Article pubs.acs.org/JAFC
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 CaliforniaDavis, 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 CaliforniaDavis, 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 transverse relaxation time (T2) of the vacuolar component and by light microscopy. The discoloration of peach slices of different 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 suggested by Cantos et al.,9 the importance of cellular integrity and accessibility of the enzyme to its substrate may be the primary factor in the development of enzymatic browning. This study was designed to investigate this hypothesis. In intact plants, PPOs are localized in plastids10 and remain physically separated from phenolic substrates, which are in the vacuole.11,12 However, once the plant loses integrity in its cell walls and membranes by either cutting, senescence, or physical stress, enzymes and substrates are allowed to mix and the browning reaction occurs as a consequence.1 The effect of HPP on PPOs has been previously studied either in the form of a partially purified extract13 or measured directly from the plant material after processing.14 One goal of the current study is to compare the effect of HPP on PPOs, both in an extract and as found in a plant food matrix. Fruit ripening is an irreversible developmental process that involves specific biochemical and physiological attributes.15 Peaches are a climacteric fruit, in which ripening is associated with the production of ethylene and a significant increase in cellular respiration. Ethylene has an important role in all stages of peach ripening; this plant hormone sets off the activity of enzymes responsible for fruit softening, ripening, color development, and sugar content.16−19 The ripening process results in elevated sugar-to-acid ratios,20 decreases in acidity21
1. INTRODUCTION High pressure processing (HPP) is a novel advanced process being extensively studied because of its ability to retain products with natural attributes while inducing destruction of microorganisms and modifying enzyme activity. HPP is a potential alternative method for peach preservation, besides canning and freezing, because it can provide a new product with novel crispy and aromatic characteristics. The quality of HPP preserved fruits can, however, change during storage due to the use of elevated pressure levels, which may induce changes in membrane permeability and trigger loss of subcellular compartmentalization. After loss of cellular integrity in fruits, substrates are able to mix with enzymes, as occurs in the case of enzymatic browning, which is undesirable to consumers. In a large number of fruit and vegetable crops, losses are a result of postharvest deteriorative reactions,1 in which enzymatic browning reaction causes the second largest quality loss.2 Enzymatic browning in fruits is initiated by the oxidation of phenolic compounds, mainly by polyphenol oxidases (PPOs)3 with a partial role of peroxidases (PODs).4 The product of this reaction is quinone, which further undergoes nonenzymatic processes to form melanins, brown products on the cut surfaces of fruit that are exposed to oxygen. Most studies of enzymatic browning have measured the activity of PPOs and the concentration of their total phenolic substrates and correlated these to the degree of browning, for example, apples5 and peaches.6−8 However, in preliminary studies it was determined they neither correlated well to the difference in lightness in stored HPP treated peaches. Therefore, as © XXXX American Chemical Society
Received: May 18, 2016 Revised: August 23, 2016 Accepted: August 24, 2016
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DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
TA.XT2 Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK): (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, M1−M3, were harvested at 312, 314, and 316 days after blossom formation, respectively. Approximately three peaches per maturity stage per processing replicate were hand peeled and cut into approximately 3 cm thick slices before being vacuum packed in polyethylene bags (4 mil vacuum pouches, Ultrasource, North Kansas City, MO, USA). Each bag contained three peach slices of the same maturity, classified by firmness, one from each of three different fruits, all of which were to be analyzed using the same analytical method. Peach extracts taken from each maturity stage were vacuum packed in polyethylene bags of approximately 2 mL. On each replicate day of processing, six packages (one for each of the five analytical methods plus a control) for each of the three maturity levels were processed at each of the three pressures: 0.1, 200, and 400 MPa. The same fruit was analyzed for all analytical parameters, for example, difference in lightness, the paramagnetic study using NMR, PPO activity, total phenols, and light microscopy. The control sample was unprocessed sliced peaches in a vacuum package (approximately 0.1 MPa). All of the packaged samples were kept at ambient temperature (22 ± 2 °C) for 30 min prior to HPP treatment. 