Minimally of Polyphenol Oxidase Activity and Controlling of Rotting and Browning of Longan Fruits cv. DAW by SO2 Treatment under Cold Storage Conditions

INTRODUCTION

Longan (Dimocarpus longan Lour.) is a tropical fruit in the Spindaceae family. In Thailand, the Longan is a most extensive production and one of the most economically important fruits that has exported fresh Longan to China, Hong Kong, Malaysia, Singapore, Indonesia and Canada (Tongdee, 1997). The cultivated areas are in the Northern region of Thailand. In the year 2008, dried and especially fresh fruit of the Longan is mostly marketed locally and export of the fruit has been increasing rapidly, the exported of fresh Longan is about 168,286 tons and frozen Longan at 346 tons (Lin el al., 2001). However, the quantity of domestic and export Longan has been limited by its highly perishable nature, short storage life and susceptibility to postharvest diseases, as a result of bacterial, yeast and fungal infections (Tongdee, 1997). Color deterioration causes the fruit to fetch a lower price at market and even be unmarketable (Smith and McWilliams, 1978). Rapid pericarp browning during storage is the main problem resulting in restrictions on the export of Longan to long-distant markets (Sardsud et al., 1994). The fresh Longan fruit could be stored for only 2-3 days at ambient temperature, which caused of discoloration and disorder by postharvest disease including chilling injury and especially on the pericarp browning (Martinez and Whitaker, 1995). Pericarp browning has been attributed to oxidation of phenolics by PPO, producing brown coloured by-products (Ferrar and Walker, 1996). The PPO has been widely studied in various fruits such as apple, grape, litchi and plum (Lin et al., 1994) but little is known about the Longan.

For many years, the recommended method to control postharvest decay and prevent pericarp browning in the Longan has been sulfur dioxide (SO₂) treatment. The use of SO₂ fumigation has been the most effective practical postharvest treatment for control of quality during storage (Deng et al., 2005). It is currently commercially used in many countries. Recently, importing countries such as China and Singapore have restricted the import of Longan product and other fruits and reduced the maximum permitted residual level of SO₂. Longan consumers are becoming cautious regarding SO₂ residues, due to allergenic symptoms and caused of off-tasted (Whangchai et al., 2006).

The storage of Longan fruit under cold condition and the treatment of fresh Longan by using sulfur dioxide is very effective application in browning prevention on the pericarp of the fruits (Jiang et al., 2004). However, due to the restriction of the import countries, sulfur dioxide is less use due to allergic to humans (Underhill et al., 1997). There is a need to find out the suitable of sulfur dioxide concentration and the storage conditions which are the effective and could be prevented the browning and prolonged the storage shelf life of fresh Longan. Thus, the aim of this study was to evaluate the effect of sulfur dioxide treatment and storage conditions on browning prevention and prolonged the storage shelf life of fresh the Longan fruit to provide the better appearance and safe for consumers.

MATERIALS AND METHODS

Longan (Dimocarpus longan Lour.) fruit cv. Daw was harvested from Chiang Mai province, Thailand in the year 2007. The fruits were then separated into bunches with selected homogenous size and grading. The fruit bunches without defects and spoilage was used for the experiments. The experimental design in this study was laid out in 2x2x5 Factorial in CRD with 4 replications. Treatments were including with and without SO₂treat Then, the treated fruits were store at 2±2 and 7±2°C. Finally, fruits were stored for 0, 2, 4, 6 and 8 weeks. The fruits were sampling immediately after SO₂treatment at the rate 4.50 tons per 2.5 kg SO₂ and then every twice month after stored under various storage temperatures as discussed above.

Weight Loss Percentage
The fresh weight of the fruit was determined for all treatments as an index of desiccation rate. Weight loss was calculated as following;

Weight loss = (W_f - Weight of sample)/W_f x 100

where, W_f was weight of fresh fruit and Ws was weight of sample.

