The Impact of Maturity Stage on Cell Membrane Integrity

Article

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The Impact of Maturity Stage on Cell Membrane Integrity and Enzymatic Browning Reactions in High Pressure Processed Peaches (Prunus persica) † § ‡ † ⊥ † Chukwan Techakanon, , Thomas M. Gradziel, Lu Zhang, , and Diane M. Barrett*, † Department of Food Science and Technology, University of CaliforniaDavis, One Shields Avenue, Davis, California 95616, United States § Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, 31 Makham Tia, Muang Surat Thani, Suratthani 84000, Thailand ‡ Department of Pomology, University of CaliforniaDavis, One Shields Avenue, Davis, California 95616, United States ⊥ Department of Applied Biology & Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China

ABSTRACT: Fruit maturity is an important factor associated with final product quality, and it may have an effect on the level of browning in peaches that are high pressure processed (HPP). Peaches from three different maturities, as determined by firmness (M1 = 50−55 N, M2 = 35−40 N, and M3 = 15−20 N), were subjected to pressure levels at 0.1, 200, and 400 MPa for 10 min. The damage from HPP treatment results in loss of fruit integrity and the development of browning during storage. Increasing pressure levels of HPP treatment resulted in greater damage, particularly in the more mature peaches, as determined by shifts in ff transverse relaxation time (T2) of the vacuolar component and by light microscopy. The discoloration of peach slices of di erent maturities processed at the same pressure was comparable, indicating that the effect of pressure level is greater than that of maturity in the development of browning. KEYWORDS: high pressure, 1H NMR, maturity, peaches, enzymatic browning

1. INTRODUCTION suggested by Cantos et al.,9 the importance of cellular integrity High pressure processing (HPP) is a novel advanced process and accessibility of the enzyme to its substrate may be the being extensively studied because of its ability to retain primary factor in the development of enzymatic browning. This study was designed to investigate this hypothesis. In intact products with natural attributes while inducing destruction of 10 microorganisms and modifying enzyme activity. HPP is a plants, PPOs are localized in plastids and remain physically separated from phenolic substrates, which are in the potential alternative method for peach preservation, besides 11,12 canning and freezing, because it can provide a new product with vacuole. However, once the plant loses integrity in its cell novel crispy and aromatic characteristics. The quality of HPP walls and membranes by either cutting, senescence, or physical stress, enzymes and substrates are allowed to mix and the preserved fruits can, however, change during storage due to the 1 ff use of elevated pressure levels, which may induce changes in browning reaction occurs as a consequence. The e ect of HPP on PPOs has been previously studied either in the form of a membrane permeability and trigger loss of subcellular fi 13 compartmentalization. After loss of cellular integrity in fruits, partially puri ed extract or measured directly from the plant material after processing.14 One goal of the current study is to substrates are able to mix with enzymes, as occurs in the case of ff enzymatic browning, which is undesirable to consumers. compare the e ect of HPP on PPOs, both in an extract and as In a large number of fruit and vegetable crops, losses are a found in a plant food matrix. 1 Fruit ripening is an irreversible developmental process that result of postharvest deteriorative reactions, in which fi 15 enzymatic browning reaction causes the second largest quality involves speci c biochemical and physiological attributes. 2 Peaches are a climacteric fruit, in which ripening is associated loss. Enzymatic browning in fruits is initiated by the oxidation fi of phenolic compounds, mainly by polyphenol oxidases with the production of ethylene and a signi cant increase in 3 4 cellular respiration. Ethylene has an important role in all stages (PPOs) with a partial role of peroxidases (PODs). The ff product of this reaction is quinone, which further undergoes of peach ripening; this plant hormone sets o the activity of enzymes responsible for fruit softening, ripening, color nonenzymatic processes to form melanins, brown products on − development, and sugar content.16 19 The ripening process the cut surfaces of fruit that are exposed to oxygen. Most 20 21 studies of enzymatic browning have measured the activity of results in elevated sugar-to-acid ratios, decreases in acidity PPOs and the concentration of their total phenolic substrates and correlated these to the degree of browning, for example, Received: May 18, 2016 − apples5 and peaches.6 8 However, in preliminary studies it was Revised: August 23, 2016 determined they neither correlated well to the difference in Accepted: August 24, 2016 lightness in stored HPP treated peaches. Therefore, as Published: August 24, 2016

