Abstract: The experiment of Longan fruit cv. Biew Kiew, untreated (control) and treated with SO2 treatment were stored under 2±2 and 7±2°C for 0, 2, 4, 6 and 8 weeks were studied. The treatment of fresh longan fruit with SO2 fumigation combined with the suitable storage condition improved the overall longan fruit quality, especially on inner and outer peel tissue and aril color than no SO2 treatment. Treatment stabilizes peel color with no subsequent loss of color during storage (fruit color were bright-yellowish color). When the fruit showed during SO2 treatment, increasing of storage duration and temperatures, the dark color of inner and outer peel of longan fruit was appeared, this was correlated with the increasing of PPO activity. The activity of PPO enzyme in control fruit (no SO2 treatment) gradually lower than SO2 treatments. Fruit exposed to cool storage temperature (2°C) exhibited a lower PPO enzymatic activity compared to those kept in high storage temperature (7°C). Moreover, PPO enzymatic activity significantly increased over the storage durations The additional SO2 treatment no subsequent loss of weight of longan fruit during storage. However, the sulphite residues could detect immediately after SO2 treatment in all part of longan fruit, especially on aril tissue. The SEM evaluation found that 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. The injured cell would accelerate the oxidation of phenolic substances and the oxidative products resulted in dark color of inner and outer peel.
INTRODUCTION
Longan fruit (Dimocarpus longan Lour.) is a non-climacteric subtropical fruit, which was an evergreen tree of the Sapindaceae family. Longan fruit was grown commercially in many countries, including China, India, Vietnam and Thailand (Huang, 1995; Campbell and Campbell, 2001). In Thailand, dried and especially fresh fruit of longan is mostly marketed locally and export of the fruit has been increasing rapidly in recent years (Lin et al., 2001). Longan fruit contain a relatively large black or brown seed at maturity. The fruit are conical, heart-shaped or spherical with a thin, leathery and indehiscent pericarp. The pericarp can vary in color from yellowish to light brown and the skin is smooth (Wong and Ketsa, 1991). The edible portion of the longan fruit is a fleshy, translucent-white aril. The aril is an extension of the funiculus of seed stalk that arises from the placenta and surrounds to seed. One of the most important problems in marketing longan fruit is rapid pericarp browning a few days after harvest (Wu et al., 1999). Color deterioration causes the fruit to fetch a lower price at market and even be unmarketable. Browning can be associated with desiccation and/or heat stress, senescence, chilling injury and pest or pathogen attack (Pan, 1994). Browning has been attributed to enzymatic oxidation of phenolics by polyphenol oxidase (PPO) (Liu, 1999; Tian et al., 2002a). PPO is activated by moisture loss from the fruit and treatments to reduce desiccation also reduce browning (Su and Yang, 1996). PPO activity was relatively low at harvest, decreased initially, during low temperature storage and increased reached a peak after long term of storage (Wu et al., 1999). Sulfur dioxide (SO2) is an effective inhibitor of PPO and also effectively reduces fruit browning (Zhang et al., 1999). However, evidence for the role of PPO in longan fruit pericarp browning in correlative and the underlaying biochemistry and physiology, which response with storage temperature and duration is require further investigation. Thus, the objective of this study was to investigate the effects of sulfur dioxide treatment integrate with storage temperature on the changing of physiology and biochemistry of longan fruit cv. Biew Kiew during storage.
MATERIALS AND METHODS
Longan (Dimocarpus longan Lour.) fruit cv. Biew Kiew 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 were used for the experiments. The experiment design in this study was raid out in 2x2x5 Factorial in CRD with 4 replications. Treatments were including with no SO2 treatment and SO2 treating. Then, the treated fruits were stored 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 SO2 treatment at the rate 4.50 tons per 2.5 kg-SO2 and then every month after stored under various storage temperatures as discussed above.
