Inhibitory Effects of Na-Hypochlorite and Heating on the Mycobiota Associated with Fruits or Juice of Passion (Passiflora edulis Sims) in Uganda.

A total of 34 species belonging to 21 genera of fungi were recorded on passion fruits of both pure and hybrid origin in Uganda, however, the pure type exhibited wider spectrum (28 species and 16 genera) than the hybrid type (21 & 15). Also, yeasts (unidentified and Rhodotorula mucilaginosa) were also encountered in high numbers. Moreover, the mean count of all mycobiota obtained from the pure type was higher than that of hybrid, despite the bigger size of the later. Members of yeasts and Cladosporium followed by Phoma, Penicillium, Fusarium and Alternaria species dominated on passion fruits of pure origin, while only C. cladosporioides, F. solani and yeasts dominated on the hybrid type. Treatment with Na-hypochlorite exhibited inhibitory effects on the total mycobiotic propagules as well as the dominant species from fruits of both types. The current results, therefore, suggest the use of Na-hypochlorite to control the post-harvest mycobiota associated with passion fruits. Regarding the mycobiota contaminating passion juice, yeasts were found to be the major contaminants with Candida parapsilosis being the most common. Moulds constituted only a minor proportion with Acremonium strictum followed by Fusarium chlamydosporum, F. moniliforme, F. acuminatum and F. solani as the most dominant species. In the heat-treated juice samples, the counts of the most commonly encountered mycobiota (both yeasts and molds) were significantly inhibited or completely eliminated. Some unidentified Bacillus species were also recovered from the juice, however, their counts in the heated samples were increased but insignificantly.

A characteristic shared by most fruits is their high acidity and a pH of under 3.5 is not uncommon (Splittstoesser, 1987; Saenz et al., 1998). Various organic acids are responsible for this acidity. pH is the single most important factor with respect to the type of microorganisms that can spoil this class of food. While most species of bacteria are inhibited by the hyrdogen ion concentrations for various fruits, yeasts and molds are more aciduric, therefore they are the principal spoilage microorganisms of fruits and fruit products (Splittstoesser, 1987; Deak and Beuchat, 1993; Tournas, 1994; Eseigbe and Bankole, 1996; Dugan and Roberts, 1997; Prada and Pagnocca, 1997).

Although the surfaces of fresh fruits harbor large numbers of both yeasts and molds, yeasts generally lack the mechanisms to invade and infect plant tissue and therefore are secondary rather than primary agents of spoilage (Dennis, 1983; Splittstoesser, 1987; Fleet and Heard, 1992). Some of the molds responsible for spoilage are true plant pathogens in that they can invade and cause an infection of intact, formerly healthy tissue. Others are saprophytic species whom become established after the fruit has been infected by a pathogenic organism or has been damaged by some physical or physiological cause. Growth of saprophytic molds generally is restricted to dead plant tissues (Splittstoesser, 1987; Mehrotra, 1998).

In passion fruits, brown spot is caused by either Alternaria alternata (Fullerton, 1982) or A. passiflorae (Inch, 1978; Emechebe and Mukibi, 1975; Snowdon, 1990). Fruit infection by Septoria spot developed in leaves (caused by Septoria passiflorae) can occur at any stage of growth of fruits (Snowdon, 1990). A number of other fungi associated with decaying passion fruits include Aspergillus niger, Cladosporium sp., Colletotrichum gloeosporioides, Fusarium oxysporum, Penicillium expansum, Phytophthora nicotianae var. parasitica and Rhizopus stolonifer (Snowdon, 1990).

Molds and yeasts are important in fruit processing, however, in that, unless removed or destroyed, they may be introduced in large numbers into juice and other products (Splittstoesser, 1987). Some of the postharvest treatments used to destroy fungal spores are chemical (Abdel-Mallek et al., 1995; Dugan and Roberts, 1997; Schirra et al., 1997; Mehrotra, 1998), biological (Janisiewicz et al., 1998; Mari and Guizzardi, 1998) or a combination of chemical and heat treatments (Johnson et al., 1990; Schirra et al., 1997; Obeta and Ugwuanyi, 1997).

On the other hand, most aciduric microorganisms were reported to possess little heat resistance and, as a result, fruits and fruit products usually require only a relatively mild thermal process to achieve commercial stability (Stokes, 1971; Splittstoesser, 1987; Schirra et al., 1997). However, others can survive after heat-treatments and can grow and spoil the products during storage, which results in great economic losses (Tournas, 1994).