(due to decreases in malic and citric acid), changes in ground TA.XT2 Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK): color of the skin, and an increase in volatile compounds.22 (1) maturity 1 (M1), which was 50−55 N; (2) maturity 2 (M2), 35− 40 N; and (3) maturity 3 (M3), 15−20 N. The three maturity stages, Measurement of respiration rate, ethylene, sugar, and acid all − involve destructive evaluation; however, the use of firmness as a M1 M3, were harvested at 312, 314, and 316 days after blossom formation, respectively. Approximately three peaches per maturity maturity indicator can be nondestructive. All of these changes stage per processing replicate were hand peeled and cut into create desirable fruit characteristics and make peaches more approximately 3 cm thick slices before being vacuum packed in palatable to the consumer. Fruit maturity is one of the most polyethylene bags (4 mil vacuum pouches, Ultrasource, North Kansas important factors associated with the quality of the final City, MO, USA). Each bag contained three peach slices of the same processed product; therefore, selecting the right maturity stage maturity, classified by firmness, one from each of three different fruits, is critically important and processors need to be concerned with all of which were to be analyzed using the same analytical method. this. However, very few researchers have focused on the effect Peach extracts taken from each maturity stage were vacuum packed in 23,24 polyethylene bags of approximately 2 mL. On each replicate day of of fruit maturity on the quality of HPP treated products. fi This study, therefore, explores methods to measure changes in processing, six packages (one for each of the ve analytical methods plus a control) for each of the three maturity levels were processed at cell integrity of fruits from different initial maturities following 1 each of the three pressures: 0.1, 200, and 400 MPa. The same fruit was the high pressure process. Nuclear magnetic resonance ( H analyzed for all analytical parameters, for example, difference in NMR) water proton relaxometry is a nondestructive measure- lightness, the paramagnetic study using NMR, PPO activity, total ment that detects physiological changes of water in a sample. phenols, and light microscopy. The control sample was unprocessed This method has undergone continuous development and has sliced peaches in a vacuum package (approximately 0.1 MPa). All of been applied in a wide range of plant studies, for example, plant the packaged samples were kept at ambient temperature (22 ± 2 °C) freezing,25 HPP treated strawberries,26 tomato pericarp for 30 min prior to HPP treatment. ripening,24 and identification of black heart in pomegranates.27 2.2. High Pressure Processing (HPP). The packages containing − three different maturity stages of peaches were processed at 0.1 The proton spin spin (T2) relaxation time is usually in the range of seconds to milliseconds on the 1T aspect system. The (control group was at a standard atmosphere, 101.3 kPa or 0.1013 ff MPa), 200, and 400 MPa for 10 min in a high pressure processing unit value is related to properties of water in di erent locations in (2L-700 Lab system, Avure Technologies Inc., Kent, WA, USA). The the tissue, to total water content in both free and bound form, pressure levels used in this experiment were justified on the basis of 28 and to the interaction of water with macromolecules. In plant the rupture of the plant cellular membranes at 200 MPa and the cells, the plasmalemma (membrane surrounding the cytoplasm) inactivation of microorganisms at 400 MPa, both of which were and tonoplast (membrane surrounding the vacuole) provide observed in preliminary experiments. The high pressure unit had a 2.0 the primary control of permeability between their compart- L vessel, and 600 MPa was the maximum pressure level; the pressurizing medium was water. The initial high pressure unit ments. Once cell damage occurs as a consequence of high ° pressure treatment, decompartmentalization of membranes is temperature (Ti) was approximately 23 C. The maximum temper- 29 ature in the high pressure chamber depended on the target pressure triggered, leading to an increase in cell permeability. This ° ff and was 28 and 32 C for the treatment at 200 and 400 MPa, induces an exchange of water between di erent cell compart- respectively. In each operation, there will be a come-up (approximately ments. The increased water exchange rate will facilitate 2 and 4 min for 200 and 400 MPa, respectively) stage, a constant intercellular water transport. The degree of membrane damage pressure stage for 10 min, and a decompression stage. At the end of can therefore be determined by observing changes in the water the holding period, pressure is released to atmospheric pressure within proton relaxation behavior (T2) of the vacuolar and other a few seconds. The entire high pressure process was carried out three components. Because the browning reaction is a consequence times, on three separate days. 2.3. Nuclear Magnetic Resonance (NMR) Relaxometry. After of membrane rupture, the T2 relaxation time of the vacuolar high pressure treatment, the NMR relaxometry analysis was carried out compartment may be a useful tool for predicting browning ° potential of a product. In addition to 1H NMR water proton at room temperature (22 C) within 1 h. Each sample was cut into one cylindrical piece that was 15 mm in diameter with a height of 15 mm relaxometry, it is possible to use light microscopy and viable cell using a cork borer. The piece was immersed in 5 mL of a 50 mM staining to identify when membrane rupture occurs as a result MnCl2 solution for 300 min, during which it was taken out for of high pressure processing. Hence, the loss in tonoplast monitoring T2 relaxation time every 30 min. The samples were blotted integrity leads to interaction between the enzyme and substrate, dry and then placed into a covered NMR tube, and then NMR and the application of vacuolar staining may be a useful tool for relaxometry measurements were performed using an NMR spec- predicting enzymatic browning. trometer (Aspect AI, Industrial Area Havel Modi’in, Shoham, Israel) fi The purpose of this study is to determine whether maturity with a magnetic eld of 1.02 T and a frequency of 43.5 MHz. The T2 ff relaxation decay curve was obtained using the Carr−Purcell− has an e ect on enzymatic browning reactions in peaches − following high pressure processing at different pressure levels. Meiboom Gill (CPMG) sequence with an echo time of 0.5 ms and a range of 7500−15000 echoes. The measurement was monitored in Parameters involved in the browning reaction, which include 1 the samples every 30 min for 300 min. The T2 spectrum, which cell damage measured using both H NMR and microscopic determines the change in each plant cell compartment, was acquired observation of peach cells, PPO activity, total phenol content, by non-negative least squares algorithm using Prospa (Magritek, and degree of browning as difference in lightness, will be Wellington, New Zealand). quantified. 2.4. Microscopic Study. Following HPP, peach samples were cut into small rectangular shapes approximately 1.0 × 0.5 × 0.3 cm and 2. MATERIALS AND METHODS placed in the sample holder of a Vibratome1000 Plus (The Vibratome Co., St. Louis, MO, USA). From each peach sample, three section 2.1. Raw Materials. All of the fruit from three trees of the specimens of approximately 200 μm in thickness were prepared and clingstone peach variety ‘Loadel’ were harvested by hand from then submerged into a staining solution, 0.5% neutral red in acetone orchards of the Foundation Plant Services, University of California, filtered twice with Whatman no. 1 paper and then diluted to 0.04% in Davis, CA, USA, and stored in a dark room at 4 °C and a relative 0.55 M mannitol−0.01 M N-(2-hydroxyethyl)piperazine-N′-2-ethane- humidity of 84% for approximately 2 days until processed. Peaches sulfonic acid (HEPES) buffer, pH 7.8. After 2 h of soaking, the were classified into three maturity stages by firmness using the specimens were rinsed for 0.5 h in the 0.55 M mannitol−0.01 M

