Abstract
This research explores the cell wall composition and polyphenol oxidase activity of two pawpaw (Asimina triloba) fruit varieties, Susquehanna and Green River Belle, that were subjected to high pressure processing and 45 days of refrigerated storage. We hypothesize that high pressure processing may inhibit enzymatic action responsible for pawpaw's deleterious postharvest tissue softening and browning. Glycome profiling uses mAb groupings that recognize 19 groups of glycan epitopes present in most major classes of cell wall glycans and was used to determine cell wall composition. Results show that both varieties have typical type I primary cell walls of flowering dicots. However, differences in the fine cell wall structure between the varieties can be inferred and the varieties behaved differently during refrigerated storage, likely indicating of a difference in cell wall-modifying enzymes present in the primary cell walls. High pressure processing treatment does not seem to be effective at eliminating polyphenol oxidase activity.
Introduction
Pawpaw (Asimina triloba L. Dunal) is a climacteric tree fruit from the tropical Annonaceae family grown in eastern United States (Archbold and Pomper, 2003). It is unusual in that it is the only Annonaceous fruit that grows in temperate climates and its flavor resembles a combination of banana and mango (Brannan et al., 2012). Pawpaw fruit pulp is susceptible to rapid decline in postharvest shelf life due to increases in soluble solids content, tissue browning, and softening (McGrath and Karahadian, 1994). Research on the physiology and biochemistry of pawpaw fruit associated with ripening and postharvest storage has been slow to emerge. What is known is that during fruit ripening and postharvest handling, increases have been reported for respiration, ethylene generation (Archbold and Pomper, 2003), headspace volatiles, and soluble solids (Archbold and Pomper, 2003; McGrath and Karahadian, 1994).
Tissue softening in fruit during ripening and postharvest storage is attributed mainly to modifications in cell wall polymers such as hemicelluloses, pectins, and cellulose, which result in plant cell wall loosening. Cell wall-modifying enzymes are the main contributors to the loosening of fruit cell walls during storage. These enzymes work in concert to induce alterations in the fine structures of cell wall polymers, such as loss of neutral or acidic sugars and de-esterification, which promote de-polymerization and solubility of the polymers (Prasanna et al., 2007). Hemicelluloses are a diverse set of heteropolymers of polysaccharides and include xylans, glucuronoxylans, arabinoxylans, glucomannan, and xyloglucans (XyGs) (Faik, 2013; Scheller and Ulvskov, 2010). Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes that have been shown to play an important role in the control of plant cell wall loosening (Rose et al., 2002). Pectins are classified as rhamnogalacturonan I (RG-I), RG-II, and homogalacturonan (HG). RG-I consists of a backbone of alternating β-
Degradation of pectins during ripening appears to be associated with tissue softening in many fruits such as melon, papaya, watermelon, peach, tomato, mango, and avocado (Brummell, 2006). As an example, the ripening of mango seems to be the result of a progressive depolymerization of pectic and hemicellulosic polysaccharides with loss of galactose, arabinose, and mannose residues observed at the ripe stage (Yashoda et al., 2007). In tomatoes, the pectin depolymerization enzyme endo-polygalacturonase is implicated in decreased cell wall integrity and the associated decrease in shelf life during long-term storage (Langley et al., 1994).