Little is known about the mycobiota associated with fruits and juice of passion (Lutchmeah, 1993). Thus, this investigation was designed to throw light on the mycobiota associated with both fruits and juice of post-harvest marketed purple passion in Uganda (both pure and hybrid types). Postharvest treatments were also assessed for their beneficial to control these mycobiota.

Materials and Methods

Isolation of mycobiota associated with passion fruits and the effectiveness of Na-hypochlorite on these mycobiota

Purple passion (Passiflora edulis Sims, Family: Passifloraceae) fruit samples of both pure (Passiflora edulis Sims forma edulis, has round or egg-shaped fruits, 4~5 cm in diameter, pH range = 4.2~5.0) and hybrid (presumbly it originated as a mutation from the pure type, it has somewhat larger fruits 7~8 cm in diameter, pH range = 4.2~4.8, with about double weight of that of the pure type) origins were collected from different shops and kiosks at Nakasero and Wandegeya in Kampala, Uganda during February 2000. Fruits of similar size, appearance and freedom from decay and injury were selected (50 from each passion type). Furthermore, each group of samples was subdivided into two sub-groups (25 samples each). One subgroup from each passion type acted as control-, and the other as chlorine treated-samples. For the treatment, fruit samples were washed thoroughly with 2.5% sodium hypochlorite solution (approximately equal to 62.5% of the household chlorine, Jik, bleach made by Reckitt Colman, Nairobi, Kenya which was used) for two minutes, then left for 48 hours at room temperature (25°~28℃).

The technique used for enumeration and isolation of mycobiota was adopted from Abdel-Mallek et al. (1995). Individual samples were put in clean polyethylene bags and aliquots of 100 ml of sterilized tap water were added. The bags were then gently shaken by hand for 5 minutes. One-hundred microlitre ml of washing water was transferred aseptically onto the surface of agar plates. Czapek yeast autolysate agar (CYA) of Pitt (1973) supplemented per litre with 250 mg chloramphenicol, 25 mg rose bengal and 2 mg dichloran was used as the isolation medium. Three inoculated plates for each fruit sample were incubated at 25° ± 2℃ for 7~10 days during which the growing fungi were counted, isolated and identified.

Isolation of mycobiota from passion fruit juice and the effectiveness of heating on these mycobiota

Twenty random samples of the freshly prepared passion juice (pH = 4.4~5) marketed for human consumption (sold within 1 or 2 days of preparation) were collected from different shops and kiosks in Kisugu, Kampala during April 2000. Each sample was subdivided into two sub-samples. One of the subsamples was heated in a water bath at 80℃ for 30 minutes as recommended by Murdock and Hatcher (1978) and Pitt and Hocking (1997). For the isolation of mycobiota, 1 ml from each juice sample (heated or not heated) was then distributed on the surfaces of 4 CYA agar plates of the same composition mentioned above. Plates were incubated at 25 ± 2℃ for 10~15 days during which the developing colonies were counted, isolated and identified.

Identification of isolated fungi

Fungi were identified on the basis of their macroscopic and microscopic features using the keys of Raper and Fennell (1965), Booth (1970), Ellis (1971), Pitt (1979), Pitt and Hocking (1997).

Statistical analysis

The data obtained were subjected to student T-test and significant difference at 0.01 and 0.05 level of probability was subsequently determined following the procedure of Snedecor and Cochrane (1967).

Results and Discussions

Mycobiota associated with passion fruits and the effectiveness of Na-hypochlorite on these mycobiota

Data presented in Table 1 showed that the total number of propagules found associated with non-treated pure passion fruits ranged between 2.73 × 104 to 32.3 × 104 c.f.u/fruit, while with those of hybrid origin between 3.87 × 104 to 23.0 × 104 with the mean for the hybrid type being lower (12.02 × 104) than that of the pure type (17.29 × 104).This suggests that the hybrid type is more resistant to be attacked with fungal species than the pure type. On the other hand, treatment of both passion types with 2.5% Na-hypochlorite solution showed statistically significant (at P = 0.01) inhibitory effects on the total mycobiotic propagules. In this respect, the population of pathogenic fungi on the surface of citrus fruits and peaches arriving from the orchard have been reported to be greatly reduced by surface treatment with a solution of hypochlorite or sodium O-phenyl phenate (Smith and Redit, 1968; Murdock, 1972).

Table 1

Minimum, maximum and mean of fungal propagules in control and Na-hypochlorite - treated passion fruit samples of both pure and hybrid origin

Figures (× 104) are mean of the total fungal propagules found in washing water of 100 individuals of passion fruit (calculated from 25 in each case, 100 ml washing water each). The asterisks ** indicate statistical significance at P = 0.01.