7217 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224 Journal of Agricultural and Food Chemistry Article

Figure 1. T2 relaxation spectrum of maturity 1 stage peaches: (a) control (unprocessed) sample; (b) following HPP at 200 MPa; (c) following HPP at 400 MPa.

HEPES buffer solution, mounted on a microscope slide, and covered with 100 mL of cold 0.1 M sodium phosphate dibasic anhydrous with a coverslip. A microscopic observation was done at 40× (Fisher, Fair Lawn, NJ, USA), pH 7.3, 20 mM EDTA (Fisher), and 6% magnification with a light microscope (Olympus System Microscope, (w/v) of the surfactant Triton X-114 (Sigma-Aldrich, St. Louis, MO, model BHS, Shinjuku-ku, Tokyo, Japan) on the same day as high USA). After homogenization, the mixture was refrigerated at 4 °C for pressure processing. A digital color camera (Olympus MicroFire, 60 min before centrifuging at 28374g at 4 °C for 45 min with a Olympus, Tokyo, Japan) attached to the microscope provided color refrigerated Sorvall-RC5 centrifuge (E. I. DuPont Co, Wilmington, photomicrographs at 800 × 600 pixel resolution in captured images DE, USA). Triton X-114 8% (w/v) was added to the supernatant, and (Olympus software, Olympus America, Melville, NY, USA). the mixture was incubated in a 40 °C water bath for 15 min. At this 2.5. Partial Purification of Peach Polyphenol Oxidase (PPO). step, an opaque yellowish color was observed due to an increase in A partially purified crude enzyme extract was obtained following a temperature inducing the production of a micellar mass, and this modification of the method described by Espıń et al.30 A 200 g peach caused the onset of turbidity. A clear supernatant was obtained of each maturity stage was homogenized at room temperature using a following centrifugation at 578.6g (Centra CL2 tabletop centrifuge, small blender (Waring, Conair Corp., Stamford, CT, USA) for 2 min IEC, Needham, MA, USA) for 10 min at 25 °C, and a second phase