Polyphenol Oxidase Activity and Determination of Pericarp pH (unit)
Three fruits per treatment were thawed and peeled. And 2 g of pericarp tissue was homogenized in 0.1 M phosphate buffer, pH 6.6 and 0.5 g of insoluble polyvinyl pyrollidone (Merck) for 30 sec with a polytron homogenizer (Kinematica GmbH, Kreins, Luzern, Switzerland; probe diameter, 20 mm). The homogenate was centrifuged for 10 min at 8000x g in a Sorvall rotor SS-34 at 4°C. The supernatant was collected and centrifuged repeated in 1.5 mL tubes at 20 000x g for 10 min at 4°C. The supernatant was collected into a fresh tube and 0.75 mL was used for the PPO assay in duplicate. The PPO assay was conducted by adding 0.12 mL 4-methyl catechol (Sigma, St. Louis, MO, USA) freshly dissolved (0.25 g) in 2 mL of ethanol and 10 mL of distilled water (final concentration, 23 mM). A contron spectrophotometer was used to follow changes in absorbance at 410 nm over 2 min and the linear progress of the reactions was recorded between 30 and 90 sec. Protein content was determined according to Bradford with bovine serum protein as the standard. Results were calculated as in Δactivity mg^-1 protein x1000. The change in the pH of the buffer was determined in duplicate. To determine the pH of pericarp and aril tissue, extraction was carried out as described, but without PVP and with distilled water instead of phosphate buffer.

Peel and Aril Color
The pericarp (peel) and aril color of the Longan fruit were analyzed initially and after various storage duration. The color was measured on opposite sides of the fruit using (colourQuest XE, Hunter Associates Laboratory, Inc., New York, USA) Minolta chromameter (model CR-200; Minolta, Ramsey, NJ) which provided CIE L^*, C^*, H^*, A^* and B^* values.

Preparation of Longan Pericarp for Scanning Electron Microscope (SEM)
The Longan pericarps were cut into 5 mm squares for SEM evaluation. The pericarps were cut in a dish of 0.1 M phosphate buffer pH 7.3. The pieces were transferred immediately after they were cut into a primary fixative. The Longan pericarp pieces were fixed in a fixative solution as described by Bozzola and Russell (1999) with slight modification for anatomical study. The pericarp specimens were fixed with a primary fixative containing 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.3 at 4°C for 2 h. After that the tissue was usually washed in the same buffer vehicle used in the glutaraldehyde fixation step. Next, the specimens were post-fixed in 1% osmium tetroxide in the same buffer for 2 h. Then, the specimens were dehydrated stepwise by exposure to ethanol-buffer mixture (30, 50, 70, 80, 90 and 100%) allowing 15 min in each and critical point drying with liquid CO₂. This is a critical drying technique, as it achieves a phase change from liquid to dry gas without the effects of surface tension and is, therefore, suitable for delicate biological specimens for removal of water from the specimens. For SEM, the dried specimen was mounted on specimen studs and sputter coated with gold. Finally, the specimens were viewed with a scanning electron microscope (JEOL, JSM-5910LV, JEOL Ltd., Tokyo, Japan) at 15 kV.

Sulphite Residual (mg kg^-1)
A sample of 50 g from the whole fruit, aril and peel was obtained from a minimum of 30 fruits and stored overnight at -70°C. Sample were then examined in duplicate for sulphite residual according to De Vries et al. (1987).

Statistical Analysis
The statistical analysis was carried out using a statistical software Statistic version released 8.0 and Least significant different test at 95% was used to determine significant difference among the treatments.

RESULTS AND DISCUSSIONS

The ANOVA analysis indicated that SO₂treatment changed pH value of peel tissue significantly (Table 2). The pH value of peel tissue decreased significantly after treated the Longan fruit with SO₂(4.30), when compared with non SO₂treatment (5.36). However, SO₂treatment did not affected on pH value of aril. The storage temperature did not effect on pH value of peel and aril changed. On the other hand, the storage duration was the main factor that affect on the change of peel and aril pH. The pH value of both part of the Longan fruit increased significantly in the long term of storage (Table 1). Additionally, ANOVA analysis indicated that the storage duration did not affect only pH value of peel and aril changed but also affected on weight loss of the Longan fruit (Table 2), while the weight loss increased significantly during the long term of storage (Table 1). However, weight loss of the Longan fruit did not affected by SO₂treatment and storage temperatures (Table 1). Table 1 and 2 indicated that all treatments not affected on the change of PPO activity.