Weight loss: The fresh weight of the fruit was determined for all treatments as an index of desiccation rate. Weight loss was calculated as following:
Polyphenol oxidase activity and determination of pericarp pH: 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 8000 x g in a Sorvall rotor SS-34 at 4°C. The supernatant was collected and centrifuged repeated in 1.5 mL tubes at 20 000 x 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 (1976) with bovine serum protein as the standard. Results were calculated as in Δ activity mg-1 protein x 1000. 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 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 Date) Minolta chromameter (model CR-200; Minolta, Ramsey, Date 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. Washing is extremely important because it eliminates any free unreacted glutaraldehyde that remains within the tissue. If aldehydes remaining from the primary fixation are oxidized by osmium tetroxide they may generate a peppery spot background and interfere in the specimens. 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. Coated samples were stored in desiccator until assessed. Finally, the specimens were viewed with a scanning electron microscope (JEOL, JSM-5910LV, JEOL Ltd., Tokyo, Japan) at 15 kV.
Sulphite residual: 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. (1986).
Statistical analysis: The statistical analysis was carried out using a statistical program Statistic version released 8.0 and least significant different test at 95% was used to determine significant difference among the treatments.
RESULTS AND DISCUSSION
The ANOVA results indicated that peel tissue pH and aril pH was affected by SO2 treatment, storage temperatures, storage durations and interaction between them (Table 2). Initially, peel tissue pH was lower than the aril pH (5.12 and 6.75, respectively) (Table 1). After SO2 treatment, peel tissue pH significantly decreased. However, aril pH was significantly increased (4.32 and 6.88, respectively). These changes stated similarly with the effects of storage temperatures. Peel tissue pH significantly decreased when storage temperature increasing from 2 to 7°C. On the other hand, pH of aril increased significantly when storage temperature increased. Moreover, both peel tissue pH and aril pH increased gradually over the storage durations (8 weeks) in all treatments, the decrease being greater in SO2 treatments. In pH measurement of peel tissue was low which was agreed with Underhill and Critchley (1992) measured a pH of 2.5 in the peel of longan after SO2 treatments. Presumably, the diffusion between peel and aril was responsible for the elevated pH observed for longan fruit. According to Jiang (1999), a pH value lesser than 4.2 in the longan peel may abolish of PPO activity.
The ANOVA results showed that the percentage of weight loss was significantly affected by all treatments. The percentage of weight loss generally low in SO2 treatments (Table 1). The main factors affected the percentage of weight loss were storage temperatures and storage durations. The percentage of weight loss gradually increased when the storage temperatures and storage duration increase (Table 1). The fruit stored in low temperature (7°C) combined with SO2 treatment could maintained the lower percentage of weight loss than no SO2 treatment (Table 2). The high percentage of weight loss of longan fruit may cause wilt and the reduction of freshness, then resulted in browning on the peel and finally fruits were rotten or decay. The browning reaction on the longan peel was caused by oxidation of phenolic compounds by PPO enzyme (Tian et al., 2002b; Liu, 1999) and also from the loss of moisture on the fruit hence the enzyme is activated (Su and Yang, 1996; Lu et al., 1992). The activity of PPO enzyme in control fruit (no SO2 treatment) gradually lower than SO2 treatments. Fruit exposed to cool storage temperature (2°C) exhibited a lower PPO enzymatic activity compared to those kept in high storage temperature (7°C). Moreover, PPO enzymatic activity significantly increased over the storage durations (Table 1). Peel browning and decay are the main factors influencing postharvest quality and storage life of longan fruit. The fact that the browning index of longan peel increased along with PPO activity during storage time and the SO2 treatment particularly with storage temperatures significantly inhibited PPO activity and effectively prevented peel browning, further indicates that browning of longan peel is related to PPO activity. However, PPO activity is not a unique limiting factor in enzymic browning, since pH, fruit decay and senescence can also influence browning (Larrigaudiere et al., 1998; Tian et al., 2002b).