Certain enzymes that have the potential to degrade cell walls include polygalacturonase, endo-(1,4)-β-
Pawpaw fruit pulp is also known to be susceptible to brown discoloration. Polyphenol oxidase (PPO, EC 1.10.31) has been implicated in browning (Fang et al., 2007) and its possible substrates identified (Brannan et al., 2015). PPO is responsible for enzymatic browning in fruits by catalyzing mono- and o-diphenol conversion to o-quinones during ripening and postharvest handling, storage, and processing. Antioxidant content (including mono- and o-diphenols) of pawpaw from several species has been analyzed and shown change during ripening (Brannan et al., 2015; Brannan and Wang, 2017; Chaves et al., 2011; Prieto et al., 2007). In other Annonaceous fruits, PPO activity has been determined for atemoya (Chaves et al., 2011), cherimoya (Prieto et al., 2007), soursop (Deoliviera et al., 1994; Falguera et al., 2012), and sugar apple (Wu, 2000). The first study on the activity of PPO in pawpaw (Fang et al., 2007) identified two isoforms of pawpaw PPO of 28.2 and 38.3 kDa, with greatest activity at pH 6.5–7.0 and 5–20 ℃. Subsequently, PPO activity and kinetic parameters have been determined in different pawpaw varieties (Brannan, 2016). PPO activity assessed during frozen storage showed that ascorbic acid and n-acetylcysteine were effective PPO activity inhibitors in pawpaw (Brannan and Wang, 2017).
It has been shown that high pressure processing (HPP) of fresh products can be very effective for destruction of pathogenic organisms at relatively low pressures (Guerrero-Beltran et al., 2005) but complete or even partial inactivation of many enzymes has not been assured at commercially feasible processing conditions (Terefe et al., 2014). HPP occurs in flexible containers that are loaded into high-pressure vessels filled with a liquid that exerts the pressure. The pressure is applied by forcing additional liquid into the sealed vessel until the desired processing pressure is reached. Pasteurization treatments use pressures in the range of 600 MPa for several minutes at chilled or ambient temperatures. HPP treatment decreased PPO activity in pawpaw and PPO activity was observed to decline after only one day of refrigerated storage (Zhang et al., 2017).
Identification of cell wall polysaccharides in pawpaw and their structural changes during postharvest storage have not been undertaken. Because HPP has been commercially effective in fruits that are susceptible to rapid tissue browning and softening, e.g. avocado, it may prove beneficial toward inhibition of enzymatic action responsible for these deleterious effects on pawpaw. Therefore, the objective of this study is to determine the change in the composition of cell wall polysaccharides and the activity of PPO as affected by HPP in pawpaw fruit pulp from two varieties during refrigerated storage.
Materials and methods
Materials
All chemicals and reagents were obtained from Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St Louis, MO). Pawpaws (A. triloba) from two varieties (Green River Belle (GRB), Susquehanna (SQ)) were donated by Fox Paw Ridge Farm (Cincinnati, OH).
Sample preparation
Pawpaws from each variety were identified as ripe by feel, picked from the tree, and then transported to the laboratory on ice (∼3 h). Each whole pawpaw was weighed. Pulp was separated from skin by hand and seeds removed using a 6 mm mesh food strainer (Pragotrade USA, Inc., Strongsville, OH). Fruits from each variety were stored in polyethylene/nylon 27.94 cm bags (FoodSaver, Jarden Corp., Rye, NY) with an oxygen transmission rate of 6.7 cc/m2/24 h/23 ℃/0% RH. Bags were vacuum sealed and held at 4 ℃ overnight before HPP processing.
Six matched pawpaws of similar weight and shape from both varieties were left whole. These fruits were vacuum packaged in individual packages as described above and stored refrigerated (4 ℃) overnight before HPP processing.
HPP and refrigerated storage of pawpaw pulp
HPP was conducted in a commercial HPP unit (NC Hyperbaric 420, Hyperbaric USA, Miami, FL) located at Sandridge Food Corporation, Medina, OH. The morning after harvest, vacuum packed samples (250 g) and whole fruits were transported to the HPP facility on ice. The samples were placed in a high pressure vessel (380 mm internal diameter), filled with water at 4 ℃ and subjected to 600 MPa for 5 min. After processing, the samples were transported back to the laboratory on ice. Pulp from day 0 samples was immediately stored frozen (−40 ℃) until analyzed. Pulp selected as day 15, 30, and 45 was stored at 4 ℃ for the designated storage time and then held at −40 ℃ until analyzed.