Table 1:	The effects of sulphur dioxide treatments, storage temperatures and storage durations on Longan cv. Daw fruit quality changes

The different letters indicate the statistically significant difference by LSD at 5% level. Polyphenol enzymatic activity* (PPO) was described in Δactivity mg^-1 proteinx10³

Table 2:	Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of peel and aril pH and weight loss of Longan cv. Daw

The different letters indicate the statistically significant difference by LSD at 5% level. ns: not significantly different : Polyphenol enzymatic activity* was described in Δactivity mg^-1protein x 10³

The ANOVA analysis indicated that SO₂did not affect on aril color changed. However, the storage duration was the main factor affected on the change of aril color (Table 3) (Fig. 1a-d). The aril was bright orange-yellow color after stored for 8 weeks (C^*, H^*, L^*, A^* and B^*were increased significantly) (Table 1). Moreover, the storage temperature, interaction between SO₂ and storage duration and interaction between all treatment significantly increased C^* and B^* values (Table 3), which was indicated that the aril became dull yellow color (Fig. 1g, h). The ANOVA results indicated that SO₂treatment, storage temperature and storage duration significantly affected on H^*, L^* and A^* values (Table 4). The SO₂treatment and storage temperature significantly increased H^* and L^* values, while A^* value significantly decreased (Table 1). The results showed that inner part of peel color of no SO₂ treatment and stored under 7°C was more darkened than that SO₂ treatment and stored under 2°C (L^* value decreased), which was more bright green-yellow color (A^*decreased) (Table 4) (Fig. 1c, d). For the storage duration, H^* and A^* values significantly increased, while L^* value decreased after stored for 8 weeks (Table 1). This results indicated that the inner part of peel color was became orange-yellow darkness color after stored for 8 weeks (Fig. 1e-g). Interestingly, all treatments were SO₂ treatment, storage temperature and duration was the main factors affected on the outer peel color (Table 5). The outer peel red color of no SO₂ treatment were scarlet than orange-red (H^*decreased), became darkened (L^* decreased), less intensely red (C^* decreased) and blue-yellowish (B^* decreased) color (Table 1).

Table 3:	Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of aril color of Longan cv. Daw

The different letters indicate the statistically significant difference by LSD at 5% level. ns: Not significantly different

Moreover, under high storage temperature (7°C) and long term of storage (8 weeks), the outer peel color became blue-yellowish (B^* decreased), darkened (L^* decreased) and more scarlet than orange-red (hue angle; H^*, decreased) or changed to cloudy and dark or scarlet, which was showed in browning (Table 1) (Fig. 1e, h). Pericarp browning increased with increasing of storage period. Fruit fumigated with SO₂ did not show any pericarp browning throughout this investigation. According to Duan et al. (2004) the major factors reducing the storage life and marketability of the Longan fruit are microbial decay and pericarp browning. Low temperature storage at 1-5°C is used to reduce pathological decay, but has only a limited role in reducing pericarp browning. In this study, the SO₂ treatment inhibited browning and decreased PPO activity of Longan pericarp during storage. Low PPO activity correlated with low browning appearance. According to Jiang and Fu (1998), the sulfur dioxide application gave better results in controlling litchi browning and 80-85% inhibition of PPO (Jiang, 1999). Moreover, the fruit deteriorates rapidly when removed from cold storage. It was observed that under the refrigeration conditions Longan fruits have a storage life of approximately 30 days. Pulp quality and disease development are generally stable during cold storage until such time as fruits become visually unacceptable from pericarp browning (Jiang and Li, 2001). Sulfur dioxide fumigation has been the most effective postharvest treatment for control of pericarp browning in the Longan fruit and is used extensively in commercial situations at present. However, there is increasing consumer and regulatory resistance to the use of this chemical (Jiang et al., 2002).

