Polygalacturonase and Pectin Methylesterase Activities of CaCl2 Treated Red-Fleshed Dragon Fruit (Hylocereus polyrhizus) Harvested at Different Maturity

Fruits harvested at different maturity possess different biochemical constituents and physiological properties that make the fruits may react somewhat differently to the postharvest treatment. A study to examine the activity of Polygalacturonase (PG) and Pectin Methylesterase (PME) enzymes during storage in dragon fruit (Hylocereus polyrhizus) harvested at 28 days (Index 3) and 34 days (Index 5) after anthesis and postharvest treated with 0, 2.5, 5.0 and 7.5 g L −1 CaCl2 was performed. The PG activity was lower in younger fruit and vice-versa for PME activity. Increasing concentration of CaCl2 effectively reduced the activity of both enzymes. PG activity for fruit treated with 0, 5 and 7.5 g L −1 CaCl2 increased linearly with the time of storage while its activity for the fruit treated with 2.5 g L −1 CaCl2 was lower at the beginning of storage. PG activity of Index 5 fruits increased almost linearly during storage while its activity in Index 3 fruits was low at the early days of storage and later continued to increase until day seven. At both maturity indices, the PME activity was low at the early days of storage and later continued to increase until day seven. Overall, results obtained indicated that CaCl2 postharvest treatment reduced both PME and PG activities thus slowing down the softening process giving an evident that calcium possess a distinguishable role in the reducing softening of fruit, regardless of maturity index.


INTRODUCTION
Red-fleshed dragon fruit (Hylocereus polyrhizus) is a climbing cacti and has gained popularity due to its highly nutritious and delicious fruits. The fruit is normally harvested at its full maturity stage (33-35 days after anthesis) and at this stage, the fruit is characterized by its bright red skin with dark red flesh. Fruits harvested at full maturity may have a short storage life, which is partially associated with the disintegration of cell wall that bring about changes in fruit firmness which is largely linked with the activity of cell wall degrading enzymes, including Polygalacturonase (PG), Pectin Methylesterase (PME) and β-galactosidase.
Dragon fruit is a fast developing fruit. Its maturation and ripening are coupled with the increasing concentration of soluble solids and ascorbic acid contents, alongside with decreasing in fruit firmness. The fruit pH is generally decrease with the advancement of fruit ripening, followed by an increase in the pH as the ripening progressed (Novita, 2008) and this make the fruit more palatable. Therefore, harvesting the fruit at varying maturity would give fruits of different physicochemical and organoleptic quality. Younger fruits would be firmer, contains higher concentration of acids but generally having a lower sugar content. More mature fruit would contain relatively lower acids with higher sugar concentration but it could have a soft texture. The

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Previous research has shown that calcium plays a significant role in reducing mechanical damage of both climacteric and non-climacteric fruits (Lamikanra and Watson, 2004). If the activities of PG and PME can be reduced in the whole fruit via application of calcium in dragon fruit as observed in the fresh-cut dragon fruit (Chuni et al., 2010), the longevity of the fruit can be extended. By treating the fruit with calcium, the rate of firmness loss is expected to be reduced as calcium would reduce the cell wall degrading enzymes activity. In this study, changes in the activities of polygalacturonase and pectin methylesterase were investigated on dragon fruit harvested at two different maturity indices (Index 3 and 5) treated with varying levels of CaCl 2 during 7 days of storage.

Plant Material and Post-Harvest CaCl 2 Treatment
Dragon fruits (Hylocereus polyrhizus) of uniform size with maturity indices 3 and 5 (corresponding to 28 and 34 days after anthesis, respectively) were harvested from a commercial farm in Nilai, Negeri Sembilan, Malaysia. The fruits were then rinsed with tap water, air-dried and soaked in Tween-20 for 5 min and left to dry at room temperature (25 o C). Once dried, the fruits were dipped into four levels of CaCl 2 concentration (0, 2.5, 5.0 and 7.5 g L −1 ) for two hours and air-dried. Zero (0 min) duration of dipping is referred to as a very quick dip of less than 5 sec. The flesh of the fruit was cut into small cubes (~1 cm 3 ) for analysis.

Enzyme Extraction
Extraction of PG and PME enzymes was performed as described in Chuni et al. (2010). Ten g of tissues were homogenized using a domestic blender in 20 mL of a buffer solution containing 0.1 M sodium citrate, 1 M NaCl, 13 mM EDTA, 10 mM β-mercaptoethanol and 2% (w/v) polyvinylpyrrolidone (PVP-40) at pH 4.6. The extracts were left for 30 min with occasional stirring. The supernatants were recovered by centrifugation at 29000×g for 30 min and kept at 4 o C in order to extend the shelf life of the enzymes (up to one month) and prevent significant loss in their activity.

Assay of Enzymes
Polygalacturonase (PG, EC 3.2.1.67) activity was measured by the 2-cyanoacetamide method based on the spectrophotometric determination of reducing groups released (Gross, 1982). The enzyme was assayed in a solution made using 0.75 mL of 1.5% (w/v) polygalacturonic acid, 0.1 ml sodium acetate (0.1 M) and 1.0 mL supernatant at pH 5.2, adjusted using HCl. The mixture was incubated for 1 hour at 37°C. The absorbance was measured by using a spectrophotometer (Model PRIM Light 230V) at 276 nm. Monogalacturonic acid was used as the standard to establish the calibration. PG activity was expressed as nkatal/g Fresh Weight (FW). One nkatal was defined as the amount of enzyme that releases in mol of reducing group (monogalacturonic acid) per one hour.