HPP, color, hardness, and percent sugar of whole fruit
Six whole fruits from each variety were transported to the HPP facility where three were subjected to HPP with the conditions described above and three matching fruits were not. After returning from the HPP facility, a portion of the skin was removed and pulp color, fruit hardness, and percent sugar (°Brix) were measured on the exposed pulp. Pulp color was measured using a Konica-Minolta BC-10 colorimeter and reported as L*, a*, and b*. Hardness (kg force) was performed by penetrometry using cylindrical probe (10 mm) at a crosshead speed of 5 mm/s to a depth of 10 mm. °Brix recorded directly from juice (∼1 ml) that was extracted directly from the pulp using a refractometer.
Preparation of alcohol insoluble residue (AIR) extracts and fractionation
Plant cell wall glycan-directed mAbs used for glycome profiling of pawpaw pulp.
Antibodies were grouped according to a hierarchical clustering of the predominant polysaccharides they recognize and are listed in the WallMabDB plant cell wall-directed mAb database (www.wallmabdb.net).
Determination of PPO in pawpaw
PPO activity was accomplished at pH 6.5 according to a published method (Fang et al., 2007) that was optimized for pawpaw pulp and used subsequently (Brannan, 2016; Brannan and Wang, 2017; Zhang et al., 2017). The crude extract of PPO was mixed with 0.4 ml of 0.2 M catechol and absorbance at 420 nm was determined immediately and at 15 s intervals up to 600 s. A PPO sample blank contained all the reagents except that the diluted PPO solution was replaced by the same volume of 0.1 M sodium phosphate buffer (pH 6.5). Soluble protein was determined on the crude extract using the Lowry protein assay and quantified based on an albumin standard curve. PPO activity was measured as the increase in absorbance over time and reported as ΔABS/min/g protein.
Statistics
Analysis of variance was used to determine significant differences between PPO activities. Means (n = 6) were generated from three distinct crude PPO extractions were performed for each variety, processing treatment, and storage time combination, and duplicate PPO analysis was performed on each extraction. Duncan's Multiple Range test was used to separate means. From four whole fruits, data for percent sugar, pH, color, and hardness for control fruits and fruits subjected to HPP were analyzed by t-tests to determine differences.
Results and discussion
This study reports for the first time the primary cell wall composition of pawpaw pulp using the glycome profiling technique. Glycome profiling is a powerful method that provides a semi-quantitative measure of most major noncellulosic cell wall glycan epitopes in the cell walls of pawpaw and, in this paper, how they are affected by HPP and refrigerated storage. This ELISA-based method provides detailed information about specific cell wall glycan epitopes using more than 150 monoclonal antibodies specific to more than 30 groups of glycan epitopes covering most major noncellulosic matrix glycans in higher plant cell walls (listed in Table 1). The output shown is presented as a heat map with antibodies grouped according to their glycan specificity. AIRs from both pawpaw varieties were prepared and then subjected to four sequential extractions using increasingly harsh reagents, first an oxalate extraction, followed in order by three alkaline extractions, carbonate, 1 M NaOH, and 4 M NaOH. Oxalate binds Ca2+ from cell walls, releasing the most loosely associated pectic polymers, while carbonate treatment extracts relatively tightly bound pectins as well as certain type II AGPs. NaOH solutions of different strengths extract mostly hemicelluloses and solubilize very tightly bound pectins and AGPs. This fractionation does not necessarily separate cell wall polysaccharides into pure fractions such that epitopes recognized by the same monoclonal antibodies may be present on different wall polymers, thus these results are only semi-quantitative. The strength of the signals is indicative of the abundance of the corresponding epitopes in those particular fractions. Importantly, the sequential extraction would show how easily the epitopes are extracted, which can be used to infer changes to the cell wall polymers during storage or between cultivars.