2.2. High Pressure Processing (HPP). The packages containing three different maturity stages of peaches were processed at 0.1 (control group was at a standard atmosphere, 101.3 kPa or 0.1013 MPa), 200, and 400 MPa for 10 min in a high pressure processing unit (2L-700 Lab system, Avure Technologies Inc., Kent, WA, USA). The pressure levels used in this experiment were justified on the basis of the rupture of the plant cellular membranes at 200 MPa and the inactivation of microorganisms at 400 MPa, both of which were observed in preliminary experiments. The high pressure unit had a 2.0 L vessel, and 600 MPa was the maximum pressure level; the pressurizing medium was water. The initial high pressure unit temperature (Ti) was approximately 23 °C. The maximum temperature in the high pressure chamber depended on the target pressure and was 28 and 32 °C for the treatment at 200 and 400 MPa, respectively. In each operation, there will be a come-up (approximately 2 and 4 min for 200 and 400 MPa, respectively) stage, a constant pressure stage for 10 min, and a decompression stage. At the end of the holding period, pressure is released to atmospheric pressure within a few seconds. The entire high pressure process was carried out three times, on three separate days. 2.3. Nuclear Magnetic Resonance (NMR) Relaxometry. After high pressure treatment, the NMR relaxometry analysis was carried out 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 using a cork borer. The piece was immersed in 5 mL of a 50 mM MnCl2 solution for 300 min, during which it was taken out for monitoring T2 relaxation time every 30 min. The samples were blotted dry and then placed into a covered NMR tube, and then NMR relaxometry measurements were performed using an NMR spectrometer (Aspect AI, Industrial Area Havel Modi’in, Shoham, Israel) with a magnetic field of 1.02 T and a frequency of 43.5 MHz. The T2 relaxation decay curve was obtained using the Carr−Purcell− Meiboom−Gill (CPMG) sequence with an echo time of 0.5 ms and a range of 7500−15000 echoes. The measurement was monitored in the samples every 30 min for 300 min. The T2 spectrum, which determines the change in each plant cell compartment, was acquired by non-negative least squares algorithm using Prospa (Magritek, Wellington, New Zealand). 2.4. Microscopic Study. Following HPP, peach samples were cut into small rectangular shapes approximately 1.0 × 0.5 × 0.3 cm and placed in the sample holder of a Vibratome1000 Plus (The Vibratome Co., St. Louis, MO, USA). From each peach sample, three section specimens of approximately 200 μm in thickness were prepared and then submerged into a staining solution, 0.5% neutral red in acetone filtered twice with Whatman no. 1 paper and then diluted to 0.04% in 0.55 M mannitol−0.01 M N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer, pH 7.8. After 2 h of soaking, the specimens were rinsed for 0.5 h in the 0.55 M mannitol−0.01 M
(due to decreases in malic and citric acid), changes in ground color of the skin, and an increase in volatile compounds.22 Measurement of respiration rate, ethylene, sugar, and acid all involve destructive evaluation; however, the use of firmness as a maturity indicator can be nondestructive. All of these changes create desirable fruit characteristics and make peaches more palatable to the consumer. Fruit maturity is one of the most important factors associated with the quality of the final processed product; therefore, selecting the right maturity stage is critically important and processors need to be concerned with this. However, very few researchers have focused on the effect of fruit maturity on the quality of HPP treated products.23,24 This study, therefore, explores methods to measure changes in cell integrity of fruits from different initial maturities following the high pressure process. Nuclear magnetic resonance (1H NMR) water proton relaxometry is a nondestructive measurement that detects physiological changes of water in a sample. This method has undergone continuous development and has been applied in a wide range of plant studies, for example, plant freezing,25 HPP treated strawberries,26 tomato pericarp ripening,24 and identification of black heart in pomegranates.27 The proton spin−spin (T2) relaxation time is usually in the range of seconds to milliseconds on the 1T aspect system. The value is related to properties of water in different locations in the tissue, to total water content in both free and bound form, and to the interaction of water with macromolecules.28 In plant cells, the plasmalemma (membrane surrounding the cytoplasm) and tonoplast (membrane surrounding the vacuole) provide the primary control of permeability between their compartments. Once cell damage occurs as a consequence of high pressure treatment, decompartmentalization of membranes is triggered, leading to an increase in cell permeability.29 This induces an exchange of water between different cell compartments. The increased water exchange rate will facilitate intercellular water transport. The degree of membrane damage can therefore be determined by observing changes in the water proton relaxation behavior (T2) of the vacuolar and other components. Because the browning reaction is a consequence of membrane rupture, the T2 relaxation time of the vacuolar compartment may be a useful tool for predicting browning potential of a product. In addition to 1H NMR water proton relaxometry, it is possible to use light microscopy and viable cell staining to identify when membrane rupture occurs as a result of high pressure processing. Hence, the loss in tonoplast integrity leads to interaction between the enzyme and substrate, and the application of vacuolar staining may be a useful tool for predicting enzymatic browning. The purpose of this study is to determine whether maturity has an effect on enzymatic browning reactions in peaches following high pressure processing at different pressure levels. Parameters involved in the browning reaction, which include cell damage measured using both 1H NMR and microscopic observation of peach cells, PPO activity, total phenol content, and degree of browning as difference in lightness, will be quantified.