Fig. 1:

The effects of SO₂ treatments, storage temperature and storage duration on the changing of inner and outer peel tissue and aril color of Longan cv. daw. (a) No SO₂ treatment at the initially of storage, (b) SO₂ treatment at the initially of storage, (c) inner and outer peel tissue and aril color changing by no SO₂ treatment at the initially of storage, (d) inner and outer peel tissue and aril color changing by SO₂ treatments at the initially of storage, (e) and (f) the changing of inner and outer peel tissue and aril color by no SO₂ treatments stored at 2 and 7°C for 8 weeks, respectively, (g) and (h) the changing of inner and outer peel tissue and aril color by SO₂ treatments stored at 2 and 7°C for 8 weeks, respectively

The pearson correlation coefficients analysis showed that SO₂ treatment and storage temperature factors resulted weight loss, the changing of peel and aril tissue pH and polyphenol enzymatic activity were had the positive correlation between them (Table 6, 7). Moreover, the effect of storage duration had positive correlation between peel tissue pH-PPO enzymatic activity and weight loss-aril tissue pH but stated the negative correlation between aril pH and peel tissue pH (Table 8). Meanwhile, the experiment found that a lower pH in the peel kept in SO₂ treatment might be beneficial in preventing browning. The rapid increase in the browning index of the Longan fruit stored in SO₂ treatment after long term of storage may be due to the senescence and fruit decay, indicated by increases in pH value, which was agreed with Tian et al. (2002). According to Solomon et al. (1992) reported that PPO catalyzed browning of fruit could be prevented by several application such as; heat inactivation of enzymes, exclusion or removal of one or both of the substrates (O₂ and phenols), adding compounds that inhibit PPO or prevent melanin formation and especially on controlling the pH to be lowering to 2 or more units below the pH optimum, by reaction-inactivation of the browning enzyme.

Table 4:	Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of inner part of peel color of Longan cv. Biewkaew

The different letters indicate the statistically significant difference by LSD at 5% level. ns: not significantly different

However, experimental results indicated that non-treated and treated Longans fruit with SO₂ provided the pH about 4.30-5.36 in peel and about 6 in arils tissue. This results congruence to Wong (1995) reported that the pH optima to most PPO’s activity are near 6. Under this condition, PPO was activated and accelerated the browning of Longan fruits. Moreover, Underhill and Critchley (1992) found that the pericarp browning was correlated with moisture loss. Likewise, it is every likely that the natural cracking of Longan peel facilitates rapid moisture loss and cause surface browning during harvest and storage. The surface cracking was also impair the physiological function of the cuticle and increasing water permeability, which may cause water soaking at the inner side of the peel (Medeira et al., 1999). The injured cell would accelerate the oxidation of phenolic substances and the oxidative products resulted in dark color of inner and outer peel (Abe, 1990). The PPO and peroxidase (POD) catalyze the oxidation of phenolics to quinines and then condense tannins to brown polymers. The initiation of the enzymatic browning depends largely on the loss of compartmentation of enzymes and substrates. In this study, there were high activities of PPO and POD in the Longan fruit at harvest, but no skin browning occurred while high ATP production and low malondialdehyde (MDA) content were observed, which further supports the hypothesis that the loss of compartmentation of enzymes and substrates was the key factor for the enzymatic browning reaction of plant tissues. Thus, reduced skin browning of the Longan fruit by pure oxygen treatment could be accounted for maintenance of compartmentation of enzymes and substrates by enhanced respiration and ATP production.

Table 5:	Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of outer part of peel color of Longan cv. Daw

The different letters indicate the statistically significant difference by LSD at 5% level. ns: Not significantly different

Table 6:	Pearson correlation coefficients of SO₂ treatment on the change of polyphenoloxidase enzymatic activity (PPO), Weight Loss (WL) and pH value of peel and aril of Longan fruits cv. DAW

Table 7:	Pearson correlation coefficients of storage temperatures on the change of polyphenoloxidase enzymatic activity (PPO), Weight Loss (WL) and pH value of peel and aril of Longan fruits cv. DAW

Table 8:	Pearson correlation coefficients of storage duration on the change of polyphenoloxidase enzymatic activity (PPO), Weight Loss (WL) and pH value of peel and aril of Longan fruits cv. DAW

The result suggests that SO₂ treatment may be suitable for keeping of the Longan’s fruit over a relatively short period, which skin ultrastructure played a role in its storability. However, the suitable concentration and fumigation time is also necessary to point out. The Longan pericarp was thick about 630-700 μm and composed of three layers. The outer layer is exocarp consisted of natural opening and cracking on the surface.