Cell wall glycan composition of the two varieties of fresh pawpaw pulp
As a first step, we sought to determine whether the pulp from the two pawpaw cultivars, GRB and SQ, have a typical type I cell wall composition and whether the glycome technology can identify structural differences between the two cultivars. To this end, untreated, fresh pawpaw pulp cell wall glycans from the two pawpaw varieties were analyzed (Figure 1). The presence of XyG epitopes (both fucosylayed and non-fucosylated) was clearly detected by this method. Other hemicelluloses such as xylans were also detected, especially epitopes detected by XYLAN 1/XG group of mAbs, in both pawpaw varieties. However, xylan epitopes recognized by XYLAN 2 and XYLAN 5–7 groups of mAbs were detected more in SQ. In general, xylan epitopes were much weaker in abundance compared to XyG epitopes. Acetylated glucomannan epitopes were weakly detected in both pawpaw varieties. As expected, epitopes corresponding to galactomannan (composed of mannose and galactose, which are associated with seed gums) or β-glucans (commonly referred to as mixed-linkage glucans found mostly in grass cell walls) were not observed in both varieties. Type II arabinogalactans (AGPs) are heteropolymers of Heat map showing cell wall glycan epitopes from alcohol insoluble residues from fresh, untreated pawpaw pulp (A. triloba) from variety GRB and SQ. Glycome profiling involves an ELISA screening using 155 plant cell wall glycan-directed monoclonal antibodies. The detailed list of the antibodies used is shown on the right panel, color-coded as groups based on a hierarchical clustering of the cell wall glycans recognized by each antibody group. The ELISA signal strength is displayed by a black-blue-red-yellow scale, with black indicating the absence of binding and yellow showing strongest binding. Labels at the bottom indicate the reagents used for each extraction step. AG: arabinogalactan; GRB: Green River Belle; RG: rhamnogalacturonan; SQ: Susquehanna.
It is interesting to note the extractability differences of some epitopes between the two varieties, from which can infer some structural differences in their cell walls. For example, shown in Figure 1, oxalate treatment extracted more XyG epitopes (both fucosylated and non-fucosylated) from AIR of SQ compared to GRB. This may suggest the presence of more loosely associated XyG in SQ cell walls. Also, our analysis suggests that SQ may have some XyGs that are more tightly associated with cellulose because strong signals were observed in harsher treatment (4 M NaOH) compared to GRB. Since all the extracts were generated from similar fresh weight of pulp and the treatments produced comparable amounts of cell wall materials, it strongly suggests that cell wall materials account for the same amount of pulp fresh weight in both varieties. The differences in the extractability of the polymers are an indication of differences in the fine cell wall structure in the two pawpaw varieties. Further microscopic analyses are needed to confirm this hypothesis. These subtle structural differences can be used to develop strategies to improve postharvesting shelf life and storage of pawpaw.
Changes in cell wall glycans from pawpaw pulp affected by HPP processing
Next, we sought to determine what changes in cell wall glycans occur in pawpaw pulp during 45 days of refrigerated storage. To this end, pulps from both varieties were tested with and without HPP processing. As indicated in Figure 2 for GRB pulp, no major difference in the extractability of XyG epitopes between HPP processed and unprocessed pulp was observed. However, oxalate extractions, which would be expected to release the most loosely associated pectic polysaccharides as well as arabinogalactan-proteins (recognized by AG 1–4 groups of mAbs), indicated the presence of epitopes recognized by HG Backbone 1, but not HG Backbone 2. HG Backbone 1 extractability appears to intensify in HPP-processed pulp. The extractability of AG 1–4 epitopes seems to increase during refrigerated storage of both the control and HPP processed pawpaw pulp. Alkali solutions of different strengths appear to extract more hemicellulose and more tightly bound pectins and AGP proteins during refrigerated storage in both the control (unprocessed) and HPP-processed pulp. The major effect was observed with the extractability of AG epitopes that seems to correlate with the length of refrigerated storage. In both varieties, carbonate and 1 M NaOH extracted more RG-1c and RG1/AG recognized epitopes after 45 days of refrigerated storage, with an increased effect due to HPP processing. These extracts confirm that longer refrigerated storage could affect the structure of AG-1–4 proteins, which increases their extractability (with carbonate and 1 M NaOH) after long period of refrigerated storage in both control and HPP-treated samples. A similar effect of 4 M NaOH on the extractability of xylan epitopes (detected by XYLAN 5–7 groups of mAbs) was observed for both the control and HPP-processed samples, which appear to increase during refrigerated storage.