Data in Table 2 showed that the most commonly encountered mycobiota from non-treated passion fruits of pure origin were yeasts (55.83% of the total propagules) and Cladosporium (34.91%) followed by Phoma (4.0%), Penicillium (2.08%), Fusarium (1.63%) and Alternaria (0.95%). Yeasts were also commonly reported from fruits from 20 different species of angiosperms in a tropical rain forest (Prada and Pagnocca, 1997), banana fruits (Postmaster et al., 1997), as well as from apple wounds (Mercier and Wilson, 1994). C. cladosporioides, unidentified Phoma species, P. expansum, F. solani, F. moniliforme and A. alternata were the most dominant species. F. moniliforme has been reported earlier to cause dry rot of passion fruits (Lutchmeah, 1993), while F. solani was reported from banana (Jimenez et al., 1993) and guavas (Majumdar and Pathak, 1989). A. alternata was earlier isolated most commonly from brown spot of passion fruit in the Pacific Islands (Fullerton, 1982), from apple fruits and the majority of its isolates were pathogenic to apples (Wojtas-Koziel and Borecka, 1990), while P. expansum was predominant from rotting apples and pears (Snowdon, 1990). The remaining species (P. chrysogenum, F. subglutinans and Alternaria passiflorae) were infrequently encountered (Table 2). P. chrysogenum was one of the primary rot causing fungi of black plum in Nigeria (Eseigbe and Bankole, 1996), while F. subglutinans is a pathogen of pineapples (Bolkan et al., 1979; Rohrbach and Taniguchi, 1984) and bananas (Jimenez et al., 1993; Wade et al., 1993). A. passiflorae is reported most commonly to cause brown spot of passion fruit in Uganda and Australia (Emechebe and Mukibi, 1975; Inch, 1978). Moreover, of the species reported in the current study, Penicillium expansum, Cladosporium sp., F. oxysporum, A. niger, Colletotrichum gloeosporioides were found associated with decaying passion fruits (Snowdon, 1990).

Table 2

Associated mycobiota with passion fruits and the effectiveness of Na-hypochlorite on their total propagules

TFP = Total fungal propagules, caculated per 25 samples in each case [0.3 ml of washing water from each sample (out of 100 ml) was used].

%TFP = Percentage total fungal propagules, calculated per total fungi.

%F = Percentage frequency; calculated per total number of samples investigated.

*Species isolated occasionally (having less than 10 TFP) were omitted from the table and these were either reported from pure passion fruits (Allescheriella crocea, Aspergillus niger, A. ochraceus, A. tamarii, A. versicolor, Microascus cinereus, Mucor sp., Neurospora crassa), or hybrid passion fruits (Colletotrichum gloeosporioides, Humicola grisea, Setosphaeria rostrata) or from both (Epicoccum nigrum, Fusarium dimerum, and Pestalotiopsis guepinii).

The yeast-like fungus, Geotrichum candidum, was isolated in moderate frequency (36% of the samples), while other species of 8 genera (Acremonium, Aspergillus, Epicoccum, Microascus, Mucor, Pestalotiopsis, Rhodotorula and Trichoderma) were reported infrequently and accounted collectively a minor proportion of the total propagules (Table 2). Some of these were reported as significant pathogens of fruits during postharvest storage such as Pestalotiopsis sp. and Acremonium strictum on banana (Wallbridge, 1981), Geotrichum candidum on citrus (Hall and Scott, 1977) and tomatoes (Okali and Erinle, 1989), Epicoccum nigrum on tomatoes, apples and pears (Bruton et al., 1993), T. harzianum on apples (Penrose et al., 1984), and species of Mucor on guavas (Ito et al., 1979) and mangoes (Johnson et al., 1990).

Statistical analysis revealed that, of the most commonly encountered fungi, yeasts, C. cladosporioides, P. expansum and A. alternata were highly significantly (at P = 0.01) inhibited with Na-hypochlorite - treatment. On the other hand, F. moniliforme, F. solani and Phoma sp. were also significantly inhibited but at P = 0.05 (Table 3). Also, the total fungal propagules were strongly inhibited (at P = 0.01) by about 84.6%. In this respect, the use of Na-hypochlorite has been proved to be effective for the control of passion fruit rot-development caused by fungal species most commonly encountered from passion fruits (Ismail, 2001). Also, Na-hypochlorite is reported to be lethal to fungal propagules and fungi in vitro (Mehrotra, 1998) and to have good curative properties against tomato fruit rots especially those caused by Alternaria alternata and Aspergillus niger (Abdel-Mallek et al., 1995). Control of brown spots of passion fruits (caused by A. alternata and A. passiflorae) is by pruning and destroying the debris and by spraying the passion trees (vines) regularly with fungicide (Snowdon, 1990).