Fig. 2:

Transverse sectional micrographs of the Longan fruit pericarps cv. Daw affected by SO₂treatment and various storage condition. (a, b) Longan pericarps after no SO₂and SO₂treatment at the initially of storage, (c, d) longan pericarps affected by no SO₂treatment after stored at 2 and 7°C for 8 weeks and (e, f) Longan pericarps affected by SO₂treatment after stored at 2 and 7°C for 8 weeks

It was covered by thin discontinuous layer of cuticle and brown epidermal hair. The mesocarp, main part of the pericarp consisted of about 70% of the pericarp tissue. It contained elliptical in shape with thick cell walls (Fig. 2a, b). The vascular bundles were tubular and consisted of one layer cell. When the fruit showed during SO₂ treatment, increasing of storage duration and temperatures, the dark color of inner and outer peel of the Longan fruit was appeared. The SEM observation showed a layer of injured cell in the pericarp was fibrous tissues disappeared (Fig. 2c, d). Wax that covered the pericarp and epidermal hair also damaged. The mesocarp cell were also damaged and had collapsed. The destruction of cell membrane was also observed (Fig. 2e, f). Underhill and Critchley (1992) found that the pericarp browning was correlated with moisture loss. Likewise, it is every likely that the natural cracking of Longan peel facilitates rapid moisture loss and cause surface browning during harvest and storage. The surface cracking was also impair the physiological function of the cuticle and increasing water permeability, which may cause water soaking at the inner side of the peel (Medeira et al., 1999). The injured cell would accelerate the oxidation of phenolic substances and the oxidative products resulted in dark color of inner and outer peel (Abe, 1990).


Fig. 3:	The effect of storage duration on SO₂ contamination in aril of Longan cv. Daw


Fig. 4:	The effect of storage duration on SO₂ contamination in peel of Longan cv. Daw

At the prior of storage, the contamination of sulphite residue was found highest in both aril and peel tissue. On the other hand, the contamination of sulphite significantly decreased along the storage durations (Fig. 3, 4). However, sulphite contamination still high concentration in peel tissue (350 mg kg^-1) after stored for 8 weeks (Fig. 4), while sulphite contamination was not found in aril after stored for 4 weeks (Fig. 3). The fumigation time and concentration are the most important factors affecting the SO₂ residues. Higher concentration and longer fumigation time resulted in higher SO₂ residue (Ye and Ge, 1996) which was mainly located in the peel and much less in the aril and gradually decreased with prolonged storage (Lemmer et al., 2000). Han et al. (2001) reported that most of the SO₂ residue was located in the pericarp. Appropriate SO₂ treatment lowered the SO₂ residue level in the pulp to as low as 10 μg g^-1. The eating quality was maintained during the early stage of storage and the shelf life was extended as compared with the control fruit. If SO₂ concentration and fumigation time were strictly controlled, lower residue and longer storage life could be achieved.

CONCLUSIONS

In conclusion, the combined application of SO₂ treatment and cold storage temperature stored Longan fruits under the cold condition significantly prevented pericarp browning of harvested the Longan fruits. Exposure of the Longan fruits to those conditions enhanced high color quality, reduced weight loss percentage, prevented cell wall cracking and delayed the activity of PPO and the decompartmentation of PPO and POD and their substrates.

ACKNOWLEDGMENTS

The authors wish to thank for Ministry of Agriculture and Cooperation, Department of Agriculture, Royal Thai government for financial support provided is gratefully acknowledged, Assistant Prof Dr. Vicha Sardsud, Postharvest Technology Research Institute, Chiang Mai University and Dr. Pitipong Thobunluepop, Department of Agricultural Technology, Faculty of Technology, Maha Sarakham University for comment on the manuscript.