7218 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224 Journal of Agricultural and Food Chemistry Article partitioning step with 8% (w/v) Triton X-114 and incubation at 40 °C of the tissue, because 1H NMR signal intensity is directly for 15 min was performed. The peach extract was collected from the proportional to the proton density.34 Water protons are the supernatant after it was centrifuged at 578.6g for 10 min at 25 °C and 35 − ° major proton signals in plant tissues. In this experiment, the stored at 10 C until use. T distributions from a CPMG experiment for unprocessed 2.6. PPO Assay. The enzyme assay was performed using the 2 spectrophotometric method described by Espıń et al. with some (control) maturity stage 1 peaches, as well as those following modifications.30 Samples were analyzed within 1 h, directly after the HPP treatment at 200 and 400 MPa, are shown as examples in HPP process. The assays started by mixing 10 μL of the crude peach Figure 1, panels a, b, and c, respectively. The peach samples had extract with 1.0 mL of a medium containing 0.6 mL of 100 mM acetate three compartments, which were generated from different buffer (pH 5.5), 0.2 mL of 25.0 mM dihydroxyhydrocinnamic acid proton environments within the sample. In previous work, 28 (DHCA) (Sigma-Aldrich), and 0.2 mL of 2.5 mM 3-methyl-2- Snaar and Van As submerged apple tissue in 50 mM MnCl2 benzothiazolinone (MBTH) (Sigma-Aldrich). A reddish color adduct in an isotonic mannitol solution and monitored the uptake of formed from this reaction was monitored at 500 nm for 120 s using 2+ fi Mn in each compartment to assign each relaxation time peak. UV spectrophotometry (UV2101PC, Shimadzu Scienti c Instruments The authors also observed three main populations of water and Inc., Columbia, MD, USA). Each sample requires approximately 5 min for the preparation of crude extract and the PPO assay. PPO activity assigned these compartments to the vacuole (the highest peak was calculated from the following equation, where Abs(0) is the initial and longest T2), the cytoplasm (the second compartment), and absorbance and Abs(1) is the absorbance at the end of linearity: the cell wall or extracellular water (the shortest T2). In the current study, the T of the vacuolar compartment of =− · 2 PPO activity (units/mL) Abs(1) Abs(0)/min mL of juice the unprocessed (control) sample had a mean value for nine 2.7. Analysis of Total Phenols. Peach samples were preserved at peaches of 0.80 s (Figure 1a). The cytoplasm and cell wall −80 °C after HPP until analysis. Samples were thawed at room compartments had mean values of 0.30 and 0.14 s, respectively. temperature (22 °C) before analysis of total phenols according to the fi After HPP, there was a signi cant shift in the T2 compared to Folin−Ciocalteu method as described by Waterhouse.31 Peach the unprocessed sample. The T2 of the vacuolar compartment samples (20 g) were blended with 30 mL of deionized water for 2 decreased to 0.62 s following pressure treatment at 200 MPa min. A 6.4 g mixture was vortexed with 27.6 mL of 76% (v/v) aqueous acetone for 2 min. The tube containing the solution was allowed to and declined further to 0.52 s following the 400 MPa treatment. further homogenize in a shaker for 10 min. Cell wall particles were The other two compartments (cytoplasm and cell wall) showed discarded after centrifugation at 578.6g for 10 min at room the same declining trend in T2 relaxation time following HPP. temperature (Centra CL2 tabletop centrifuge, IEC). Supernatant (1 High pressure processing causes disruption of the subcellular mL) was transferred to a new tube containing 0.36 mL of 2 N Folin structure; hence, this damage results in increasing permeability reagent (Sigma-Aldrich, Buchs, Switzerland), and then the solution of the membranes. Because the plasmalemma and tonoplast was vortexed and allowed to stand for 5 min. Sodium carbonate (6 provide primary control of the permeability into their mL) was added and mixed well before 2.64 mL of deionized water was ± ° respective compartments, changes in proton interaction added. The solution was vortexed and incubated in a 50 0.1 C would directly correlate to the damage of these membranes. water bath for 5 min and cooled to room temperature for 1 h. The absorbance of a blue complex product was determined at 760 nm In the present experiment, results illustrated in Figure 1b,c were using UV spectrophotometry. The standard curve was prepared using as expected, in that T2 relaxation time decreases as pressure gallic acid (Arcos Organics, Geel, Belgium) at concentrations of 0−500 levels increase, because the damage to the membranes is of a mg/L. The total phenols results were expressed as gallic acid greater degree with increasing pressure. When pressurizing, air equivalents (GAE) per gram of fresh weight of peaches. in the tissue is compressed, and then it expands on pressure 2.8. Degree of Browning. The browning of peach samples was release, causing the rupture of cell membranes.26 Damage to reported as the difference in lightness (DL*), which is the difference * * the cell membrane can occur both during pressurization and between the initial L and the L of the same sample after 2 weeks of also during depressurization. We would assume that, in more storage at 4 °C and 84% relative humidity. Lightness was determined using a Minolta CR-400 colorimeter (Minolta Camera Co, Ltd., mature fruit, the membranes would be more susceptible to both ° pressurization- and depressurization-induced changes. Japan). The beam diameter was 11 mm with a viewing angle of 0 .A 36 white calibration plate was used for calibration (L* = 96.88, a* = 0.02, Gonzalez et al. also observed a decrease in T2 relaxation b* = 2.05). The values were expressed in the CIE L*a*b* system. The time of the vacuolar compartment in onion samples processed initial lightness of peach samples was measured after HPP, and then at 200 MPa for 5 min. When considering the NMR peak, the samples were stored at 4 °C for 2 weeks before the lightness was merging of cytoplasm and vacuolar compartments was observed measured. in peach samples following the 400 MPa treatment (Figure 1c). 2.9. Statistical Analysis. This experiment was performed in three fi ff A similar nding was reported in potato samples following high replicate processing runs on three separate days. The e ects of pressure application at 300 MPa, and the merging was even maturity and the level of high pressure processing on the difference in 37 lightness, PPO activity of the intact fruit, PPO activity of the extract, more obvious in 500 MPa treated samples. This information and total phenols were analyzed using analysis of variance (ANOVA) suggests a rise in membrane permeability following pressure for each maturity and processing level. The plots present the mean treatments and an exchange of water between the vacuolar and with its standard deviation for each determination. Tukey’s test was cytoplasm compartments by diffusion and differences in used to compare means of each condition at P < 0.05 (SAS version 9.4, osmotic potential during processing at higher pressure levels, Cary, NC, USA). for example, ≥300 MPa. Considering the maturity effect, the least mature peaches 3. RESULTS AND DISCUSSION (M1) showed relatively stable T2 values in the vacuolar T ff 3.1. 2 Relaxation of Peach Samples of Di erent compartment of the unprocessed samples (0.1 MPa) during Maturities following HPP. Measurement of NMR relaxation 300 min of immersion in MnCl2 (Figure 2a); however, a time is a useful technique widely applied in plant studies to significant decline occurred in the samples processed at 200 and investigate the physical properties of water in various 400 MPa. The same pattern in T2 relaxation time was observed tissues.32,33 The signals are generally an average over the in the more mature peaches at stages M2 (Figure 2b) and M3 whole sample, leading to information on the water relationships (Figure 2c), but at even more rapid rates. When the rates of

