA), total anthocyanin compounds (TAC), and DPPH, ABTS antioxidant activity (AA) in an attempt to establish the optimal treatment condition to reach optimum nutritional properties.
Chemicals LC-MS grade water, methanol and acetonitrile were obtained from Merck (Darmstadt, Germany). Formic acid (99%, purity) was from Acros (Morris Plains, NJ). Folin-Ciocalteau's phenol reagent, ascorbic acid (99%, crystalline), DTT (1,4-dithiothreitol), ABTS, DPPH, and catechin were from Sigma-Aldrich (St. Louis, MO). Cyanidin 3-O-glucoside (Cyn-Glu) was purchased from Polyphenols Laboratories (Hanaveien, Norway). KCl, K2HPO4, Na2CO3, gallic acid, C2H3NaO2, K2S2O8, HCl, NaOH and NaHSO3 were analytical grade from Damao chemicals (Tianjin, China).
Fruit characterization Plums of four cultivars were harvested at commercial harvest maturity from a commercial orchard in Wengyuan County, Guangdong Province of China. Fruits were packed into fiberboard cartons, transferred to the pilot plant of Shenzhen Boston Flavored & Fragrances co., LTD. in 3 h. Fruit maturity was judged on the basis of soluble solids content (SSC) and fruit firmness. Upon arrival, the fruits were screened based on similarity in color, shape and size. Therefore, fruits of uniform size, free from disease or blemishes were used for the experiment. The pH was measured using a pH meter (pHS-3C, Shanghai precise scientific equipment corp., China). Fruit weight was measured using a digital balance (TXB622L, Shimadzu, Japan). Ten fruits constituted one replication.
Fruit color determination The flesh color parameters including L (lightness), a (redness/greenness) and b (yellowness/blueness) were measured with a Hunter Color Meter (PCM™, Colortec, Clinton, NJ, USA) using the 15 mm aperture. The hue angle (°h) and chrome value (C) was calculated from value a and b as reported early by McGuire (1992). Ten fruits constituted one replication.
Fruit firmness determination Fruit firmness was measured using a hand pressure tester (GY-3, Mingrui electronic corp., Guangzhou, China) fitted with an 11 mm cylinder probe. Ten fruits per replication were subjected to firmness testing with each fruit punctured on both the sides at equatorial region. The firmness was expressed as Pa.
Soluble solids content SSC of the plum juice was determined by an Atago PR-101 digital refractometer at 20°C. Results were expressed as °Brix.
Pretreatments Fig.1 illustrates the step for the production of plum juice at laboratory scales. The surface of fruits was rinsed with tap water, cool by dried air. The flesh and stone of plums were separated using a knife and cut into 2 cm thick small pieces. The effect of 3 pretreatments (MB, SO2, and HB) was evaluated. Here control, MB, SO2, or HB samples were assigned as Sc, Sm, Ss and Sh. For Sm samples, the following conditions were applied: every 400 g plum pieces were microwave blanched for 45, 90 or 150 s individually at 700 W by a microwave-oven (P70D20TL-D4, Galanz, Shunde, China). For Ss samples, every 400 g plum pieces were mixed with 0.009%, 0.018% and 0.036% NaHSO3. For Sh samples, every 400 g plum pieces was firstly packaged into a vacuum bag and immersed into the 95°C hot water for 1, 3 or 5 min. For HB and MB, samples were cooled with ice to room temperature after treatments. All of samples after 3 treatments were manufactured using a domestic blender for 1 min at max velocity (WF-A4000, Xuzhong food machinery, China). 10 g plums purée were separated for PPO and POD analysis. The left plum purée were further pressed through bag press under 3 bar to obtain the raw plum juice. The raw plum juice was kept at –20°C for further analysis.
Diagrams of steps for the production of “Sanhua” plum juice.
Enzymatic clarification and thermal treatments The obtained raw juice through MB (700 W – 150 s) was subject to enzymatic clarification with commercial pectinase (0.1 mL/kg, Klerzyme150, DSM Food Specialties, Allonne, France) for 2 h at 45°C followed by centrifuging at 3500 rpm (KDC-12, USTC Chuangxin Co., Hefei, China) for 10 min to obtain raw clarified juice. The enzymatic clarification condition was chosen according to the negative alcohol precipitation test. The clarified and unclarified raw plum juice was subjected to pasteurization (95°C – 90 s) or UHT treatments (125°C – 15 s). Pasteurization was operated by a plate heat-exchanger (model Junior, APV Iberica S.A., Madrid) fed at 1 L/min. UHT was sterilized with a unit (FT74 UHT/HTST, Armfield Corporation, Ringwood, England) fed at 20 L/h. After thermal treatment, juice was hot-filled in the sterilized polyethylene containers. The juice-filled bottles were kept upside down immediately after filling for sterilization of the cap and then turned to their proper position after cooling to room temperature in ice.
Mass balance and phytochemicals recovery during juicing Plums were microwave blanched, crushed, depectinized and centrifuged to obtain clarified raw plum juice as above description. The raw juice and separated pulp was kept –20°C for further analysis.