Data Analysis
The experiment was conducted in a Complete Randomized Design (CRD) with three replications. Data obtained were subjected to Analysis of Variance (ANOVA) and means comparisons were performed by using Least Significance Difference (LSD) at p≤0.05 level with SAS package (version 9.0, Cary, NC, USA). Regression analysis was also carried out to examine the trend of the response of the enzymes vs. time of storage for different concentrations of CaCl 2 .

Activity of Polygalacturonase
Changes in the PG activity in fruit at two different stages of maturity as affected by post-harvest calcium treatment were shown in Table 1   Analysis of variance shows that PG activity differed significantly (p<0.05) between maturity indices. PG activity was higher in more ripen fruit (Index 5) compared to Index 3 fruit (p<0.05), a result paralleled to the one obtained in previous study (result not shown). Result in Table 1 also indicated that different CaCl 2 concentration significantly affected the PG activity for both maturity indices (p<0.05). Fruits treated with 7.5 g L −1 CaCl 2 gave the lowest PG activity, followed by fruit treated with 5, 2.5 and 0 g L −1 CaCl 2 for both maturity indices. There was also an interaction found between maturity indices and different calcium concentration (p<0.01). It can be stated that dragon fruit with different maturity and treated with varying levels of CaCl 2 concentration acted together to altered the PG activity. Figure 1 shows that activity of PG for fruit treated with varying levels of CaCl 2 concentration differed significantly and behave differently over time as indicated by significant interaction between CaCl 2 concentration and storage time (p<0.001). The lowest PG activity for fruit with maturity index 3 (Fig. 1a) after seven days occurred in fruit treated with 7.5 g L −1 CaCl 2 followed by fruit treated with 5 g L −1 CaCl 2 with their respective values of 2.22 and 2.77 nkat g −1 FW. Similar result was obtained for fruit with maturity index 5 (Fig.  1b). For both maturities, the trend of the response curves was almost similar. The PG activity increased with the storage time but the value was decreased as the CaCl 2 concentration increased. Low PG activity obtained from these two treatments reflected the depressing effect of varying CaCl 2 concentration and maturity indices on PG activity of dragon fruit.
Post-harvest application of CaCl 2 has been proven to reduce the enzymes levels and increase the neutral sugar in fruits (Manganaris et al., 2005). Exogenously applied calcium binds the negative charges of deesterified uranic acid residues generated by PME during ripening and therefore enhancing the tissue's mechanical strength (Magee et al., 2003). Previous research done on freshly cut cantaloupes has shown that CaCl 2 treatment improves the fruit firmness and quality (Luna-Guzman et al., 1999). These results might indicate that CaCl 2 has reduced the PG activity thus slowing down the softening process. It is therefore evident that calcium possess a distinguishable role in the reducing softening of fruit. Table 1 and Fig. 2 show the changes in PME activity of fruit with two maturity indices that has been given a post-harvest CaCl 2 treatment. Like PG, there was a significant difference (p<0.05) in PME activity between maturity indices in PME. Contrary from PG activity, PME activity was higher in unripe fruit (Index 3) compared to more mature fruit (Index 5). Beside maturity index, varying levels of CaCl 2 concentration also contributed to the significant effect of PME activity for both maturity indices (p<0.05). High CaCl 2 concentration reduced the PME activity. At 0 g L −1 CaCl 2, the activity of PME was the highest, followed by fruit treated with 2.5, 5 and 7.5 g L −1 CaCl 2 . Maturity index and calcium concentration itself altered the PME activity markedly (p<0.001). The effect of CaCl 2 on PME activity was different for fruits of varying maturity stages as shown by a significant interaction between maturity index and calcium concentration (p<0.001). Response curve for PME (Fig. 2) show that over storage time, there was a significant difference (p<0.001) for PME activity of fruit treated with different level of CaCl 2 for both maturity indices. The trends of the curve were similar for both maturity indices. There was a sharp increase in PME activity after four days of storage for fruit treated with 0 g L −1 CaCl 2 . Like PG, PME activity for all treatments increased over time but when the Ca concentration increased, the PME activity was decreased.

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Post-harvest dips in CaCl 2 solutions allows the formation of COOgroups from the pectin content of the fruits with which Ca 2+ ions can form salt-bridge cross-links (Saftner et al., 2003). This makes the cell wall less accessible to the softening enzymes. Since PG only hydrolyses homogalacturonan regions whose uranic acid residues have been previously demethylated by PME (De Assis et al., 2001) and since pectins are synthesized and deposited on the cell wall (Staehelin and Moore, 1995), the negative charges generated by PME are necessary for calcium binding to the cell wall and to bring out calcium's firming effects. Thus, the calcium application to the fruit can significantly contribute to the texture retention in fruit. Furthermore, increasing Ca concentration in dragon fruit has been beneficial in Science Publications AJABS reducing incidence of postharvest diseases (Awang et al., 2011;2013).

CONCLUSION
PG and PME acted differently depend on the maturity index of the fruit. PG activity was higher in more mature fruit (Index 5) while PME activity was higher in more unripe fruit (Index 3). Clear evidence on the depressing effect of CaCl 2 on PG and PME activities suggests that the salt could be used as a post-harvest treatment on dragon fruit to prolong the shelf-life of the fruit. Higher concentration of CaCl 2 (7.5 g L −1 ) can effectively reduce the PG and PME activities compared to 0, 2.5 and 5 g L −1 CaCl 2 . By giving the pre-harvest calcium treatment in early stage (Index 3), perhaps the PME activity can be reduced further and this can lead to the reduction of PG activity as well.

ACKNOWLEDGEMENT
This study was financially supported by the Ministry of Science, Technology and Innovation, Malaysia (Sciencefund-05-01-04-SF0174) granted to corresponding author.