7219 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224 Journal of Agricultural and Food Chemistry Article

labile protons and water protons, which reduces the T2 relaxation time of water. Musse et al.40 also observed higher concentrations of sugar in the tissue at the later ripening stages, because tomatoes store starch and convert it to sugar when they mature. As a consequence, T2 relaxation times were reported to decrease. In another study, Clark and MacFall42 used NMR to follow persimmon development and ripening and also observed that T2 decreased during fruit development. In the case of peach fruit, sugar is produced and transported from leaves to the fruit without starch production, and the sugar level is unchanged after harvest. Therefore, initial T2 values of the vacuolar compartment of the three peach maturities in this study were not significantly different, indicating similar sugar ff and chemical composition. The di erence in T2 shift of the three maturities following HPP at different pressure levels is therefore most likely due to differences in membrane permeability caused by cell damage during the HPP treatment, and the level of damage may have been influenced by maturity. 3.2. Microscopic Evaluation of Peach Tissues. Viable cells were visually identified as an intense red area concentrated within smooth round cells, resulting from the uptake of neutral red dye through the intact tonoplast in the acidic environment of the vacuole.43 Intact vacuoles were visible only in unprocessed (control) samples at all levels of maturity, but interestingly, maturity stage 3 had the greatest number of intact vacuoles (Figure 3a). Following pressure treatment at either

Figure 2. T2 relaxation times of the vacuolar compartment of peach samples from maturity stages (a) 1, (b) 2, and (c) 3 after processing at 0, 200, and 400 MPa. The samples were submerged in MnCl2 solution for 300 min, and the T2 was monitored every 30 min.

decrease in T2 were compared between the three maturities, it was clear that the T2 of the most mature fruit decreased the most rapidly, indicating the most severe membrane rupture and loss of cell compartmentation. With regard to general effects of maturity on cell damage during fruit ripening, metabolic changes typically include alteration of the cell structure, changes in cell walls correlated with depolymerization of matrix glycan,38 increased permeability of the plasma membrane, decreases in structural integrity, and increases in intracellular spaces.39 Decreases in T relaxation time observed in this study are highly correlated 2 × to an increase in the permeability of the membrane, which Figure 3. Light micrographs (40 ) of peach cells from maturity stages resulted from the combined effect of physical changes during (M) 1, 2, and 3 following treatment at pressure levels (a) 0.1 MPa (unprocessed control), (b) 200 MPa, and (c) 400 MPa and staining fruit maturation and damage to the cell during high pressure with neutral red dye. processing. Musse et al.40 studied the changes that occurred during postharvest tomato fruit ripening using MRI and NMR 200 or 400 MPa there was complete loss of cell integrity; fi relaxometry and observed signi cant changes in T2 relaxation therefore, no viable cells were observed in peaches of any of the time in the vacuolar compartment of the core, placenta, and three maturity stages (Figure 3b,c). Any time pressure is outer pericarp. All three tissue types started to show a decrease applied, there will be a come-up stage, a constant pressure in T2 on day 7 after harvest and declined the most by day 16. stage, and a decompression. At this time it is not possible to The authors found an approximately 25% decline in T2 evaluate microstructural changes during each phase separately; relaxation time of the vacuolar compartment after 21 days of rather, the change is observed after the entire process is storage, as compared to the initial T2. Changes in T2 relaxation completed. signal were reported to correlate with water content, sugar As membranes lose their integrity due to pressure-induced concentration, chemical composition, cell dimension, and damage, the vacuoles lose their ability to maintain an acidic pH membrane permeability.41 Sugar content was suggested to environment, resulting in a lack of ionization and accumulation play a crucial role in the shift in T2 relaxation time because of red dye in the vacuole. Acids are stored in the vacuole so ff sugar has a lower T2 than water. Sugar in general has labile when the tonoplast ruptures, the acids di use out due to protons (−OH), and proton exchange will occur between the osmotic differences. The neutral red dye changes from yellow