2. MATERIALS AND METHODS 2.1. Raw Materials. All of the fruit from three trees of the clingstone peach variety ‘Loadel’ were harvested by hand from orchards of the Foundation Plant Services, University of California, Davis, CA, USA, and stored in a dark room at 4 °C and a relative humidity of 84% for approximately 2 days until processed. Peaches were classified into three maturity stages by firmness using the B
DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
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 a coverslip. A microscopic observation was done at 40× magnification with a light microscope (Olympus System Microscope, model BHS, Shinjuku-ku, Tokyo, Japan) on the same day as high pressure processing. A digital color camera (Olympus MicroFire, Olympus, Tokyo, Japan) attached to the microscope provided color photomicrographs at 800 × 600 pixel resolution in captured images (Olympus software, Olympus America, Melville, NY, USA). 2.5. Partial Purification of Peach Polyphenol Oxidase (PPO). A partially purified crude enzyme extract was obtained following a modification of the method described by Espı ́n et al.30 A 200 g peach of each maturity stage was homogenized at room temperature using a small blender (Waring, Conair Corp., Stamford, CT, USA) for 2 min
with 100 mL of cold 0.1 M sodium phosphate dibasic anhydrous (Fisher, Fair Lawn, NJ, USA), pH 7.3, 20 mM EDTA (Fisher), and 6% (w/v) of the surfactant Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA). After homogenization, the mixture was refrigerated at 4 °C for 60 min before centrifuging at 28374g at 4 °C for 45 min with a refrigerated Sorvall-RC5 centrifuge (E. I. DuPont Co, Wilmington, DE, USA). Triton X-114 8% (w/v) was added to the supernatant, and the mixture was incubated in a 40 °C water bath for 15 min. At this step, an opaque yellowish color was observed due to an increase in temperature inducing the production of a micellar mass, and this caused the onset of turbidity. A clear supernatant was obtained following centrifugation at 578.6g (Centra CL2 tabletop centrifuge, IEC, Needham, MA, USA) for 10 min at 25 °C, and a second phase C
DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry partitioning step with 8% (w/v) Triton X-114 and incubation at 40 °C for 15 min was performed. The peach extract was collected from the supernatant after it was centrifuged at 578.6g for 10 min at 25 °C and stored at −10 °C until use. 2.6. PPO Assay. The enzyme assay was performed using the spectrophotometric method described by Espı ́n et al. with some modifications.30 Samples were analyzed within 1 h, directly after the HPP process. The assays started by mixing 10 μL of the crude peach extract with 1.0 mL of a medium containing 0.6 mL of 100 mM acetate buffer (pH 5.5), 0.2 mL of 25.0 mM dihydroxyhydrocinnamic acid (DHCA) (Sigma-Aldrich), and 0.2 mL of 2.5 mM 3-methyl-2benzothiazolinone (MBTH) (Sigma-Aldrich). A reddish color adduct formed from this reaction was monitored at 500 nm for 120 s using UV spectrophotometry (UV2101PC, Shimadzu Scientific Instruments Inc., Columbia, MD, USA). Each sample requires approximately 5 min for the preparation of crude extract and the PPO assay. PPO activity was calculated from the following equation, where Abs(0) is the initial absorbance and Abs(1) is the absorbance at the end of linearity:
of the tissue, because 1H NMR signal intensity is directly proportional to the proton density.34 Water protons are the major proton signals in plant tissues.35 In this experiment, the T2 distributions from a CPMG experiment for unprocessed (control) maturity stage 1 peaches, as well as those following HPP treatment at 200 and 400 MPa, are shown as examples in Figure 1, panels a, b, and c, respectively. The peach samples had three compartments, which were generated from different proton environments within the sample. In previous work, Snaar and Van As28 submerged apple tissue in 50 mM MnCl2 in an isotonic mannitol solution and monitored the uptake of Mn2+ in each compartment to assign each relaxation time peak. The authors also observed three main populations of water and assigned these compartments to the vacuole (the highest peak and longest T2), the cytoplasm (the second compartment), and the cell wall or extracellular water (the shortest T2). In the current study, the T2 of the vacuolar compartment of the unprocessed (control) sample had a mean value for nine peaches of 0.80 s (Figure 1a). The cytoplasm and cell wall compartments had mean values of 0.30 and 0.14 s, respectively. After HPP, there was a significant shift in the T2 compared to the unprocessed sample. The T2 of the vacuolar compartment decreased to 0.62 s following pressure treatment at 200 MPa and declined further to 0.52 s following the 400 MPa treatment. The