Oxidative enzymes assay PPO and POD were extracted and assayed by the description by Terefe et al. (2010). Extraction buffer was consisted of 0.2 M sodium phosphate buffer (pH = 6.5) containing of 4% (w/v) PVPP, 1 M NaCl and 1% (v/v) Triton X-100. Crude enzyme extract was prepared by homogenizing 10 g plum purée with 20 mL extraction buffer in ice for 10 min at 9000 rpm (Polytron PT 6100 homogenizer, Kinematica). Supernatant was recovered by centrifuging at 6000 rpm for 5 min at 4°C.
For determining PPO activity, 40 µL crude enzyme extract or extraction buffer as blank was added into 200 µL 70 mM catechol solutions, mixed well with plate shaker. The reaction plate was programmed to read once per minute for 10 min at 420 nm by Victor X3 Multilabel Plate Reader (Perkin-Elmer, Turku, Finland). The operation was carried out at 25°C. 70 mM catechol as substrate was prepared in 50 mM sodium phosphate buffer (pH = 6.5) solution.
For determining POD activity, 30 µL crude enzyme extract or extraction buffer as blank, 200 µL 50 mM phosphate buffer (pH = 6.5), 30 µL of 1% p-phenylenediamine, and 30 µL of 1.5% H2O2, mixed well with plate shaker. 1% p-phenylenediamine was prepared in 50 mM phosphate buffer (pH = 6.5). The reaction plate was programmed to read once per minute for 10 min at 485 nm by Plate Reader. Residual PPO and POD activity is calculated as the ratio between treated and untreated sample.
Determination of nutrition characteristics Extract was prepared with the following procedures for determination of TF, TP, TAC and AA.
For plum purée, 5 g samples were mixed with 10 mL 90% methanol containing 0.5% formic acid, sonicated (Xinyi sonic corp., Ningbo, China) for 10 min, and the supernatant was recovered by centrifuging at 6000 rpm for 5 min. The solid was re-extracted three times under the same conditions until the solution became colorless. The combined supernatants were evaporated with a Rotavapor (RE-52AAA, Jiapeng tech., Shanghai, China) at 40°C to evaporate methanol. The remaining aqueous extract was recovered in 3 mL 100% methanol.
Juice was centrifuged at 10000 rpm for 10 min to obtain the supernatant. The supernatant or the extract passed through a 0.45 nm PTFE syringe filter (Whatman, Clifton, NJ) and kept –20°C for further analysis.
Total flavonoids TF were measured by a spectrophotometric assay as described by Dewanto et al. (2002). The absorbance was determined at 510 nm on a UV-Vis spectrophotometer (UV-1800, Ruili corp., Beijing, China). The TF content was expressed as mg (+)-catechin equivalents per liter juice.
Total phenolics TP were determined as described by Waterhouse (2001). The absorbance was read at 765 nm on a UV-Vis spectrophotometer. TP were expressed as mg of gallic acid equivalents (GAEs) per liter juice.
Identification and quantification of anthocyanins The identification and quantification of anthocyanins were achieved by UPLC-PDA-MSn consisted with a Thermo LTQ XL mass spectrometer with an Accela 1250 binary pump, an autosampler, an Accela PDA detector and an Accela column compartment. Chromatographic separation of anthocyanins was achieved by reversed phase on a Symmetry C18 column (150 mm x 4.6 mm i.d.3.5 µm, Waters Corp, Milford, MA) at a flow rate of 1 mL/min. The mobile phase consisted of a combination of A (5% formic acid in water) and B (5% formic acid in acetonitrile). The gradient elution was applied as follows: 0 – 30 min, 0 – 40%B; 30 – 32 min, 40 – 100%B; 32 – 38 min, re-equilibration to initial conditions. The injection volume was 25 µL and the autosampler kept at 4°C. The column temperature was held at 40°C, and UV-Vis absorption spectra were recorded on-line from 200 – 700 nm using a PDA. UPLC eluate was split approximately 1:5 by a micro splitter valve and interfaced with a Thermo LTQ XL mass spectrometer operated in positive ion electrospray mode. Helium was used as sheath gas and nitrogen as auxiliary gas. Data was collected over an m/z range of 150 – 2000. Data dependent MS2 analysis was performed with a normalized collision energy of 20% to obtain the MS2 spectra of the screened anthocyanins. Most settings were optimized using “tune plus” (Xcalibur 2.07, Thermo Scientific) via automatic tuning by Cyn-Glu standard solution (1 mg/mL in 0.5% formic acid in 90% methanol). The LTQ XL conditions were set as follows: shealth gas: 70; auxiliary gas: 15; spray voltage: 3.5 kV; capillary temperature: 350°C; capillary voltage: 48 V; tube lens: 170 V. Peak tentatively determination was performed by UV-visible characteristics and mass spectral data (m/z, fragmentation patterns). Each anthocyanin compound was expressed as mg Cyn-Glu equivalents per liter juice.
Ascorbic acid TAA content was determined by description of Garzon et al. (2009). For plum purée, 10 g samples were homogenated with 40 mL 0.1% DTT in a Warring blender and for juice, sample was added with 0.1% DTT. The mixture was centrifuged at 6000 rpm for 10 min. The supernatant was collected and pH was adjusted to 5 ∼ 5.2 with 0.1 M NaOH. The reaction was kept at room temperature for 2 h to allow complete reduction of AA and then passed through a 0.45 nm PTFE syringe filter for further analysis.