7220 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224 Journal of Agricultural and Food Chemistry Article

(basic) to red (acidic) as it enters the intact vacuole. When the tonoplast ruptures, the dye is still red, but because it is no longer retained in the intact vacuole, but rather diluted in the uncolored liquid from the rest of the cell, it is not an intense red but more light red or pink. Changes in cell structure during peach maturation also have an impact on cell viability. The middle lamella, the pectin-rich layer between cells, which serves as a glue binding adjacent cells, generally undergoes degradation with maturity, leading to a loss of intercellular adhesion. Observations with an electron microscope indicate dissolution of the middle lamella occurs 44−47 46 ff * − * during fruit ripening. Crookes and Grierson found that Figure 4. Di erence in lightness (L after 2 weeks L initial) of peach dissolution of the middle lamella and disruption of the primary samples taken from different maturity stages after treatment at cell wall occurred during ripening of tomato fruit. These pressure levels of 0.1, 200, and 400 MPa. Values with the same letter authors suggested that these changes were correlated with the are not significantly different across maturity and treatment variables at fi synthesis of the pectin-degrading enzyme, polygalacturonase a signi cance level of P < 0.05. (PG). In the unprocessed (control) peaches, the most mature following the same pressure treatment as illustrated in Figure 4. fruits, which were stage M3, had the greatest number of intact ff vacuoles (Figure 3a, M3). This may be due to the ripening- This may imply that the e ect of HPP pressure level on peach discoloration is greater than that of the effect of maturity. The related breakdown of the middle lamella and reduction in ff intercellular adhesion, which loosens the matrix the cells are di erence in lightness of samples treated at 200 MPa was held in. It may be that the liberation of peach cells from their comparable to those processed at 400 MPa. There is a strong cellular matrix allows them to resist the strain of high pressure possibility that the loss of cell integrity may reach a threshold application. As a consequence, the unprocessed (control) M3 after HPP at 200 MPa, which is in agreement with observations peaches exhibited more viable cells, compared to maturity from the light microscopy (absence of viable cells at 200 and stages 1 and 2. 400 MPa in Figure 3). The decrease in T2 relaxation time is 3.3. Degree of Browning in Peach Samples of consistent in HPP treated samples and can be related to the Different Maturities following HPP. The discoloration of loss of cell integrity during HPP (Figure 1b,c), which occurred peaches was not observed immediately after HPP treatment; simultaneously with the loss of tonoplast integrity, as observed rather, enzyme-catalyzed browning induced the development of using light microscopy (Figure 3b,c). The previous study in our color in treated peach samples after 2 weeks of refrigerated group of onion cell integrity after high pressure processing supports the finding that membrane decompartmentalization is storage. Preliminary experiments determined that browning was 36 not visible until after the first week of storage and that color was triggered by HPP. 3.4. PPO Activity of Peach Samples and Partially constant by 2 weeks; therefore, the measurement was obtained Purified Peach Extracts from Different Maturities then. Because oxygen is required for the reaction, it may be that fi fi following HPP. PPO activity in the peach tissues taken from the packaging lm permeability allowed for slow in ltration fi during storage and that surface browning reactions were maturity stages 1 and 2 decreased signi cantly as a result of the complete by 2 weeks. The unprocessed samples taken from the 200 MPa treatment (Figure 5) and decreased further after they three different peach maturities had a low level of difference in lightness during storage, and there was no significant difference among the three maturities. A sharp increase in degree of browning was observed in all of the HPP samples. Samples processed at 200 MPa had a mean difference in lightness of 7.19, 7.89, and 5.38 for maturity levels 1, 2, and 3, respectively; however, they were not significantly different at P < 0.05. Browning reactions occur as a consequence of decompartmentalization of membranes in plant cells either by cutting, senescence, or physical stress.1 In the control samples, which did not incur much damage, the difference in lightness remained at a low level (<2.00) during storage in all maturity ff stages (Figure 4). After the damage in the membranes was Figure 5. PPO activity of peach homogenates taken from di erent initialized by HPP, there was a higher probability of interaction maturity stages and processed at 0.1, 200, and 400 MPa. Values with the same letter are not significantly different across maturity and between the enzyme, which was originally located in the treatment variables at a significance level of P < 0.05. cytoplasm, and its substrate, originally located in the vacuole. This difference in lightness in high pressure treated samples increased significantly following HPP treatment. The same were processed at 400 MPa. This trend was different in the observation was reported by Guerrero-Beltrań et al. in HPP maturity stage 3 peaches, for which mean PPO values were 402, treated peach puree.48 Development of browning was reported 424, and 277 (×100 unit/mL) for the control and 200 and 400 as the difference in color (ΔE*) by these authors, and this value MPa treatments, respectively. Only the 400 MPa application increased in samples following HPP at 103 MPa; the difference resulted in PPO activity that was significantly lower than that of was even greater in 207 MPa treated samples. the control or 200 MPa peach tissues. However, the initial PPO With regard to the maturity effect, there were no significant activity in the control samples at maturity stage 3 was differences in discoloration among the three maturity stages significantly lower than that of the unprocessed controls