The peel color in both site; inner and outer, of no SO2 treatments were more scarlet than orange-red (hue angle; H*, decreased), became darkened (L* decreased) and less intensely red (chroma; C*, decreased) in comparison to the SO2 treatments (Table 3 and 4). For SO2 treatments, inner peel color was more green (A* decreased). On the other hand, outer peel color was blue-yellowish (B* decreased) (Table 3).
| Table 1: | The effects of sulphur dioxide treatments, storage temperatures and storage durations on longan cv. Biew Kiew fruit quality changes |
| The different letters indicate the statistically significant difference by LSD at 5% level. *: Polyphenol enzymatic activity was described in Δactivity mg-1 proteinx1000 | |
| 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. Biew Kiew |
| The different letters indicate the statistically significant difference by LSD at 5% level. ns: Not significant | |
| Table 3: | Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of outer part of peel color of longan cv. Biewkaew |
| The different letters indicate the statistically significant difference by LSD at 5% level. ns: Not significant | |
| Table 4: | Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of aril color of longan cv. Biew Kiew |
| The different letters indicate the statistically significant difference by LSD at 5% level. ns: Not significant | |
Moreover, the ANOVA results indicated that storage temperature was a main factor that affected the change of outer peel color (Table 4). Under high storage temperature (7°C), the outer peel color was less intensely color (C*, decreased), more purple-red (H*, decreased), became darkened (L* decreased) and showed blue-yellowish (B* decreased) (Table 4). Additionally, the storage temperatures affected on the changing of inner peel color, which appeared less intensely color (C*, decreased), became darkened (L* decreased) and showed blue-yellowish (B* decreased) (Table 3). Extremely changes also were observed by storage durations factor. The peel color in both inner and outer became dark brown color when the storage duration increased (Table 3, 4). For aril color, SO2 treatments resulted more yellow and bright appearances (significantly increased in H*, L* and B* value) (Table 5). The storage duration also the main factor affected aril color. The aril color was changed to yellowing and cloudy fruit (L*, A* and B* value decreased, but C* and H* increased) (Table 5). Moreover, this factor also affected on the change of peel color (Table 4), inner and outer part of peel was changed to cloudy and dark red or scarlet, which was showed in browning (Table 3, 4). For the pearson correlation coefficients analysis showed that SO2 treatment and storage temperature factors resulted positive correlation between weight loss and the changing of peel and aril tissue pH (Table 6, 7). Moreover, the effect of storage duration had positive correlation between peel tissue pH, but stated the negative correlation between aril pH and peel tissue pH (Table 8). SO2 treatment affected both external and internal quality. It changed fruit color from the original dim brown or green-brown to bright yellow-green or light yellow and prevented longan fruit from browning throughout storage duration (Fig. 1a-h). This effect was due to the phenol-quinone browning reaction catalyzed by PPO being inhibited by SO2 treatment (Wu et al., 1999). Meanwhile, anthocyanin was fixed by SO2 treatment through the bleaching reaction and the formation of colorless sulfo-compounds (Huang and Scott, 1985). It has been proved that sulphur-fumiganted longan fruit were bleached immediately and anthocyanin content decreased markedly to a stable but low level during storage. The small amount of SO2 treatment could be released from the sulfo-compounds (Huang, 1995). In the later stage of storage, the release of SO2 from sulfo-compounds might have probably caused a slight increase of anthocyanin in the fumigated fruit peel. The acidity condition affects the ratio between the various forms of the pigments, i.e. red flavylium cation (AH+), the blue quinonoidal base (A), the colorless carbinol (B) and pale yellow chalcones (C). The pKa of the reaction between the flavylium ion and the colorless carbinol from of Pg 3-G was found to be 2.98 (Sondheimer, 1953).