HPLC analysis was performed in a Shimadzu LC-20AD/T HPLC equipped with a SPD-M20A detector (Kyoto, Japan). The separation was carried out using a Symmetry C18 column. Flow rate is 0.5 mL/min. The running conditions were isocratic elution of 2% KH2PO4 (pH = 2.5) containing 0.1% DTT for 15 min. The injection volume was 20 µL and the autosampler kept at 25°C. The column temperature was held at 40°C, and UV-Vis absorption spectra were recorded on-line at 243 nm.
Antioxidant activity ABTS was determined according to the method of Re et al. (1999). ABTS•+ was produced by mixing 7 mM ABTS stock solution with 2.45 mM K2S2O8. After the K2S2O8 was fully dissolved, the solution was held at room temperature shielded for 16 h before use. ABTS•+ working solution was prepared by diluting ABTS•+ stock solution 7.5 times with 95% ethanol. 25 µL plum juice, ascorbic acid (6.25 mM) with 7 serial dilutions and 50% acetone as blank were added into 200 µL ABTS•+ working solution, mixed well with plate shaker and the absorbance at 734 nm was immediately recorded by Plate Reader. EC50 is expressed as ascorbic acid equivalent (g AA/L).
The DPPH was determined according to the method of Cheng et al. (2006). The DPPH stock solution (0.625 mM) was prepared monthly and kept at 4°C in dark. The 0.208 mM fresh DPPH working solution was made daily by diluting the stock solution in 50% acetone. 100 µL 0.208 mM fresh DPPH solution was mixed with 100 µL plum juice, ascorbic acid (6.25 mM) with 7 serial dilutions and 50% acetone as blank and stand at ambient temperature for 40 min at dark. The absorbance at 515 nm was recorded by Plate Reader. EC50 is expressed as ascorbic acid equivalent (g AA/L).
Physico-chemical parameters of plums from 4 cultivars Physico-chemical parameters including pH, average fruit weight, SSC, fruit firmness and color were analyzed. Table 1 showed different cultivars differed in quality variables, with a range of 10.3 to 12.1% SSC, 45.6 – 58.8 g fruit weight, 3.4 – 4.0 N firmness, and 24.9 – 27.0 hue values. Fruits from 4 cultivars showed very similar pH.
| Dami | Xiaomi | Jima | Zaoshi | |
|---|---|---|---|---|
| L | 21.3a | 20.0a | 22.5b | 21.8a |
| a | 26.9a | 24.6b | 27.2a | 24.7b |
| b | 12.7a | 11.4b | 13.5c | 12.6a |
| C | 29.7a | 27.1b | 30.4a | 27.7b |
| °h | 25.3a | 24.9a | 26.4a | 27.0a |
| pH* | 3.3a | 3.2a | 3.5a | 3.3a |
| Firmness (N) | 4.0a | 3.4b | 3.9a | 3.6a |
| Soluble solids content (°Brix )* | 10.3a | 11.2a | 11.7a | 12.1b |
| Fruit weight (g) | 58.8a | 45.6b | 48.6b | 55.4a |
*: pH and soluble solids content was determined with plum juice. The same letter in the same parameter indicates homogeneous groups established by the ANOVA (P < 0.05) C = (a*2 + b*2) 1/2; °h = arctangent (b/a)
Characterization of anthocyanins A representative HPLC chromatogram of “Sanhua” plum juice anthocyanins of “Dami” was shown in Fig.2. Complete baseline separation was achieved. Acquired at 520 nm, 2 distinct anthocyanin peaks were detected.
HPLC chromatogram of “Sanhua” plum juice anthocyanins which was acquired at 520 nm. Mobile phase: A-5% formic acid in water; B-5% formic acid in acetonitrile. The gradient elution was applied as follows: 0 – 30 min, 0 – 40%B; 30 – 32 min, 40 – 100%B; 32 – 38 min, re-equilibration to initial conditions. 1- cyanidin 3-O-glucoside; 2 - cyanidin 3-O rutinoside.
Peak 1 revealed a molecular ion at m/z 449 and a fragment ion at m/z 287, indicating it was a cyanidin hexose. Comparing to the standard, peak 1 was confirmed as Cyn-Glu. Peak 2 revealed molecular ions at m/z 595 and major fragment at m/z 287. The neutral loss of 308 corresponded to one molecule of rutinoside. Thus peak 2 was identified as cyanidin 3-O-rutinoside (Cyn-Rut). It was previously reported that purple plum cultivar contained significant amount of Cyn-Glu and Cyn-Rut and Cyn-Rut was a major form (Tomás-Barberán et al., 2001, Raynal et al., 1989).