7221 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224 Journal of Agricultural and Food Chemistry Article taken from maturity stages 1 and 2. Only a few studies have histidine residue that binds the two coppers at the active site of provided information on changes in PPO activity during peach the enzyme. As a consequence, inactivation of PPO occurs maturation. Flurkey and Jen reported very high PPO activity because of loss of histidine and release of copper at the active − during the early developmental stage of the freestone peach site.57 59 Inactivation of mushroom PPO was calculated to cultivar ‘Redhaven’, but found that this level declined to a fairly occur at the rate of approximately 1 in 5000 turnovers of the constant activity after the pit-hardening stage.49 A study of substrate to product.59 The results determined for inactivation postharvest ripening of the freestone cultivar ‘Premier’ found an of PPO, which was HPP processed within a peach tissue increasing trend in PPO activity, with a maximum activity at 8 system, are therefore a combination effect of the HPP itself, as days of storage.8 From these earlier studies, it appears that PPO well as the influence of the phenolics on both product activity is relatively high in the growing peach to the point of pit inhibition and turnover of the enzyme. hardening, which is the most active growing period, when the In contrast, a different trend in PPO activity was observed production of protein and total solids content reaches its peak. when the enzyme was extracted and partially purified from In the current study it was found that PPO activity was not peach tissues and then analyzed following HPP treatment. Our significantly different in maturity stages 1 and 2, although there study found that the most mature peach (M3) had the highest was a declining trend, but by maturity stage 3 the activity had levels of PPO activity (Figure 6). It is possible that the significantly declined. PPO activity in peaches is therefore degradation of the middle lamella during maturation, which dependent on both cultivar and the maturity at harvest. would have been the most advanced in the M3 peaches, makes Figure 6 illustrates the PPO activity in peach extracts, in enzyme extraction from the fruit easier. In the least mature which the trend was somewhat different from that in the fruit peach, M1, there was a significant decrease in activity after both 200 and 400 MPa applications, and the final level of PPO activity in all samples was lower than that in M2 and M3 extracts after the same treatment. Day60 suggested that peach immaturity was associated with the development of flesh discoloration. There were no significant differences in PPO activity in the extracts from maturity stages 2 and 3 after the 200 MPa treatments, as compared to the controls, but there was a significant decline in activity after the application of 400 MPa. A similar decrease in PPO activity was also reported in HPP treated banana puree, in which a 21% reduction followed pressure treatment at 689 MPa 14 ff for 10 min. The results obtained from the PPO extract, which Figure 6. PPO activity of peach extracts taken from di erent maturity contains no phenolic compounds capable of reacting with the stages and processed at 0.1, 200, and 400 MPa. Values with the same fi ff letter are not significantly different across maturity and treatment enzyme, may represent the speci ceect of HPP alone on variables at a significance level of P < 0.05. denaturation of PPO. In this case, the phenolic substrates of the enzyme were removed during the partial purification step and therefore would not provide a second avenue for PPO tissues. PPO activity decreased significantly in the extracts denaturation through either product inhibition or enzyme following pressure treatment at 400 MPa in all three maturity turnover. stages, and in the maturity 1 extracts there was also a significant The activation/inactivation of PPO after the HPP process is reduction after the 200 MPa application. The PPO activity of controversial because the change in enzyme configuration has maturity stages 1 and 2 was significantly lower than that of been studied only in purified mushroom PPO at an ultrahigh maturity stage 3, which was the opposite of what was found in pressure level of ∼800−900 MPa.61 The decrease in PPO the peach tissues. activity after lower pressure treatments may be explained by the One focus of this study was to compare the activity of the reaction of the enzyme and the substrate once compartmen- extracted PPO and that of PPO still present in its cellular talization is destroyed by high pressure, resulting in turnover of matrix within the fruit tissues themselves following HP the enzyme and quinone product inhibition. Hsu et al.62 treatment, because the fruit matrix may act as a barrier, suggested that phenolic compounds must be removed before protecting the enzyme from damage. A reduction in PPO an assay of PPO activity, because oxidized phenols can inhibit activity in the fruit tissues may arise from two factors: (1) the PPO catalysis. In our study, the inactivation of PPO activity effect of high pressure, which may cause enzyme denaturation; varied depending on maturity, degree of HPP processing, and and (2) the presence of phenolic compounds, which are known type of sample (fruit tissue or extract) as presented in Figures 5 to participate in product inhibition and turnover of the enzyme. and 6. o-Quinones, which are oxidation products of phenolic 3.5. Total Phenols of Peach Samples of Different compounds, have been reported to have an effect on PPO Maturities following HPP. One of the advantages of HPP is activity because these reactive compounds can interact with the ability to retain the nutritional attributes of fresh food. In − proteins and produce covalent condensation.50 54 The −SH the present experiment, the total phenol content of peaches did − and NH2 groups of amino acids have high potential to not change in the maturity range studied, and it was also condense with quinones; therefore, the protein structure and unaffected by high pressure processing at 200 and 400 MPa function may change as a consequence.55 In addition, quinones (data not shown). Peach samples had a mean value of total can generate free radicals through redox-recycling, which can phenols content in the range from 4.4 to 5.2 mg/g sample. damage proteins, amino acids, or lipids.56 In the case of enzyme Patras et al.63 also found that no significant changes in total turnover, PPOs are irreversibly inactivated during the oxidation phenols were observed in strawberry and blackberry pureeś process due to a free radical-catalyzed fragmentation of the pressure treated at 400 and 500 MPa (P < 0.05). Another study