other two compartments (cytoplasm and cell wall) showed the same declining trend in T2 relaxation time following HPP. High pressure processing causes disruption of the subcellular structure; hence, this damage results in increasing permeability of the membranes. Because the plasmalemma and tonoplast provide primary control of the permeability into their respective compartments, changes in proton interaction would directly correlate to the damage of these membranes. In the present experiment, results illustrated in Figure 1b,c were as expected, in that T2 relaxation time decreases as pressure levels increase, because the damage to the membranes is of a greater degree with increasing pressure. When pressurizing, air in the tissue is compressed, and then it expands on pressure release, causing the rupture of cell membranes.26 Damage to the cell membrane can occur both during pressurization and also during depressurization. We would assume that, in more mature fruit, the membranes would be more susceptible to both pressurization- and depressurization-induced changes. Gonzalez et al.36 also observed a decrease in T2 relaxation time of the vacuolar compartment in onion samples processed at 200 MPa for 5 min. When considering the NMR peak, the merging of cytoplasm and vacuolar compartments was observed in peach samples following the 400 MPa treatment (Figure 1c). A similar finding was reported in potato samples following high pressure application at 300 MPa, and the merging was even more obvious in 500 MPa treated samples.37 This information suggests a rise in membrane permeability following pressure treatments and an exchange of water between the vacuolar and cytoplasm compartments by diffusion and differences in osmotic potential during processing at higher pressure levels, for example, ≥300 MPa. Considering the maturity effect, the least mature peaches (M1) showed relatively stable T2 values in the vacuolar compartment of the unprocessed samples (0.1 MPa) during 300 min of immersion in MnCl2 (Figure 2a); however, a significant decline occurred in the samples processed at 200 and 400 MPa. The same pattern in T2 relaxation time was observed in the more mature peaches at stages M2 (Figure 2b) and M3 (Figure 2c), but at even more rapid rates. When the rates of
PPO activity (units/mL) = Abs(1) − Abs(0)/min · mL of juice
2.7. Analysis of Total Phenols. Peach samples were preserved at −80 °C after HPP until analysis. Samples were thawed at room temperature (22 °C) before analysis of total phenols according to the Folin−Ciocalteu method as described by Waterhouse.31 Peach samples (20 g) were blended with 30 mL of deionized water for 2 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 further homogenize in a shaker for 10 min. Cell wall particles were discarded after centrifugation at 578.6g for 10 min at room temperature (Centra CL2 tabletop centrifuge, IEC). Supernatant (1 mL) was transferred to a new tube containing 0.36 mL of 2 N Folin reagent (Sigma-Aldrich, Buchs, Switzerland), and then the solution was vortexed and allowed to stand for 5 min. Sodium carbonate (6 mL) was added and mixed well before 2.64 mL of deionized water was added. The solution was vortexed and incubated in a 50 ± 0.1 °C 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 using UV spectrophotometry. The standard curve was prepared using gallic acid (Arcos Organics, Geel, Belgium) at concentrations of 0−500 mg/L. The total phenols results were expressed as gallic acid equivalents (GAE) per gram of fresh weight of peaches. 2.8. Degree of Browning. The browning of peach samples was reported as the difference in lightness (DL*), which is the difference between the initial L* and the L* of the same sample after 2 weeks of storage at 4 °C and 84% relative humidity. Lightness was determined using a Minolta CR-400 colorimeter (Minolta Camera Co, Ltd., Japan). The beam diameter was 11 mm with a viewing angle of 0°. A white calibration plate was used for calibration (L* = 96.88, a* = 0.02, b* = 2.05). The values were expressed in the CIE L*a*b* system. The initial lightness of peach samples was measured after HPP, and then samples were stored at 4 °C for 2 weeks before the lightness was measured. 2.9. Statistical Analysis. This experiment was performed in three replicate processing runs on three separate days. The effects of maturity and the level of high pressure processing on the difference in lightness, PPO activity of the intact fruit, PPO activity of the extract, and total phenols were analyzed using analysis of variance (ANOVA) for each maturity and processing level. The plots present the mean with its standard deviation for each determination. Tukey’s test was used to compare means of each condition at P < 0.05 (SAS version 9.4, Cary, NC, USA).