Effect of pretreatments on POD and PPO The inactivation of PPO and POD in plums was studied with the HB (95°C, 1 –5 min), MB (700 W, 45 s – 150 s) or SO2 (0.009% – 0.036%). Results were shown in Table 2. Pretreatments had various influence on the residual activity of PPO and POD in 4 cultivars. At the longest treatment time of HB and MB, POD and PPO activity could be almost completely inactivated.
| Cultivar | Treatments | PPO residual activity (%) | POD residual activity (%) | |
|---|---|---|---|---|
| Heat Blanching |
“Dami” | 95°C-1 min | 44 | 48 |
| 95°C-3 min | 17 | 18 | ||
| 95°C-5 min | 5 | 4 | ||
| “Xiaomi” | 95°C-1 min | 45 | 39 | |
| 95°C-3 min | 16 | 11 | ||
| 95°C-5 min | 3 | 1 | ||
| “Jima” | 95°C-1 min | 27 | 31 | |
| 95°C-3 min | 15 | 16 | ||
| 95°C-5 min | 6 | 7 | ||
| “Zaoshi” | 95°C-1 min | 44 | 40 | |
| 95°C-3 min | 21 | 16 | ||
| 95°C-5 min | 8 | 4 | ||
| Microwave Blanching |
“Dami” | 45s | 32 | 37 |
| 90s | 21 | 15 | ||
| 150s | 3 | 0 | ||
| “Xiaomi” | 45s | 41 | 40 | |
| 90s | 25 | 16 | ||
| 150s | 5 | 3 | ||
| “Jima” | 45s | 36 | 30 | |
| 90s | 26 | 10 | ||
| 150s | 3 | 0 | ||
| “Zaoshi” | 45s | 26 | 41 | |
| 90s | 9 | 13 | ||
| 150s | 5 | 0 | ||
| SO2 | “Dami” | 0.009% | 79 | 50 |
| 0.018% | 36 | 24 | ||
| 0.036% | 19 | 12 | ||
| “Xiaomi” | 0.009% | 58 | 43 | |
| 0.018% | 34 | 29 | ||
| 0.036% | 18 | 15 | ||
| “Jima” | 0.009% | 64 | 43 | |
| 0.018% | 21 | 24 | ||
| 0.036% | 19 | 13 | ||
| “Zaoshi” | 0.009% | 54 | 46 | |
| 0.018% | 24 | 20 | ||
| 0.036% | 17 | 20 |
For 3 types of treatments, it was observed that PPO and POD residual activity decreased with the extending of blanching time or the increasing of SO2 concentration. For “Dami” and “Zaoshi”, the order of PPO and POD residual activity is SO2 (0.036%) > heat (95°C – 5 min) > microwave (150s). For “Xiaomi”, the order is SO2 (0.036%) > microwave (150s) > heat (95°C – 5 min). For “Jima”, the order of PPO is SO2 (0.036%) > microwave (150s) > heat (95°C – 5 min) while the order of POD is SO2 (0.036%) > heat (95°C – 5 min) > microwave (150s). These results indicate that 3 treatments had a significant effect on PPO and POD activity and this effect was higher with HB or MB.
Early studies have investigated the inactivation of PPO and POD in plums, however, these 4 cultivars have never been studied. As we know, thermostability of PPO and POD depends on the plant species and cultivar. For instance, Siddiq et al. (1994) have found plum PPO could be completely inactivated at 65°C – 20 min. The same author also found PPO in Stanley plums could be completely inactivated at 75°C – 5 min (Siddiq et al., 1992). From the report of Weemaes et al. (1998), PPO from plums seemed to have at least two isoenzymes and thermal inactivation followed first-order decay. HB is always considered as an efficient means for enzyme inactivation; however, product quality is significantly destroyed. MB as a minimal processing is an interesting alternative. Early studies have compared HB and MB (Soysal and Soylemez, 2005; Matsui et al., 2007, Matsui et al., 2008) and the authors concluded MB could be better than HB due to high TAA retention. From our study, MB shortened processing time when achieving the same effect as HB. SO2 was used to inhibit fruit and vegetables enzymatic browning by inhibiting PPO and POD activity (Rossi et al., 2003; Lee et al., 2002, Sayavedra-Soto et al., 1986).
Changes in nutrition qualities during pretreatments Table 3 shows the change in AA, TP, TF, TAC and TAA from plum juice of 4 cultivars during 3 types of pretreatments. TP, TAC and TF were increased with the treatment time or increasing of SO2 concentration. The highest phenolics, anthocyanins, flavonoids were found in the MB for 150s. The following description of value was in the order of “Dami”, “Xiaomi”, “Jima” and “Zaoshi”.