Heat map showing cell wall glycan epitopes from alcohol insoluble residues from pawpaw pulp (A. triloba variety GRB) with and without HPP that was stored refrigerated (4 ℃) for 45 days. Glycome profiling involves an ELISA screening using 155 plant cell wall glycan-directed monoclonal antibodies. The detailed list of the antibodies used is shown on the right panel, color-coded as groups based on a hierarchical clustering of the cell wall glycans recognized by each antibody group. The ELISA signal strength is displayed by a black-blue-red-yellow scale, with black indicating the absence of binding and yellow showing strongest binding. Labels at the bottom indicate the reagents used for each extraction.
As indicated in Figure 3 for SQ pulp, the storage effect on untreated samples appears to follow a general trend consisting in a decrease in the intensity of almost all signals after 30 days of storage. For example, XyG epitopes' extractability with oxalate increased after 15 days of storage (compared to fresh pulp) but drastically decreased at 30 and 45 days of storage. A similar trend was observed for pectic epitopes (detected by RG-I/AG, GR-1a, RG-1b, and RG-1c, HG BACKBONE 1 groups of mAbs) and xylan epitopes (Figure 3). Theoretically, this could be explained by possible degradation/solubilization of the cellulose, XyGs, xylans, and pectin epitopes by cell wall enzymes such as cellulases (GH9 family, EC 3.2.1.4), XTHs (GH16 family, EC 3.2.1.151), and endo-polygalacturonases (GH28, EC 3.2.1.15). Faik et al. (1998) showed that cellulase and XTH activities remain strongly attached to residual cell wall of tomato and required the use of salt to extract them from the cell wall (Faik et al., 1998). Our hypothesis is also supported by the fact that HPP treatment reduced the decrease in signals in oxalate extracts and NaOH extracts, but not in carbonate extracts (Figure 3). Cellulases could modify cell walls not only by degrading the paracrystalline sites of cellulose microfibrils, but also by releasing XyG tethers between the microfibrils. Most plant cellulases use as substrates XyG, xylans, and glucomannan. Thus, cellulases could be a good target for manipulation to improve pawpaw pulp storage.
Heat map showing cell wall glycan epitopes from alcohol insoluble residues from pawpaw pulp (A. triloba variety SQ) with and without HPP that was stored refrigerated (4 ℃) for 45 days. Glycome profiling involves an ELISA screening using 155 plant cell wall glycan-directed monoclonal antibodies. The detailed list of the antibodies used is shown on the right panel, color-coded as groups based on a hierarchical clustering of the cell wall glycans recognized by each antibody group. The ELISA signal strength is displayed by a black-blue-red-yellow scale, with black indicating the absence of binding and yellow showing strongest binding. Labels at the bottom indicate the reagents used for each extraction.
Overall, similar cell wall glycans were present in both GRB and SQ, namely glycan epitopes recognized by HG Backbone 1, AG 1–4, RG-1c, RB1/AG, and Xylans 5, 6, 7, but the two varieties behaved differently during refrigerated storage. This is most likely indicative of a difference in cell wall-modifying enzymes present in the primary cell walls of these two varieties. However, HPP treatment seems to reduce depolymerization of cell wall polymers to some degree in GRB but not as efficiently in SQ. Therefore, genetic manipulation of some of these enzymes may be necessary to extend shelf life storage of pawpaw pulp.