3. RESULTS AND DISCUSSION 3.1. T2 Relaxation of Peach Samples of Different Maturities following HPP. Measurement of NMR relaxation time is a useful technique widely applied in plant studies to investigate the physical properties of water in various tissues.32,33 The signals are generally an average over the whole sample, leading to information on the water relationships D
DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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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 and chemical composition. The difference 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 T2 relaxation time observed in this study are highly correlated to an increase in the permeability of the membrane, which resulted from the combined effect of physical changes during fruit maturation and damage to the cell during high pressure processing. Musse et al.40 studied the changes that occurred during postharvest tomato fruit ripening using MRI and NMR relaxometry and observed significant changes in T2 relaxation time in the vacuolar compartment of the core, placenta, and outer pericarp. All three tissue types started to show a decrease in T2 on day 7 after harvest and declined the most by day 16. The authors found an approximately 25% decline in T2 relaxation time of the vacuolar compartment after 21 days of storage, as compared to the initial T2. Changes in T2 relaxation signal were reported to correlate with water content, sugar concentration, chemical composition, cell dimension, and membrane permeability.41 Sugar content was suggested to play a crucial role in the shift in T2 relaxation time because sugar has a lower T2 than water. Sugar in general has labile protons (−OH), and proton exchange will occur between the
Figure 3. Light micrographs (40×) of peach cells from maturity stages (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 with neutral red dye.
200 or 400 MPa there was complete loss of cell integrity; therefore, no viable cells were observed in peaches of any of the three maturity stages (Figure 3b,c). Any time pressure is applied, there will be a come-up stage, a constant pressure stage, and a decompression. At this time it is not possible to evaluate microstructural changes during each phase separately; rather, the change is observed after the entire process is completed. As membranes lose their integrity due to pressure-induced damage, the vacuoles lose their ability to maintain an acidic pH environment, resulting in a lack of ionization and accumulation of red dye in the vacuole. Acids are stored in the vacuole so when the tonoplast ruptures, the acids diffuse out due to osmotic differences. The neutral red dye changes from yellow E
DOI: 10.1021/acs.jafc.6b02252 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry (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 during fruit ripening.44−47 Crookes and Grierson46 found that dissolution of the middle lamella and disruption of the primary cell wall occurred during ripening of tomato fruit. These authors suggested that these changes were correlated with the synthesis of the pectin-degrading enzyme, polygalacturonase (PG). In the unprocessed (control) peaches, the most mature fruits, which were stage M3, had the greatest number of intact vacuoles (Figure 3a, M3). This may be due to the ripeningrelated breakdown of the middle lamella and reduction in intercellular adhesion, which loosens the matrix the cells are held in. It may be that the liberation of peach cells from their cellular matrix allows them to resist the strain of high pressure application. As a consequence, the unprocessed (control) M3 peaches exhibited more viable cells, compared to maturity stages 1 and 2. 3.3. Degree of Browning in Peach Samples of Different Maturities following HPP. The discoloration of peaches was not observed immediately after HPP treatment; rather, enzyme-catalyzed browning induced the development of color in treated peach samples after 2 weeks of refrigerated storage. Preliminary experiments determined that browning was not visible until after the first week of storage and that color was constant by 2 weeks; therefore, the measurement was obtained then. Because oxygen is required for the reaction, it may be that the packaging film permeability allowed for slow infiltration during storage and that surface browning reactions were complete by 2 weeks. The unprocessed samples taken from the 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 (