| Cultivar | Treatments | Total Flavonoids (mg catechin/L) | Total Phenolics (mg GAEs/L) | Total Anthocyanins (mg Cyn-Glu/L) | Total Ascorbic Acid (mg/L) | |||
|---|---|---|---|---|---|---|---|---|
| Cyn-Glu | Cyn-Rut | Total | ||||||
| Heat Blanching | “Dami” | Untreated | 221 | 2687 | 18 | 61 | 79 | 10.3 |
| 95°C-1 min | 278 | 3709 | 30 | 89 | 119 | 11.9 | ||
| 95°C-3 min | 323 | 3924 | 26 | 98 | 124 | 9.8 | ||
| 95°C-5 min | 351 | 3973 | 38 | 114 | 152 | 8.1 | ||
| “Xiaomi” | Untreated | 272 | 2161 | 15 | 53 | 67 | 13.6 | |
| 95°C-1 min | 340 | 3451 | 29 | 91 | 120 | 13.9 | ||
| 95°C-3 min | 377 | 3817 | 29 | 99 | 128 | 11.8 | ||
| 95°C-5 min | 422 | 3743 | 37 | 101 | 138 | 11.6 | ||
| “Jima” | Untreated | 283 | 2454 | 22 | 67 | 89 | 15.0 | |
| 95°C-1 min | 354 | 2839 | 31 | 92 | 123 | 17.3 | ||
| 95°C-3 min | 383 | 3068 | 36 | 101 | 137 | 15.5 | ||
| 95°C-5 min | 469 | 3833 | 45 | 119 | 164 | 14.7 | ||
| “Zaoshi” | Untreated | 251 | 2741 | 22 | 63 | 85 | 10.0 | |
| 95°C-1 min | 312 | 2980 | 31 | 82 | 113 | 8.6 | ||
| 95°C-3 min | 401 | 3032 | 33 | 96 | 129 | 7.6 | ||
| 95°C-5 min | 391 | 3808 | 43 | 124 | 167 | 6.7 | ||
| Microwave Blanching | “Dami” | 45s | 336 | 3278 | 32 | 91 | 123 | 12.6 |
| 90s | 376 | 3798 | 39 | 110 | 149 | 11.9 | ||
| 150s | 452 | 4296 | 46 | 127 | 173 | 10.3 | ||
| “Xiaomi” | 45s | 338 | 3214 | 35 | 97 | 132 | 14.3 | |
| 90s | 380 | 3068 | 41 | 111 | 152 | 12.8 | ||
| 150s | 468 | 4029 | 46 | 123 | 169 | 12.2 | ||
| “Jima” | 45s | 395 | 3310 | 37 | 104 | 141 | 17.8 | |
| 90s | 463 | 3487 | 43 | 120 | 163 | 17.0 | ||
| 150s | 506 | 4134 | 47 | 132 | 179 | 15.5 | ||
| “Zaoshi” | 45s | 367 | 3272 | 34 | 94 | 128 | 10.0 | |
| 90s | 439 | 3559 | 32 | 104 | 136 | 9.9 | ||
| 150s | 512 | 4104 | 47 | 140 | 187 | 9.9 | ||
| SO2 | “Dami” | 0.009% | 230 | 2659 | 27 | 102 | 129 | 14.0 |
| 0.018% | 225 | 3016 | 35 | 103 | 138 | 12.5 | ||
| 0.036% | 254 | 3211 | 31 | 112 | 143 | 16.1 | ||
| “Xiaomi” | 0.009% | 251 | 2102 | 29 | 91 | 120 | 13.0 | |
| 0.018% | 291 | 2232 | 29 | 98 | 127 | 15.3 | ||
| 0.036% | 316 | 2409 | 37 | 103 | 140 | 15.7 | ||
| “Jima” | 0.009% | 247 | 2625 | 33 | 101 | 134 | 18.5 | |
| 0.018% | 289 | 2668 | 35 | 104 | 139 | 20.1 | ||
| 0.036% | 318 | 2883 | 40 | 112 | 152 | 21.2 | ||
| “Zaoshi” | 0.009% | 263 | 2581 | 33 | 87 | 120 | 9.5 | |
| 0.018% | 218 | 3085 | 32 | 93 | 125 | 9.1 | ||
| 0.036% | 301 | 3060 | 39 | 103 | 142 | 10.7 | ||
Cyn-Glu, cyanidin 3-O-glucoside; Cyn-Rut, cyanidin 3-O-rutinoside.
Comparing 5 min HB to control, TP was increased about 48%, 73%, 56% and 39%. Comparing 150 s MB to control, TP were increased about 60%, 86%, 68% and 50%. However, the improvement of TP after SO2 treatment was not as much as after blanching. Comparing the samples treated with 0.036% NaHSO3 to control, TP were increased only about 20%, 11%, 17% and 12%.
Comparing 95°C – 5 min to control, TAC was increased about 92%, 106%, 84% and 96%. Comparing 150 s MB to control, TAC was increased about 119%, 152%, 101% and 120%. TAC was increased about 81%, 109%, 71% and 67% after 0.036% NaHSO3 treatment. Although HB, MB and SO2 treatment extracted more anthocyanins, the profile of anthocyanin almost remained unchanged that Cyn-Rut was the predominant compound accounting for 72% – 79%.
Comparing 95°C – 5 min to control, TF were increased about 59%, 55%, 66% and 56%. Comparing 150 s MB to control, TF were increased about 105%, 72%, 79% and 104%. However, TF were only increased about 12% to 20% after 0.036% NaHSO3 treatment. This observation is similar with TP which was also minor increase after SO2 treatment.
The effect of pretreatments on DPPH and ABTS antioxidant activity was shown in Table 4. The average increase of DPPH is 59%, 36%, 17% while the average increase of ABTS is 38%, 45% and 23% for HB, MB and SO2. Overall, regardless of cultivars and AA assay method, 3 types of pretreatments could significantly improve AA especially HB and MB produced higher DPPH and ABTS activity.