| Table 5: | Effects of sulphur dioxide treatment, storage temperatures and storage durations on the changes of inner part of peel color of longan cv. Biew Kiew |
| The different letters indicate the statistically significant difference by LSD at 5% level. ns: not significant | |
| Table 6: | Pearson correlation coefficients of sulphur dioxide treatments on the changes of polyphenol enzymatic activity, weight loss and pH of peel and aril of longan cv. Biewkaew fruit |
| Table 7: | Pearson correlation coefficients of storage temperatures on the changes of polyphenol enzymatic activity, weight loss and pH of peel and aril of longan cv. Biewkaew fruit |
| Table 8: | Pearson correlation coefficients of storage durations on the changes of polyphenol enzymatic activity, weight loss and pH of peel and aril of longan cv. Biewkaew fruit |
Therefore, assuming that vacuolar pH is close to fruit pH, much of the anthocyanin pigment will have converted into colorless carbinol and chalcone and some irreversible degradation may have taken place. So, SO2 treatment reduced the pH value in the peel, causing the redder peel color. Moreover, with regard to the mechanism by which enzymic discoloration is inhibited by SO2 treatment, Day (1996) hypothesized that SO2 treatment might cause substrate inhibition of PPO or alternatively, high level of colorless quinones subsequently formed might cause feed back product inhibition of PPO (Han et al., 1999). Moreover, the mechanisms of sulphur dioxide, which inhibited enzymatic skin browning during storage were reduction of pH in pericarp cytoplasm, inhibition of PPO activity, increase of free and total phenolic contents and reduction of ascorbic acid content (Wu et al., 1999). 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. (2002b). The result suggests that SO2 treatment may be suitable for keeping longan fruit over a relatively short period, which skin ultrastructure played a role in longan storability. However, the suitable concentration and fumigation time is also necessary to point out. The normal longan fruit 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. 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. Biewkeaw. (a): No SO2 treatment at the
initially of storage, (b): SO2 treatment at the initially of
storage, (c): inner peel tissue and aril color changing by no SO2
treatment at the initially of storage, (d) inner 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°C 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°C and 7°C for 8 weeks, respectively |
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. 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 longan fruit was appeared. SEM observation showed a layer of injured cell in the pericarp was fibrous tissues disappeared. 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. 2a-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).
The whole fruit and peel tissue sulphite residue under cold storage temperature (2°C) was highest (Fig. 3). Moreover, the contamination of sulphite residue was found highest immediately after treatment. On the other hand, the contamination of sulphite significantly decreased along the storage durations.
| Fig. 2: | Transverse sectional micrographs of longan fruit pericarps
cv. Biew Kiew affected by SO2 treatment and various storage condition.
(a and b): Longan pericarbs after no SO2 and SO2 treatment
at the initially of storage, (c and d): Longan pericarbs affected by no
SO2 treatment after stored at 2 and 7°C for 8 weeks, (e and
f): Longan pericarbs affected by SO2 treatment after stored at
2 and 7°C for 8 weeks |
| Fig. 3: | Effects of storage temperatures on sulphur dioxide contamination in the composition of longan cv. Biewkaew fruit |
However, sulphite contamination still high in peel tissue (900.20 mg kg-1) and whole fruit (127.73 mg kg-1), while found in aril only 0.17 mg kg-1 (Table 9). The fumigation time and concentration are the most important factors affecting the SO2 residues.
| Table 9: | Effects of storage durations on the changes of sulphur dioxide contamination in the composition of longan cv. Biewkaew fruit |
| ns: Not significant | |
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. If SO2 concentration and fumigation time were strictly controlled, lower residue and longer storage life could be achieved.
CONCLUSIONS
The treatment of fresh longan fruit with SO2 fumigation combined with the suitable storage condition improved the overall longan fruit quality, especially on inner and outer peel tissue and aril color than no SO2 treatment. Treatment stabilizes peel color with no subsequent loss of color during storage (fruit color were bright-yellowish color). The additional SO2 treatment no subsequent loss of weight of longan fruit during storage. However, the sulphite residues could detect immediately after SO2 treatment in all part of longan fruit, especially on aril tissue. Thus, if SO2 concentration and fumigation time were strictly controlled, lower residue and longer storage life could be achieved.
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.