7222 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224 Journal of Agricultural and Food Chemistry Article by Landl et al.64 also reported that total phenols in ‘Granny (7) Cheng, G. W.; Crisosto, C. H. Browning potential, phenolic Smith’ apple pureé were unaffected by pressure treatment at composition, and polyphenoloxidase activity of buffer extracts of peach 400 MPa. In section 3.4 above, it was stated that there were two and nectarine skin tissue. J. Am. Soc. Hortic. Sci. 1995, 120, 835−838. factors affecting PPO inactivation in the fruit tissues, for (8) Brandelli, A.; Lopes, C. H. Polyphenoloxidase activity, browning example, HPP treatment and potential product inhibition from potential and phenolic content of peaches during postharvest ripening. − the phenolic substrates. The fact that the level of phenolics did J. Food Biochem. 2005, 29, 624 637. not change significantly with maturity level or HPP treatment (9) Cantos, E.; Tudela, J. A.; Gil, M. I.; Espín, J. C. Phenolic indicates that either product inhibition by phenolics does not compounds and related enzymes are not rate-limiting in browning development of fresh-cut potatoes. J. Agric. Food Chem. 2002, 50, occur in peach tissues or the concentration of phenolic 3015−3023. compounds required to illicit this response is so low that it (10) Vaughn, K. C.; Duke, S. O. Function of polyphenol oxidase in does not result in a significant reduction in the concentration. − fi fi higher plants. Physiol. Plant. 1984, 60, 106 112. Fruit maturity has a signi cant impact on nal product (11) Vamos-Vigyá zó,́ L.; Haard, N. F. Polyphenol oxidases and fi quality, because development involves speci c biochemical and peroxidases in fruits and vegetables. Crit. Rev. Food Sci. Nutr. 1981, 15, physiological changes, which result in different degrees of cell 49−127. damage during processing. 1H NMR is an effective method to (12) Walker, J. R.; Ferrar, P. H. Diphenol oxidases, enzyme-catalysed compare the levels of damage due to loss of peach integrity at browning and plant disease resistance. Biotechnol. Genet. Eng. Rev. different maturities following HPP. The information obtained 1998, 15, 457−498. from light micrographs, difference in lightness, and PPO (13) Butz, P.; Koller, W.; Tauscher, B.; Wolf, S. Ultra-high pressure activity provides insight on the changes occurring during fruit processing of onions: chemical and sensory changes. LWT−Food Sci. 1994 − maturation. A sharp decrease in the T2 relaxation time plot of Technol. , 27, 463 467. the M3 sample following 400 MPa treatment indicated the (14) Palou, E.; Lopez-Malo, A.; Barbosa-Canovas, G.; Welti-Chanes, highest permeability, compared to other maturities and J.; Swanson, B. Polyphenoloxidase activity and color of blanched and fi ff high hydrostatic pressure-treated banana puree. J. Food Sci. 1999, 64, pressure levels. However, there was no signi cant di erence − in discoloration among the three maturities following the same 42 45. pressure treatment. This implies that the effect of HPP pressure (15) Giovannoni, J. Molecular biology of fruit maturation and ripening. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 725−749. level on peach discoloration is greater than that of maturity and (16) Bonghi, C.; Ferrarese, L.; Ruperti, B.; Tonutti, P.; Ramina, A. that processors do not need to sort fruit by maturity prior to β ff Endo- -1, 4-glucanases are involved in peach fruit growth and processing. While high pressure processing o ers an alternative ripening, and regulated by ethylene. Physiol. Plant. 1998, 102, 346− to traditional canning and freezing and has the potential to 352. produce novel peach products with crisp texture and aromatic (17) Downs, C. G.; Brady, C. J.; Gooley, A. 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7224 DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. 2016, 64, 7216−7224