| Cultivar | Treatments | ABTS (g VC/L) | DPPH (g VC/L) | |
|---|---|---|---|---|
| Heat Blanching | “Dami” | Untreated | 4.7 | 2.7 |
| 95°C-1 min | 5.8 | 3.2 | ||
| 95°C-3 min | 6.2 | 3.1 | ||
| 95°C-5 min | 6.5 | 3.6 | ||
| “Xiaomi” | Untreated | 4.3 | 2.0 | |
| 95°C-1 min | 5.6 | 4.5 | ||
| 95°C-3 min | 5.4 | 4.7 | ||
| 95°C-5 min | 5.6 | 4.5 | ||
| “Jima” | Untreated | 3.9 | 2.5 | |
| 95°C-1 min | 4.2 | 2.7 | ||
| 95°C-3 min | 5.0 | 2.6 | ||
| 95°C-5 min | 5.1 | 3.3 | ||
| “Zaoshi” | Untreated | 6.5 | 3.5 | |
| 95°C-1 min | 8.3 | 4.6 | ||
| 95°C-3 min | 8.8 | 4.9 | ||
| 95°C-5 min | 10.0 | 5.1 | ||
| Microwave Blanching | “Dami” | 45s | 5.7 | 2.8 |
| 90s | 6.0 | 2.7 | ||
| 150s | 6.1 | 3.7 | ||
| “Xiaomi” | 45s | 4.8 | 2.4 | |
| 90s | 5.0 | 4.0 | ||
| 150s | 5.5 | 4.3 | ||
| “Jima” | 45s | 4.2 | 2.5 | |
| 90s | 4.3 | 2.8 | ||
| 150s | 4.6 | 3.2 | ||
| “Zaoshi” | 45s | 7.2 | 3.8 | |
| 90s | 7.5 | 4.1 | ||
| 150s | 8.9 | 4.4 | ||
| SO2 | “Dami” | 0.009% | 5.4 | 2.2 |
| 0.018% | 5.8 | 3.1 | ||
| 0.036% | 5.5 | 3.1 | ||
| “Xiaomi” | 0.009% | 5.0 | 2.7 | |
| 0.018% | 5.0 | 2.8 | ||
| 0.036% | 5.5 | 2.6 | ||
| “Jima” | 0.009% | 4.1 | 2.8 | |
| 0.018% | 4.5 | 2.9 | ||
| 0.036% | 4.7 | 3 | ||
| “Zaoshi” | 0.009% | 6.6 | 3.8 | |
| 0.018% | 7.4 | 4.1 | ||
| 0.036% | 7.1 | 3.8 |
The increase of phenolics, flavonoids, anthocyanins and AA after HB or MB treatment could correlate with the inactivation of PPO and POD. As we know, PPO and POD are mainly responsible for browning reactions which involved the conversion of phenolics to quinones and further polymerization to produce browning pigments (Kader et al., 1997). Phytochemicals always bound with other compounds or in cell structures and heat not only destroy oxidative enzyme but also disrupt the cell membranes or change cell texture to increase the release of phytochemicals into medium. Previous studies have found heat could increase the extractability of phytochemicals (Howard et al., 1999; Leong and Oey, 2012). In this study, TAC was improved remarkably after SO2 treatment. This finding agreed with early studies who found SO2 could preserve anthocyanin and improve color quality of final products (Skrede et al., 2000; Lee et al., 2002; Rommel et al., 1992; Rossi et al., 2003; Fang et al., 2006).
Compared to control, HB slightly decreased while MB and SO2 slightly increase or maintain TAA. Early studies have reported TAA is sensitive to pH, heat, oxygen, metal and enzymes (Gregory, 1996). TAA can be rapidly oxidized to dehydroascorbic acid and further hydrolyzed to diketogulonic acid with oxygen or ascorbic acid oxidase. This study agreed with the report of Igual et al. (2010) who found greater retention of TAA after MB than heat treatment. Thus it would be preferable to apply MB as pretreatment method.
Changes in nutrition qualities during enzymatic clarification and thermal treatments Table 5 and Table 6 showed the changes in AA, TP, TF, anthocyanins and TAA during enzymatic clarification and thermal treatments.
| Cultivar | Treatments | Total Flavonoids (mg catechin/L) | Total Phenolics (mg GAEs/L) | Total Anthocyanins (mg Cyn-Glu/L) | Total Ascorbic Acid (mg/L) | |||
|---|---|---|---|---|---|---|---|---|
| Cyn-Glu | Cyn-Rut | Total | ||||||
| Unclarified Juice | “Dami” | Untreated | 452 | 4296 | 46 | 127 | 173 | 10.3 |
| Pasteurization | 557 | 5045 | 46 | 139 | 185 | 9.4 | ||
| UHT | 413 | 3896 | 33 | 116 | 149 | 9.7 | ||
| “Xiaomi” | Untreated | 468 | 4029 | 46 | 123 | 169 | 12.2 | |
| Pasteurization | 569 | 648 | 47 | 128 | 175 | 11.3 | ||
| UHT | 410 | 3876 | 36 | 116 | 152 | 11.6 | ||
| “Jima” | Untreated | 506 | 4134 | 47 | 132 | 179 | 15.5 | |
| Pasteurization | 603 | 4852 | 48 | 146 | 194 | 14.8 | ||
| UHT | 499 | 4018 | 37 | 115 | 152 | 14.0 | ||
| “Zaoshi” | Untreated | 512 | 4104 | 47 | 140 | 187 | 9.9 | |
| Pasteurization | 631 | 4856 | 52 | 149 | 202 | 9.3 | ||
| UHT | 480 | 4011 | 41 | 128 | 169 | 10.1 | ||
| Clarified Juice | “Dami” | Untreated | 624 | 5637 | 50 | 165 | 214 | 9.5 |
| Pasteurization | 790 | 6370 | 57 | 173 | 230 | 9.1 | ||
| UHT | 573 | 5274 | 36 | 138 | 174 | 10.2 | ||
| “Xiaomi” | Untreated | 604 | 5440 | 58 | 168 | 226 | 11.2 | |
| Pasteurization | 754 | 5783 | 52 | 185 | 237 | 10.7 | ||
| UHT | 614 | 5110 | 45 | 148 | 193 | 10.2 | ||
| “Jima” | Untreated | 664 | 5244 | 52 | 181 | 233 | 15.4 | |
| Pasteurization | 741 | 6031 | 64 | 173 | 237 | 15.2 | ||
| UHT | 632 | 4925 | 45 | 140 | 185 | 14.6 | ||
| “Zaoshi” | Untreated | 633 | 5525 | 60 | 177 | 237 | 9.6 | |
| Pasteurization | 778 | 6627 | 64 | 182 | 246 | 10.7 | ||
| UHT | 650 | 5285 | 55 | 149 | 204 | 9.8 | ||
Cyn-Glu, cyanidin 3-O-glucoside; Cyn-Rut, cyanidin 3-O-rutinoside.