PPO activity in GRB and SQ pawpaw pulp
In another set of experiments, we investigated PPO activity in fresh pulp from the two pawpaw varieties. Overall, there was no difference in PPO activity between pawpaw pulp from either variety for the control samples (p = 0.777) or the HPP-processed samples (p = 0.865). PPO activity was significantly reduced over the storage period for HPP-processed pulp compared to unprocessed pulp (1.8 versus 2.4 ΔABS/min/mg protein, respectively) in GRB pulp but not in SQ pulp (1.9 versus 2.0 ΔABS/min/mg protein, respectively). In both varieties, refrigerated storage significantly affected PPO activity irrespective of processing, with the PPO at 0 days of storage being significantly higher than the PPO activity for remainder of the storage time.
As shown in Figure 4, results for the two-way interactions indicate similar findings. In GRB pulp, control pulp was significantly higher in PPO activity than HPP-processed pulp at day 0, but there was no change in either control pulp or HPP-processed pulp thereafter. This is consistent with the inactivation of some PPO by HPP treatment. However, HPP treatment does not seem to be effective at eliminating PPO activity. This suggests that pawpaw pulp may contain various types of PPO enzymes. Similarly, in SQ pulp, PPO activity was significantly higher in the control pulp at day 0 compared to HPP-processed pulp, which is consistent with inactivation of PPO activity by HPP treatment. Again, there was no difference between control pulp or HPP-processed pulp at any of the storage points, suggesting the presence of residual PPO activity that was not inactivated by HPP treatment. These results agree with a recent study that shows that HPP reduced but did not completely inhibit PPO activity in pawpaw pulp from the variety Shenandoah (Zhang et al., 2017).
PPO activity in two pawpaw varieties (GRB, upper; SQ, lower) with and without HPP subjected to 45 days of refrigerated storage. Different letters indicate significant differences (p < 0.05).
Effect of HPP and refrigerated storage on color, hardness, percent sugar, and cell wall glycans
Percent sugar (°Brix), pH, CIE chromaticity coordinates (L*, a*, b*), and hardness for whole pawpaw fruit from two varieties with or without high pressure processing (HPP).
Letters a,b significantly different at p < 0.001.
Letters x,y significantly different at p < 0.001.
Storage also caused changes to the pawpaw pulp. AG 1–4 was indicated in both varieties, but an increase during refrigerated storage of both the control and HPP pawpaw pulp was observed only in GRB pulp. These glycans have been shown to increase during fruit ripening and mechanical wounding (Fragkostefanakis et al., 2012). Xylans 5, 6, 7 are indicated in both varieties, and they have been indicated as substrates for in enzymatic cell wall degradation in papaya. It is interesting that they increase during refrigerated storage of GRB pulp but decrease during refrigerated storage of SQ pulp. In any event, it is likely that pawpaw tissue softening is related at least in part to xylan degradation during storage. Both of the cell wall glycan changes observed during refrigerated storage could be related to the observation that refrigerated storage significantly reduced PPO activity.
Pawpaw ripening after harvest happens very quickly (Archbold and Pomper, 2003) and is a challenge to commercialization of the fruit. Currently, whole pawpaw fruit and fresh pawpaw pulp are difficult to distribute long distances due to rapid browning and textural changes. Commercial pawpaw pulp is available frozen with ascorbic acid added, which has been shown to be an effective browning inhibitor in pawpaw (Brannan and Wang, 2017). Due to the extreme softening of the pawpaw pulp and the cracked, seeping surface (data not shown) of whole HPP-processed fruits from this study, cold storage (Galli et al., 2008) remains the best option for whole pawpaw fruit. For pawpaw pulp, results from this study and a recent study from our laboratory indicate that HPP processing and storage time led to reduced PPO activity and HPP processing does not affect the initial sensory qualities (color, texture, taste, or flavor) of pawpaw pulp from variety Shenandoah (Zhang et al., 2017). This study, which provides a characterization of pawpaw cell wall glycans and PPO and some information about how they are affected during refrigerated storage, indicates that specific study on the degradation of several cell wall polymers (i.e. XyG, pectins, and AGPs) could warrant further investigation as it relates to pawpaw tissue softening and PPO-mediated tissue browning.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant from the Ohio University Research Committee (RC1005214).