Table 3

The inhibitory effect of sodium hypochlorite on the mycobiota most commonly associated with pure and hybrid passion fruits

Figures are the means ± standard deviations of the total fungal propagules (TFP), out of 25 passion fruit samples.

The asterisks * and ** indicate statistical significance at P = 0.05 and P = 0.01, respectively.

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On the other hand, the most commonly encountered species on hybrid passion fruits were C. cladosporioides (66.17% of the total propagules), F. solani (11.91%) and yeasts (19.5%). Moderately encountered species included A. alternata and F. moniliforme while the remaining fungi were infrequent (Table 2).

The treatment with Na-hypochlorite strongly inhibited the total mycobiotic propagules as well as the most common three taxa (at P = 0.01) (Table 3).

Noteworthy to mention that to be certain fungal species, as shown in Table 2, appeared after Na-hypochlorite treatment from either or both passion types (Aspergillus sydowii, Cladosporium sphaerospermum, Cochliobolus lunatus, Fusarium oxysporum, F. stilboides, Myrothecium roridum) or increased their numbers (Acremonium strictum, F. moniliforme and Penicillium chrysogenum). Of these, F. oxysporum was reported earlier as a postharvest pathogen of bananas (Wallbridge, 1981; Jimenez et al., 1993), citrus, pome fruits, tomatoes and melons (Snowdon, 1990) and of black plum (Eseigbe and Bankole, 1996). The results presented here suggest that higher concentrations of Na-hypochlorite or other chemical compounds may be used to control such fungi (most of them were rarely reported on either or both passion types, Table 2).

Mycobiota inhabiting passion fruit juice

Yeasts, which were commonly isolated from passion fruits, were the major contaminants of passion juice, constituting about 91.3% of the total microflora. Two species (Candida parapsilosis and Rhodotorula mucilaginosa) could be identified while some others could not, with C. parapsilosis being the most common. It was isolated in all samples, accounting for 57.77% of all yeast propagules. R. mucilaginosa was less frequent giving rise to only a small proportion (0.78%) (Table 4). These two species were reported earlier from fruit juice concentrates (Deak and Beuchat, 1993).

Table 4

The inhibitory effect of heating on the counts (represented by TFP and mean ± SD, out of 20 samples analysed, 1 ml each) of the microflora contaminating passion fruit juice

TFP: Total fungal propagules, calculated per 20 ml in all juice samples examined (1 ml/sample).

NCS: Number of contaminated samples, out of 20 examined.

The asterisks * and ** indicate statistical signifance at P = 0.05 and P = 0.01, respectively.

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The counts of C. parapsilosis and the unidentified yeasts as well as the total yeasts were significantly (at P = 0.01) reduced by heat-treatment. Moreover R. mucilaginosa was completely eliminated from the heated juice (Table 4).

Molds constituted a minor proportion (1.87%) of the total microflora of passion juice (Table 4). The most commonly encountered fungi from non-treated samples were Acremonium strictum (48.62% of the total fungal propagules, in 17 samples out of 20 analyzed), followed by Fusarium chlamydosporum (13.76%, 2), F. moniliforme (12.84%, 5), F. acuminatum (7.65%, 8) and F. solani (4.59%, 5). A. strictum, F. moniliforme (Teleopmorph: Gibberella fujikuroi), F. solani (Teleomorph: Nectria haematococca) were reported in small numbers from mango juice or natural apple drink, however the most commonly encountered fungi from fruit juices and drinks were members of Aspergillus, Penicillium and Eurotium (Abdel-Sater et al., 2001).

A. strictum, the most dominant fungus, was completely eliminated by heating. On the other hand, Fusaria (all species) were significantly inhibited (at P = 0.05) by 97.6% (Table 4). Most fusaria are known to produce chlamydospores which could resist heating, that is why were not completely eliminated.

Unidentified species of Bacillus were also recovered from both heated and non-heated juices, however their counts were increased by about 41.2% in the heated ones (Table 4).

Conclusions

From the current results, it could conclude that the numbers and composition of mycobiota (both yeasts and molds) tend to be more associated with passion fruits of pure origin than that of hybrid origin. However, these mcycobiota were significantly inhibited on both passion types by Na-hypochlorite - treatment. So, we recommend the use of Na-hypochlorite (2.5%) to control these postharvest associated mycobiota by washing passion fruits for two minutes. On the other hand, the most commonly encountered mycobiota from passion juice were either significantly inhibited or completely eliminated on heat-treatment. These findings also suggest the use of heat-treatment (at least at 80℃ for 30 minutes) to minimize or eliminate those mycobiota contaminating passion juice.