| Cultivar | Treatments | ABTS (g VC/L) | DPPH (g VC/L) | |
|---|---|---|---|---|
| Unclarified Juice | “Dami” | Untreated | 6.1 | 3.7 |
| Pasteurization | 7.1 | 4.3 | ||
| UHT | 6.1 | 3.8 | ||
| “Xiaomi” | Untreated | 5.5 | 4.3 | |
| Pasteurization | 5.3 | 6.3 | ||
| UHT | 4.9 | 4.3 | ||
| “Jima” | Untreated | 4.6 | 3.2 | |
| Pasteurization | 7.7 | 5.0 | ||
| UHT | 5.3 | 3.7 | ||
| “Zaoshi” | Untreated | 8.9 | 4.4 | |
| Pasteurization | 9.8 | 5.1 | ||
| UHT | 8.8 | 3.9 | ||
| Clarified Juice | “Dami” | Untreated | 9.1 | 5.1 |
| Pasteurization | 11.2 | 6.0 | ||
| UHT | 9.6 | 4.8 | ||
| “Xiaomi” | Untreated | 7.0 | 6.5 | |
| Pasteurization | 9.7 | 9.6 | ||
| UHT | 8.5 | 7.9 | ||
| “Jima” | Untreated | 6.6 | 5.0 | |
| Pasteurization | 8.1 | 5.7 | ||
| UHT | 7.3 | 4.7 | ||
| “Zaoshi” | Untreated | 12 . 8 | 5.6 | |
| Pasteurization | 17 . 9 | 6.6 | ||
| UHT | 14.3 | 4.6 |
After pasteurization, TP from four cultivars were slightly higher than the raw juice which was enhanced around 17%, 14% for unclarified and clarified samples. At UHT condition, TP in all treated samples remained no significant different from control regardless of cultivars. This result agreed with Fang et al. (2006), who found the UHT had no effect on TP in bayberry juice. Clarification significant increased TP around 31%, 35%, 27% and 35% for 4 cultivars. Similar results were found by Rinaldi et al. (2003) that about two-fold increase of phenolics in pomegranate juice after clarification.
Clarification also significantly improved TAC around 24%, 34%, 30% and 27% for 4 cultivars. At pasteurization condition, all heat-treated samples contained no more anthocyanins than the control. At UHT condition, there were substantial losses of 12% and 17% for unclarified and clarified juice. This result was similar with the findings of Fang et al. (2006), who reported only 6% anthocyanin losses after UHT operation in bayberry juice. In addition, heat did not change the profile of anthocyanin compounds.
Clarification significantly improved TF around 38%, 29%, 31% and 24% for 4 cultivars. At pasteurization condition, TF were significantly higher than control, which were enhanced around 22% and 21% for unclarified and clarified samples. At UHT, TF in all samples remained no significant difference from control. Our findings agreed with previous report that pasteurization had no effect on TF for tomato (Dewanto et al., 2002).
2 High-throughput assays (ABTS and DPPH) were applied here to determine the AA. As we know, different AA assay may influence the experimental results, and thus more radical systems are required to investigate the AA. From Table 6, after pasteurization treatments, AA was significantly higher than control regardless of assay methods and various cultivar exhibited different degree of increasing. Our findings were in accordance with early report that thermal processing significantly improved the AA of tomato juice (Dewanto et al., 2002). However, for UHT treatment, this treatment slightly increased or maintained AA regardless of AA assay method. Following the similar trend as TF, TAC and TP, clarification improved the AA that the average of increase is 41% and 43% for ABTS and DPPH. According to previous reports, phenolics including flavonoids and anthocyanins are the most important contributor to AA (Floegel et al., 2011; Fiore et al., 2005). The explanation of the increase of phytochemicals and AA after clarification is that pectinase helped to decrease pectin content thus increase yield and liberate antioxidants from matrix to make it more accessible during extraction. Also samples after pasteurization contained higher phytochemicals and AA than control. As we early discussed, thermal processing helped to release more bound phenolics from the cellular constituents as well as inactivated the oxidative and hydrolytic enzymes. UHT may achieve the similar goal in terms of releasing the bound phenolics and inactivate the enzymes, however, antioxidant compounds loss may also accompany this process due to the extreme treatment. The results were in agreement with early report that pasteurized samples conserved a higher AA than sterilized ones (Fiore et al., 2005).
UHT and pasteurization treatment did not cause significant loss of TAA in clarified or unclarified juice (Table 5). This result was similar with Iversen (1999), who found minor loss of TAA in black currant after pasteurization. However, great losses of TAA were found in tomato juice (Dewanto et al., 2002), grapefruit juice (Igual et al., 2010) and fruit beverages (Cilla et al., 2012) during thermal treatments. This difference could be due to the different heat treatment conditions and different food matrix (Cilla et al., 2012). The stability of TAA depended on food matrix, pH, oxygen, other antioxidant compounds and heat treatment conditions (Cilla et al., 2012; Pérez-Vicente et al., 2002; Cilla et al., 2011). Ascorbic acid is commonly used as a nutritional marker in industry to ensure the high quality of final product. Actually, ascorbic acid shows great thermal stability in this study possible due to the low pH of plums juice. Clarification has no effect on TAA in juice which was in accordance with the finding by Kaur et al. (2011).
Mass balance and phytochemical recovery during juice processing The juice yield was 81 – 85% of the starting material of 4 cultivars plums. Table 7 presents TP, flavonoids, anthocyanins, TAA, DPPH and ABTS distribution in juice and pulp. Significant loss of phytochemical was found during juice processing including 19 – 33% of phenolics, 27 – 38% of flavonoids, 14 – 28% of anthocyanins, 19 – 32% of TAA, 18 – 38% of DPPH and 22 – 32% of ABTS. The low recovery was also observed by previous studies (Skrede et al., 2000; Lee et al., 2002; Fang et al., 2006; Iversen, 1999). Possible explanation could be that residual oxidative enzyme in press cake degraded the phytochemicals. MB could break down the pulp and could have increased extraction of phytochemicals, but still a substantial amount remained in the press cake. Another possible explanation could be the low recovery of phytochemicals from press cake during extraction for further analysis. In other words, the actual phytochemicals content in presscakes could have been higher. Further study needed to validate the phytochemical extraction method from press cake. Press cake contained significant amounts of phytochemicals which could be a rich source of natural antioxidant.
| Cultivar | Portion | Total Phenolics (%) | Total Flavonoids (%) | Total Anthocyanins (%) | Total Ascorbic Acid (%) | DPPH (%) | ABTS (%) |
|---|---|---|---|---|---|---|---|
| “Dami” | Juice | 68 | 60 | 64 | 65 | 56 | 66 |
| Pulp | 26 | 22 | 24 | 27 | 26 | 22 | |
| Loss | 26 | 28 | 16 | 29 | 18. | 22 | |
| “Xiaomi” | Juice | 63 | 62 | 65 | 66 | 60 | 65 |
| Pulp | 28 | 24 | 21 | 26 | 22 | 22 | |
| Loss | 24 | 34 | 14 | 32 | 27 | 32 | |
| “Jima” | Juice | 78 | 64 | 71 | 59 | 65 | 77 |
| Pulp | 16 | 19 | 20 | 23 | 17 | 11 | |
| Loss | 19 | 27 | 28 | 19 | 38 | 26 | |
| “Zaoshi” | Juice | 75 | 68 | 65 | 72 | 61 | 55 |
| Pulp | 18 | 14 | 23 | 20 | 21 | 34 | |
| Loss | 33 | 38 | 22 | 28 | 23 | 31 |
From our study, one could conclude that pretreatment could significantly increase the TAC, TF, TP and DPPH, ABTS of plum juice. MB resulted in better retention of phytochemicals than other two means. PPO and POD residual activities were significantly decreased with the extending treatment time of HB and MB and increasing of SO2 concentration. The high content of phytochemicals and AA could correlate with the inactivation of PPO and POD. Enzymatic clarification increased nutrition value by extracting more anthocyanins (29%), flavonoids (31%), phenolics (32%) and improving the antioxidant activity (DPPH of 43% and ABTS 41%). Pasteurization treatment only slightly increased and UHT treatment remained unchanged or slightly degraded the nutrition qualities. Significant loss of phenolics (19 – 33%), flavonoids (27 – 38%), anthocyanins (14 – 28%), ascorbic acid (19 – 32%), DPPH (18 – 38%) and ABTS (22 – 31%) were found during juicing process. Our study contributed to the understanding of the relationships between pretreatment, heat treatment, enzymatic clarification and nutrition value of “Sanhua” plum juice. Further study also needed to develop a new technique for higher recovery of phytochemicals into juice to improve the nutrition quality of final product.
Acknowledgments This study was financially supported by the “the Fundamental Research Funds for the Central Universities” of China (Grant No. 21612315) and Natural Science Foundation of Guangdong Province (Grant No, S2012040006809).
Heat blanching
MBMicrowave blanching
PPOPolyphenoloxidase
PODPeroxidase
AAAntioxidant activity
TPTotal phenolics
TFTotal flavonoids
TAATotal ascorbic acid
TACTotal anthocyanin compounds
DTT1,4-Dithiothreitol
Cyn-GluCyanidin 3-O-glucoside
SSCSoluble solids content
GAEsGallic acid equivalents
Cyn-RutCyanidin 3-O-rutinoside
